Genome editing for resistance against plant pests and pathogens

Abstract

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

Full text

GENOMEEDITINGINPLANTS Review
Genome editing for resistance against plant pests
and pathogens
Cla
´udia Rato .Miguel F. Carvalho .Cristina Azevedo .Paula Rodrigues Oblessuc
Received: 30 September 2020 / Accepted: 27 May 2021
ÓThe Author(s), under exclusive licence to Springer Nature Switzerland AG 2021
Abstract The conventional breeding of crops strug-
gles to keep up with increasing food needs and ever-
adapting pests and pathogens. Global climate changes
have imposed another layer of complexity to biolog-
ical systems, increasing the challenge to obtain
improved crop cultivars. These dictate the develop-
ment and application of novel technologies, like
genome editing (GE), that assist targeted and fast
breeding programs in crops, with enhanced resistance
to pests and pathogens. GE does not require crossings,
hence avoiding the introduction of undesirable traits
through linkage in elite varieties, speeding up the
whole breeding process. Additionally, GE technolo-
gies can improve plant protection by directly targeting
plant susceptibility (S) genes or virulence factors of
pests and pathogens, either through the direct edition
of the pest genome or by adding the GE machinery to
the plant genome or to microorganisms functioning as
biocontrol agents (BCAs). Over the years, GE tech-
nology has been continuously evolving and more so
with the development of CRISPR/Cas. Here we
review the latest advancements of GE to improve
plant protection, focusing on CRISPR/Cas-based
genome edition of crops and pests and pathogens.
We discuss how other technologies, such as host-
induced gene silencing (HIGS) and the use of BCAs
could benefit from CRISPR/Cas to accelerate the
development of green strategies to promote a sustain-
able agriculture in the future.
Keywords Genome editing Pathogen resistance 
Pest resistance Susceptibility genes Resistance
factors Crop protection
Abbreviations
BCA Biocontrol agent
Cas9 CRISPR-associated protein 9
CRISPR Clustered regularly interspaced short
palindromic repeats
DAMPs Damage-associated molecular patterns
dsRNA Double-stranded RNA
ET Ethylene
ETI Effector-trigged immunity
ETS Effector-trigger susceptibility
GE Genome editing
gRNA Guide RNA
HIGS Host-induced gene silencing
HR Hypersensitive response
JA Jasmonic acid
MAPK Mitogen activated protein kinase
NB-
LRR
Nucleotide-binding leucine-rich repeat
protein
C. Rato P. R. Oblessuc (&)
InnovPlantProtect Collaborative Laboratory, Department
of Protection of Specific Crops, Elvas, Portugal
e-mail: poblessuc@iplantprotect.pt
M. F. Carvalho C. Azevedo (&)
InnovPlantProtect Collaborative Laboratory, Department
of New Biopesticides, Elvas, Portugal
e-mail: cazevedo@iplantprotect.pt
123
Transgenic Res
https://doi.org/10.1007/s11248-021-00262-x(0123456789().,-volV)(0123456789().,-volV)

PAMPs Pathogen-associated molecular patterns
PCD Programmed cell death
PR Pathogenesis-related
PRR Pattern recognition receptor
PTI Pattern-triggered immunity
R Resistance
RLK Receptor-like kinase
RNAi RNA interference
RNP Ribonucleoprotein
S Susceptibility
SA Salicylic acid
sRNA Small RNA
TAL Transcription activator-like
TALEN Transcription activator-like effector
nuclease
TF Transcription factor
TIR Toll/interleukin-1 receptor
VIGS Virus-induced gene silencing
ZFN Zinc finger nuclease
Significance statement
Genome editing is increasingly being used to protect
plants against pests and diseases. This review dis-
cusses recent progress on the editing of plant suscep-
tibility genes, and pests and pathogen virulence
factors. It offers a comprehensive overview of funda-
mental studies and proof of concept investigations
which demonstrate the potential for commercializa-
tion. It also shows how genome editing may be
combined with other technologies to develop new
solutions for environmentally-friendly and sustainable
plant protection. The strategies and successes here
described are particularly relevant when considering
disease outbreaks that may compromise food security
worldwide.
M. Margarida Oliveira, ITQB NOVA, Instituto de
Tecnologia Quı
´
mica e Biolo
´gica Anto
´nio Xavier,
Universidade Nova de Lisboa, Av. da Repu
´blica,
2780–157, Oeiras, Portugal.
Supriya Chakraborty,School of Life Sciences,
Jawaharlal Nehru University, New Delhi—110,067,
India.
Introduction
Global food security depends on innovative solutions
that reconcile an increase in crop production and
sustainability, i.e. without expanding agricultural land
and agrochemical use. This great challenge is further
aggravated by climate change. The increase in global
temperatures and changes in atmospheric composition
contribute to the emergence of pests and pathogens on
new crops and locations (Fones and Gurr 2017). Pests
and pathogens can quickly adapt to the new environ-
mental conditions acquiring new virulence genes
through mutation, hybridization or horizontal gene
transfer, becoming resistant to disease control mea-
sures and/or more aggressive to hosts (Tre˛bicki and
Finlay 2018; Fones et al. 2020). Pests and pathogens of
plants are diverse, ranging from intracellular virus to
extracellular bacteria, fungi, oomycetes, and insects,
and depending on lifestyle they can be classified as
biotrophs (feed from live cells) or necrotrophs (feed
from dead cells). Their virulence factors are also
diverse, including proteins, toxins or RNA molecules
with distinct modes of action that can inhibit the plant
immune responses, modulate hormone levels, or
facilitate the acquisition of nutrients, allowing the
pests and pathogens to exploit plants’ susceptibility
(S) genes to feed from and colonize the host (reviewed
by Wang and Wang 2018). On the counter part, plants
use resistance genes to activate the immune system to
defend themselves from pests and pathogens (Jones
and Dangl 2006). Food security depends on the host’s
success on winning this ceaseless arms race. There-
fore, improving plant resistance either by boosting
resistance genes or blocking susceptible ones can shift
this arms race towards plant health.
The first layer of plant immunity is known as
pattern-triggered immunity (PTI; Jones and Dangl
2006). PTI is triggered when host pattern recognition
receptors (PRRs) perceive pathogen-associated
molecular patterns (PAMPs) or damage-associated
molecular patterns (DAMPs). PAMPs are highly
conserved molecular signatures shared among
microbes, derived from flagellin, chitin, elongation
factor-Tu, and others. DAMPs are plant self-mole-
cules that are released from damaged cells or secreted
by intact cells undergoing pathogen invasion, includ-
ing cytosolic proteins, peptides, amino acids, and
nucleotides. PAMPs and DAMPs are perceived by
PRR proteins at plant cell membrane. Classical
123
Transgenic Res

examples are the epitopes flg22, elf18 and the
peptidoglycan chitin, which are perceived by the
receptor-like kinases (RLKs) FLAGELLIN-INSEN-
SITIVE 2 (FLS2), elongation factor-Tu (EFR), and
RECEPTOR KINASE 1 (CERK1), respectively
(Go
´mez-Go
´mez et al. 2001; Zipfel et al. 2006; Miya
et al. 2007). Similarly, plant elicitor peptides (Pep) and
components of plant cell wall matrix have as receptors
members of the PERCEPTION OF THE ARABI-
DOPSIS DANGER SIGNAL PEPTIDE (PEPR) and
WALL-ASSOCIATED KINASEs (WAKs) families,
respectively (Krol et al. 2010; Kohorn and Kohorn
2012). Upon perception of these elicitors, a burst of
reactive oxygen species (ROS) occurs rapidly via the
NADPH (nicotinamide adenine dinucleotide phos-
phate) oxidase RESPIRATORY BURST OXIDASE
HOMOLOG PROTEIN D (RBOHD) at the plant cell
membrane, followed by an influx of extracellular Ca
2?
into the cytosol, which results in the activation of other
ion fluxes (influx of H
?
and efflux of K
?
,Cl
-
and
NO
3
-
), promoting extracellular alkanization and
depolarization of the plasma membrane. After these
first responses, RLKs activation also induce a cascade
of signal transduction events mediated by mitogen
activated protein kinases (MAPKs), leading to phos-
phorylation of several transcription factors (TFs) that
regulate the expression of pathogenesis-related (PR)
proteins and modulate plant hormone responses (re-
viewed by Nishad et al. 2020). PTI outcome for non-
host plants is the elimination of the microbe and
absence of disease (Fig. 1).
Pathogens can overcome the first layer of plant
defence by blocking PTI through the quick action of a
collection of virulence proteins, known as effector-
trigger susceptibility (ETS; Jones and Dangl 2006).
Effectors have different sites of action, functioning
either in the plant apoplast or cytoplasm. Apoplastic
effectors can interfere with PAMP and DAMP
perception, apoplastic ROS production, and act as
inhibitors of host proteases. Cytoplasmic effectors are
translocated into different cell compartments and
interfere with various plant physiological processes,
including ROS burst, MAPK activation, expression or
secretion of PR proteins, or yet with the plant hormone
biosynthesis and signaling (reviewed by Wang and
Wang 2018). Whilst the primary function of pest and
pathogen effectors is to promote infection and plant
susceptibility, plants have evolved proteins capable of
recognizing these virulence factors to promote
effector-trigged immunity (ETI; Jones and Dangl
2006). ETI is activated when nucleotide-binding
leucine-rich repeat proteins (NB-LRRs) or their
guardees or decoy proteins interact with a pest/patho-
gen effector. These are major resistance (R) genes that
share downstream signalling pathways with PTI, but
result in programmed cell death (PCD), a stronger and
long-lasting response. Downstream the R-genes, the
ETI hypersensitive response (HR), a primary compo-
nent of the PCD in which a strong ROS burst is
observed, have ENHANCED DISEASE SUSCEPT-
IBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4
(PAD4), and SENESCENCE-ASSOCIATED GENE
101 (SAG101) as the most important hubs. ETI
signalling through EDS1/PAD4 will activate salicylic
acid (SA)-mediated responses, whilst EDS1/SAG101
primarily mediate HR (reviewed by Thordal-Chris-
tensen 2020). Recently, an alternative R-gene mode-
of-action was described. The NB-LRR gene ZAR1
(HOPZ-ACTIVATED RESISTANCE 1) was shown to
assemble into a complex ring-shaped pentamer,
hypothetically disrupting the cell membrane integrity
with a pore-forming channel (Wang et al. 2019a). This
‘‘resistosome’’ would also result in ETI-mediated PCD
and consequent plant resistance to the pest or pathogen
(Fig. 1).
One of the most environmentally-friendly and
sustainable crop protection approaches is to breed
cultivars for resistance to pests and diseases. Poten-
tially all plant resistance/susceptibility layers can be
manipulated to improve crop protection. Breeding in
favour of PTI is possibly a broader, durable, and
sustainable resistance, although its quantitative char-
acteristic makes it more difficult to be selected and less
effective in the short run than ETI. For these reasons,
breeders prefer to select for ETI-related major race-
specific R genes. Nonetheless, pathogens can over-
come this type of resistance in a short period of time
due to its qualitative characteristic. Thus, a combina-
tion of PTI and ETI boosting would be ideal.
Conventional breeding is time-consuming and labour
intensive whereas, molecular breeding can be faster,
creating more flexible solutions that can be adapted
and applied as soon as problems arise. Crop improve-
ment is performed by genetic selection or modification
of the DNA sequence known to be involved in the trait
of interest, and includes molecular marker-assisted
selection, genomic selection, and genetic engineering,
123
Transgenic Res

in which targeted genome editing (GE) techniques
have gained great importance over the last few years.
Targeted GE techniques induce double-strand DNA
breaks resulting in targeted modifications when the
breaks are repaired by non-homologous end joining
(NHEJ) or homology-dependent repair (HDR). The
major four classes of sequence specific nucleases
responsible for the double-strand breaks used for GE
are the meganucleases, zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases
(TALENs), and clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated
protein 9 (Cas9; reviewed by Kaur et al. 2021;
Ghogare et al. 2021). In brief, meganucleases are
endodeoxyribonucleases with a large recognition site
that induce a staggered cut with overhangs within the
target site. The most commonly used is the naturally
occurring I-SceI, although hybrid enzymes can be
developed, depending on the specificity required by
the selected target. ZFNs are endonucleases that
contain multiple zinc-finger DNA-binding domains
and several zinc fingers as a tandem array, recognizing
a 3-bp module. Hybrid FokI restriction enzyme fused
to zinc finger DNA-binding domain is a common ZFN
example, with specificity directed by the zinc fingers
combination. TALENs are similar in principle to
ZFNs, although the endonuclease domain is now
paired with multiple transcription activator-like
(TAL) effector domains that recognize single base
pairs. Therefore, the specificity of the TALENs target
site reflects the number of TAL effector domains
included in the nuclease. CRISPR/Cas9 is a natural
bacterial adaptive immune defence system against
invasive DNA sequences, where a guide RNA (gRNA)
MAPK
cascade
Dying or infected cell
ROS
ROS
Defence genes
Ca2+ Cl-/H+/NO3
-
K+
Eectors
Alkalinization
PAMPs
DAMPs
ROBHD
Pests and Pathogens
PRR
Chitinases
Phytoalexins
PR proteins
HR and PCD
Rgene
Sgene
Host
factors
R gene
TF/Hormone
Fig. 1 Defence and attack strategies deployed during plant-
pathogen interactions. Plant disease resistance responses are
induced upon recognition of PAMPs and/or effectors from pests
and pathogens by plant PRR proteins. This recognition leads to
the reprograming of transcriptional regulation of defence genes
and of plant hormonal responses. Some effectors specifically
bind to, induce and/or decrease gene expression of target genes
or protein activity. Pathogens can also negatively impact plant
growth and developmental-associated processes (e.g.
transcriptional expression of genes and negative regulation of
signalling pathways). See text for further details. Abbreviations:
HR, Hypersensitive Response; PCD, Programmed Cell Death;
PR, Pathogenesis-Related; R gene, Resistance gene; S gene,
Susceptibility gene; ROS, Reactive Oxygen Species; PAMPs,
Pathogen-Associated Molecular Patterns; PRR, Pattern Recog-
nition Receptors; RBOHD, Respiratory Burst Oxidase Homo-
logue D; TF, Transcriptional Factor
123
Transgenic Res

aligns with the target sequence, directing the double-
strand break induced by the endonuclease Cas9.
Synthetic gRNA can be designed to target a specific
plant or pest/pathogen gene to induce deletions, point
mutations or insertions (reviewed by Kaur et al. 2021;
Ghogare et al. 2021).
There are several examples on GE in plants to
knockout the function of S genes to overcome virus,
bacterial and fungal diseases. Here we describe the
most recent studies and the ones with more impact in
crops. Plant GE to improve protection against pests is
still an underdeveloped field (reviewed by Bisht et al.
2019). The use of small RNAs (sRNAs), i.e. short,
noncoding RNA molecules that guide silencing of
genes, is an emergent field to improve plant protection
that has gained the interest of scientists and agrono-
mists. RNA silencing has been shown to regulate PTI
and ETI against bacteria, oomycetes, and viruses.
Additionally, cross-kingdom sRNAs have been rec-
ognized as a new plant defence response, where pests
and pathogens virulence factors are silenced by sRNA
produced in the plant and translocated to the pathogen,
a natural form of host-induced gene silencing (HIGS;
reviewed by Hou and Ma 2020). GE approaches can
facilitate the manipulation of plant genomes to
promote HIGS and confer resistance to pests and
pathogens. The genome of pests and microbes can also
be edited to improve plant protection. Several studies
show that CRISPR/Cas-based gene drive techniques
(i.e. genetic engineering of super-Mendelian inherited
genes) are effective to control pests in a sustainable
manner (Tyagi et al. 2020). BCAs are also a very
sustainable alternative to control pests and pathogens
in the field, and GE of phages, bacteria and fungi can
facilitate BCAs’ use by repressing possible plant
virulence factors or environmental/animal-induced
detrimental traits, or yet by adding traits of interest
(reviewed by Huss and Raman 2020; Leung et al.
2020). All these are relatively new applications of GE
techniques and will be reviewed here.
In the last decades, genome sequencing innovations
and other molecular biology tools have enabled great
advances in our understanding of gene function in
crops and pests/microbes, particularly in the plant-
pathogen interactions field. All this knowledge com-
bined with GE techniques, can now be used to improve
plant health. Breeding efforts could potentially be
reduced from the traditional 7–25 years to approxi-
mately 2–3 years. CRISPR/Cas9, for instance, can be
designed in a matter of days and be implemented in
weeks, for as little as €10 (Friedrichs et al. 2019).
Depending on the crop, edited plants that are free of
exogenous DNA/RNA, can be obtained in one year,
and improvements are enduring. Therewith, GE has
been successfully used in agricultural crops for the
control of pests and diseases. This review will focus on
the GE achievements to boost plant protection, at both
plant and pest/pathogen levels, focusing on CRISPR/
Cas technology as it is one of the most powerful and
promising GE techniques shaping the field of plant
biotechnology.
Plant genome editing to enhance plant immunity
Successful enhancement of plant immunity by GE
requires information about the host target gene
sequence and molecular function, and ideally the host
whole genome sequence as well to monitor and
minimize off-target events. Fortunately, the number
of plant species, including many crops, that have been
fully sequenced is steadily increasing, as is the
knowledge on the genetic and molecular details of
plant immunity, particularly on negative regulators of
plant defence (e.g., host S genes). Pests and pathogens
use host S genes for successful nutrition, infection,
establishment, and proliferation (Pavan et al. 2010).
These can be involved in assisting penetration,
providing support for pathogen subsistence, or in the
negative regulation of the plant immune signalling
(van Schie and Takken 2014). S genes knockout is a
straightforward way to develop plant disease resis-
tance against a specific pathogen (Zaidi et al. 2018)
but it may also result in broad-spectrum resistance, if it
induces a prolonged or constitutive defence response
(Lapin and Van den Ackerveken 2013). Indeed, GE
technologies have been exploited to develop plant
resistance against pests and pathogens through tar-
geted mutagenesis of S genes (Fig. 2; Tables 1,2,3,
4). However, it is important to note that S genes are
frequently involved in plant growth and development,
with mutations having the potential to cause undesir-
able effects which could compromise this approach.
Fungal and oomycete pathogens: genome editing
of plant S genes to enhance crop resistance
Fungal pathogens are accountable for numerous
diseases, including mildew, rust, and rot, causing
123
Transgenic Res

dramatic losses in crop yields and seriously compro-
mise quality (Savary et al. 2019). This class of
pathogens possesses diverse lifestyles and pathogenic-
ity mechanisms, as well as high genetic flexibility.
This means that they can adapt and break R gene-
mediated resistance, develop resistance to fungicides,
and even invade new hosts, representing a major
challenge in disease control (Doehlemann et al. 2017).
GE has been successfully used to address this
challenge (Fig. 2) and improve plant resistance
against fungal and oomycete pathogens (Table 1).
Among the diverse classes of S genes, the ones
involved in plant immune signalling are specially
targeted to incorporate resistance by GE. A classic
example is the calmodulin-binding MILDEW RESIS-
TANCE LOCUS O (MLO) gene, whose loss-of-func-
tion mutations protect virtually every plant species
from infection by the powdery mildew fungi (Kusch
and Panstruga 2017). Powdery mildew, a global fungal
disease that infects a wide range of plants, traditionally
has been controlled by breeding resistant cultivars.
Wheat (Triticum aestivum) was the first crop to have
the MLO genes edited. Targeted mutations introduced
in three TaMLO homoeoalleles by TALEN technology
conferred heritable broad-spectrum resistance to pow-
dery mildew caused by Blumeria graminis f. sp. tritici
(Wang et al. 2014). Tomato (Solanum lycopersicum)
Slmlo mutants also showed resistance against powdery
mildew, in this case caused by the fungal pathogen
Oidium neolycopersici (Nekrasov et al. 2017).
SlMLO1 edition by CRISPR/Cas9 took less than a
year and resulted in plants free of foreign DNA, with
mutations indistinguishable from those naturally
occurring, and without off-target mutations as shown
by whole-genome sequencing. RNA interference
(RNAi)-mediated silencing of the grapevine (Vitis
vinı
´
fera) gene VvMLO-7 reduced susceptibility to
powdery mildew caused by Erysiphe necator (Pessina
et al. 2016), one of the most damaging grapevine
diseases that is traditionally controlled by highly toxic
sulfur-based and synthetic fungicides. Based on this
finding, purified CRISPR/Cas9 ribonucleoproteins
Promoter
S gene
Rfactors
Rfactors
PG
PG
PG
Plant genes
(e.g. Mlo, EDR1, ERF922,
Sec3A, BSR-D1, WRKY52,
WRKY70, PMR4, 14-3-3d,
NPR3, PSK1, SWEET, eIF4,
BSV)
(e.g. JAZ2,
P450 CYP71A1)
Cas9/13a/13d,Rx,gRNA
viP-Cas9, gRNA
PG
PatG
(e.g. CR, Rep, and IR, CP of
TYLCV; TIR, CP/MP and Rep/C4
of ChiLCV; IR,CP/MP, Rep/RepA
of WDV; genome of SRBSDV
and RSMV; P3/CI/Nib and CP of
PVY)
PG
VG
Cas9
gRNA
+atVG
se
n
egn
eg
ohtaP
(e.g.TYLCCNV, TbCSV,
TYLCV, ChiLCV, CMV,
TMV, SRBSDV, RSMV)
Fig. 2 Summary of GE strategies to boost plant protection. The
different strategies used to modulate plant immunity or
pathogen virulence are indicated, as well as examples of the
genes edited by the respective approach. Abbreviations: S gene,
Susceptibility gene; PG, Plant genome; R factors, Resistance
factors; viP, virus-inducible promoter; gRNA, single guide
RNA; gRNA, guide RNA; PatG, Pathogen genome; VG, Viral
genome; atVG, attenuated virus genome; see text for genes
nomenclature
123
Transgenic Res

(RNPs) targeting VvMLO-7 was delivered to grape
Chardonnay cultivar protoplasts in order to increase
resistance to powdery mildew in this important
cultivar. This methodology not only enabled to
improve on-target and reduced off-target activities
(Malnoy et al. 2016), but also prevented some of the
drawbacks associated with the plasmid-mediated
delivery of GE components to plant cells, such as
the random integration of plasmid sequences in the
host genome. These advantages allowed the genera-
tion of DNA-free genome edited plants, potentially
bypassing the current GMO regulations. Nonetheless,
plant regeneration from the protoplasts and inocula-
tion experiments are needed to confirm the efficiency
of this method for GE resistance, as protoplast regen-
eration is not straightforward for most crop species,
which represents a bottleneck for the application of
this methodology.
Another strategy to improve powdery mildew
resistance in wheat by knocking out susceptibility
components is editing the ENHANCED DISEASE
RESISTANCE 1 (EDR1) gene. Arabidopsis thaliana
EDR1 is a MAPKKK that plays a negative role in the
defence response against powdery mildew (E. cichor-
acearum), and like MLO, is highly conserved across
plant species (Frye et al. 2001). TaEDR1 knockdown
by virus-induced gene silencing (VIGS) and RNAi
resulted in enhanced resistance to powdery mildew,
suggesting that TaEDR1 negatively regulates powdery
mildew resistance in wheat (Zhang et al. 2017a). As
expected, wheat mutant plants with CRISPR/Cas9-
mediated multiplex GE of the three TaEDR1
Table 1 Engineering fungi and oomycetes resistance through genome editing of susceptibility genes in crops
Plant species Targeted
gene(s) (function
a
)
Outcome (pathogen species) GE
method
References
Triticum
aestivum
TaMLO-A1/-B1/-D1
(calmodulin binding)
Broad-spectrum resistance to powdery mildew
(Blumeria graminis f. sp. tritici strains)
TALEN Wang et al. (2014)
TaEDR1-1A/-1B/-1D
(MAPKKK)
Enhanced resistance to powdery mildew (Blumeria
graminis f. sp. tritici)
CRISPR/
Cas9
Zhang et al.
(2017a)
TaNFXL1 (transcription
factor)
Enhanced resistance to Fusarium head blight
(Fusarium graminearum)
CRISPR/
Cas9
Brauer et al.
(2020)
Oryza sativa OsERF922 (transcription
factor)
Enhanced resistance to rice blast (Magnaporthe
oryzae)
CRISPR/
Cas9
Wang et al.
(2016a)
OsSEC3A (exocyst
subunit)
Enhanced defence response to rice blast
(Magnaporthe oryzae)
CRISPR/
Cas9
Ma et al. (2018)
OsBSR-D1 (transcription
factor)
Broad-spectrum resistance to rice blast
(Magnaporthe oryzae strains)
CRISPR/
Cas9
Zhu et al. (2020)
Vitis vinifera VvMLO-7 (calmodulin
binding)
Possible enhanced resistance to powdery mildew
(Erysiphe necator)
CRISPR/
Cas9
Malnoy et al.
(2016)
VvWRKY52 (transcription
factor)
Increased resistance to grey mould (Botrytis cinerea) CRISPR/
Cas9
Wang et al. (2018)
Solanum
lycopersicum
SlMLO1 (calmodulin
binding)
Resistance to powdery mildew (Oidium
neolycopersici)
CRISPR/
Cas9
Nekrasov et al.
(2017)
SlPMR4 (callose
synthase)
Enhanced resistance to powdery mildew (Oidium
neolycopersici)
CRISPR/
Cas9
Santilla
´n Martı
´nez
et al. (2020)
Brassica
napus
BnWRKY70 (transcription
factor)
Enhanced resistance to stem rot (Sclerotinia
sclerotiorum)
CRISPR/
Cas9
Sun et al. (2018)
Gossypium
hirsutum
Gh14-3-3d (14–3-3
signalling)
Enhanced resistance to verticillium wilt (Verticillium
dahlia)
CRISPR/
Cas9
Zhang et al. (2018)
Theobroma
cacao
TcNPR3 (SA receptor) Enhanced resistance to black pod (Phytophthora
tropicalis)
CRISPR/
Cas9
Fister et al. (2018)
Citrullus
lanatus
ClPSK1 (Phytosulfokine
peptide hormone)
Enhanced resistance to Fusarium wilt (Fusarium
oxysporum f. sp. niveum)
CRISPR/
Cas9
Zhang et al.
(2020a,b)
a
MAPKKK mitogen-activated protein kinase kinase kinase, SA salicylic acid
123
Transgenic Res

homologues showed resistance to powdery mildew
(Zhang et al. 2017a). The stress adaptative signalling
hub 14-3-3 is known to contribute to cotton (Gossyp-
ium hirsutum) susceptibility to Verticillium dahliae,
the causal agent of verticillium wilt, a destructive
disease difficult to control due to the lack of isolated
resistant genes and poor germplasm resources in
cotton. Silencing of 14-3-3 by RNAi in G. barbadense
showed reduced sensitivity to V. dahliae infection as
compared to wild type plants (Gao et al. 2013). Cotton,
being an allotetraploid species is difficult to edit.
Nonetheless, CRISPR/Cas9 edited Gh14-3-3d gene
was stably transmitted to the next generations and
conferred high resistance to verticillium wilt in Cas9/
gRNA transgene-free cotton plants. Resistance was
linked to the induction of genes involved in brassi-
nosteroid and jasmonic acid (JA) signalling (Zhang
et al. 2018). The subunit of the exocyst complex
OsSEC3A participates in rice (Oryza sativa) suscep-
tibility to rice blast, caused by Magnaporthe oryzae.
Plants harbouring OsSEC3A mutations introduced by
CRISPR/Cas9 displayed enhanced resistance against
rice blast associated to increased defence responses
(Ma et al. 2018).
Durable broad-spectrum resistance has the poten-
tial to be achieved by loss-of-function mutations in
immune signalling genes such as MLO,EDR1,14-3-3
and SEC3 in virtually every plant species, as is clearly
the case for MLO genes (Wang et al. 2014; Malnoy
et al. 2016; Nekrasov et al. 2017). Atedr1 mutants are
resistant to the oomycete Phytophthora infestans
(Geissler et al. 2015), whereas OsEDR1 RNAi rice
plants display enhanced resistance to the bacterial
pathogen Xanthomonas oryzae pv. oryzae (Shen et al.
2011), suggesting that similarly to mlo mutants, edr1
and possibly 14-3-3 and sec3a mutant plants might
also be resistant to other pathogens. However, mod-
ifications in such genes could also result in increased
susceptibility to other pathogens, as it happens for M.
oryzae,Fusarium graminearum and Ramularia collo-
cygni, which are known to be more virulent in a mlo
background (Jarosch et al. 1999; Jansen et al. 2005;
McGrann et al. 2014). This enhanced susceptibility
may be particularly critical in wheat, in which blast
disease caused by M. oryzae pv. Triticum is an
emerging disease with a potentially catastrophic
outcome (Islam et al. 2019). Moreover, disruption of
S genes with signalling roles could result in growth
defects and/or yield loss (Zaidi et al. 2018). There are
several such examples: the pleiotropic effects of mlo
loss-of-function are often associated with impaired
yield and quality (Wang et al. 2014; Acevedo-Garcia
et al. 2017; Kusch and Panstruga 2017); T0 Gh14-3-3-
edited plants showed dwarf phenotype and fewer
flowers (Zhang et al. 2018); and rice plants with
mutations in OsSEC3A showed dwarf stature plus
lesion-mimic phenotypes (Ma et al. 2018). Interest-
ingly, Taedr1 mutant showed no obvious growth and
developmental defects (Zhang et al. 2017a). Further
studies in this direction would be of great value for
wheat breeders and ultimately to grain producers.
Plant immunity is a consequence of PAMP/DAMP
(PTI) or effector recognition (ETI), which leads to
activation of a signal transduction cascade and regu-
lation of TFs that activate the expression of genes
involved in plant defence (Fig. 1). TFs have been the
target of GE to enhance plant resistance against fungi.
WRKY transcription factors for instance are key
regulators, both positive and negative, of plant
immunity (Eulgem and Somssich 2007). In Arabidop-
sis, AtWRKY11 and AtWRKY70 are involved in JA-
and SA-induced resistance to pathogens, and in
rapeseed (Brassica napus), BnWRKY11 and
BnWRKY70 are differentially expressed upon inocu-
lation with the pathogenic fungus Sclerotinia sclero-
tiorum that causes stem rot disease (Sun et al. 2018). In
rapeseed, two Bnwrky11 and Bnwrky70 mutants were
obtained by CRISPR/Cas9. In T1, most of Bnwrky70
mutants showed editing in three BnWRKY70 copies
and exhibited enhanced resistance to stem rot, while
BnWRKY11 mutants showed no significant differ-
ence (Sun et al. 2018). The grape TF VvWRKY52 is
involved in biotic stress responses (Wang et al. 2017).
CRISPR/Cas9 edited grapes harbouring biallelic
mutations in VvWRKY52 showed increased resistance
to Botrytis cinerea in the first generation and no off-
target events (Wang et al. 2018). Interestingly, in
Arabidopsis, WRKY52 is a Toll/interleukin-1 recep-
tor (TIR)-NB-LRR protein containing a WRKY
domain that confers resistance against the bacterial
pathogen Ralstonia solanacearum (Deslandes et al.
2002), probably acting against fungal and bacterial
pathogens (Narusaka et al. 2009). Therewith, this is
another example of a possible broad resistance gene
that would be interesting to investigate.
The mycotoxin virulence factor deoxynivalenol is
known to promote the growth of Fusarium gramin-
earum in wheat. The deoxynivalenol-induced
123
Transgenic Res

TaNFXL1 TF was edited by CRISPR/Cas9, resulting
in enhanced resistance to Fusarium head blight and
suggesting that TaNFXL1 can promote susceptibility
to F. graminearum (Brauer et al. 2020). Another
example is the C2H2-type TF from rice OsBSR-D1,
which had its natural allele bsr-d1 identified by
genome-wide association to confer non-race-specific
resistance to blast (Li et al. 2017). A single nucleotide
change in the BSR-D1 promoter resulted in enhanced
binding of a repressive MYB transcription factor,
hence suppressing BSR-D1 expression. OsBSR-D1
promotes peroxidase expression in rice that degrades
H
2
O
2
and suppresses resistance to M. oryzae. Based on
this observation, CRISPR/Cas9 was used to knockout
OsBSR-D1 to develop rice plants with broad-spectrum
resistance to blast by eliciting HR (Li et al. 2017; Zhu
et al. 2020).
Plant hormones play an important role in plant
defence and susceptibility (Fig. 1). SA-mediated
defence response is elicited against hemi- and
biotrophic pathogens whilst JA and ethylene (ET)
are elicited in defence signalling mainly against
necrotrophs. The NON-EXPRESSOR OF PATHO-
GENESIS-RELATED 3 (NPR3) is a paralogue of
Arabidopsis NPR1, both being SA receptors. Knock-
down of the cacao (Theobroma cacao)TcNPR3 by
RNAi enhanced resistance to Phytophthora tropicalis,
the causal agent of black pod (Shi et al. 2013). Cacao is
a tropical tree crop with limited sources of genetic
resistance and slow generation time, hence TcNPR3 is
a good GE target. CRISPR/Cas9-edited Tcnpr3
showed improved resistance to P. tropicalis infection
and increased expression of downstream defence
genes, confirming the function of TcNPR3 as a
repressor of cacao immunity (Fister et al. 2018).
Furthermore, sequencing analysis of predicted off-
targets revealed no additional mutation sites. PMR4,
encoding a callose synthase, is another SA responsive
gene. In Arabidopsis, PMR4 plays an important role in
callose deposition triggered by PTI in a NPR1-
dependent manner (Nishimura et al. 2003; Dong
et al. 2008), although it also negatively regulates
SA-associated defence (Nishimura et al. 2003). A
recent effort to increase powdery mildew resistance in
tomato involved the knockout of POWDERY MIL-
DEW RESISTANCE 4 (SlPMR4) by targeted mutage-
nesis using CRISPR/Cas9 technology (Santilla
´n
Martı
´nez et al. 2020). All mutant tomato plants
showed reduced but not complete loss of susceptibility
to Oidium neolycopersici, confirming previous obser-
vations where SlPMR4 knockdown by RNAi
enhanced resistance against powdery mildew (Huibers
et al. 2013). The NPR3 and PMR4 function as SA
response repressors, suggesting that both NPR3- and
PMR4-edited plants could show broad resistance
against other pathogens, hence testing this hypothesis
could bring important information to the field of crop
protection.
As previously mentioned, JA and ET are involved
in plant resistance or susceptibility and have antago-
nist function with SA responses. The ETHYLENE
RESPONSE FACTOR (ERF) gene family play pivotal
roles in plant adaptation to multiple biotic and abiotic
stresses (Phukan et al. 2017). In rice, for instance, the
expression of OsERF922 is strongly induced by both
virulent and avirulent strains of M. oryzae. Knock-
down of OsERF922 by RNAi leads to increased
resistance to M. oryzae, suggesting that OsERF922 is a
negative regulator of rice blast resistance (Liu et al.
2012). Building on this observation, targeted modifi-
cation of OsERF922 using CRISPR/Cas9 generated
knockout mutants with enhanced resistance to rice
blast, without affecting major agronomic traits (Wang
et al. 2016a). ClPSK1 encodes a precursor of the plant
hormone phytosulfokine involved in plant immunity
repression (Hammes 2016). Recently, CRISPR/Cas9-
mediated knockout of ClPSK1 resulted in enhanced
watermelon (Citrullus lanatus) resistance to fusarium
wilt, caused by F. oxysporum f. sp. niveum (Zhang
et al. 2020a). In line with this result, Clpsk1 transcript
was significantly induced upon this fungus infection,
and application of exogenous phytosulfokine
increased pathogen growth. Studying the effects of
GE in other genes regulating plant hormone responses
is another interesting approach to achieve broad
resistance, although it is important to be aware of
possible defence-growth trade-off problems.
Bacterial pathogens: genome editing of plant S genes
to enhance crop resistance
Plant pathogenic bacteria are challenging to control
primarily due to asymptomatic infections, lack of
effective agrochemicals, large diversity, quick multi-
plication rate, and easy spread amongst hosts and
geographical regions. Consequently, the most efficient
strategies to control bacterial pathogens are mainly
based on plant genetic resistance, good agronomic
123
Transgenic Res

practices, and biocontrol agents. The increasing
knowledge in the molecular mechanisms of host–
pathogen interactions opens new opportunities to
engineer plant resistance against bacteria. As for other
pathogens, GE of S genes is becoming popular for
breeding crops for bacterial resistance (Fig. 2). Sev-
eral studies have been published on the application of
GE strategies to counteract various bacterial diseases
(Table 2).
Bacterial pathogens produce effectors that can bind
to the promoter’s region of S genes promoting its
expression and consequent ETS (Fig. 1). TAL effec-
tors of Xanthomonas oryzae pv. oryzae contribute to
pathogen virulence by binding to effector-binding
elements in the promoter region of specific rice S
genes, such as the SUGAR WILL EVENTUALLY BE
EXPORTED TRANSPORTER (SWEET) and the
LATERAL ORGAN BOUNDARIES (LOB), in a
sequence-specific manner, resulting in its transcrip-
tional activation (Antony et al. 2010). The SWEET
family genes encode sugar transporters, many of
which involved in plant-pathogen interactions (Chen
et al. 2010; Streubel et al. 2013). SWEET transporters
facilitate the exploitation of the host resources by the
pathogen by exporting sucrose from the host cells,
making it available to the pathogen hence conferring
disease susceptibility (Chen et al. 2010,2012). Early
on, TALEN was used to mutate predicted TAL
effector-binding sites within the OsSWEET14
(Os11N3) promoter to engineer heritable broad-
Table 2 Genome editing of crop susceptibility genes for engineering resistance to bacteria and broad-spectrum resistance to bacteria,
fungi and oomycetes
Plant species Targeted
gene(s) (function)
Outcome (pathogen species) GE
method
References
Oryza sativa OsSWEET14 (Os11N3)
(sugar transporter)
Broad-spectrum resistance to bacterial blight
(Xanthomonas oryzae pv. oryzae strains)
TALEN Li et al. (2012);
Blanvillain-
Baufume
´et al.
(2017)
CRISPR/
Cas9
Zafar et al. (2020);
Zeng et al. (2020)
OsSWEET13 (Xa25)
(sugar transporter)
Resistance to bacterial blight (Xanthomonas oryzae
pv. oryzae)
CRISPR/
Cas9
Zhou et al. (2015)
OsSWEET11 (Xa13 or
Os8N3) (sugar
transporter)
Resistance to bacterial blight (Xanthomonas oryzae
pv. oryzae)
CRISPR/
Cas9
Kim et al. (2019); Li
et al. (2020b)
OsSWEET11/13/14
(sugar transporter)
Broad-spectrum resistance to bacterial blight
(Xanthomonas oryzae pv. oryzae strains)
CRISPR/
Cas9
Oliva et al. (2019);
Xu et al. (2019)
OsBSR-K1 (TPRs-
domain; RNA-
binding)
Broad-spectrum resistance to bacterial blight
(Xanthomonas oryzae pv. oryzae) and rice blast
(Magnaporthe oryzae)
CRISPR/
Cas9
Zhou et al. (2018)
Solanum
lycopersicum
DMR6 (2-oxoglutarate
Fe(II) oxygenases;
SA responses)
Broad-spectrum resistance to bacterial speck
(Pseudomonas syringae), Xanthomonas spp. and
leaf blight (Phytophthora capsici)
CRISPR/
Cas9
de Toledo
Thomazella et al.
(2016)
SlJAZ2 (JA co-
receptor)
Resistance to bacterial speck (Pseudomonas syringae
pv. tomato)
CRISPR/
Cas9
Ortigosa et al. (2019)
Malus pumila DIPM-1/-2/-4 (RLK) Increased resistance to fire blight disease (Erwinia
amylovora)
CRISPR/
Cas9
Malnoy et al. (2016)
Citrus
paradisi
CsLOB1 (transcription
factor)
Enhanced resistance to citrus canker (Xanthomonas
citri subsp. citri)
CRISPR/
Cas9
Jia et al.
(2016,2017)
Citrus sinensis CsLOB1 (transcription
factor)
Resistance to citrus canker (Xanthomonas citri subsp.
citri)
CRISPR/
Cas9
Peng et al. (2017)
CsWRKY22
(transcription factor)
Increased resistance to citrus canker (Xanthomonas
citri subsp. citri)
CRISPR/
Cas9
Wang et al. (2019b)
RLK receptor-like kinase, TPRs tetratricopeptide repeats, SA salicylic acid JA jasmonic acid
123
Transgenic Res

spectrum resistance to X. oryzae pv. oryzae strains (Li
et al. 2012). Mutant rice plants exhibited enhanced
resistance due to lack of OsSWEET14 induction by the
bacterial pathogen. These results were later confirmed
by similar approaches using TALEN (Blanvillain-
Baufume
´et al. 2017) and CRISPR/Cas9 technologies
(Zafar et al. 2020; Zeng et al. 2020). Despite the
apparent success of the mutations introduced in the
OsSWEET14 promoter in the rice cv. Kitaake
(Blanvillain-Baufume
´et al. 2017), the same mutation
showed only moderate resistance or even susceptibil-
ity to African X. oryzae pv. oryzae strains (Oliva et al.
2019). Therefore, in an effort to broaden the resis-
tance, CRISPR/Cas9 was used to knockout OsS-
WEET14 by targeting its coding region in rice cv.
Zhonghua 11. The resulting mutant plants showed
broad-spectrum resistance to Asian strains and at least
one African strain. Additionally, these Ossweet14
plants showed increased plant height and no detri-
mental effects on yield, despite the known role of
OsSWEET14 in plant development (Oliva et al. 2019).
Similar results were obtained by Zhou et al. (2015), by
generating rice harbouring mutations in the coding
region of the S gene OsSWEET13 (Xa25) using
CRISPR/Cas9. The mutants exhibited resistance to
bacterial blight, with no detectable morphology
defects under normal growth conditions.
Recently, the coding region of OsSWEET11 (also
known as Xa13 and Os8N3) was also edited by
CRISPR/Cas9 (Li et al. 2020b). OsSWEET11 regu-
lates rice bacterial blight resistance but it also
participates in pollen development (Chu et al.
2004,2006). Constitutive interference or knockdown
of OsSWEET11 enhances bacterial blight resistance
but reduces pollen fertility, compromising its use in
breeding (Chu et al. 2006). Indeed, edition of
OsSWEET11 coding sequence improved resistance
to X. oryzae pv. oryzae but also caused changes in
important agronomic traits and a sterile phenotype (Li
et al. 2020b). To minimise any effect in pollen
development, a CRISPR/Cas9 specific deletion was
engineered in the promoter region of OsSWEET11 that
contains a pathogen-induced element (Ro
¨mer et al.
2010; Yuan et al. 2011). Homozygous mutant Oss-
weet11 plants displayed significantly enhanced bacte-
rial blight resistance, and no significant effects in the
agronomic traits analysed, including fertility (Kim
et al. 2019). In another attempt to engineer broad-
spectrum bacterial blight resistance in rice, CRISPR/
Cas9 technology was used to disrupt the TAL effector-
binding sites of two S genes, OsSWEET11 and
OsSWEET14, in rice cv. Kitaake, which already
harbours a recessive resistance allele of OsSWEET13
(Xu et al. 2019). The resulting triple mutant line
exhibited broad-spectrum resistance to most X. oryzae
pv. oryzae strains, as a result of loss of ETS. Strong,
broad-spectrum resistance was also obtained when
OsSWEET11,OsSWEET13 and OsSWEET14 were
simultaneously edited by CRISPR/Cas9 to introduce
mutations in all promoters (Oliva et al. 2019).
Importantly, the triple mutant lines showed no alter-
ation in agronomic traits, suggesting their potential use
in agriculture.
The citrus (Citrus sinensis) transcription factor
CsLOB1 is another S gene induced by TAL effectors
of Xanthomonas citri subsp. citri, the causal agent of
the citrus canker (Hu et al. 2014). This is a devastating
disease that affects the majority of the commercial
citrus cultivars worldwide and is responsible for
significant economic losses. The most effective and
sustainable approach to control this pathogen, is the
generation of resistant citrus varieties. Conventional
citrus breeding is challenging mainly due to poly-
ploidy, polyembryony, and prolonged crossing cycles.
To overcome these, several GE-based attempts have
been made to generate canker-resistant citrus in Citrus
paradisi and C. sinensis. The CsLOB1 promoter
contains the effector-binding element of the effector
PthA4. Building on these observations, the effector-
binding element in the CsLOB1 promoter and its
coding region were independently targeted via
CRISPR/Cas9 (Jia et al. 2016,2017; Peng et al.
2017). Both approaches resulted in plants resistant to
X. citri subsp. citri, without obvious effects on both
growth and off-target mutations (Jia et al. 2017),
opening the opportunity to generate CsLOB1-edited
citrus varieties to fight citrus canker.
Bacterial effectors also interact with plant proteins
to promote ETS (Fig. 1). The enterobacterium Er-
winia amylovora is the bacterial pathogen behind fire
blight disease in apple (Malus pumila) and other
commercially valuable plants (Malnoy et al. 2016).
The E. amylovora pathogenicity effector DspA/E
interacts with four apple RLKs from the DSPE-
INTERACTING PROTEINS OF MALUS (DIPM-1/-
2/-3/-4) family (Borejsza-Wysocka et al. 2006).
Simultaneous targeted mutagenesis of DIPM-1,
DIPM-2, and DIPM-4 by direct delivery of purified
123
Transgenic Res

CRISPR/Cas9 RNPs to apple protoplasts resulted in
the generation of DNA-free genome edited apple cells
(Malnoy et al. 2016). Similarly, to the VvMLO-7
edited grape protoplasts previously mentioned, this
methodology is promising by reducing off-targets and
generating transgene-free plants. Nonetheless, as only
protoplasts were evaluated, future inoculation exper-
iments are needed to confirm the efficiency of DIPM
edition to increase apple resistance to fire blight.
Contrary to the TAL effectors mentioned above,
effectors can indirectly regulate gene expression
(Fig. 1). WRKY22 is involved in resistance to the
bacterial pathogen Pseudomonas syringae in Ara-
bidopsis (Hsu et al. 2013) and to the fungal pathogen
M. oryzae in rice (Abbruscato et al. 2012). There is a
negative correlation between CsWRKY22 expression
and C. sinensis immune response to canker. Therefore,
citrus plants harbouring mutations in CsWRKY22
showed reduced susceptibility to citrus canker (Wang
et al. 2019b). Gene transcription can also be modu-
lated at the mRNA level. BROAD-SPECTRUM
RESISTANCE KITAAKE-1 (BSR-K1) encodes a tetra-
tricopeptide repeats-containing protein, that binds to
mRNAs of multiple defence-related OsPAL genes,
promoting their turnover in rice. PAL is required for
the biosynthesis of secondary metabolites, including
lignin and SA, and OsPAL1 overexpression confers
resistance to M. oryzae. CRISPR/Cas9-derived knock-
out of OsBSR-K1 led to OsPAL accumulation and
enhanced broad-spectrum resistance to both M. oryzae
and X. oryzae pv. oryzae without major yield penalty
(Zhou et al. 2018). Cultivars harbouring broad-spec-
trum resistance loci offer the ultimate approach to
control pathogens as these typically confer resistance
to diverse races or strains of the same pathogen or even
to multiple pathogens species (reviewed by Li et al.
2020c).
The successful examples of GE to engineer plant
defence against pathogens may not translate under
field conditions, where plants are exposed to varied
conditions, including numerous pests, pathogens, and
abiotic stresses. Broad-spectrum resistance against
pathogenic bacteria and fungi was also obtained
through GE of the S gene DOWNY MILDEW
RESISTANCE 6 (SlDMR6-1) in tomato. This crop is
severely affected by the bacteria P. syringae and
Xanthomonas spp., and Phytophthora spp. oomycetes
(Schwartz et al. 2015). In Arabidopsis, DMR6 was
shown to convert SA to 2,3-dihydroxybenzoic acid
(2,3-DHBA), reducing SA levels hence suppressing
SA-mediated plant immunity (Zhang et al. 2017b). In
tomato plants, SlDMR6-1, an orthologue of the
Arabidopsis DMR6, is upregulated in response to P.
syringae pv. tomato and P. capsica infections (de
Toledo Thomazella et al. 2016). CRISPR/Cas9 dis-
ruption of SlDMR6-1 active site resulted in plants with
resistance against these pathogens and to Xan-
thomonas spp., without detrimental effects on plant
growth and development (de Toledo Thomazella et al.
2016). These results suggest that knocking out
SlDMR6-1 may potentially confer broad-spectrum
disease resistance to various others plant pathogens.
Recently, enhanced resistance to the rice blast fungus
and to bacterial blight was achieved by CRISPR/Cas9
multiplex GE, creating Cas9/gRNA transgene-free
homozygous triple mutants with specific mutations int
OsTMS5,PYRICULARIA ORYZAE RESISTANCE 21
(OsPi21), and OsXa13 genes (Li et al. 2019). TMS5 is
a protein involved in thermosensitive male sterility,
OsPi21 is a S gene that slows plant defence against M.
oryzae, and as described above, Xa13 is a SWEET
family member that favours pathogen growth. These
examples highlight the potential of CRISPR/Cas9
genome edition of plants with the so desired durable,
broad-spectrum resistance, against not only a diverse
range of pathogen species but also from divergent
kingdoms.
Plant response to pathogens is under the tight
regulation of hormone signalling (Fig. 1; reviewed by
Glazebrook 2005). Recently, the trade-off between
plant defences against pathogens with opposing
infection strategies (i.e. biotrophs and necrotrophs)
was solved by spatially uncoupling the antagonism
between JA and SA plant hormone-mediated defence
pathways at stomata, the entry gates for some
pathogens (Ortigosa et al. 2019). P. syringae pv.
tomato, which causes bacterial speck disease in
tomato, produces the JA mimic coronatine that
induces stomatal opening, facilitating bacterial viru-
lence and allowing bacterial colonization (Melotto
et al. 2006). In Arabidopsis, this stomatal response to
coronatine is dependent on JASMONATE ZIM-
DOMAIN-2 (JAZ2). In an attempt to improve resis-
tance to bacterial speck disease, the tomato ortholog of
AtJAZ2,SlJAZ2, was targeted by CRISPR/Cas9 to
prevent coronatine-induced stomatal opening (Or-
tigosa et al. 2019). Sljaz2 mutant plants displayed
bacterial speck resistance, without compromising
123
Transgenic Res

resistance to the necrotroph B. cinerea. JA-signalling
outside stomata remained unaffected, enabling JA-
mediated responses against B. cinerea, demonstrating
the successful uncoupling of JA- and SA-mediated
defence pathways towards necrotrophs and biotrophs,
respectively. This uncoupling represents a major
breakthrough to obtain broad-spectrum resistance
against distinct classes of pathogens.
Viral pathogens: genome editing of plant S genes
to enhance crop resistance
Once a virus gains access to plant cells or vasculature,
the development of incompatible interactions between
pathogen and plant can be determined by the presence
of R genes or by the absence of critical factors for
completion of their life cycles, encoded by the so-
called S genes (Fig. 1). During plant-virus interaction
S genes assist in viral replication, translation and
movement. Some are essential for host survival while
others are not, therefore providing opportunities for
intervention through GE to enhance plant resistance
(Garcia-Ruiz 2018). Identification of these factors has
relied on different screening approaches and showed
that at times, some host proteins play a part in the life
cycle of viruses belonging to very different families.
That was the case for plasmodesmal-located proteins
involved in the movement of Grapevine fanleaf virus
(GFLV) and Cauliflower mosaic virus (CaMV; Amari
et al. 2010), and also endosomal SYNAPTOTAGMIN
A (SYTA) for the movement of Cabbage leaf curl
virus (CaLCuV), Turnip vein clearing virus (TVCV),
and Turnip mosaic virus (TuMV; Lewis and Lazarow-
itz 2010; Uchiyama et al. 2014). Nevertheless, com-
pelling evidence for the lack of universal susceptibility
factors came from large, comparable genetic screens
(Kushner et al. 2003; Panavas et al. 2005), indicating
the need to search for new plant S genes required for
virus proliferation. Presently, the best studied S genes
for viral infection belong to the EUKARYOTIC
TRANSLATION INITIATION FACTOR 4 (eIF4) fam-
ily, which are critical for the life cycle of multiple
viruses (reviewed by Jaafar and Kieft 2019). Based on
this evidence, most GE strategies described to date are
focused on eIF4E members (Table 3).
The efficiency of eIF4E GE in plant resistance
against viruses was first validated in Arabidopsis.
eIF(iso)4E CRISPR/Cas9 mutations conferred resis-
tance to TuMV, with transgene-free lines showing no
visible changes in growth and development (Pyott
et al. 2016). Later, the technology was applied to crop
GE. CRISPR/Cas9 edition of eIF4E in cucumber
(Cucumis sativus) introduced modifications directed at
its N and C termini, resulting in eif4e plants resistant to
members of the Potyviridae family Cucumber vein
yellowing virus (CVYV), Zucchini yellow mosaic
virus (ZYMV) and Papaya ringspot mosaic virus-W
(PRSV-W), but not to the cucumovirus Cucumber
mosaic virus (CMV) or tobamovirus Cucumber green
mottle mosaic tobamovirus (CGMMV;
Table 3 Engineering virus resistance through genome editing of susceptibility genes in crops
Plant species Targeted gene(s) Outcome GE method References
Cucumis sativus eIF4E Resistance to CVYV, ZYMV, PRSV-
W
CRISPR/
Cas9
Chandrasekaran et al.
(2016)
Oryza sativa eIF4G Resistance to RTSV CRISPR/
Cas9
Macovei et al. (2018)
Manihot esculenta nCBP-1/-2
(eIF4E)
Enhanced resistance to CBSV CRISPR/
Cas9
Gomez et al. (2019)
Musa balbisiana (e)BSV Resistance to BSV CRISPR/
Cas9
Tripathi et al. (2019)
Solanum
lycopersicum
eIF4E1 Resistance to PVY
N
, CMV CRISPR/
Cas9
Atarashi et al. (2020)
SlPelo Resistance to TYLCV CRISPR/
Cas9
Pramanik et al. (2021)
eIF4 eukaryotic translation initiation factor 4, (e)BSV endogenous virus sequences, CVYV Cucumber vein yellowing virus, ZYMV
Zucchini yellow mosaic virus, PRSV-W Papaya ring spot mosaic virus-W, RTSV Rice tungro spherical virus, CBSV Cassava brown
streak virus, BSV Banana streak virus, PVY
N
Potato virus Y-N, CMV Cucumber mosaic virus, TYLCV Tomato yellow leaf curl virus
123
Transgenic Res

Chandrasekaran et al. 2016). Interestingly, these
plants were phenotypically normal. Segregation of
mutated eIF4E and Cas9/gRNA transgene allowed the
selection of non-transgenic eif4e mutant lines, and
sequence analysis of putative off-target sites showed
no evidence of unwanted GE (Chandrasekaran et al.
2016). GE of rice plants was also used to generate
resistance to Rice tungro spherical virus (RTSV) by
introducing mutations within the eIF4G coding
sequence adjacent to a motif critical for resistance
(Macovei et al. 2018). Segregation analysis led to
Cas9/gRNA transgene-free lines showing resistance to
RTSV, and normal agronomic parameters. Candidate
off-target genes were sequenced, with no mutations
detected. Importantly, only in-frame homozygous
mutations in critical residues (but not truncations)
were viable, suggesting that eIF4G is essential for
plant survival (Macovei et al. 2018). Another report
described the edition of eIF4E isoforms NOVEL CAP-
BINDING PROTEIN (NCBP)-1 and NCBP-2 in cas-
sava (Manihot esculenta; Gomez et al. 2019). These
proteins interact with a viral component of the
Potyviridae family member of Cassava brown streak
virus (CBSV), which is involved in cassava brown
streak disease. Genome edited ncbp-1/-2 double
mutant lines showed a delay and weaker symptoms
development, but off-targets GE were also detected
(Gomez et al. 2019).
Recently, eIF4E1 was also edited in tomato by
CRISPR/Cas9. While a single nucleotide insertion in
the gene led to a lower accumulation of Potato virus Y
strain N (PVY
N
), but not PVY
O
, a 9-nucleotide
deletion reduced the levels of CMV strain yellow
(CMV-Y) and associated symptoms. Also, CMV-O
aphid transmission, which is more efficient than
CMV-Y transmission, was decreased in half (Atarashi
et al. 2020). Another example of GE in tomato for
virus resistance was described for the susceptibility
factor Ty-5/SlPelo, likely involved in ribosome recy-
cling during protein synthesis. Plant lines harbouring
single nucleotide insertions led to frameshifts and
premature stop codon, which correlated with resis-
tance to Tomato yellow leaf curl virus (TYLCV), as
well as reduced viral accumulation (Pramanik et al.
2021).
Some plant viruses are known to integrate their host
genome, as first reported in banana (Musa balbisiana)
for Banana streak virus (BSV; Harper et al. 1999).
This represents a major problem for breeders as under
stress conditions endogenous (e)BSVs become viru-
lent. CRISPR/Cas technology was used to target all
three ORFs of the locus comprising the infectious
integrated viral sequences (Tripathi et al. 2019).
Following water stress, from a total of 8 lines
analysed, 2 showed moderate symptoms while 6
remained symptomless along the experiment. Further
analysis indicated that the former 2 were edited at
ORF1 and 2 whereas the symptomless lines had been
modified at all three ORFs. Off-target GE events
analysis indicated a very low frequency of non-
specific genome modifications (Tripathi et al. 2019).
Insect resistance: plant genome editing to enhance
crop resistance
There is currently very limited information on the use
of GE to introduce plant resistance to insects. In the
only example we are aware of, CRISPR/Cas9 was
used to verify the inactivation of rice cytochrome P450
CYP71A1, a tryptamine 5-hydroxylase that catalyses
the conversion of tryptamine to serotonin (Lu et al.
2018). As a result, serotonin plant production stopped,
leading to an increase in SA levels and higher
resistance to brown plant hoppers and stripped stem
borers (Table 4). In a different strategy, CRISPR/Cas9
was used to target six loci involved in tomato yield and
productivity in wild tomato S. pimpinellifolium
(Zso
¨go
¨n et al. 2018). This wild tomato has the
advantage of being resistant to a wide range of
arthropod pests including spider mites, but it has low
yields (Rakha et al. 2017). In one generation, the
productivity and quality of the edited tomato lines
were enhanced. Although neither R or S genes were
targeted in this study, the de novo domestication to this
wild tomato improved agronomic traits whilst would
keep pest resistance, and therefore enhancing yield,
although the de novo domesticated plants still need to
be challenged with pests to confirm this.
CRISPR/Cas-based genome editing for targeted
suppression of pests and pathogens
Engineering viral sequences has been the focus of
many studies in the last decades, with great advances
mainly in the field of RNA silencing and GE. On the
contrary, despite its importance in crop production,
manipulation of pests genomes by GE approaches is
still in its infancy. CRISPR/Cas is naturally used by
123
Transgenic Res

bacteria and Archaea to edit and fight foreign genetic
elements such as phages, plasmids and transposons
(Hille et al. 2018). Direct manipulation of pests and
viral genomes by GE approaches has been used in both
plant protection and to study gene function (Tables 4
and 5; Fig. 2). Cas9 and gRNA sequences can be
introduced in plants to reduce viral load by targeting
genes important for replication and expression of
structural components. Studies on insect gene function
using CRISPR/Cas9 have been slowly advancing the
knowledge on the potential targets that could be used
to fight pests in crops using a similar strategy.
Insect genome editing to inhibit pest proliferation
The control of insects through genetics has been
exploited for several decades. An example is the
release of mutant populations unable to generate
offspring, obtained by the sterile insect technique. The
concept relies on having an overwhelming population
of sterile insects competing for resources and mating
opportunities. As female receptivity may be reduced
or turned off, under favourable circumstances, a
systematic decrease in the number of descendants
can be expected (Table 4). A classic example of
success in using this approach is the eradication of the
cattle New World screwworm, as well as the ongoing
efforts to control the Mediterranean fruit fly Ceratitis
capitata and the malaria vectors Anopheles sp. (Dunn
and Follett 2017). Although radiation mutagenesis is
the method traditionally used, novel technologies such
as GE are being explored, with multiple proof-of-
concept examples in the literature, including for plant
pests. Being Drosophila sp. an excellent genetic
system, it is not surprising that early applications of
GE technologies emerged within this genus. D. suzukii
is a major problem for soft-skinned fruit growers. Two
Drosophila sp. genes have been targeted by injecting
plasmid DNA encoding Cas9 and gRNAs into
embryos, the white gene (modulating eye pigmenta-
tion) and the Sex lethal gene (controlling female
development; Li and Scott 2016). Progeny with white
eyes or females showing altered genitalia and ovaries
were recovered with either intervention. Also, the
transcription factor Abdominal-A (Abd-A), that regu-
lates proper segment development during embryoge-
nesis in D. melanogaster, was targeted in Spodoptera
litura, a worm responsible for great losses worldwide.
Cas9 mRNA and gRNAs targeting Abd-A were
microinjected into embryos, and larvae with anoma-
lous segmentation were recovered (Bi et al. 2016). C.
capitata was also validated for interventions with
CRISPR/Cas9 through the disruption of the white (we)
and the paired (Ccprd; required for segmentation of
the developing embryo) genes. Injection of Cas9 and
gRNA RNPs complexes into embryos led to flies with
white eyes (we) or embryonic developmental malfor-
mations (Ccprd; Meccariello et al. 2017). Another
example came from the brown planthopper Nila-
parvata lugens, one of the most serious pests of rice
which is also a vector for viral diseases. CRISPR/Cas9
was employed with gRNAs targeted at two genes
Table 4 Engineering insect resistance through genome editing of crop and insect sequences
Target
organism
Species Targeted gene(s) Outcome GE
method
References
Plant Oryza sativa CYP71A1
cytochrome P450
Enhanced resistance to hoppers and stripped
stem borers
CRISPR/
Cas9
Lu et al. (2018)
Insect Drosophila
suzukii
White and sex lethal White eyes or females showing altered
genitalia and ovaries
CRISPR/
Cas9
Li and Scott
(2016)
Spodoptera
litura
Abdominal-A Anomalous segmentation of larvae CRISPR/
Cas9
Bi et al. (2016)
Ceratitis
capitata
White and paired White eyes or embryonic developmental
malformations
CRISPR/
Cas9
Meccariello et al.
(2017)
Nilaparvata
lugens
Cinnabar and white Altered eye pigmentation CRISPR/
Cas9
Xue et al. (2018)
Bemisia
tabaci
White Altered eye pigmentation CRISPR/
Cas9
Heu et al. (2020)
123
Transgenic Res

involved in eye pigmentation, cinnabar and white.
Following embryo injection with RNPs, insects
showing heritable altered eye were recovered (Xue
et al. 2018). CRISPR/Cas9 was also used to generate
mutants with an edited white ABC transporter gene in
the silverleaf whitefly Bemisia tabaci, an insect that
causes great damage to crops across the world, not
only through its feeding activity but also as a viral
vector (Heu et al. 2020). As expected, injection of
Cas9 and gRNAs RNPs into vitellogenic adult females
led to heritable altered eye colours.
The possibility of introducing targeted lethal
mutations into insects’ genomes opens interesting
prospects for their control. In that regard, the concept
of gene drive is especially appealing: a gene that when
inserted into a chromosome propagates itself faster
than would be expected through standard inheritance
(Table 4). A proof-of-concept was presented for the
malaria vector Anopheles gambiae encoding the
highly specific homing endonuclease gene I-SceI and
reporter fluorescent proteins. As the nuclease is
transmitted to the progeny, it induces a double strand
break repair in the homologous chromosome inherited
from the non-carrier parent. Because the template for
repair is I-SceI itself, the rate at which it is transmitted
is biased (Windbichler et al. 2011). In another report,
the same principle was applied to insert CRISPR/Cas9
gene drive constructs at three A. gambiae loci whose
disruption led to recessive female sterility, with
transmission rates as high as 99.6% for the Drosophila
sp. ortholog of nudel (Hammond et al. 2016). A note of
caution was presented by the same group, which
showed that after an early increase in transmission
peaking at the 6
th
generation, the gene drive was
resistant to transmission at later generations due to the
selection of nuclease-induced mutations at the target
site which were detected as early as the 2
nd
generation
(Hammond et al. 2017). However, further work with
CRISPR/Cas9 targeting of the A. gambiae double sex
gene (which controls sexual differentiation) indicated
that careful choice of the target may overcome those
limitations. Females exhibited complete sterility, egg
output fell, and the populations collapsed entirely after
8–12 generations, with no mutant alleles resistant to
the gene drive having an impact on the transmission
process (Kyrou et al. 2018). Also, a group proposed an
approached termed precision guided sterile insect
technique in which one line expresses Cas9 and
another expresses gRNAs to disrupt genes required for
female survival (sex lethal,transformer,doublesex)
and male fertility (b-Tubulin 85D,fuzzy onions,
protamine A,spermatocyte arrest). Using D. melano-
gaster, they obtained descendants that were 100%
sterile males (Kandul et al. 2019).
Overall, considering the increasing number of GE
applications to a wide range of insects, similar
pertinent gene drive examples are likely to be
proposed in the future for the precise control of
agricultural pests, opening good prospects to reduce
pesticide application in the environment and minimize
unintended impacts on non-targets.
Viral genome editing to inhibit its proliferation
GE technologies can be used to edit viral genomes,
therefore interfering with their life cycles. The fact
that in the field, RNAi transgenic lines can select for
geminivirus populations that escape this mechanism,
indicates that alternative solutions are needed (Fuentes
et al. 2015; Mehta et al. 2018). The first proof-of-
concept study was performed in N. benthamiana
transiently expressing the ZFN editing system to target
genes important for Tomato yellow leaf curl China
virus (TYLCCNV) and Tobacco curly shoot virus
(TbCSV) replication and structure, namely: C1 repli-
cation-associated protein (Rep), C2 transcriptional
activator protein (TrAP), C3 replication enhancer
protein (REn), C4 RNA-silencing suppressor, V1 coat
protein (CP), and V2 precoat (Chen et al. 2014). Later,
CRISPR/Cas9 system was incorporated into N. ben-
thamiana and Arabidopsis plants to prove its effi-
ciency in editing Rep and CP genes, as well as
intergenic regions (IR) from several geminiviruses,
showing a reduction in virus titer (Baltes et al. 2015).
Furthermore, CRISPR/Cas9 components were
incorporated into crop plants to prevent virus infection
(Table 5). Transgenic stable tomato plants containing
Cas9 and gRNAs targeting CP and Rep from Tomato
yellow leaf curl virus (TYLCV) resulted in the plants
harbouring lower viral loads across generations. There
were however cases of functional edited viruses being
recovered. Efforts to generate transgenic tomato lines
targeting the IR, which could be a better target to
reduce viable edited mutants, were however unsuc-
cessful (Tashkandi et al. 2018). Cassava (Manihot
esculenta) expressing Cas9 and gRNAs targeting
TrAP and REn genes of African cassava mosaic virus
(ACMV) is another example. Nevertheless, these
123
Transgenic Res

transgenic lines showed no resistance due to the
frequency that mutant viruses emerged following
infection, suggesting that this technology may not
always produce the desired outcome for virus control
(Mehta et al. 2019). Following preliminary transient
validation, a strategy that could lead to better
outcomes might be targeting other regions of the
genome with multiple gRNAs simultaneously
expressed (Rybicki 2019). This was actually effective
to control Chilli leaf curl virus (ChiLCV) in N.
benthamiana by transiently expressing Cas9 and
double or triple gRNAs constructs in N. benthamiana
targeting combinations of IR, CP/movement protein
(MP) and Rep/C4. Effective control of symptoms and
virus accumulation, with no escape mutants, were
observed (Roy et al. 2019). The same strategy was
used in barley (Hordeum vulgare) to express Cas9 and
gRNAs directed at IR, CP/MP, Rep/RepA and Rep of
Wheat dwarf virus (WDV). Interestingly, in this case,
some gRNAs did not seem to induce changes in the
viral genome and one gRNA was not expressed. Yet,
three out of the four lines analysed exhibited no
symptoms or viral accumulation (Kis et al. 2019).
These studies were applied to ssDNA viruses from the
Geminiviridae family but CRISPR/Cas9 was also
successfully applied to the dsDNA CaMV. gRNA
constructs directed at CP sequences expressed in
transgenic Arabidopsis led mostly to full resistance
(up to 96%), although in some instances functional
viruses were detected (Liu et al. 2018).
Cas enzymes with ribonuclease activity, can also be
applied to control plant RNA viruses as shown with
the transient expression of Cas13a, FnCas9, and
Cas13d/CasRx plus gRNA in N. benthamiana and
Arabidopsis (Aman et al. 2018). Following validation
in transient N. benthamiana assays, the Cas13a system
was employed to degrade RNA viruses in transgenic
rice with gRNAs directed at the genomes of Southern
rice black-streaked dwarf virus (SRBSDV) and Rice
stripe mosaic virus (RSMV; Zhang et al. 2019).
Unfortunately, the specific sequences targeted in these
virus genomes were not described, but following
infection, plants showed lower viral load and mild or
no clear symptoms. Additionally, transgenic potato
plants expressing Cas13a/gRNAs targeting conserved
regions of the potyviral membrane protein (P3), the
cytoplasmic inclusion bodies (CI), the viral replicase
(Nib) and CP of three major Potato virus Y (PVY)
strains were evaluated for viral resistance. Upon
challenge with all PVY isolates, selected lines showed
virtually no infection symptoms and a reduced viral
load, which correlated with the level of Cas13a/gRNA
expression (Zhan et al. 2019).
A major concern associated with constitutive
overexpression of CRISPR/Cas9 systems is off-target
genome modifications. To limit this constraint, Ji et al.
Table 5 Engineering virus resistance in crops through genome editing of virus sequences
Plant species Targeted gene(s) Outcome GE method Reference
DNA viruses
Hordeum vulgare MP/CP, Rep, Rep/
RepA, IR
Resistance to WDV CRISPR/Cas9 Kis et al. (2019)
Solanum
lycopersicum
CP, Rep Enhanced resistance to TYLCV CRISPR/Cas9 Tashkandi et al.
(2018)
IR, CP Enhanced resistance to TYLCV Virus-inducible
CRISPR/Cas9
Ghorbani Faal et al.
(2020)
Manihot esculenta TrAP, REn No resistance of ACMV CRISPR/Cas9 Mehta et al. (2019)
RNA viruses
Oryza sativa Viral genomes Enhanced resistance to SRBSDV,
RSMV
CRISPR/Cas13a Zhan et al. (2019)
Solanum
tuberosum
P3, CI, Nib, CP Enhanced resistance to PVY
strains
CRISPR/Cas13a Zhan et al. (2019)
CP capsid protein, Rep replication-associated protein, IR intergenic region, TrAP transcriptional activator protein, REn replication
enhancer protein, MP movement protein, P3 membrane protein, CI cytoplasmic inclusion protein, Nib viral replicase, bTYLCV
Tomato yellow leaf curl virus, ACMV African cassava mosaic virus, WDV Wheat dwarf virus, SRBSDV Southern rice black-streaked
dwarf virus, RSMV Rice stripe mosaic virus, PVY Potato virus Y
123
Transgenic Res

(2018a) developed a virus-inducible system based on
the promoter sequences of geminivirus Beet severe
curly top virus (BSCTV). After validation in transient
N. benthamiana assays, transgenic Arabidopsis
expressing virus-inducible Cas9 and gRNAs directed
at Rep gene were infected with BSCTV. Plants
showed very low viral accumulation and no symp-
toms. Importantly, these plants did not show off-target
genome modifications (Ji et al. 2018). Similar con-
cerns led to another report describing a Cas9 expres-
sion system under the control of a virus-inducible
endogenous rgsCaM tomato promoter. Transient
assays confirmed the inducible expression of Cas9 in
tomato upon infection with Tomato yellow leaf curl
virus, as well as the reduction of viral load and
symptoms directed by gRNA aimed at IR and CP
sequences (Ghorbani Faal et al. 2020).
Pests and pathogens targeted suppression
approaches that would benefit from CRISPR/Cas
technology
Plants have evolved with a plethora of microorgan-
isms that, as a community, are often referred to as the
microbiome. The interaction between plants and their
microbiomes can have positive outcomes to the plant
by promoting growth and/or protecting them against
biotic or abiotic stresses. These BCAs properties can
arise from the direct interaction of the microbe with
the plant or with the pest or phytopathogen itself (Ko
¨hl
et al. 2019). The interaction between the BCA and the
plant can result in local or systemic induction of
defence signalling pathways, making the host plant
more resistant to infection by pathogenic microbes.
The interaction between BCA and pest or pathogen is
also possible, and can result in the BCAs production of
metabolites, such as lytic enzymes, siderophores or
hydrogen cyanide with antimicrobial properties that
reduce the pathogen growth or directly kill it, in
competition for the environmental niche and/or avail-
able nutrients or in the BCAs parasitism of the
phytopathogen (Avis et al. 2008;Ko
¨hl et al. 2019).
Besides the plant growth promoting properties, if
properly channelled, BCAs have a vast potential to be
used as biochemical alternatives (Baker 1987; Cook
1993). There is yet another level of interaction
between plants and its microbiome that happens
through sRNAs (reviewed by Cai et al. 2018), which
has been studied mainly in plants interaction with
oomycetes, fungi, nematodes, and insects. As dis-
cussed above, when plants sRNA target pests or
pathogens genes, it is called host-induced gene
silencing (HIGS). HIGS has been cleverly highjacked
by scientists to introduce sequences in the plant
designed to target a chosen region in the pathogen
genome, thus silencing targeted pathogen virulence
genes and therefore controlling the disease incidence
(Hou and Ma 2020). The use of BCAs in combination
with HIGS to control pests and pathogens in crops is
very promising, and further coupled with the emerging
GE techniques such as CRISPR/Cas could lead to
great achievements, helping to increase productivity in
more sustainable and eco-friendly agricultural
systems.
Suppression of pests and pathogens by biological
control agents
BCAs can regulate pest and plant pathogen popula-
tions, being used as alternatives to chemical insecti-
cides and fungicides. One example is the
entomopathogenic ascomycete fungi belonging to
the Metarhizium and Beauveria genera (Rohrlich
et al. 2018), which have been developed into living
formulations, registered as mycoinsecticides or
mycoacaricides, and reached field-testing stage with
reports of high efficacy in pest control (Ownley et al.
2008; St Leger and Wang 2010). However, in the field
the viability of these living organisms is compromised
due to exposure to adverse environmental conditions
such as temperature, humidity and UV radiation, as
well as fungicides applications. Moreover, BCAs have
a relatively slow kill capacity and higher cost than
conventional chemical insecticides, which makes
them more appropriate for preventative control rather
than for outbreaks (Ko
¨hl et al. 2019). BCAs genome
engineering could enable the development of microor-
ganisms more resistant to adverse environmental
conditions, to fungicides and with increased pest/-
pathogen control. Targeted gene engineering of fungi
has been slow-progressing mostly due to the low rate
of homologous recombination, consequence of the
presence of non-homologous and end joining DNA
repair mechanisms that results in the ectopic expres-
sion of the targeted DNA, and the limited availability
of adequate selectable markers. There are however
several examples in which traditional genome engi-
neering has been successfully applied to pest
123
Transgenic Res

pathogenic fungi (Table 6). Trichoderma spp., have
been widely used as antagonistic fungal agents against
several pests and as plant growth promoting microor-
ganisms (Verma et al. 2007). Disruption of the N-
acetyl-ß-D-glucosaminidase in T. hamatum by inser-
tional mutagenesis of the gene, showed increased plant
growth promotion (Ryder et al. 2012). However, this
mutation negatively impacted its success as a sapro-
trophic competitor and as an antagonist of soil-borne
pathogens. Another example is Beauveria bassiana¸ in
which exogenous overexpression of a tyrosinase gene
required for melanin formation showed increased UV-
resistance (Shang et al. 2012). In Metarhizium aniso-
pliae additional copies of the toxin cuticle-degrading
protease (Pr1) were introduced to accelerate the
fungal kill-power (St Leger et al. 1996). The consti-
tutive Pr1 production by the fungus in the Manduca
sexta hemolymph activated the prophenoloxidase
system, causing 25% reduction in time of larvae death
and 40% reduction in food consumption, hence greatly
increasing M. anisopliae virulence (St Leger et al.
1996). In another study, M. anisopliae virulence was
also increased by overexpressing the scorpion neuro-
toxin AaIT, one of the most toxic known insect-
selective peptides (Wang and St Leger 2007).
Although the AaIT gene has already passed regulatory
hurdles to field release, it is important to mention that
M. anisopliae can infect different insect classes such
as locusts, beetles, crickets and mosquitoes and
therefore it could put in risk beneficial insects.
Beside toxins, the range of fungal virulence genes
constantly being discovered and that could be
exploited to improve BCAs properties is vast. By
combining virulence genes from different pathogens
and/or creating synthetic multifunctional genes, novel
combinations of specificities and virulence can be
created (Wang and St Leger 2007). GE methodology
will have a fundamental role in facilitating and
accelerating these studies. However, despite some
developments in the GE of pathogenic fungi, to the
best of our knowledge, most are still reporting the
development of the technique itself, rather than its use
to directly improve BCA properties (Table 6; Nødvig
et al. 2015; Krappmann 2017; Deng et al. 2017). For
example, the CRISPR/Cas9 system was developed for
Trichoderma reesei to induce mutagenesis or to
introduce new genes by homologous recombination
into a target site of the fungal genome (Liu et al. 2015).
This methodology can also be used to generate
multiple genome modifications simultaneously by
Table 6 Genome engineering and editing of biocontrol agents (BCAs) to improve antagonism activity or study virulence factors
Microorganism
(BCA)
Targeted
gene(s) (function)
Outcome Genome
engineering
method
References
BCAs to control insect pests
Trichoderma
hamatum
NAG (N-acetyl-b-D-
glucosaminidase)
Reduced antagonism to several pests
and enhanced plant growth
Insertional
mutagenesis
Ryder et al. (2012)
Beauveria
bassiana
Tyr (tyrosinase) Mycoinsecticide and increased UV-
resistance
Exogenous
overexpression
Shang et al. (2012)
Pks15 (polyketide
synthesis)
Reduced virulence to diverse insects CRISPR/Cas9 Toopaang et al. (2017);
Udompaisarn et al.
(2020)
Metarhizium
anisopliae
Pr1 (cuticle-degrading
protease)
Increased virulence to Manduca
sexta hemolymph
Overexpression St Leger et al. (1996)
AaIT (scorpion
neurotoxin)
Increased virulence to wide range of
insects
Overexpression Wang and St Leger (2007)
BCAs to control fungal and oomycete pathogens
Purpureocillium
lilacinum
lcsL (transcription
factor)
Reduced virulence to the
Phytophthora infestans
CRISPR/Cas9 Jiao et al. (2019)
Bacillus subtilis
and B. mycoides
sfp (4-
phosphopantetheinyl
transferase)
Reduced virulence to Rhizoctonia
solani and Fusarium culmorum
CRISPR/Cas9 Yi et al. (2018)
123
Transgenic Res

co-transforming gRNAs and donor DNAs for different
targets (Liu et al. 2015). Similarly, CRISPR/Cas9
system for Bacillus licheniformis has recently been
developed for industrial purposes (Li et al. 2018; Zhou
et al. 2019; Zhan et al. 2020). B. licheniformis is an
important microorganism used as cell factory for the
production of biochemicals and enzymes, but some
races are also important plant growth promoters and/or
possess BCA properties. Race SA03 confers increased
saline-alkaline tolerance in Chrysanthemum plants
(Zhou et al. 2017), whereas race MH48 reduces foliar
fungal diseases in Camellia oleifera (Won et al. 2019).
Development of CRISPR/Cas9 system for B. licheni-
formis opens new avenues to improve its growth
promoting rhizobacteria and BCA traits for agricul-
tural use. Another example is the CRISPR/Cas9
method developed for Streptococcus pyogenes to
establish a highly-efficient multigene disruption
method consisting of an all-in-one knockout plasmid
with a target-gRNA, Cas9, and a homologous repair
template (Zhang et al. 2016). Soon-after, a two-
plasmid similar system was described to delete large
chromosome fragments (So et al. 2017).
CRISPR/Cas9 has been used to investigate genes
involved in BCAs virulence (Table 6). CRISPR/Cas9
coupled with blastospore-mediated transformation
was first optimized for Beauveria bassiana to pre-
cisely edit single and multiple genes (Chen et al.
2017). Very recently, a B. bassiana mutant for the
polyketide synthesis 15 (Pks15) gene, which has
crucial roles in insect virulence (Toopaang et al.
2017), was complemented through an in-cis genetic
complementation system using CRISPR/Cas9. The
authors successfully used a CRISPR/Cas9 vector
previously developed for another phylogenetically
very distant fungus (Udompaisarn et al. 2020).
CRISPR/Cas9 methodology was also developed for
Purpureocillium lilacinum (Jiao et al. 2019) based on
the same system as Chen et al. (2017). P. lilacinum is a
filamentous saprobic fungus with BCA properties
against plant parasitic nematodes, as well as against
insects and plant pathogens (Goffre
´and Folgarait
2015; Wang et al. 2016b). CRISPR/Cas9 was intro-
duced into P. lilacinum to increase homologous
recombination efficiency to disrupt lcsL, a putative
bZIP transcription factor from the lcs cluster that is
involved in the syntheses, modification, and regulation
of the lipopeptides leucinostatins, the most prominent
secondary metabolites produced by the fungus (Jiao
et al. 2019). lcsL deletion reduced the antagonistic
effect against the oomycete Phytophthora infestans
demonstrating the importance of this gene in P.
lilacinum virulence.
Bacillus spp. are soil bacteria with BCA properties
that associate and colonize different plant species
(Choudhary and Johri 2009). One of the most common
species, B. subtilis, suppresses pathogens growth by
secretion of antimicrobial compounds and/or induc-
tion of plant systemic resistance (Choudhary and Johri
2009; Ongena et al. 2007). They are interesting from a
commercial point of view as they possess specific
metabolic and physiological traits that facilitate cre-
ating formulations as fertilizers or as BCAs (Kumar
et al. 2011). Despite the benefits of using Bacillus spp.
as BCAs, the detailed molecular mechanisms involved
in the interaction with plants still lack full understand-
ing, mostly due to the difficulty in performing reverse
genetics in environmentally isolated species. CRISPR/
Cas9 system for Bacillus spp. was first developed for
the model organism B. subtilis 168, using a plasmid
with a cas9 gene controlled by a mannose inducible
promoter and a gRNA driven by a constitutive
promoter (Altenbuchner 2016). A similar system was
developed for the undomesticated strain ATCC6051a,
commonly used for the industrial production of
enzymes (Zhang et al. 2016). Despite their interest,
these methods were developed for model or undo-
mesticated strains that have natural transformability.
Recently, CRISPR/Cas9 transformation of recalcitrant
species and with phytopathogen BCA properties has
also been successful (Yi et al. 2018). In B. subtilis HS3
and B. mycoides EC18, disruption of 4’-phosphopan-
tetheinyl transferase (sfp), required for the production
of several lipopeptide antibiotics, demonstrated that
the surfactin and fengycin families of lipopeptides are
responsible for the antagonistic activity against two
fungal pathogens, Rhizoctonia solani and Fusarium
culmorum (Yi et al. 2018).
GE is starting to gain momentum to improve in field
fitness and effectiveness of microorganisms such as
BCAs. In addition to increasing the BCA kill-power,
by manipulating toxins and virulence factors, or the
BCA adaption to environmental stresses, the idea to
have a kill switch would also be of high value to field
applications. Engineering extreme susceptibility to
sprayed-on compounds would be very useful to easily
kill the BCA in case it becomes a pest or shows
123
Transgenic Res

unexpected off-target effects, therefore avoiding envi-
ronmental risks.
Suppression of pests and pathogens by host-induced
gene silencing (HIGS)
HIGS was initially reported for transgenic petunias
and relies on a regulatory mechanism that limits RNA
levels (Napoli et al. 1990; van der Krol et al. 1990).
One of the hallmarks of the canonical RNAi pathway
is the accumulation of sRNAs that have a reverse-
complement sequence to their target for degradation
by the RNA-induced silencing complex (Tabara et al.
1999; Hammond et al. 2000,2001; Song et al. 2004).
Progress in HIGS relied on technological develop-
ments in the fields of gene delivery and transgenic
plant regeneration. These were mostly based on the
naturally occurring T-DNA transfer by Agrobacterium
tumefaciens (Fraley et al. 1983; Zambryski et al. 1983;
Horsch et al. 1985) and also DNA uptake by proto-
plasts (Krens et al. 1982), electroporation (Fromm
et al. 1985), and biolistic transformation (Klein et al.
1987). The first examples came from work on plants
expressing translatable or untranslatable viral coat,
such as Papaya ringspot virus (PRSV) in papaya
(Fitch et al. 1992), Tobacco etch virus (TEV) in
tobacco (Lindbo and Dougherty 1992) and Plum pox
virus (PPV) in plum (Scorza et al. 1994; Hily et al.
2004), with several of these transgenic lines actually
reaching the market (Dong and Ronald 2019). Tech-
nologies such as homologous recombination and
recently developed GE allow for targeted insertions
into specific regions of the genome. These eliminate
the potential negative effects associated with random
disruption of important coding or non-coding regions,
whilst also allow generating plants without
selectable markers (Chen et al. 2019).
Cross-kingdom RNA trafficking enable the transfer
of sRNA from plants to fungi, oomycetes, nematodes,
and insects (reviewed by Cai et al. 2018; Wang and
Dean 2020). For example, a combination of transient
(biolistics and VIGS) and stable T-DNA insertion
systems was used to express interfering RNAs in
barley and wheat directed at development and
pathogenicity genes of Blumeria graminis (Nowara
et al. 2010). Fungal growth was affected, indicating
transfer of interfering RNA molecules from the host to
the pathogen. Oomycetes have also shown suscepti-
bility to this mechanism, as reported for transgenic
potato expressing dsRNA, particularly those directed
at the G-protein b-subunit GPB1 of P. infestans (Jahan
et al. 2015). HIGS also works for nematodes, with the
first examples coming from tobacco plants expressing
dsRNA targeting a splicing factor or an integrase from
the root-knot nematode Meloidogyne incognita (Ya-
dav et al. 2006). Both approaches effectively depleted
the nematode’s cognate mRNA and controlled the
disease. There are also successful examples of insect
control using transgenic expression of inhibitory
sequences. Transgenic tobacco and Arabidopsis plants
expressing dsRNA designed to deplete a cytochrome
P450 of the cotton bollworm Helicoverpa armigera
reduced its tolerance to the plant toxic compound
gossypol and slowed its growth (Mao et al. 2007). To
our knowledge, examples of HIGS directed at bacte-
rial diseases are limited to the control of A. tumefa-
ciens, which transfers a tumour-inducing T-DNA into
plants. Transgenic expression of dsRNA sequences
with homology to T-DNAs in Arabidopsis and tomato
led to efficient control of tumour formation (Escobar
et al. 2001).
For over 20 years, plant viruses are known to
encode proteins that act as RNAi suppressors (Kass-
chau and Carrington 1998; Anandalakshmi et al. 1998;
Brigneti et al. 1998; Voinnet et al. 1999) by interfering
with one or more steps of the pathway (reviewed by
Yang and Li 2018; Guo et al. 2019; Li and Wang
2019). More recently, this class of proteins was also
found in bacteria and oomycetes (Navarro et al. 2008;
Qiao et al. 2013), suggesting that in field conditions,
infections by viruses, oomycetes, or bacteria could
potentially reduce the efficiency of HIGS (Savenkov
and Valkonen 2001). Future combination of this
powerful approach with CRISPR/Cas9 methodology
may, however, enhance the development of plant hosts
harbouring resistance to different pests and pathogens.
Genome editing perspectives to improve plant
protection
Given the outstanding success of the CRISPR/Cas9
technology in so many diverse areas, it is pre-
dictable that plant pathology research will also
continue benefiting from the ever-improving toolbox
being validated and becoming available for a wider
range of plant species and pathogens. In doing so,
CRISPR-based technologies will continue providing
solutions to improve agriculture and general plant
123
Transgenic Res

health, to combat vector-borne diseases, and control
invasive species. Despite the many successful stories,
some aspects need to be carefully investigated and
resolved. Scientists must not be blinded by its
advantages whilst neglecting pitfalls. Studies report
a wide and sometimes unpredictable range of off-
target effects (see e.g. Ji et al. 2018), requiring
comprehensive genotyping, and for large field tests,
ideally whole genome sequencing, which is becoming
economically more accessible. Also, the selection of
traits to be introduced in plants needs to be carefully
planned to ensure no negative impacts on the food
chain. In some cases, before even start considering
GE, further research is required to identify new
sources of resistance genes. This is particularly
important for insect resistance, for instance, where
the pool of resistance genes available to be edited is
rather poor.
De novo or fine-tuning of R gene expression, allelic
replacement, total abolishment of certain undesired
functions of S genes or ‘‘weak’’ alleles are all possible
ways to explore how to improve plant immunity. For
instance, the replacement of the elicitin receptor-like
protein (ELR) of potato cultivars by the wild potato
(Solanum microdontum) allele, that recognizes a
highly conserved Phytophthora spp. elicitin, would
attain durable broad-spectrum late blight resistance
(Du et al. 2015). This approach could be extended to
many PAMP receptors in different plant species, as
shown for the flagellin receptor FLS2/3 (Hind et al.
2016) and for the elongation factor-Tu receptor EFR,
increasing resistance to R. solanacearum in tomato
(Lacombe et al. 2010) and Medicago (Pfeilmeier et al.
2019), to X. oryzae pv. oryzae and Acidovorax avenae
subsp. avenae in rice (Schwessinger et al. 2015;Lu
et al. 2015), and to P. syringae pv. oryzae in wheat
(Schwessinger et al. 2015). Similarly, the tobacco
Ngene confers resistance to Tobacco mosaic virus
(TMV) when transformed into otherwise susceptible
tomato plants (Whitham et al. 1996). Nencodes a
protein comprising NB-LRR regions (Whitham et al.
1994) that controls infection by triggering a hyper-
sensitive response (HR; Whitham et al. 1996). This R
gene transfer strategy was emulated on other occa-
sions (Bendahmane et al. 1999; Spassova et al. 2001;
Hu et al. 2015; Kim et al. 2017), suggesting that it
could be used with targeted GE techniques to
efficiently generate disease resistant and
selectable marker-free plants (Chen et al. 2019).
R genes, such as NB-LRRs are valuable resources
for transferring resistance from wild species to elite
varieties. Sometimes, the critical differences between
R genes from wild species and elite varieties are
restricted to a single nucleotide variant (Lindner et al.
2020). In this case newly developed base editors
(reviewed by Anzalone et al. 2020) could be exploited
to generate specific base changes. In addition to single
base exchanges, early studies demonstrated that
insertions of short donor sequences can be achieved
through the CRISPR/Cas9 technology (Shan et al.
2013; Li et al. 2013). The targeting of regulatory
mechanisms such as protein post-translational modi-
fication (PTMs) could also fine-tune resistance
responses. Some pathogen effectors target host cells
by ubiquitinating essential immunity proteins and host
ubiquitin ligase knockout increases resistance to
biotrophic pathogens in Arabidopsis (Trujillo et al.
2008) and to Phytophthora infestans in potato (Bos
et al. 2010). Downstream components of the resistance
process, such as MAPKs, transcription factors, and
proteases/lipases could also be targeted. This includes
negative regulators of the SA response such as the
MAP kinase MPK4, whose functional impairment
increases Arabidopsis resistance against Pseu-
domonas syringae and Peronospora parasitica (Peter-
sen et al. 2000). In crops, homologs of these genes
could be edited to confer broad resistance. Another
strategy is to express plant enzymes that neutralise
fungal toxins in otherwise susceptible plants (Johal
and Briggs 1992). For example, F. graminearum,an
important fungal pathogen of wheat, produces myco-
toxins particularly dangerous to humans and animals.
Transgenic wheat expressing a barley UDP-glucosyl-
transferase that metabolises the F. graminearum toxin
deoxynivalenol to a less toxic derivative, provides
high levels of resistance to the fungal pathogen, with
only reduced symptoms of Fusarium head blight in the
field (Li et al. 2015).
Transgenes can be constitutively or differentially
expressed, individually or multiplexed as gene stacks,
for a stronger effect in the plant. Once constructed,
gene stacks can be incorporated into different crop
species and newly discovered genes can be further
added on to the stack. Stacking of multiple R genes
using CRISPR/Cas9-mediated knock-in potentialize
downstream genetic segregation and so could be a
great advance to plant breeding. A first in line to test
this could be rice and bacterial blight and leaf streak
123
Transgenic Res

pathogens (Xanthomonas oryzae pv. oryzae and X.
oryzae pv. oryzicola, respectively), where a series of R
genes and their cognate avirulence and virulence
effector genes have been identified and characterized
(reviewed by Jiang et al. 2020). However, although
HDR-based technology is available to perform tar-
geted insertions and replacements in important crops
like rice (Lu et al. 2020), to our best knowledge, GE
was not yet used to introduce R genes and promote
resistance. Up to now, only proof of concept studies
have been performed, and replacement of an endoge-
nous gene requires a selectable variant containing a
selection marker, and so targeted replacement without
direct selection remains a challenge.
Coupled with structure-based design principles, the
function of proteins involved in pathogen resistance
can be improved, and/or novel ones developed by
fusing or splitting domains, rational design, and
directed evolution. Certain protein motifs are suffi-
cient to determine plant host resistance. Examples of
these include the highly conserved EDVID motif of
the CC domain, required for Rprotein function
(Rairdan et al. 2008) and the overexpression of
isolated TIR domains, being sufficient to trigger HR
(Swiderski et al. 2009; Maekawa et al. 2011; Bernoux
et al. 2011). Furthermore, chimeras of subdomains
from different PRRs have been used to form functional
receptors. For instance, when the extracellular LRR
domain of the EFR receptor was replaced with
corresponding parts from different families of R
receptors (like, FLS2 or XA21), a broader spectrum
of resistance against diverse pathogens was achieved
(Albert et al. 2010; De Lorenzo et al. 2011; Sch-
wessinger et al. 2015). Hence, the versatile pro-
grammability of CRISPR/Cas9-based GE could
enable modulation of these domains to achieve
broad-spectrum resistance (Andolfo et al. 2016).
The development of disease resistance by GE may
involve altering the effector-target interaction, knock-
ing out S genes, knocking in R genes, but also
uncoupling the antagonistic action of growth and
defence mechanisms. As discussed, activation of
defence mechanisms often causes plant growth inhi-
bition and yield reduction. Recently, OsALDH2B1
was shown to function as a master regulator of the
growth-defence trade-off in rice (Ke et al. 2020). Loss
of OsALDH2B1 function enhanced resistance to
multiple pathogens (fungal blast, bacterial leaf blight,
and leaf streak), but caused a reduction of rice growth
and yield. Its primary function is as a mitochondrial
aldehyde dehydrogenase involved in male fertility, but
also functions as a transcription factor with both
repressing and activating activities involved in the
regulation of a diverse range of biological processes.
These findings may have important implications in
plant breeding for resistance by using GE technologies
to modulate OsALDH2B1 (or similar genes) activity to
balance growth-defence trade-off. A recent study to
explore how a transcription factor network coordinates
growth and defence, used a high-throughput pheno-
typing approach to measure growth and flowering in a
set of Arabidopsis mutants previously linked to the
aliphatic glucosinolate defence pathway (Li et al.
2020a). This study not only provided new insights on
how these biological processes are integrated to
optimize fitness, but it also highlighted potential
candidate genes for GE to balance growth and defence
while breeding for resistance. Furthermore, the
authors observed that the capability to promote both
growth and defence was highly dependent on the
specific environment in which the phenotypes were
being measured, raising the importance of analysing
plants response to GE under different growth condi-
tions, which is particularly relevant in the design of
plants for different environments, as well as in face of
global climate change.
Technically, there is still a great scope for GE
improvement through the CRISPR/Cas system. Com-
parative analyses of the various methods available will
identify the best and most robust approaches accord-
ing to the exact needs, thus helping streamlining
processes. Structure-based improvement of Cas9 effi-
ciency for each species, through protein engineering,
is gaining momentum as Cas9 structural data becomes
available. Eventually, discovery of different CRISPR
systems and components from other bacteria will
likely identify additional Cas enzymes.
Concluding remarks
The development of new tools to improve plant
protection is critical in the context of current agricul-
tural, environmental and ecological challenges, par-
ticularly in the face of global climate change and
expanding world population. GE is an important
instrument to improve food safety and security in a
sustainable manner, and to mitigate the challenges
123
Transgenic Res

imposed by the world growing population and climate
change. Indeed, a variety of GE efforts led to plants
with potentially valuable traits that could be readily
implemented in the field. Here, we discussed studies
that showcase how the targeted knockout of negative
regulators and S genes by GE is a powerful strategy for
crop protection. GE has been shown to efficiently edit
S genes against plant pathogenic fungi and bacteria,
mainly. GE has also been successfully used to control
insect vectors of animal diseases, but more research is
required to promote plant protection against pests. The
advances in CRISPR/Cas technology will enable this
area to evolve, mainly by the application of gene
knockout approaches and using improved Cas
enzymes and techniques that, depending on the
plant-pathogen and defence approach targeted, facil-
itate the development of transgene-free plants.
GE approaches targeting viral DNA or RNA
require the continuous expression of CRISPR/Cas
system in the plant, resulting in transgenic lines. Plants
carrying this system can edit the virus genome,
conferring resistance. Similarly, HIGS plants need to
have the RNAi construct against specific genes of the
targeted pathogen integrated into the plant genome,
also resulting in transgenic plants. On the other hand,
methods involving CRISPR/Cas edition of S genes, or
yet insertion of R genes, can yield non-transgenic
plants, as after plant genome editing the CRISPR/Cas
system is not needed and can be removed. Also, in a
non-transgenic plant approach, BCAs could bear the
CRISPR/Cas system that would act on the targeted
gene(s) of the pathogen or pest. These may be taken
into consideration when selecting the GE approach
used to obtain resistant crops.
Despite the indisputable value of GE resources in
general and CRISPR/Cas in particular, scientists are
still limited by regulatory issues and by public
opinions and perceptions that entails that these tech-
nologies are some way from commercial applications,
especially in Europe. It is essential to have mecha-
nisms of governance in place to assure that this
technology is ethically and safely developed and
employed. The barrier of legislation and public
perception is slowly turning. The general public may
start trusting more these new technologies and the
scientists, realizing how important it is to respond
quickly, easily, and safely in extreme circumstances of
potentially critical plant disease outbreaks that could
compromise food security worldwide. If this is the
case, it might open the door to precise breeding being
commonplace to respond to climate change and food
scarcity. The potential impact of GE, and CRISPR
technology in particular, in synthetic plant biology to
breed resistance against pests and diseases is enor-
mous and will have a direct impact on agriculture
sustainability on a scale never before experienced.
Acknowledgements We are grateful to all the researchers
whose contributions have been cited, which have helped us to
prepare this review paper. We apologize to those authors whose
excellent work could not be cited due to space limitations.
Author contributions All authors participated in literature
gathering and manuscript writing. CA designed the figures in
collaboration with the other authors.
Funding This review was funded by the European Social
Fund (ALT20-05-3559-FSE-000036) and the Fundac¸a
˜o para a
Cie
ˆncia e Tecnologia (FCT) based on RCM 23/2018 March, 8.
Declarations
Conflict of interest The authors declare that there is no con-
flict of interest.
References
Abbruscato P, Nepusz T, Mizzi L et al (2012) OsWRKY22, a
monocot wrky gene, plays a role in the resistance response
to blast. Mol Plant Pathol 13:828–841. https://doi.org/10.
1111/j.1364-3703.2012.00795.x
Acevedo-Garcia J, Spencer D, Thieron H et al (2017) mlo-based
powdery mildew resistance in hexaploid bread wheat
generated by a non-transgenic TILLING approach. Plant
Biotechnol J 15:367–378. https://doi.org/10.1111/pbi.
12631
Albert M, Jehle AK, Mueller K et al (2010) Arabidopsis thali-
ana pattern recognition receptors for bacterial elongation
factor Tu and flagellin can be combined to form functional
chimeric receptors. J Biol Chem 285:19035–19042. https://
doi.org/10.1074/jbc.M110.124800
Altenbuchner J (2016) Editing of the Bacillus subtilis genome
by the CRISPR-Cas9 system. Appl Environ Microbiol
82:5421–5427. https://doi.org/10.1128/AEM.01453-16
Aman R, Ali Z, Butt H et al (2018) RNA virus interference via
CRISPR/Cas13a system in plants. Genome Biol 19:1.
https://doi.org/10.1186/s13059-017-1381-1
Amari K, Boutant E, Hofmann C et al (2010) A family of
plasmodesmal proteins with receptor-Like properties for
plant viral movement proteins. PLoS Pathog 6:e1001119.
https://doi.org/10.1371/journal.ppat.1001119
Anandalakshmi R, Pruss GJ, Ge X et al (1998) A viral sup-
pressor of gene silencing in plants. Proc Natl Acad Sci U S
A 95:13079–13084. https://doi.org/10.1073/pnas.95.22.
13079
123
Transgenic Res

Andolfo G, Iovieno P, Frusciante L, Ercolano MR (2016)
Genome-editing technologies for enhancing plant disease
resistance. Front Plant Sci 7:1813. https://doi.org/10.3389/
fpls.2016.01813
Antony G, Zhou J, Huang S et al (2010) Rice xa13 recessive
resistance to bacterial blight is defeated by induction of the
disease susceptibility gene Os-11N3. Plant Cell
22:3864–3876. https://doi.org/10.1105/tpc.110.078964
Anzalone AV, Koblan LW, Liu DR (2020) Genome editing with
CRISPR–Cas nucleases, base editors, transposases and
prime editors. Nat Biotechnol 38:824–844. https://doi.org/
10.1038/s41587-020-0561-9
Atarashi H, Jayasinghe WH, Kwon J et al (2020) Artificially
edited alleles of the eukaryotic translation initiation factor
4E1 gene differentially reduce susceptibility to Cucumber
mosaic virus and Potato virus Y in tomato. Front Microbiol
11:3075. https://doi.org/10.3389/fmicb.2020.564310
Avis TJ, Gravel V, Antoun H, Tweddell RJ (2008) Multifaceted
beneficial effects of rhizosphere microorganisms on plant
health and productivity. Soil Biol Biochem 40:1733–1740.
https://doi.org/10.1016/j.soilbio.2008.02.013
Baker KF (1987) Evolving concepts of biological control of
plant pathogens. Annu Rev Phytopathol 25:67–85. https://
doi.org/10.1146/annurev.py.25.090187.000435
Baltes NJ, Hummel AW, Konecna E et al (2015) Conferring
resistance to geminiviruses with the CRISPR–Cas
prokaryotic immune system. Nat Plants 1:15145. https://
doi.org/10.1038/nplants.2015.145
Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rx
gene from potato controls separate virus resistance and cell
death responses. Plant Cell 11:781–791. https://doi.org/10.
1105/tpc.11.5.781
Bernoux M, Ve T, Williams S et al (2011) Structural and
functional analysis of a plant resistance protein TIR
domain reveals interfaces for self-association, signaling,
and autoregulation. Cell Host Microbe 9:200–211. https://
doi.org/10.1016/j.chom.2011.02.009
Bi H-L, Xu J, Tan A-J, Huang Y-P (2016) CRISPR/Cas9-me-
diated targeted gene mutagenesis in Spodoptera litura.
Insect Sci 23:469–477. https://doi.org/10.1111/1744-7917.
12341
Bisht DS, Bhatia V, Bhattacharya R (2019) Improving plant-
resistance to insect-pests and pathogens: the new oppor-
tunities through targeted genome editing. Semin Cell Dev
Biol 96:65–76. https://doi.org/10.1016/j.semcdb.2019.04.
008
Blanvillain-Baufume
´S, Reschke M, Sole
´M et al (2017) Tar-
geted promoter editing for rice resistance to Xanthomonas
oryzae pv. oryzae reveals differential activities for
SWEET14-inducing TAL effectors. Plant Biotechnol J
15:306–317. https://doi.org/10.1111/pbi.12613
Borejsza-Wysocka EE, Malnoy M, Aldwinckle HS et al (2006)
The fire blight resistance of apple clones in which DspE-
interacting proteins are silenced. Acta Hortic 704:509–514.
https://doi.org/10.17660/actahortic.2006.704.80
Bos JIB, Armstrong MR, Gilroy EM et al (2010) Phytophthora
infestans effector AVR3a is essential for virulence and
manipulates plant immunity by stabilizing host E3 ligase
CMPG1. Proc Natl Acad Sci U S A 107:9909–9914.
https://doi.org/10.1073/pnas.0914408107
Brauer EK, Balcerzak M, Rocheleau H et al (2020) Genome
editing of a deoxynivalenol-induced transcription factor
confers resistance to Fusarium graminearum in wheat. Mol
Plant-Microbe Interact 33:553–560. https://doi.org/10.
1094/MPMI-11-19-0332-R
Brigneti G, Voinnet O, Li W-X et al (1998) Viral pathogenicity
determinants are suppressors of transgene silencing in Ni-
cotiana benthamiana. EMBO J 17:6739–6746. https://doi.
org/10.1093/emboj/17.22.6739
Cai Q, He B, Kogel KH, Jin H (2018) Cross-kingdom RNA
trafficking and environmental RNAi - nature’s blueprint for
modern crop protection strategies. Curr Opin Microbiol
46:58–64. https://doi.org/10.1016/j.mib.2018.02.003
Chandrasekaran J, Brumin M, Wolf D et al (2016) Development
of broad virus resistance in non-transgenic cucumber using
CRISPR/Cas9 technology. Mol Plant Pathol
17:1140–1153. https://doi.org/10.1111/mpp.12375
Chen LQ, Hou BH, Lalonde S et al (2010) Sugar transporters for
intercellular exchange and nutrition of pathogens. Nature
468:527–532. https://doi.org/10.1038/nature09606
Chen LQ, Qu XQ, Hou BH et al (2012) Sucrose efflux mediated
by SWEET proteins as a key step for phloem transport.
Science 335:207–211. https://doi.org/10.1126/science.
1213351
Chen W, Qian Y, Wu X et al (2014) Inhibiting replication of
begomoviruses using artificial zinc finger nucleases that
target viral-conserved nucleotide motif. Virus Genes
48:494–501. https://doi.org/10.1007/s11262-014-1041-4
Chen J, Lai Y, Wang L et al (2017) CRISPR/Cas9-mediated
efficient genome editing via blastospore-based transfor-
mation in entomopathogenic fungus Beauveria bassiana.
Sci Rep 7:45763. https://doi.org/10.1038/srep45763
Chen K, Wang Y, Zhang R et al (2019) CRISPR/Cas genome
editing and precision plant breeding in agriculture. Ann
Rev Plant Biol 70:667–697. https://doi.org/10.1146/
annurev-arplant-050718-100049
Choudhary DK, Johri BN (2009) Interactions of Bacillus spp.
and plants – With special reference to induced systemic
resistance (ISR). Microbiol Res 164:493–513. https://doi.
org/10.1016/j.micres.2008.08.007
Chu Z, Ouyang Y, Zhang J et al (2004) Genome-wide analysis
of defense-responsive genes in bacterial blight resistance
of rice mediated by the recessive R gene xa13. Mol Genet
Genom 271:111–120. https://doi.org/10.1007/s00438-003-
0964-6
Chu Z, Yuan M, Yao J et al (2006) Promoter mutations of an
essential gene for pollen development result in disease
resistance in rice. Genes Dev 20:1250–1255. https://doi.
org/10.1101/gad.1416306
Cook RJ (1993) Making greater use of introduced microor-
ganisms for biological control of plant pathogens. Annu
Rev Phytopathol 31:53–80. https://doi.org/10.1146/
annurev.py.31.090193.000413
De Lorenzo G, Brutus A, Savatin DV et al (2011) Engineering
plant resistance by constructing chimeric receptors that
recognize damage-associated molecular patterns
(DAMPs). FEBS Lett 585:1521–1528. https://doi.org/10.
1016/j.febslet.2011.04.043
de Toledo Thomazella DP, Brail Q, Dahlbeck D, Staskawicz BJ
(2016) CRISPR-Cas9 mediated mutagenesis of a DMR6
123
Transgenic Res

ortholog in tomato confers broad-spectrum disease resis-
tance. bioRxiv. https://doi.org/10.1101/064824
Deng H, Gao R, Liao X, Cai Y (2017) CRISPR system in fila-
mentous fungi: Current achievements and future direc-
tions. Gene 627:212–221. https://doi.org/10.1016/j.gene.
2017.06.019
Deslandes L, Olivier J, Theulie
`res F et al (2002) Resistance to
ralstonia solanacearum in Arabidopsis thaliana is conferred
by the recessive RRS1-R gene, a member of a novel family
of resistance genes. Proc Natl Acad Sci U S A
99:2404–2409. https://doi.org/10.1073/pnas.032485099
Doehlemann G, O
¨kmen B, Zhu W, Sharon A (2017) Plant
pathogenic fungi. In: The Fungal Kingdom. Eds: John
Wiley and Sons, pp. 703–776. https://doi.org/10.1128/
microbiolspec.funk-0023-2016
Dong OX, Ronald PC (2019) Genetic engineering for disease
resistance in plants: recent progress and future perspec-
tives. Plant Physiol 180:26–38. https://doi.org/10.1104/pp.
18.01224
Dong X, Hong Z, Chatterjee J et al (2008) Expression of callose
synthase genes and its connection with Npr1 signaling
pathway during pathogen infection. Planta 229:87–98.
https://doi.org/10.1007/s00425-008-0812-3
Du J, Verzaux E, Chaparro-Garcia A et al (2015) Elicitin
recognition confers enhanced resistance to Phytophthora
infestans in potato. Nat Plants 1:15034. https://doi.org/10.
1038/nplants.2015.34
Dunn DW, Follett PA (2017) The sterile insect technique (sit):
an introduction. Entomol Exp Appl 164:151–154. https://
doi.org/10.1111/eea.12619
Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM
(2001) RNAi-mediated oncogene silencing confers resis-
tance to crown gall tumorigenesis. Proc Natl Acad Sci U S
A 98:13437–13442. https://doi.org/10.1073/pnas.
241276898
Eulgem T, Somssich IE (2007) Networks of WRKY transcrip-
tion factors in defense signaling. Curr Opin Plant Biol
10:366–371. https://doi.org/10.1016/j.pbi.2007.04.020
Fister AS, Landherr L, Maximova SN, Guiltinan MJ (2018)
Transient expression of CRISPR/Cas9 machinery targeting
TcNPR3 enhances defense response in theobroma cacao.
Front Plant Sci 9:268. https://doi.org/10.3389/fpls.2018.
00268
Fitch MMM, Manshardt RM, Gonsalves D et al (1992) Virus
resistant papaya plants derived from tissues bombarded
with the coat protein gene of apaya ringspot virus. Bio/
technol 10:1466–1472. https://doi.org/10.1038/nbt1192-
1466
Fones HN, Gurr SJ (2017) NOXious gases and the unpre-
dictability of emerging plant pathogens under climate
change. BMC Biol 15:36. https://doi.org/10.1186/s12915-
017-0376-4
Fones HN, Bebber DP, Chaloner TM et al (2020) Threats to
global food security from emerging fungal and oomycete
crop pathogens. Nat Food 1:332–342. https://doi.org/10.
1038/s43016-020-0075-0
Fraley RT, Rogers SG, Horsch RB et al (1983) Expression of
bacterial genes in plant cells. Proc Natl Acad Sci U S A
80:4803–4807. https://doi.org/10.1073/pnas.80.15.4803
Friedrichs S, Takasu Y, Kearns P et al (2019) Meeting report of
the OECD conference on ‘‘genome editing: applications in
agriculture—implications for health, environment and
regulation.’’ Transgenic Res 28:419–463. https://doi.org/
10.1007/s11248-019-00154-1
Fromm M, Taylor LP, Walbot V (1985) Expression of genes
transferred into monocot and dicot plant cells by electro-
poration. Proc Natl Acad Sci U S A 82:5824–5828. https://
doi.org/10.1073/pnas.82.17.5824
Frye CA, Tang D, Innes RW (2001) Negative regulation of
defense responses in plants by a conserved MAPKK
kinase. Proc Natl Acad Sci U S A 98:373–378. https://doi.
org/10.1073/pnas.98.1.373
Fuentes A, Carlos N, Ruiz Y et al (2015) Field trial and
molecular characterization of RNAi-Transgenic tomato
plants that exhibit resistance to tomato yellow leaf curl
geminivirus. Mol Plant-Microbe Interact 29:197–209.
https://doi.org/10.1094/MPMI-08-15-0181-R
Gao W, Long L, Zhu LF et al (2013) Proteomic and Virus-
induced Gene Silencing (VIGS) analyses reveal that
gossypol, brassinosteroids, and jasmonic acid contribute to
the resistance of cotton to verticillium dahliae. Mol Cell
Proteom 12:3690–3703. https://doi.org/10.1074/mcp.
M113.031013
Garcia-Ruiz H (2018) Susceptibility genes to plant viruses.
Viruses 10:484. https://doi.org/10.3390/v10090484
Geissler K, Eschen-Lippold L, Naumann K et al (2015) Muta-
tions in the EDR1 gene alter the response of Arabidopsis
thaliana to Phytophthora infestans and the bacterial
PAMPs flg22 and elf18. Mol Plant-Microbe Interact
28:122–133. https://doi.org/10.1094/MPMI-09-14-0282-R
Ghogare R, Ludwig Y, Bueno GM, Slamet-Loedin IH, Dhingra
A (2021) Genome editing reagent delivery in plants.
Transgenic Res. https://doi.org/10.1007/s11248-021-
00239-w
Ghorbani Faal P, Farsi M, Seifi A, Mirshamsi Kakhki A (2020)
Virus-induced CRISPR-Cas9 system improved resistance
against tomato yellow leaf curl virus. Mol Biol Rep
47:3369–3376. https://doi.org/10.1007/s11033-020-
05409-3
Glazebrook J (2005) Contrasting mechanisms of defense against
biotrophic and necrotrophic pathogens. Ann Rev Phy-
topathol 43:205–227. https://doi.org/10.1146/annurev.
phyto.43.040204.135923
Goffre
´D, Folgarait PJ (2015) Purpureocillium lilacinum,
potential agent for biological control of the leaf-cutting ant
Acromyrmex lundii. J Invertebr Pathol 130:107–115.
https://doi.org/10.1016/j.jip.2015.07.008
Gomez MA, Lin ZD, Moll T et al (2019) Simultaneous CRISPR/
Cas9-mediated editing of cassava eIF4E isoforms nCBP-1
and nCBP-2 reduces cassava brown streak disease symp-
tom severity and incidence. Plant Biotechnol J
17:421–434. https://doi.org/10.1111/pbi.12987
Go
´mez-Go
´mez L, Bauer Z, Boller T (2001) Both the extracel-
lular leucine-rich repeat domain and the kinase activity of
FLS2 are required for flagellin binding and signaling in
Arabidopsis. Plant Cell 13:1155–1163. https://doi.org/10.
1105/tpc.13.5.1155
Guo Z, Li Y, Ding S-W (2019) Small RNA-based antimicrobial
immunity. Nat Rev Immunol 19:31–44. https://doi.org/10.
1038/s41577-018-0071-x
123
Transgenic Res

Hammes UZ (2016) Novel roles for phytosulfokine signalling in
plant–pathogen interactions. Plant Cell Environ
39:1393–1395. https://doi.org/10.1111/pce.12679
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An
RNA-directed nuclease mediates post-transcriptional gene
silencing in Drosophila cells. Nature 404:293–296. https://
doi.org/10.1038/35005107
Hammond SM, Boettcher S, Caudy AA et al (2001) Argonaute2,
a link between genetic and biochemical analyses of RNAi.
Science 293:1146–1150. https://doi.org/10.1126/science.
1064023
Hammond A, Galizi R, Kyrou K et al (2016) A CRISPR-Cas9
gene drive system targeting female reproduction in the
malaria mosquito vector Anopheles gambiae. Nat
Biotechnol 34:78–83. https://doi.org/10.1038/nbt.3439
Hammond AM, Kyrou K, Bruttini M et al (2017) The creation
and selection of mutations resistant to a gene drive over
multiple generations in the malaria mosquito. PLoS Genet
13:e1007039. https://doi.org/10.1371/journal.pgen.
1007039
Harper G, Osuji JO, Heslop-Harrison JS, Hull R (1999) Inte-
gration of banana Streak Badnavirus into the musa gen-
ome: molecular and cytogenetic evidence. Virology
255:207–213. https://doi.org/10.1006/viro.1998.9581
Heu CC, McCullough FM, Luan J, Rasgon JL (2020) CRISPR-
Cas9-based genome editing in the silverleaf W\whitefly
(Bemisia tabaci). Cris J 3:89–96. https://doi.org/10.1089/
crispr.2019.0067
Hille F, Richter H, Wong SP et al (2018) The biology of
CRISPR-Cas: backward and forward. Cell 172:1239–1259.
https://doi.org/10.1016/j.cell.2017.11.032
Hily J-M, Scorza R, Malinowski T et al (2004) Stability of gene
silencing-based resistance to Plum pox virus in transgenic
plum (Prunus domestica L.) under field conditions.
Transgenic Res 13:427–436. https://doi.org/10.1007/
s11248-004-8702-3
Hind SR, Strickler SR, Boyle PC et al (2016) Tomato receptor
FLAGELLIN-SENSING 3 binds flgII-28 and activates the
plant immune system. Nat Plants 2:16128. https://doi.org/
10.1038/nplants.2016.128
Horsch RB, Fry JE, Hoffmann NL et al (1985) A simple and
general method for transferring genes into plants. Science
227:1229–1231. https://doi.org/10.1126/science.227.4691.
1229
Hou Y, Ma W (2020) Natural host-induced gene silencing offers
new opportunities to engineer disease resistance. Trends
Microbiol 28:109–117. https://doi.org/10.1016/j.tim.2019.
08.009
Hsu FC, Chou MY, Chou SJ et al (2013) Submergence confers
immunity mediated by the WRKY22 transcription factor in
Arabidopsis. Plant Cell 25:2699–2713. https://doi.org/10.
1105/tpc.113.114447
Hu Y, Zhang J, Jia H et al (2014) Lateral organ boundaries 1 is a
disease susceptibility gene for citrus bacterial canker dis-
ease. Proc Natl Acad Sci 111:E521–E529. https://doi.org/
10.1073/pnas.1313271111
Hu Z, Liu G, Gao J et al (2015) Tomato Tm-22 gene confers
multiple resistances to TMV, ToMV, PVX, and PVY to
cultivated potato. Russ J Plant Physiol 62:101–108. https://
doi.org/10.1134/S1021443715010070
Huibers RP, Loonen AEHM, Gao D et al (2013) Powdery mil-
dew resistance in tomato by impairment of SlPMR4 and
SlDMR1. PLoS ONE 8:e67467. https://doi.org/10.1371/
journal.pone.0067467
Huss P, Raman S (2020) Engineered bacteriophages as pro-
grammable biocontrol agents. Curr Opin Biotechnol
61:116–121. https://doi.org/10.1016/j.copbio.2019.11.013
Islam MT, Kim KH, Choi J (2019) Wheat blast in Bangladesh:
the current situation and future impacts. Plant Pathol J
35:1–10. https://doi.org/10.5423/PPJ.RW.08.2018.0168
Jaafar ZA, Kieft JS (2019) Viral RNA structure-based strategies
to manipulate translation. Nat Rev Microbiol 17:110–123.
https://doi.org/10.1038/s41579-018-0117-x
Jahan SN, A
˚sman AKM, Corcoran P et al (2015) Plant-mediated
gene silencing restricts growth of the potato late blight
pathogen Phytophthora infestans. J Exp Bot
66:2785–2794. https://doi.org/10.1093/jxb/erv094
Jansen C, von Wettstein D, Scha
¨fer W et al (2005) Infection
patterns in barley and wheat spikes inoculated with wild-
type and trichodiene synthase gene disrupted Fusarium
graminearum. Proc Natl Acad Sci 102:16892–16897.
https://doi.org/10.1073/pnas.0508467102
Jarosch B, Kogel K-H, Schaffrath U (1999) The ambivalence of
the barley Mlo locus: mutations conferring resistance
against Ppowdery mildew (Blumeria graminis f. sp. hor-
dei) enhance susceptibility to the rice blast fungus Mag-
naporthe grisea. Mol Plant-Microbe Interact 12:508–514.
https://doi.org/10.1094/MPMI.1999.12.6.508
Ji X, Si X, Zhang Y et al (2018) Conferring DNA virus resis-
tance with high specificity in plants using virus-inducible
genome-editing system. Genome Biol 19:197. https://doi.
org/10.1186/s13059-018-1580-4
Jia H, Orbovic V, Jones JB, Wang N (2016) Modification of the
PthA4 effector binding elements in Type I CsLOB1 pro-
moter using Cas9/sgRNA to produce transgenic Duncan
grapefruit alleviating XccDpthA4: DCsLOB1.3 infection.
Plant Biotechnol J 14:1291–1301. https://doi.org/10.1111/
pbi.12495
Jia H, Zhang Y, Orbovic
´V et al (2017) Genome editing of the
disease susceptibility gene CsLOB1 in citrus confers
resistance to citrus canker. Plant Biotechnol J 15:817–823.
https://doi.org/10.1111/pbi.12677
Jiang N, Yan J, Liang Y et al (2020) Resistance genes and their
Interactions with bacterial blight/leaf streak pathogens
(Xanthomonas oryzae) in Rice (Oryza sativa L.): an
updated review. Rice 13:3. https://doi.org/10.1186/
s12284-019-0358-y
Jiao Y, Li Y, Li Y et al (2019) Functional genetic analysis of the
leucinostatin biosynthesis transcription regulator lcsL in
Purpureocillium lilacinum using CRISPR-Cas9 technol-
ogy. Appl Microbiol Biotechnol 103:6187–6194. https://
doi.org/10.1007/s00253-019-09945-2
Johal GS, Briggs SP (1992) Reductase activity encoded by the
HM1 disease resistance gene in maize. Science
258:985–987. https://doi.org/10.1126/science.1359642
Jones JDG, Dangl JL (2006) The plant immune system. Nature
444:323–329. https://doi.org/10.1038/nature05286
Kandul NP, Liu J, Sanchez CHM et al (2019) Transforming
insect population control with precision guided sterile
males with demonstration in flies. Nat Commun 10:84.
https://doi.org/10.1038/s41467-018-07964-7
123
Transgenic Res

Kasschau KD, Carrington JC (1998) A Counter defensive
strategy of plant viruses. Cell 95:461–470. https://doi.org/
10.1016/S0092-8674(00)81614-1
Kaur G, Upadhyay SK, Verma PC (2021) An overview of
genome-engineering methods. Genome Eng Crop Improv
1–21
Ke Y, Yuan M, Liu H et al (2020) The versatile functions of
OsALDH2B1 provide a genic basis for growth–defense
trade-offs in rice. Proc Natl Acad Sci 117:3867–3873.
https://doi.org/10.1073/pnas.1918994117
Kim S-B, Kang W-H, Huy HN et al (2017) Divergent evolution
of multiple virus-resistance genes from a progenitor in
Capsicum spp. New Phytol 213:886–899. https://doi.org/
10.1111/nph.14177
Kim YA, Moon H, Park CJ (2019) CRISPR/Cas9-targeted
mutagenesis of Os8N3 in rice to confer resistance to
Xanthomonas oryzae pv. oryzae. Rice 12(1):1–13. https://
doi.org/10.1186/s12284-019-0325-7
Kis A, Hamar E
´, Tholt G et al (2019) Creating highly efficient
resistance against wheat dwarf virus in barley by employ-
ing CRISPR/Cas9 system. Plant Biotechnol J
17:1004–1006. https://doi.org/10.1111/pbi.13077
Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity
microprojectiles for delivering nucleic acids into living
cells. Nature 327:70–73. https://doi.org/10.1038/327070a0
Ko
¨hl J, Kolnaar R, Ravensberg WJ (2019) Mode of action of
microbial biological control agents against plant diseases:
relevance beyond efficacy. Front Plant Sci 10:845. https://
doi.org/10.3389/fpls.2019.00845
Kohorn B, Kohorn S (2012) The cell wall-associated kinases,
WAKs, as pectin receptors. Front Plant Sci 3:88. https://
doi.org/10.3389/fpls.2012.00088
Krappmann S (2017) CRISPR-Cas9, the new kid on the block of
fungal molecular biology. Med Mycol 55:16–23. https://
doi.org/10.1093/mmy/myw097
Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982)
In vitro transformation of plant protoplasts with Ti-plasmid
DNA. Nature 296:72–74. https://doi.org/10.1038/
296072a0
Krol E, Mentzel T, Chinchilla D et al (2010) Perception of the
Arabidopsis danger signal peptide 1 involves the pattern
recognition receptor AtPEPR1 and its close homologue
AtPEPR2. J Biol Chem 285:13471–13479. https://doi.org/
10.1074/jbc.M109.097394
Kumar A, Prakash A, Johri BN (2011) Bacillus as PGPR in crop
ecosystem. Bact Agrobiol Crop Ecosyst. https://doi.org/10.
1007/978-3-642-18357-7_2
Kusch S, Panstruga R (2017) Mlo-based resistance: An appar-
ently universal ‘‘weapon’’ to defeat powdery mildew dis-
ease. Mol Plant-Microbe Interact 30:179–189. https://doi.
org/10.1094/MPMI-12-16-0255-CR
Kushner DB, Lindenbach BD, Grdzelishvili VZ et al (2003)
Systematic, genome-wide identification of host genes
affecting replication of a positive-strand RNA virus. Proc
Natl Acad Sci U S A 100:15764–15769. https://doi.org/10.
1073/pnas.2536857100
Kyrou K, Hammond AM, Galizi R et al (2018) A CRISPR–Cas9
gene drive targeting doublesex causes complete population
suppression in caged Anopheles gambiae mosquitoes. Nat
Biotechnol 36:1062–1066. https://doi.org/10.1038/nbt.
4245
Lacombe S, Rougon-Cardoso A, Sherwood E et al (2010)
Interfamily transfer of a plant pattern-recognition receptor
confers broad-spectrum bacterial resistance. Nat Biotech-
nol 28:365–369. https://doi.org/10.1038/nbt.1613
Lapin D, Van den Ackerveken G (2013) Susceptibility to plant
disease: more than a failure of host immunity. Trends Plant
Sci 18:546–554. https://doi.org/10.1016/j.tplants.2013.05.
005
Leung K, Ras E, Ferguson KB et al (2020) Next-generation
biological control: the need for integrating genetics and
genomics. Biol Rev. https://doi.org/10.1111/brv.12641
Lewis JD, Lazarowitz SG (2010) Arabidopsis synaptotagmin
SYTA regulates endocytosis and virus movement protein
cell-to-cell transport. Proc Natl Acad Sci 107:2491–2496.
https://doi.org/10.1073/pnas.0909080107
Li F, Scott MJ (2016) CRISPR/Cas9-mediated mutagenesis of
the white and Sex lethal loci in the invasive pest, Droso-
phila suzukii. Biochem Biophys Res Commun
469:911–916. https://doi.org/10.1016/j.bbrc.2015.12.081
Li F, Wang A (2019) RNA-Targeted antiviral immunity: more
Than just RNA silencing. Trends Microbiol 27:792–805.
https://doi.org/10.1016/j.tim.2019.05.007
Li T, Liu B, Spalding MH et al (2012) High-efficiency TALEN-
based gene editing produces disease-resistant rice. Nat
Biotechnol 30:390–392. https://doi.org/10.1038/nbt.2199
Li J-F, Norville JE, Aach J et al (2013) Multiplex and homol-
ogous recombination–mediated genome editing in Ara-
bidopsis and Nicotiana benthamiana using guide RNA and
Cas9. Nat Biotechnol 31:688–691. https://doi.org/10.1038/
nbt.2654
Li X, Shin S, Heinen S et al (2015) Transgenic wheat expressing
a barley UDP-glucosyltransferase detoxifies deoxyni-
valenol and provides high levels of resistance to Fusarium
graminearum. Mol Plant-Microbe Interact 28:1237–1246.
https://doi.org/10.1094/MPMI-03-15-0062-R
Li W, Zhu Z, Chern M et al (2017) A natural allele of a tran-
scription factor in rice confers broad-spectrum blast resis-
tance. Cell 170:114–126. https://doi.org/10.1016/j.cell.
2017.06.008
Li K, Cai D, Wang Z et al (2018) Development of an efficient
genome editing tool in Bacillus licheniformis using
CRISPR-Cas9 nickase. Appl Environ Microbiol
84:e02608-e2617. https://doi.org/10.1128/AEM.02608-17
Li S, Shen L, Hu P et al (2019) Developing disease-resistant
thermosensitive male sterile rice by multiplex gene editing.
J Integr Plant Biol 61:1201–1205. https://doi.org/10.1111/
jipb.12774
Li B, Tang M, Caseys C et al (2020a) Epistatic transcription
factor networks differentially modulate Arabidopsis
growth and defense. Genetics 214:529–541. https://doi.
org/10.1534/genetics.119.302996
Li C, Li W, Zhou Z et al (2020b) A new rice breeding method:
CRISPR/Cas9 system editing of the Xa13 promoter to
cultivate transgene-free bacterial blight-resistant rice. Plant
Biotechnol J 18:313–315. https://doi.org/10.1111/pbi.
13217
Li W, Deng Y, Ning Y et al (2020c) Exploiting broad-spectrum
disease resistance in crops: from molecular dissection to
breeding. Annu Rev Plant Biol 71:575–603. https://doi.
org/10.1146/annurev-arplant-010720-022215
123
Transgenic Res

Lindbo JA, Dougherty WG (1992) Pathogen-derived resistance
to a potyvirus: immune and resistant phenotypes in trans-
genic tobacco expressing altered forms of a potyvirus coat
protein nucleotide sequence. Mol Plant-Microbe Interact
5:144–153. https://doi.org/10.1094/mpmi-5-144
Lindner S, Keller B, Pal Singh S et al (2020) Single residues in
the LRR domain of the wheat PM3A immune receptor can
control the strength and the spectrum of the immune
response. Plant J N/a: https://doi.org/10.1111/tpj.14917
Liu D, Chen X, Liu J et al (2012) The rice ERF transcription
factor OsERF922 negatively regulates resistance to Mag-
naporthe oryzae and salt tolerance. J Exp Bot
63:3899–3911. https://doi.org/10.1093/jxb/ers079
Liu R, Chen L, Jiang Y et al (2015) Efficient genome editing in
filamentous fungus Trichoderma reesei using the CRISPR/
Cas9 system. Cell Discov 1:15007. https://doi.org/10.
1038/celldisc.2015.7
Liu H, Soyars CL, Li J et al (2018) CRISPR/Cas9-mediated
resistance to cauliflower mosaic virus. Plant Direct
2:e00047. https://doi.org/10.1002/pld3.47
Lu F, Wang H, Wang S et al (2015) Enhancement of innate
immune system in monocot rice by transferring the
dicotyledonous elongation factor Tu receptor EFR. J Integr
Plant Biol 57:641–652. https://doi.org/10.1111/jipb.12306
Lu H, Luo T, Fu H et al (2018) Resistance of rice to insect pests
mediated by suppression of serotonin biosynthesis. Nat
Plants 4:338–344. https://doi.org/10.1038/s41477-018-
0152-7
Lu Y, Tian Y, Shen R et al (2020) Targeted, efficient sequence
insertion and replacement in rice. Nat Biotechnol. https://
doi.org/10.1038/s41587-020-0581-5
Ma J, Chen J, Wang M et al (2018) Disruption of OsSEC3A
increases the content of salicylic acid and induces plant
defense responses in rice. J Exp Bot 69:1051–1064. https://
doi.org/10.1093/jxb/erx458
Macovei A, Sevilla NR, Cantos C et al (2018) Novel alleles of
rice eIF4G generated by CRISPR/Cas9-targeted mutage-
nesis confer resistance to rice tungro spherical virus. Plant
Biotechnol J 16:1918–1927. https://doi.org/10.1111/pbi.
12927
Maekawa T, Cheng W, Spiridon LN et al (2011) Coiled-coil
domain-dependent homodimerization of intracellular bar-
ley immune receptors defines a minimal functional module
for triggering cell death. Cell Host Microbe 9:187–199.
https://doi.org/10.1016/j.chom.2011.02.008
Malnoy M, Viola R, Jung MH et al (2016) DNA-free genetically
edited grapevine and apple protoplast using CRISPR/Cas9
ribonucleoproteins. Front Plant Sci 7:1904. https://doi.org/
10.3389/fpls.2016.01904
Mao Y-B, Cai W-J, Wang J-W et al (2007) Silencing a cotton
bollworm P450 monooxygenase gene by plant-mediated
RNAi impairs larval tolerance of gossypol. Nat Biotechnol
25:1307–1313. https://doi.org/10.1038/nbt1352
McGrann GRD, Stavrinides A, Russell J et al (2014) A trade off
between mlo resistance to powdery mildew and increased
susceptibility of barley to a newly important disease,
Ramularia leaf spot. J Exp Bot 65:1025–1037. https://doi.
org/10.1093/jxb/ert452
Meccariello A, Monti SM, Romanelli A et al (2017) Highly
efficient DNA-free gene disruption in the agricultural pest
Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein
complexes. Sci Rep 7:10061. https://doi.org/10.1038/
s41598-017-10347-5
Mehta D, Hirsch-Hoffmann M, Were M et al (2018) A new full-
length circular DNA sequencing method for viral-sized
genomes reveals that RNAi transgenic plants provoke a
shift in geminivirus populations in the field. Nucleic Acids
Res 47:e9. https://doi.org/10.1093/nar/gky914
Mehta D, Stu
¨rchler A, Anjanappa RB et al (2019) Linking
CRISPR-Cas9 interference in cassava to the evolution of
editing-resistant geminiviruses. Genome Biol 20:80.
https://doi.org/10.1186/s13059-019-1678-3
Melotto M, Underwood W, Koczan J et al (2006) Plant stomata
function in innate immunity against bacterial invasion. Cell
126:969–980. https://doi.org/10.1016/j.cell.2006.06.054
Miya A, Albert P, Shinya T et al (2007) CERK1, a LysM
receptor kinase, is essential for chitin elicitor signaling in
Arabidopsis. Proc Natl Acad Sci 104:19613–19618.
https://doi.org/10.1073/pnas.0705147104
Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a
chimeric chalcone synthase gene into Petunia results in
reversible co-suppression of homologous genes in trans.
Plant Cell 2:279–289. https://doi.org/10.1105/tpc.2.4.279
Narusaka M, Shirasu K, Noutoshi Y et al (2009) RRS1 and
RPS4 provide a dual resistance-gene system against fungal
and bacterial pathogens. Plant J 60:218–226. https://doi.
org/10.1111/j.1365-313X.2009.03949.x
Navarro L, Jay F, Nomura K et al (2008) Suppression of the
microRNA pathway by bacterial effector proteins. Science
321:964–967. https://doi.org/10.1126/science.1159505
Nekrasov V, Wang C, Win J et al (2017) Rapid generation of a
transgene-free powdery mildew resistant tomato by gen-
ome deletion. Sci Rep 7:482. https://doi.org/10.1038/
s41598-017-00578-x
Nishad R, Ahmed T, Rahman VJ, Kareem A (2020) Modulation
of plant defense system in response to microbial interac-
tions. Front Microbiol 11:1298. https://doi.org/10.3389/
fmicb.2020.01298
Nishimura MT, Stein M, Hou BH et al (2003) Loss of a callose
synthase results in salicylic acid-dependent disease resis-
tance. Science 301:969–972. https://doi.org/10.1126/
science.1086716
Nødvig CS, Nielsen JB, Kogle ME, Mortensen UH (2015) A
CRISPR-Cas9 system for genetic engineering of filamen-
tous fungi. PLoS ONE 10:e0133085. https://doi.org/10.
1371/journal.pone.0133085
Nowara D, Gay A, Lacomme C et al (2010) HIGS: Host-induced
gene silencing in the obligate biotrophic fungal pathogen
Blumeria graminis. Plant Cell 22:3130–3141. https://doi.
org/10.1105/tpc.110.077040
Oliva R, Ji C, Atienza-Grande G et al (2019) Broad-spectrum
resistance to bacterial blight in rice using genome editing.
Nat Biotechnol 37:1344–1350. https://doi.org/10.1038/
s41587-019-0267-z
Ongena M, Jourdan E, Adam A et al (2007) Surfactin and fen-
gycin lipopeptides of Bacillus subtilis as elicitors of
induced systemic resistance in plants. Environ Microbiol
9:1084–1090. https://doi.org/10.1111/j.1462-2920.2006.
01202.x
Ortigosa A, Gimenez-Ibanez S, Leonhardt N, Solano R (2019)
Design of a bacterial speck resistant tomato by CRISPR/
123
Transgenic Res

Cas9-mediated editing of SlJAZ2. Plant Biotechnol J
17:665–673. https://doi.org/10.1111/pbi.13006
Ownley BH, Griffin MR, Klingeman WE et al (2008) Beauveria
bassiana: endophytic colonization and plant disease con-
trol. J Invertebr Pathol 98:267–270. https://doi.org/10.
1016/j.jip.2008.01.010
Panavas T, Serviene E, Brasher J, Nagy PD (2005) Yeast gen-
ome-wide screen reveals dissimilar sets of host genes
affecting replication of RNA viruses. Proc Natl Acad Sci
102:7326–7331. https://doi.org/10.1073/pnas.0502604102
Pavan S, Jacobsen E, Visser RGF, Bai Y (2010) Loss of sus-
ceptibility as a novel breeding strategy for durable and
broad-spectrum resistance. Mol Breed 25:1–12. https://doi.
org/10.1007/s11032-009-9323-6
Peng A, Chen S, Lei T et al (2017) Engineering canker-resistant
plants through CRISPR/Cas9-targeted editing of the sus-
ceptibility gene CsLOB1 promoter in citrus. Plant
Biotechnol J 15:1509–1519. https://doi.org/10.1111/pbi.
12733
Pessina S, Lenzi L, Perazzolli M et al (2016) Knockdown of
MLO genes reduces susceptibility to powdery mildew in
grapevine. Hortic Res 3:16016. https://doi.org/10.1038/
hortres.2016.16
Petersen M, Brodersen P, Naested H et al (2000) Arabidopsis
MAP kinase 4 negatively regulates systemic acquired
resistance. Cell 103:1111–1120. https://doi.org/10.1016/
S0092-8674(00)00213-0
Pfeilmeier S, George J, Morel A et al (2019) Expression of the
Arabidopsis thaliana immune receptor EFR in Medicago
truncatula reduces infection by a root pathogenic bac-
terium, but not nitrogen-fixing rhizobial symbiosis. Plant
Biotechnol J 17:569–579. https://doi.org/10.1111/pbi.
12999
Phukan UJ, Jeena GS, Tripathi V, Shukla RK (2017) Regulation
of Apetala2/Ethylene response factors in plants. Front
Plant Sci 8:150. https://doi.org/10.3389/fpls.2017.00150
Pramanik D, Shelake RM, Park J et al (2021) CRISPR/Cas9-
mediated generation of pathogen-resistant tomato against
tomato yellow leaf curl virus and powdery mildew. Internat
J Mol Scie 22:1878. https://doi.org/10.3390/ijms22041878
Pyott DE, Sheehan E, Molnar A (2016) Engineering of CRISPR/
Cas9-mediated potyvirus resistance in transgene-free
Arabidopsis plants. Mol Plant Pathol 17:1276–1288.
https://doi.org/10.1111/mpp.12417
Qiao Y, Liu L, Xiong Q et al (2013) Oomycete pathogens
encode RNA silencing suppressors. Nat Genet
45:330–333. https://doi.org/10.1038/ng.2525
Rairdan GJ, Collier SM, Sacco MA et al (2008) The coiled-coil
and nucleotide binding domains of the potato Rx disease
resistance protein function in pathogen recognition and
signaling. Plant Cell 20:739–751. https://doi.org/10.1105/
tpc.107.056036
Rakha M, Bouba N, Ramasamy S et al (2017) Evaluation of wild
tomato accessions (Solanum spp.) for resistance to two-
spotted spider mite (Tetranychus urticae Koch) based on
trichome type and acylsugar content. Genet Resour Crop
Evol 64:1011–1022. https://doi.org/10.1007/s10722-016-
0421-0
Rohrlich C, Merle I, Hassani IM et al (2018) Variation in
physiological host range in three strains of two species of
the entomopathogenic fungus Beauveria. PLoS ONE
13:e0199199. https://doi.org/10.1371/journal.pone.
0199199
Ro
¨mer P, Recht S, Strauß T et al (2010) Promoter elements of
rice susceptibility genes are bound and activated by
specific TAL effectors from the bacterial blight pathogen,
Xanthomonas Oryzae Pv Oryzae. New Phytol
187:1048–1057. https://doi.org/10.1111/j.1469-8137.
2010.03217.x
Roy A, Zhai Y, Ortiz J et al (2019) Multiplexed editing of a
begomovirus genome restricts escape mutant formation
and disease development. PLoS ONE 14:e0223765. https://
doi.org/10.1371/journal.pone.0223765
Rybicki EP (2019) CRISPR–Cas9 strikes out in cassava. Nat
Biotechnol 37:727–728. https://doi.org/10.1038/s41587-
019-0169-0
Ryder LS, Harris BD, Soanes DM et al (2012) Saprotrophic
competitiveness and biocontrol fitness of a genetically
modified strain of the plant-growth-promoting fungus
Trichoderma hamatum GD12. Microbiology. https://doi.
org/10.1099/mic.0.051854-0
Santilla
´n Martı
´nez MI, Bracuto V, Koseoglou E et al (2020)
CRISPR/Cas9-targeted mutagenesis of the tomato sus-
ceptibility gene PMR4 for resistance against powdery
mildew. BMC Plant Biol 20:284. https://doi.org/10.1186/
s12870-020-02497-y
Savary S, Willocquet L, Pethybridge SJ et al (2019) The global
burden of pathogens and pests on major food crops. Nat
Ecol Evol 3:430–439. https://doi.org/10.1038/s41559-018-
0793-y
Savenkov EI, Valkonen JPT (2001) Coat protein gene-mediated
resistance to Potato virus A in transgenic plants is sup-
pressed following infection with another potyvirus. J Gen
Virol 82:2275–2278. https://doi.org/10.1099/0022-1317-
82-9-2275
Schwartz AR, Potnis N, Timilsina S et al (2015) Phylogenomics
of Xanthomonas field strains infecting pepper and tomato
reveals diversity in effector repertoires and identifies
determinants of host specificity. Front Microbiol 6:535.
https://doi.org/10.3389/fmicb.2015.00535
Schwessinger B, Bahar O, Thomas N et al (2015) Transgenic
expression of the dicotyledonous pattern recognition
receptor EFR in rice leads to ligand-dependent activation
of defense responses. PLoS Pathog 11:e1004809. https://
doi.org/10.1371/journal.ppat.1004809
Scorza R, Ravelonandro M, Callahan AM et al (1994) Trans-
genic plums (Prunus domestica L.) express the plum pox
virus coat protein gene. Plant Cell Rep 14:18–22. https://
doi.org/10.1007/BF00233291
Shan Q, Wang Y, Li J et al (2013) Targeted genome modifica-
tion of crop plants using a CRISPR-Cas system. Nat
Biotechnol 31:686–688. https://doi.org/10.1038/nbt.2650
Shang Y, Duan Z, Huang W et al (2012) Improving UV resis-
tance and virulence of Beauveria bassiana by genetic
engineering with an exogenous tyrosinase gene. J Invertebr
Pathol 109:105–109. https://doi.org/10.1016/j.jip.2011.10.
004
Shen X, Liu H, Yuan B et al (2011) OsEDR1 negatively regu-
lates rice bacterial resistance via activation of ethylene
biosynthesis. Plant Cell Environ 34:179–191. https://doi.
org/10.1111/j.1365-3040.2010.02219.x
123
Transgenic Res

Shi Z, Zhang Y, Maximova SN, Guiltinan MJ (2013) TcNPR3
from Theobroma cacao functions as a repressor of the
pathogen defense response. BMC Plant Biol 13:204.
https://doi.org/10.1186/1471-2229-13-204
So Y, Park S-Y, Park E-H et al (2017) A highly efficient
CRISPR-Cas9-mediated large genomic deletion in Bacil-
lus subtilis. Front Microbiol 8:1167. https://doi.org/10.
3389/fmicb.2017.01167
Song J-J, Smith SK, Hannon GJ, Joshua-Tor L (2004) Crystal
structure of argonaute and its implications for RISC slicer
Activity. Science 305:1434–1437. https://doi.org/10.1126/
science.1102514
Spassova MI, Prins TW, Folkertsma RT et al (2001) The tomato
gene Sw5 is a member of the coiled coil, nucleotide
binding, leucine-rich repeat class of plant resistance genes
and confers resistance to TSWV in tobacco. Mol Breed
7:151–161. https://doi.org/10.1023/A:1011363119763
St Leger RJ, Joshi L, Bidochka MJ, Roberts DW (1996) Con-
struction of an improved mycoinsecticide overexpressing a
toxic protease. Proc Natl Acad Sci U S A 93:6349–6354.
https://doi.org/10.1073/pnas.93.13.6349
St Leger RJ, Wang C (2010) Genetic engineering of fungal
biocontrol agents to achieve greater efficacy against insect
pests. Appl Microbiol Biotechnol 85(901):907. https://doi.
org/10.1007/s00253-009-2306-z
Streubel J, Pesce C, Hutin M et al (2013) Five phylogenetically
close rice SWEET genes confer TAL effector-mediated
susceptibility to Xanthomonas oryzae pv. oryzae. New
Phytol 200:808–819. https://doi.org/10.1111/nph.12411
Sun Q, Lin L, Liu D et al (2018) CRISPR/Cas9-mediated
multiplex genome editing of the BnWRKY11 and
BnWRKY70 genes in Brassica napus L. Int J Mol Sci
19:2716. https://doi.org/10.3390/ijms19092716
Swiderski MR, Birker D, Jones JDG (2009) The TIR domain of
TIR-NB-LRR resistance proteins is a signaling domain
involved in cell death induction. Mol Plant-Microbe
Interact 22:157–165. https://doi.org/10.1094/MPMI-22-2-
0157
Tabara H, Sarkissian M, Kelly WG et al (1999) The rde-1 gene,
RNA interference, and transposon silencing in C. elegans.
Cell 99:123–132. https://doi.org/10.1016/S0092-
8674(00)81644-X
Tashkandi M, Ali Z, Aljedaani F et al (2018) Engineering
resistance against Tomato yellow leaf curl virus via the
CRISPR/Cas9 system in tomato. Plant Signal Behav
13:e1525996. https://doi.org/10.1080/15592324.2018.
1525996
Thordal-Christensen H (2020) A holistic view on plant effector-
triggered immunity presented as an iceberg model. Cell
Mol Life Sci. https://doi.org/10.1007/s00018-020-03515-
w
Toopaang W, Phonghanpot S, Punya J et al (2017) Targeted
disruption of the polyketide synthase gene pks15 affects
virulence against insects and phagocytic survival in the
fungus Beauveria bassiana. Fungal Biol 121:664–675.
https://doi.org/10.1016/j.funbio.2017.04.007
Tre˛ bicki P, Finlay K (2018) Pests and diseases under climate
change; its threat to food security. In: Food security and
climate change. Chichester: John Wiley and Sons Ltd.
229–249. https://doi.org/10.1002/9781119180661.ch11
Tripathi JN, Ntui VO, Ron M et al (2019) CRISPR/Cas9 editing
of endogenous banana streak virus in the B genome of
Musa spp. overcomes a major challenge in banana breed-
ing. Commun Biol. 2:46. https://doi.org/10.1038/s42003-
019-0288-7
Trujillo M, Ichimura K, Casais C, Shirasu K (2008) Negative
regulation of PAMP-triggered immunity by an E3 ubiquitin
ligase triplet in Arabidopsis. Curr Biol 18:1396–1401.
https://doi.org/10.1016/j.cub.2008.07.085
Tyagi S, Kesiraju K, Saakre M et al (2020) Genome editing for
resistance to insect pests: an emerging Tool for crop
improvement. ACS Omega 5:20674–20683. https://doi.
org/10.1021/acsomega.0c01435
Uchiyama A, Shimada-Beltran H, Levy A et al (2014) The
Arabidopsis synaptotagmin SYTA regulates the cell-to-
cell movement of diverse plant viruses. Front Plant Sci
5:584. https://doi.org/10.3389/fpls.2014.00584
Udompaisarn S, Toopaang W, Sae-Ueng U et al (2020) The
polyketide synthase PKS15 has a crucial role in cell wall
formation in Beauveria bassiana. Sci Rep 10:12630.
https://doi.org/10.1038/s41598-020-69417-w
van der Krol AR, Mur LA, Beld M et al (1990) Flavonoid genes
in petunia: addition of a limited number of gene copies may
lead to a suppression of gene expression. Plant Cell
2:291–299. https://doi.org/10.1105/tpc.2.4.291
van Schie CCN, Takken FLW (2014) Susceptibility genes 101:
how to be a good host. Annu Rev Phytopathol 52:551–581.
https://doi.org/10.1146/annurev-phyto-102313-045854
Verma M, Brar SK, Tyagi RD et al (2007) Antagonistic fungi,
Trichoderma spp.: Panoply of biological control. Biochem
Eng J 37:1–20. https://doi.org/10.1016/j.bej.2007.05.012
Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of
gene silencing: a general strategy used by diverse DNA and
RNA viruses of plants. Proc Natl Acad Sci U S A
96:14147–14152. https://doi.org/10.1073/pnas.96.24.
14147
Wang M, Dean RA (2020) Movement of small RNAs in and
between plants and fungi. Mol Plant Pathol 21:589–601.
https://doi.org/10.1111/mpp.12911
Wang C, St Leger RJ (2007) A scorpion neurotoxin increases the
potency of a fungal insecticide. Nat Biotechnol
25:1455–1456. https://doi.org/10.1038/nbt1357
Wang Y, Wang Y (2018) Trick or treat: Microbial pathogens
evolved apoplastic effectors modulating plant susceptibil-
ity to infection. Mol Plant-Microbe Interact 31:6–12.
https://doi.org/10.1094/MPMI-07-17-0177-FI
Wang Y, Cheng X, Shan Q et al (2014) Simultaneous editing of
three homoeoalleles in hexaploid bread wheat confers
heritable resistance to powdery mildew. Nat Biotechnol
32:947–951. https://doi.org/10.1038/nbt.2969
Wang F, Wang C, Liu P et al (2016a) Enhanced rice blast
resistance by CRISPR/ Cas9-Targeted mutagenesis of the
ERF transcription factor gene OsERF922. PLoS ONE
11:e0154027. https://doi.org/10.1371/journal.pone.
0154027
Wang G, Liu Z, Lin R et al (2016b) Biosynthesis of antibiotic
leucinostatins in bio-control fungus Purpureocillium
lilacinum and their inhibition on Phytophthora revealed by
genome mining. PLoS Pathog 12:e1005685. https://doi.
org/10.1371/journal.ppat.1005685
123
Transgenic Res

Wang X, Guo R, Tu M et al (2017) Ectopic expression of the
wild grape WRKY transcription factor vqWRKY52 in
Arabidopsis thaliana enhances resistance to the biotrophic
pathogen powdery mildew but not to the necrotrophic
pathogen botrytis cinerea. Front Plant Sci 8:97. https://doi.
org/10.3389/fpls.2017.00097
Wang X, Tu M, Wang D et al (2018) CRISPR/Cas9-mediated
efficient targeted mutagenesis in grape in the first genera-
tion. Plant Biotechnol J 16:844–855. https://doi.org/10.
1111/pbi.12832
Wang J, Hu M, Wang J et al (2019) Reconstitution and structure
of a plant NLR resistosome conferring immunity. Science
364:eaav5870. https://doi.org/10.1126/science.aav5870
Wang L, Chen S, Peng A et al (2019b) CRISPR/Cas9-mediated
editing of CsWRKY22 reduces susceptibility to Xan-
thomonas citri subsp. citri in Wanjincheng orange (Citrus
sinensis (L.) Osbeck). Plant Biotechnol Rep 13:501–510.
https://doi.org/10.1007/s11816-019-00556-x
Whitham S, Dinesh-Kumar SP, Choi D et al (1994) The product
of the tobacco mosaic virus resistance gene N: similarity to
toll and the interleukin-1 receptor. Cell 78:1101–1115.
https://doi.org/10.1016/0092-8674(94)90283-6
Whitham S, McCormick S, Baker B (1996) The N gene of
tobacco confers resistance to tobacco mosaic virus in
transgenic tomato. Proc Natl Acad Sci 93:8776–8781.
https://doi.org/10.1073/pnas.93.16.8776
Windbichler N, Menichelli M, Papathanos PA et al (2011) A
synthetic homing endonuclease-based gene drive system in
the human malaria mosquito. Nature 473:212–215. https://
doi.org/10.1038/nature09937
Won S-J, Kwon J-H, Kim D-H, Ahn Y-S (2019) The Effect of
Bacillus licheniformis MH48 on control of foliar fungal
diseases and growth promotion of Camellia oleifera
seedlings in the coastal reclaimed land of Korea. Pathogens
8:6. https://doi.org/10.3390/pathogens8010006
Xu Z, Xu X, Gong Q et al (2019) Engineering broad-spectrum
bacterial blight resistance by simultaneously disrupting
variable TALE-binding elements of multiple susceptibility
genes in rice. Mol Plant 12:1434–1446. https://doi.org/10.
1016/j.molp.2019.08.006
Xue W-H, Xu N, Yuan X-B et al (2018) CRISPR/Cas9-medi-
ated knockout of two eye pigmentation genes in the brown
planthopper, Nilaparvata lugens (Hemiptera: Delphaci-
dae). Insect Biochem Mol Biol 93:19–26. https://doi.org/
10.1016/j.ibmb.2017.12.003
Yadav BC, Veluthambi K, Subramaniam K (2006) Host-gen-
erated double stranded RNA induces RNAi in plant-para-
sitic nematodes and protects the host from infection. Mol
Biochem Parasitol 148:219–222. https://doi.org/10.1016/j.
molbiopara.2006.03.013
Yang Z, Li Y (2018) Dissection of RNAi-based antiviral
immunity in plants. Curr Opin Virol 32:88–99. https://doi.
org/10.1016/j.coviro.2018.08.003
Yi Y, Li Z, Song C, Kuipers OP (2018) Exploring plant-microbe
interactions of the rhizobacteria Bacillus subtilis and
Bacillus mycoides by use of the CRISPR-Cas9 system.
Environ Microbiol 20:4245–4260. https://doi.org/10.1111/
1462-2920.14305
Yuan T, Li X, Xiao J, Wang S (2011) Characterization of
Xanthomonas oryzae-responsive cis-acting element in the
promoter of rice race-specific susceptibility gene Xa13.
Mol Plant 4:300–309. https://doi.org/10.1093/mp/ssq076
Zafar K, Khan MZ, Amin I et al (2020) Precise CRISPR-Cas9
mediated genome editing in super basmati rice for resis-
tance against bacterial blight by targeting the major sus-
ceptibility gene. Front Plant Sci 11:575. https://doi.org/10.
3389/fpls.2020.00575
Zaidi SSA, Mukhtar MS, Mansoor S (2018) Genome editing:
targeting susceptibility genes for plant disease resistance.
Trends Biotechnol 36:898–906. https://doi.org/10.1016/j.
tibtech.2018.04.005
Zambryski P, Joos H, Genetello C et al (1983) Ti plasmid vector
for the introduction of DNA into plant cells without alter-
ation of their normal regeneration capacity. EMBO J
2:2143–2150. https://doi.org/10.1002/j.1460-2075.1983.
tb01715.x
Zeng X, Luo Y, Vu NTQ et al (2020) CRISPR/Cas9-mediated
mutation of OsSWEET14 in rice cv. Zhonghua11 confers
resistance to Xanthomonas oryzae pv. oryzae without yield
penalty. BMC Plant Biol. 20:313. https://doi.org/10.1186/
s12870-020-02524-y
Zhan X, Zhang F, Zhong Z et al (2019) Generation of virus-
resistant potato plants by RNA genome targeting. Plant
Biotechnol J 17:1814–1822. https://doi.org/10.1111/pbi.
13102
Zhan Y, Xu Y, Zheng P et al (2020) Establishment and appli-
cation of multiplexed CRISPR interference system in
Bacillus licheniformis. Appl Microbiol Biotechnol
104:391–403. https://doi.org/10.1007/s00253-019-10230-
5
Zhang K, Duan X, Wu J (2016) Multigene disruption in undo-
mesticated Bacillus subtilis ATCC 6051a using the
CRISPR/Cas9 system. Sci Rep 6:27943. https://doi.org/10.
1038/srep27943
Zhang Y, Bai Y, Wu G et al (2017a) Simultaneous modification
of three homoeologs of TaEDR1 by genome editing
enhances powdery mildew resistance in wheat. Plant J
91:714–724. https://doi.org/10.1111/tpj.13599
Zhang Y, Zhao L, Zhao J et al (2017b) S5H/DMR6 Encodes a
salicylic acid 5-hydroxylase that fine-tunes salicylic acid
homeostasis. Plant Physiol 175:1082–1093. https://doi.org/
10.1104/pp.17.00695
Zhang Z, Ge X, Luo X et al (2018) Simultaneous editing of two
copies of GH14-3-3D confers enhanced transgene-clean
plant defense against Verticillium dahliae in allotetraploid
upland cotton. Front Plant Sci 9:842. https://doi.org/10.
3389/fpls.2018.00842
Zhang T, Zhao Y, Ye J et al (2019) Establishing CRISPR/
Cas13a immune system conferring RNA virus resistance in
both dicot and monocot plants. Plant Biotechnol J
17:1185–1187. https://doi.org/10.1111/pbi.13095
Zhang M, Liu Q, Yang X et al (2020a) CRISPR/Cas9-mediated
mutagenesis of Clpsk1 in watermelon to confer resistance
to Fusarium oxysporum f.sp. niveum. Plant Cell Rep
39:589–595. https://doi.org/10.1007/s00299-020-02516-0
Zhang Y, Pribil M, Palmgren M, Gao C (2020b) A CRISPR way
for accelerating improvement of food crops. Nat Food
1:200–205. https://doi.org/10.1038/s43016-020-0051-8
Zhou J, Peng Z, Long J et al (2015) Gene targeting by the TAL
effector PthXo2 reveals cryptic resistance gene for
123
Transgenic Res

bacterial blight of rice. Plant J 82:632–643. https://doi.org/
10.1111/tpj.12838
Zhou C, Zhu L, Xie Y et al (2017) Bacillus licheniformis SA03
confers increased saline–alkaline Tolerance in chrysan-
themum plants by induction of abscisic acid accumulation.
Front Plant Sci 8:1143. https://doi.org/10.3389/fpls.2017.
01143
Zhou X, Liao H, Chern M et al (2018) Loss of function of a rice
TPR-domain RNA-binding protein confers broad-spec-
trum disease resistance. Proc Natl Acad Sci
115:3174–3179. https://doi.org/10.1073/pnas.1705927115
Zhou C, Liu H, Yuan F et al (2019) Development and applica-
tion of a CRISPR/Cas9 system for Bacillus licheniformis
genome editing. Int J Biol Macromol 122:329–337. https://
doi.org/10.1016/j.ijbiomac.2018.10.170
Zhu Z, Yin J, Chern M et al (2020) New insights into bsr-d1-
mediated broad-spectrum resistance to rice blast. Mol Plant
Pathol 21:951–960. https://doi.org/10.1111/mpp.12941
Zipfel C, Kunze G, Chinchilla D et al (2006) Perception of the
bacterial PAMP EF-Tu by the receptor EFR Restricts
Agrobacterium-mediated transformation. Cell
125:749–760. https://doi.org/10.1016/j.cell.2006.03.037
Zou G, Ying SH, Shen ZC, Feng MG (2006) Multi-sited
mutations of beta-tubulin are involved in benzimidazole
resistance and thermotolerance of fungal biocontrol agent
Beauveria bassiana. Environ Microbiol 8:2096–2105.
https://doi.org/10.1111/j.1462-2920.2006.01086.x
Zso
¨go
¨nA,C
ˇerma
´k T, Naves ER et al (2018) De novo domes-
tication of wild tomato using genome editing. Nat
Biotechnol 36:1211–1216. https://doi.org/10.1038/nbt.
4272
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.
123
Transgenic Res