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VdNop12, containing two tandem RNA recognition motif
domains, is a crucial factor for pathogenicity and cold
adaption in Verticillium dahliae
Jun Zhang,
†
Weiye Cui,
†
Hafiz Abdul Haseeb and
Wei Guo *
Key Laboratory of Agro-products Quality and Safety
Control in Storage and Transport Process, Ministry of
Agriculture and Rural Affairs, Institute of Food Science
and Technology, Chinese Academy of Agricultural
Sciences, Beijing, China.
Summary
Previous studies have reported the ability of fungi to
overwinter in soil or on crop debris under different
environmental conditions, but how fungi adapt to
chilling is still largely unknown. In this study, we
have identified and characterized the RNA binding
protein (RBP) (VdNop12) by screening an
Agrobacterium tumefaciens-mediated transformation-
mediated insertional mutational library of Verticillium
dahliae. We determined that this protein was essen-
tial to the pathogen for virulence on cotton plants.
VdNop12 contains two tandem RNA recognition motif
domains, and its orthologs are widely distributed in
filamentous fungi. Mutants produced by disruption of
VdNop12 showed defects in vegetative growth, con-
idiation and cell wall integrity. The mutant also
showed an increase in sensitivity to low temperature,
as compared to the wildtype and complementation
strains. Yeast complementation assay showed that
VdNop12 could functionally restore the growth phe-
notype of ΔScNop12 mutant of Saccharomyces
cerevisiae at 15C. We demonstrated that the
VdNop12 is localized in the nucleus, and its loss
resulted in the downregulated expression of several
genes related to cAMP-PKA and MAPK pathways in
V. dahliae. Our results demonstrated a crucial role of
RBPs in the regulation of morphology, cold adaption,
and pathogenic development in V. dahliae.
Introduction
Verticillium wilt is a widespread vascular fungal disease
of many economically important crops, including cotton,
tomato, potato, olive, sunflower, fruit trees, shrubs and
ornamental plants (Klosterman et al., 2011). The causa-
tive agents of this disease are soil-borne species of the
genus Verticillium, mainly Verticillium dahliae,V. albo-
atrum and V. longisporum. Among them, V. dahliae has
the broadest host range, with the capacity to infect more
than 200 dicotyledonous plant species (Klosterman
et al., 2009; Inderbitzin and Subbarao, 2014). This fungus
differs from other Verticillium species because of its abil-
ity to form microsclerotia. Microsclerotia are the main
infective inoculum of the pathogen and can survive in soil
for more than 10 years (Wilhelm, 1955). They can germi-
nate multiple times in response to root exudates under
favourable soil environmental conditions and form
hyphae to penetrate into the plant roots and reach the
xylem vessels (Calderón et al., 2014). Once they enter
the xylem vessels, the conidia of V. dahliae are trans-
ported upwards through them. The growth of V. dahliae
is favoured by cool spring weather, but the disease can
also develop at temperatures up to 30C. This phenome-
non indicates that V. dahliae can adapt to a broad range
of temperature changes under various environmental
conditions. We surmise this wide range of temperature
adaptability is one of the primary reasons why V. dahliae
has a worldwide distribution, present in both temperate
and tropical regions. However, despite its ubiquity, very
little research has been done on the mechanisms by
which this fungus adapt to cold stress.
Most studies on cold responses in fungi have focused
on yeast. These studies have demonstrated that RNA
binding proteins (RBPs) play critical roles in mediating
yeasts’ability to adapt to the cold. RBPs are character-
ized as containing one or more specific RNA binding
domains (RBDs). Common examples include the RNA
recognition motif (RRM) domain, glycine-rich domain
(GRD), K homology domain, RGG (Arg-Gly-Gly) box, zinc
fingers and cold shock domains (CSDs) (Mackereth and
Sattler, 2012). RBPs have critical functions in the regula-
tion of gene expression in eukaryotic cells, such as
Received 19 November, 2019; revised 15 September, 2020;
accepted 28 September, 2020. *For correspondence. E-mail
guowei01@caas.cn; Tel. (+86) 10 6281 5925; Fax (+86) 10 6281 5925.
†
These authors contributed equally to this work.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd
Environmental Microbiology (2020) 00(00), 00–00 doi:10.1111/1462-2920.15268
alternative splicing, RNA editing, mRNA transport and
modulation of mRNA translation and decay (Zhang
et al., 2016b).
Research on RBPs in filamentous fungi is still infancy,
below are several of the relatively few examples of their
effect on biological functions in various pathogens. In rice
blast fungus (Magnaporthe oryzae), the loss of RBP35
caused the defects in mycelial growth, infection-related
development and pathogenicity via alternative processing
of the 30-end of the target pre-mRNAs (Franceschetti
et al., 2011). Similarly, MoGrp1 of M. oryzae is a glycine-
rich protein and has been reported to function as a novel
splicing factor with poly(U) binding activity to regulate fun-
gal virulence, development, and stress responses (Gao
et al., 2019). In Fusarium graminearum (the primary
causal agent of wheat head blight), FgSRP1, FgSRP2
and FgSPK of serine/arginine-rich proteins containing
RRMs and serine/arginine-rich domains were found to be
essential for plant infection and in pre-mRNA processing
(Zhang et al., 2017; Wang et al., 2018; Zhang
et al., 2020). For the corn smut pathogen Ustilago
maydis, the loss of Kdh1 (a K homology protein) resulted
in a cold-sensitive phenotype. Furthermore, the deletion
of Khd4 led to abnormal cell morphology and reduced vir-
ulence (Becht et al., 2005). In Metarhizium anispliae
(a naturally occurring pathogen of mosquitoes), CRP1
containing CSD and CRP2 containing GRD was shown
to contribute to cold and freezing tolerance (Fang and St
Leger, 2010). While limited in number, these studies indi-
cate that RBPs may have crucial roles in the develop-
ment of fungal diseases.
In this study, we have identified an RBP
(VDAG_03303, designated as VdNop12) in V. dahliae,
which is required for pathogenicity, vegetative growth,
conidiation and cell wall stress response. We also dem-
onstrated that VdNop12 is a nuclear-localized protein that
not only affects low-temperature responses but also regu-
lates expression of genes involved in cAMP-PKA and
MAPK pathways in V. dahliae. Yeast complementation
assays showed that VdNop12 could functionally restore
the growth phenotype of ΔScNop12 mutant of Saccharo-
myces cerevisiae at 15C. Taken together, these results
demonstrated that RBPs affect morphological develop-
ment, stress response and infection-related processes in
V. dahliae.
Results
Identification of M11H01 mutant with defects in virulence
caused by a single T-DNA insertion
In the first round of screening, we used three cotton
seedlings to evaluate the change in virulence for each of
288 transformants through root-dip assays. Of these, five
mutants were found to be potentially defective for viru-
lence on cotton plants. To verify these results, each of
the five mutants was re-assessed for virulence using
16 cotton seedlings followed by statistical analysis. One
mutant (M11H01) consistently displaying significantly
reduced virulence on cotton plants compared to wildtype
strain Vd991 was selected for further investigations. At
25 days post-inoculation (dpi), plants inoculated with the
wildtype strain Vd991 showed severe necrosis and
wilting of leaves. Whereas, plants inoculated with
M11H01 showed substantially attenuated symptoms
(Fig. 1A). After 9 dpi, the disease index (DI) of cotton
inoculated with M11H01 was also significantly lower than
that of the plants inoculated with wildtype strain. At
25 dpi, the DI of plants inoculated with M11H01 was
approximately 55%, much less as compared to the >90%
of the plants inoculated with wildtype strain (Fig. 1B). To
confirm the copy number of the T-DNA insertion in
M11H01 mutant, southern hybridization was performed
using a randomly DIG-labelled HPH probe after geno-
mics DNA digestion by BamH I, which does not cut within
the T-DNA. Only a single band was observed in the
mutant strain. In contrast, no band was observed for the
wildtype strain, indicating that the M11H01 mutant has a
single T-DNA insertion (Fig. 2A). The insertion locus was
identified using a high-efficiency thermal asymmetric
Fig. 1. The V. dahliae T-DNA mutant (M11H01) shows pathogenicity
defects on cotton plants.
A. Symptoms of susceptible cotton plants (G. hirsutum cv. ‘Junmian
No.1’) infected with the wildtype Vd991, and T-DNA mutant
M11H01 at 25 days after inoculation.
B. The disease index of plants was evaluated after inoculation with
Vd991 and M11H01. The values are mean ± sd, n=3.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
2J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
interlaced PCR (hiTAIL-PCR) with degenerate primers as
previously described (Liu and Chen, 2007). Flanking
sequence analysis of T-DNA insertion sites revealed that
the functional gene (VdNop12, 100% identity with
VDAG_03303) was disrupted by the insertion events.
Further analysis of gene structure revealed that
VDAG_03303 (VdNop12) contains four exons and
encodes a protein consisting of 550 amino acids (http://
fungi.ensembl.org). The T-DNA insert was located in the
third exon of VdNop12. The insertion pattern was con-
firmed by PCR amplification with combinations of outside
primer pairs Y0042_F/R. From these results, a schematic
diagram of the T-DNA insertion pattern in M11H01 was
configured based on V. dahliae VdLs.17 genome infor-
mation (Fig. 2B). Junction sequences between the T-
DNA and the V. dahliae genome revealed that a single
base A on the wildtype genome was squeezed out
(Fig. 2C). RT-PCR was further used to compare the
expression of VdNop12 in the M11H01 mutant and the
wildtype strain Vd991. The expression of VdNop12 was
completely aborted by the T-DNA insertion (Fig. 2D).
Structural organization and phylogenetic analysis of
VdNop12
To predict the biochemical function of VdNop12, the
amino acid sequence was analysed using HMMSCAN
(https://www.ebi.ac.uk). The results showed that VdNop12
contains a coiled-coil region, an RRM1_Nop12p_like
domain and an RRM_SF domain (Fig. 3A). RRM
1_Nop12p_like domain represents the RRM 1 domain of
yeast nucleolar protein 12 (Nop12p). RRM_SF means this
domain belonging to RRM superfamily. Structural-model
analysis of VdNop12 has been performed using the
I-TASSER software tool (https://zhanglab.ccmb.med.
umich.edu/I-TASSER/). The best-predicted model (C-scor
e=−2.95) suggests that most of the amino acids of the
two tandem RRM domains were buried inside the protein
for dimerization. The model also suggests that the
N-terminal region of VdNop12 forms a negative charge
platform flanking the RRM1_Nop12p_like domain,
whereas the C-terminal region forms a positively charged
platform adjacent to the RRM_SF domain (Fig. 3B) for
binding to the RNA. All orthologues of VdNop12 were
predicted to possess at least one RRM domain. Phyloge-
netic analysis also revealed a broad distribution of
VdNop12 orthologues in ascomycetes from nonpatho-
genic to pathogenic fungi, including Nop12p from Saccha-
romyces cerevisiae with 31% similarity (Fig. 3C).
VdNop12 plays an important role in virulence to cotton
In order to confirm that the phenotypic alternations in the
mutant (M11H01) were caused by the disruption of this
gene, we generated ectopic complementation strains of
M11H01 (EC-M11H01), along with VdNop12 deletion
mutant (ΔVdNop12) and its ectopic complementation
strain EC-ΔVdNop12. Susceptible cotton seedlings
(Gossypium hirsutum cv. ‘Junmian No.1’) were inocu-
lated with the spore suspension from Vd991, M11H01,
EC-M11H01, ΔVdNop12 and EC-ΔVdNop12 strains.
Each strain was used to infect 16 cotton seedlings. The
Fig. 2. Analysis of T-DNA tagged gene in the mutant M11H01.
A. Southern blot hybridization was used to analyse the T-DNA copy number in M11H01. Total genomic DNA of the wildtype Vd991 (lane 1) and
mutant M11H01 (lane 2) were digested with BamH I and were then hybridized by a randomly DIG-labelled HPH probe.
B. Schematic diagram of T-DNA insertion in mutant M11H01. LB, left border region of T-DNA. RB, right border region of T-DNA. The small black
arrows demonstrated the position of primer pairs Y0042_F/R, which amplify the junction sequences between the T-DNA and the V. dahliae
genome.
C. Sequences of the T-DNA junction sites. Sequences of both junctions between T-DNA and the V. dahliae genome are indicated. Typical left-
border cleavage site (5) and a filter DNA (red) are indicated.
D. The transcriptional expression of VdNop12 (VDAG_03303) in M11H01 was determined by RT-PCR. Lane 1, the wildtype Vd991; lane 2, the
mutant M11H01.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 3
cotton plants inoculated with the Vd991 and complemen-
tation strains (EC-M11H01 or EC-ΔVdNop12) showed
severe wilting and necrosis of the leaves and even defoli-
ation at 25 dpi. In marked contrast, seedlings inoculated
with the M11H01 or ΔVdNop12 strain showed substan-
tially reduced symptoms (Fig. 4A). Disease index analy-
sis revealed that there was no noticeable difference
among all the strains before 7 dpi. However, after 9 dpi
the DI values of plants inoculated with wildtype strain
Vd991 and the two complementation strains (EC-
M11H01 or EC-ΔVdNop12) were much higher than that
of plants infected with the M11H01 or ΔVdNop12
mutants. At 25 dpi, the DI values for M11H01 and
ΔVdNop12 mutants were approximately 55% and 47%,
markedly lower than that for the wildtype and complemen-
tation strains (>90%) (Fig. 4B). These results indicated that
VdNop12 plays a vital role in virulence of the pathogen to
cotton plants. Fungal outgrowth assays were also per-
formed at 25 dpi by plating of the stem sections above coty-
ledons on Potato Dextrose Agar (PDA) (Fig. 4C). The
assay demonstrated that the M110H1 or ΔVdNop12
mutants were still able to colonize cotton plants. Real-time
PCR confirms that the content of fungal biomass was signif-
icantly reduced in M11H01 (88.10%) and ΔVdNop12
mutants (90.90%) in comparison with the wildtype strains.
However, in ectopic complementation transformants EC-
M11H01 and EC-ΔVdNop12, the biomass was reduced by
25.17% and 19.22% respectively (Fig. 4D).
To investigate the roles of VdNop12 during the initial
colonization of V. dahliae, the penetration abilities of all
the strains were also tested on a cellophane membrane
laid on minimal medium (MM). The MM is used for the
induction of appressoria formation in V. dahliae (Zhao
et al., 2016). All the strains including Vd991, M11H01,
Fig. 3. Structural organization and phylogenetic analysis of VdNop12.
A. Domain architecture of VdNop12.
B. Structure modelling of VdNop12 based on ITASSER prediction analysis.
C. Phylogenetic analysis of the VdNop12 orthologues in Ascomycota. The tree was constructed using the maximum-likelihood method. Branches
with bootstrap values >70% are shown in the phylogenetic tree. The size of the blue sphere indicates bootstrap values. Protein domain arrange-
ments of VdNop12 and its homologues are visualized by schematic representations on the right. Different colours represent different protein
domains. The colours of RRM1_Nop12p_Like, RRM_SF, Mpp10, RRM and RRM2_Nop12p_Like domains are shown on the upper side of the fig-
ure. Straight lines represent total protein length.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
4J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
EC-M11H01, ΔVdNop12 and EC-ΔVdNop12 were able
to penetrate through the cellophane membrane and
showed growth on MM (Fig. S3). These data demon-
strated that VdNop12 is not required for penetration to
the host in V. dahliae, indicating the loss of virulence in
ΔVdNop12 mutants was independent of the colonization
of cotton plants.
Deletion of VdNop12 results in the reduction of radial
growth and conidiation in V. dahliae
To analyse the function of VdNop12 in fungal growth and
conidia production, Vd991 (wildtype strain), M11H01, EC-
M11H01, ΔVdNop12 and EC-ΔVdNop12 were grown on
PDA for 7 days. The colony diameter of M11H01
(25.36 ± 0.48 mm) and ΔVdNop12 (24.25 ± 0.23 mm)
was significantly decreased as compared to the wildtype
Vd991 (41.01 ± 1.92 mm) at 7 days after inoculation.
However, the colony diameter of the wildtype and com-
plemented strains were not significantly different (Fig. 5A
and B).
To evaluate the production of conidia, 5 mm agar plugs
were individually collected from fungal colony’s edge of
Vd991 (wildtype strain), M11H01, EC-M11H01,
ΔVdNop12 and EC-ΔVdNop12 at 7 days after inocula-
tion. The number of conidia was calculated by a
haemocytometer after the agar plugs had been shaken in
1 ml of sterile water. In comparison with the wildtype, the
conidiation of M11H01 and ΔVdNop12 mutants was
Fig. 4. The loss of VdNop12 resulted in defects in pathogenicity on cotton plants.
A. Symptoms of susceptible cotton plants (G. hirsutum cv. ‘Junmian No.1’) infected with the wildtype (Vd991), M11H01, ΔVdNop12, EC-M11H01
and EC-ΔVdNop12 at 25 days after inoculation.
B. The DI curves of cotton plants inoculated with wildtype (Vd991), M11H01, ΔVdNop12, EC-M11H01 and EC-ΔVdNop12. The DI value was cal-
culated once every 2 days up to 25 days after inoculation.
C. Fungal recovery assay on plates was successful from plants infected with the wildtype (Vd991), M11H01, ΔVdNop12, EC-M11H01 and EC-
ΔVdNop12, but not from plants infected with H
2
O (Mock). Each stem section was from different plants.
D. Relative fungal biomasses of the wildtype (Vd991), M11H01, ΔVdNop12, EC-M11H01 and EC-ΔVdNop12 on cotton plants were determined
by qPCR. Error bars represent standard error, and high statistical significance (p< 0.01) was indicated with double asterisks (**).
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 5
reduced by 23.5% and 28.4% respectively (Fig. 5C).
Whereas, both complementation strains (EC-M11H01
and EC-ΔVdNop12) produced similar conidiation as the
wildtype strain (Fig. 5C). Take together, these results
indicated that VdNop12 has a significant role in the regu-
lation of vegetative growth and conidiation of V. dahliae.
The ΔVdNop12 mutant increases sensitivity to cell wall
stress rather than osmotic and oxidative stress
To test whether VdNop12 is involved in the abiotic
stress response, we investigated the relative growth
inhibition (RGI) of mutant strains compared to the wil-
dtype on CM plates. The plates were supplemented
with 30 μg/ml Calcofluor white (CFW), 75 μg/ml Congo
Red (CR) and 0.01% sodium dodecyl sulphate (SDS,
cell wall stress inducer), 0.7 M NaCl and 1 M sorbitol
(osmotic pressure inducers), as well as 15 mM H
2
O
2
(oxidative stress inducer). The results indicated that
VdNop12 has a negligible impact on the response to
osmotic and oxidative stresses, and the vegetative
growth rate and RGI values were not significantly dif-
ferent among all the strains under NaCl, sorbitol, or
H
2
O
2
treatments. However, in the presence of CFW,
CR or SDS, the vegetative growth of M11H01 and
ΔVdNop12 strains were significantly inhibited com-
pared to the wildtype and complementation strains
(Fig. 6A). Similarly, in the presence of CFW, CR or
SDS, the RGI values of M11H01 were increased by
8.2%, 13.5%, and 14.3%; whereas the RGI values of
ΔVdNop12 were increased by 10.3%, 16.2% and
15.3% respectively (Fig. 6B). Furthermore, the
M11H01 and ΔVdNop12 strains formed deformity in
hyphae, whereas the wild-type and complementation
strains did not (Fig. 6C). These results suggest that
VdNop12 is involved in response to cell wall perturbing
agents.
Deletion of VdNop12 increases sensitivity to cold stress
in V. dahliae
Since the Nop12p of Saccharomyces cerevisiae, the
orthologue of VdNop12, is required for normal cellular
growth at low temperature, we performed growth rate
inhibition assays at different temperatures to determine
whether VdNop12 affects the response of V. dahliae to
low temperature in an analogous manner
(Wu et al., 2001). Vegetative growth of the M11H01 and
ΔVdNop12 mutants was slower than that of the wildtype
and complementation mutants at 4, 10, 15, 20, and 25
C (Fig. 7A). Furthermore, we measured the colony diam-
eters of all the strains under different temperature condi-
tions to calculate the RGI of mycelia. The mycelial
growth of each strain at 25 C was set as the control.
Statistical analysis indicated that both the M11H01 and
ΔVdNop12 mutants enhanced the RGI of mycelia as
compared to the wildtype colonies at all temperatures
tested. At 15 C, the highest increase in RGI for M11H01
and ΔVdNop12 was recorded at 28.3% and 35.0%
respectively (Fig. 7B).
To further verify the essential impact of VdNop12 on
cold sensitivity, we introduce VdNop12 into the
Fig. 5. The loss of VdNop12
leads to the reduction of mycelial
growth and conidiation.
A. The growth phenotype of wil-
dtype (Vd991), M11H01,
ΔVdNop12, EC-M11H01 and EC-
ΔVdNop12 strains on PDA at
7 days post inoculation.
B. Statistical analysis of the col-
ony diameters of wildtype
(Vd991), M11H01, ΔVdNop12,
EC-M11H01 and EC-ΔVdNop12.
C. Conidiation was compared
among the wildtype (Vd991),
M11H01, ΔVdNop12, EC-
M11H01 and EC-ΔVdNop12.
Error bars indicate standard error.
Single asterisk (*) indicates a sig-
nificant difference of p< 0.05.
Double asterisks (**) indicate a
highly significant difference
of p< 0.01.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
6J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
temperature-sensitive Nop12p mutant of S. cerevisiae
BY4741 (ΔScNop12) through an expression vector
(pYES2). The ΔScNop12 mutant transformed with an
empty vector pYES2 served as the negative control. It is
evident from Fig. 8A, VdNop12 can functionally restore
growth defects of ΔScNop12 mutant grown on YPgal
plates for 11 days at 15 C. However, the ΔScNop12
mutant transformed with VdNop12 showed growth
defects on YPG plates at 15 C due to the suppression
of VdNop12 by glucose (Fig. 8B). These results indicate
that VdNop12 is responsible for the growth of V. dahliae
at low temperatures.
Subcellular localization of VdNop12
The YLoc web server (https://abi-services.informatik.uni-
tuebingen.de/yloc/webloc.cgi) predicted that VdNop12
was located in the nucleus with a probability of 99.6%.
To confirm the subcellular localization of VdNop12, we
generated a VdNop12-mCherry fusion construct driven
by a fungi-specific constitutive TrpC promoter. The con-
struct was introduced into the ΔVdNop12 mutant by
homologous recombination. As expected, mCherry sig-
nals were found in the nucleus of VdNop12-mCherry
transformants. The localization was confirmed by co-
Fig. 6. Deletion of VdNop12 impairs fungal growth under cell wall stress.
A. Colony morphology of the wildtype (Vd991), M11H01, EC-M11H01, ΔVdNop12 and EC-ΔVdNop12 on CM plates supplemented with 30 μg/ml
CFW, 75 μg/ml CR or 0.01% SDS at 7 dpi.
B. Relative growth inhibition (RGI) of fungal colony growth in response to different stress agents. Error bars indicate standard error. Double aster-
isks (**) indicate a highly significant difference (p< 0.01).
C. Hyphal morphology of the five above mentioned strains. The arrows indicate the deformity of hyphal morphology in M11H01 and ΔVdNop12
strain. DE: deformity, scale bar: 20 μm.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 7
staining with nuclear targeting 4, 6-diamidino-
2-phenylindole (DAPI) (Fig. 9). The colocalization of
VdNop12-mCherry signals and DAPI indicates VdNop12
may play critical roles in the nucleus of V. dahliae.
cAMP-PKA and MAPK pathways are affected in the
ΔVdNop12 mutant
Previous studies have shown that the PKA pathway and
the MAPK signalling pathways are involved in integrating
multiple extracellular and intracellular signals to regulate
the transcription of genes that help eukaryotic cells adapt
to environmental conditions (Turrà et al., 2014). Due to
the increased sensitivity to cell wall stress and low-
temperature conditions in ΔVdNop12 mutant, reverse
transcription quantitative PCR (RT-qPCR) analyses were
performed to compare the expression of five genes
(VdMsb, VdHog1,VdPbs2,VdPKAC1 and VdPKAC2)
involved in the pathways mentioned above between wil-
dtype Vd991 and ΔVdNop12.VdMsb encodes a trans-
membrane mucin that is involved in the MAPK signal
pathway and is required for fungal virulence in V. dahliae
(Tian et al., 2014). VdHog1 and VdPbs2 are key regula-
tors of stress responses, asexual development and viru-
lence to host plants in V. dahliae (Tian et al., 2016; Wang
et al., 2016b). VdPbs2 performs its function upstream of
VdHog1 (Tian et al., 2016). VdPKAC1 and VdPKAC2 are
two PKA catalytic subunit genes in V. dahliae. Deletion of
VdPKAC1 results in a decrease of virulence, conidiation
and ethylene production in V. dahliae (Tzima
et al., 2010). RT-qPCR analysis indicated that the
expression of these five genes involved in PKA and
MAPK pathways were downregulated by the deletion of
VdNop12 (Fig. 10). In ΔVdNop12 mutant, the expression
of VdPbs2 was significantly reduced. Conversely, the
expression of VdPKAC2 was decreased minimally
(Fig. 10). These results indicate that cAMP-PKA and
MAPK pathways are affected considerably by the dele-
tion of VdNop12.
Discussion
In order to survive in changing environmental conditions,
fungi have developed sophisticated mechanisms to
sense and respond to a number of environmental factors.
Among these factors, temperature is one of the most criti-
cal factors. When the ambient temperature fluctuates
above or below certain thresholds, fungal cells mount
heat or cold shock responses. One of the best-studied
examples of heat shock response in soil fungi is Histo-
plasma capsulatum, which undergoes dramatic changes
in cell shape and virulence gene expression in response
to the elevated host temperature. A study by Beyhan
et al. (2013) demonstrated that Ryp1, Ryp2 and Ryp3
are responsible for yeast-phage growth and directly regu-
late the expression of virulence genes at 37Cin
H. capsulatum.
Conversely, cold is another unfavourable temperature
condition that fungi frequently encounter in the nature.
The physiological problems caused by cold temperature
are entirely different from the high temperature stress
(Robinson, 2001; Tiwari et al., 2015). Yet, morphological
changes and expression of virulence genes of fungi dur-
ing cold temperature conditions have been rarely
Fig. 7. The loss of VdNop12 affects cold adaption in V. dahliae.
A. Growth phenotype of the wildtype (Vd991), M11H01, ΔVdNop12, EC-M11H01 and EC-ΔVdNop12 on CM plates at the different temperature
at 7 days after inoculation.
B. Relative growth inhibition (RGI) of fungal colony growth of Vd991, M11H01, EC-M11H01, ΔVdNop12 and EC-ΔVdNop12 at 4C, 10C, 15C
and 20C. After incubation for 7 days, colony diameter in each plate was measured in two perpendicular directions without the original mycelial
plug diameter (10 mm). For each plate, the average of the colony diameters was used to calculate the percentage of growth inhibition. The colony
diameter of each strain at 25C was set as the control. Error bars indicate standard error. Double asterisks (**) indicate a highly significant differ-
ence (p< 0.01).
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
8J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
analysed. Most of the knowledge about cells response to
cold stress have been originated form prokaryotic organ-
isms and plant studies (Panoff et al., 1998; Capozzi
et al., 2011; Miura and Furumoto, 2013; Wang et al.,
2016a). The effects of cold stress on cells include
reduced enzymatic activity, decreased membrane fluidity
Fig. 8. VdNop12 restores the
growth defects of temperature-
sensitive Nop12p mutant of
S. cerevisiae at 15C.
A,B. Cells of the mutant con-
taining empty vector pYES2 or
pYES2-VdNop12 were grown for
2 or 11 days at indicated temper-
atures on (A) YPgal medium
plates or (B) YPG medium plates.
The wildtype BY4741 transformed
with an empty vector pYES2 was
used as a control. Serial 10-fold
dilutions of yeast cells were
indicated.
Fig. 9. Subcellular localization of
VdNop12 in V. dahliae. Hyphae
expressing mCherry tagged
VdNop12 were observed using a
Zeiss laser confocal microscope.
The hyphae were stained with
DAPI to observe the nucleus.
Merged images of both mCherry
and DAPI show clear nuclear
localization of VdNop12. Scale
bars: 10 μm.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 9
and stable RNA structures that interfere with RNA trans-
lation (Klinkert and Narberhaus, 2009).
RNA plays many critical roles in the regulation of gene
expression. In cells, RNA is almost always accompanied
by RBPs to regulate its cellular function and protect it
from nucleolytic degradation (Maris et al., 2005). To our
knowledge, the impact of RBPs in cold acclimation has
not been previously reported for V. dahliae. In this study,
we have identified and characterized an RBP (VdNop12)
in V. dahliae, which is required for cold adaption, the cell
wall stress response, and the induction of disease symp-
toms in cotton plants. VdNop12 contains two tandem
RRM domains, and structural simulation predicts that
VdNop12 has the capacity to bind RNA. However, we did
not directly demonstrate this binding of VdNop12 with
RNA. Previous studies have shown that the deletion of
Nop12p from S. cerevisiae resulted in the mutated strain
behaving like a traditional cold-sensitive mutant. The
mutant has a similar growth rate compared to the wil-
dtype at 30C, but the growth of the mutant was much
slower when cells were shifted to 15C (Wu et al., 2001).
Although VdNop12 only shares 31% similarity with
ScNop12p, VdNop12 can restore the growth phenotype
of ΔScNop12 strain at 15C. In S. cerevisiae, ScNop12p
is required for pre-25S rRNA processing in addition to
maintaining the normal growth of the cell at low
temperatures (Wu et al., 2001). Therefore, it is reason-
able to presume that similar defects in pre-rRNA
processing may have occurred in ΔVdNop12 strain.
Plate growth assays showed that the ΔVdNop12 strain
is only hypersensitive to cell wall stress rather than
osmotic pressure or oxidative stress, indicating that the
deletion of VdNop12 impaired cell wall integrity. cAMP-
PKA and MAPK pathways have been found to play an
essential role in signal transductions of cells when fungi
adapt to environmental changes (Klinkert and
Narberhaus, 2009). In this study, we showed that the
expression of five genes related to cAMP-PKA and
MAPK pathway was downregulated in ΔVdNop12 strain.
In fission yeast Schizosaccharomyces pombe, Rnc1, a
K-homology-type RBP, directly affects the expression of
MAPK phosphatase through binding and stabilizing
Pmp1 mRNA (Sugiura et al., 2003). Interestingly, Rnc1
was directly phosphorelated at T50 by Pmk1 MAPK to
affect its binding and stabilizing Pmp1 mRNA (Sugiura
et al., 2003). Recently, Prieto-Ruiz and colleagues
showed that Rnc1 downregulates the activity of Sty1, the
MAPK of the stress-activated MAPK pathway (SAPK),
during control of cell length at division and recovery in
response to acute stress (Prieto-Ruiz et al., 2020). These
studies suggest a functional link between MAPK signal-
ling and an RBP through mRNA stabilization of MAPK
phosphatase. Herein, it is intriguing to speculate
VdNop12 directly or indirectly responsible for the mRNA
stability of the MAPK pathway components in V. dahliae.
However, the mechanism for the downregulation of
MAPK related genes in the ΔVdNop12 strain needs to be
explored in future research.
Deletion of VdNop12 from V. dahliae resulted in the
mutant that was less virulant to cotton plants, and has
less vegetative growth and conidiation. Nevertheless, the
ΔVdNop12 strain was still able to colonize cotton plants
and can systemically spread through the xylem. This is
the first study that describes the role of an RBP in the
regulation of morphology, stress response and the patho-
genic development in V. dahliae as well as identifies and
characterizes the gene responsible for its regulation.
Experimental procedures
Identification of the mutant M11H01 with defects in
pathogenicity
For the identification of the mutant M11H01, 288 T-DNA
insertional mutants from a T-DNA random insertional
library of virulent defoliating V. dahliae strain Vd991 were
randomly selected to test the pathogenicity of each
mutant in two-round screening experiments using sus-
ceptible cotton plants (G. hirsutum cv. ‘Junmian No.1’)as
described previously with slight modifications (Zhang
Fig. 10. The ΔVdNop12 mutant impairs expression of the genes related
to cAMP-PKA and MAPK pathways. The expression level of cAMP-PKA
pathway-related genes (VdPKAC1 and VdPKAC2) and MAPK pathway
associated genes (VdMsb,VdHog1 and VdPbs2) were assayed by RT-
qPCR using RNA isolated from mycelium of ΔVdNop12 mutant and the
wildtypeVd991culturedonCMplatesfor7days.Theexpressionlevel
of each gene in Vd991 was set to 1.0. Means and standard errors were
calculated with data performed in triplicate. Single asterisk (*) indicates a
significant difference at p< 0.05. Double asterisks (**) indicate a highly
significant difference at p<0.01.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
10 J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
et al., 2016a). The pathogenicity of each mutant was
evaluated via the DI with five grades according to previ-
ously described methods (Zhu et al., 2013). The M11H01
mutant, with obvious defects in pathogenicity to the cot-
ton plants, was selected for further analysis.
Identification of sequences flanking the T-DNA
insertional sites and VdNop12 gene cloning
The M11H01 mutant was cultured in liquid CM for 8 days
under 200 r.p.m. shaking, at 25C, in the dark. The
resulting mycelia were harvested to extract genomic DNA
following the cetyl-trimethylammonium bromide method
(Doyle, 1991). For hybridization, genomic DNA was
digested by BamH I, separated on a 1% agarose gel,
transferred to Hybond-N
+
membranes (GE Healthcare),
and cross-linked with UV illumination for 1 min.
Hygromycin B phosphotransferase (HPH) gene was used
as the hybridization probe. Probe labelling and Southern
hybridization analysis were performed according to
instructions of DIG-High Prime DNA Labelling and Detec-
tion Starter Kit II (Roche Diagnostics; Mannheim, Ger-
many). Hybridization signals were visualized by
ChemiScope 6000 (Clinx, Shanghai, China). A hiTAIL-
PCR was used to clone the flanking sequences of the T-
DNA insertion sites using degenerate primers (Table S1)
as has been described previously (Liu and Chen, 2007).
Resulting sequences were compared with the VdLs.17
genome database (http://fungi.ensembl.org/Verticillium_
dahliae/info/index) to identify the T-DNA insertion sites.
Based on the sequence information, the coding region of
VdNop12 (VDAG_03303) was cloned from strain Vd991
using specific primers (Table S1).
Bioinformatics and phylogenetic analysis of VdNop12
Sequences data were downloaded from the online data-
base (http://fungi.ensembl.org). The structure model of
VdNop12 was predicted by I-TASSER software tool
(https://zhanglab.ccmb.med.umich.edu/I-TASSER) (Yang
et al., 2015; Yang and Zhang, 2015). Putative VdNop12
orthologues were identified with reciprocal protein–
protein BLAST analyses (BLASTP) as described by
Altschul et al. (1997). Clustal omega program was used
to globally align the protein sequences and to determine
the sequence identity (https://www.uniprot.org). A phylo-
genetic tree was constructed using the neighbour-joining
method of MEGA X software package (Kumar
et al., 2018). Domains of the protein were detected using
HMMSCAN (https://www.ebi.ac.uk) and the HMM profile
collection from the Pfam database (Finn et al., 2015; Pot-
ter et al., 2018). Domain architectures were analysed in
combination with the phylogenetic trees using Interactive
Tree Of Life (https://itol.embl.de/) (Letunic and
Bork, 2019). The YLoc web server (https://abi-services.
informatik.uni-tuebingen.de/yloc/webloc.cgi) was used to
predict the subcellular localization of VdNop12
(Briesemeister et al., 2010).
Targeted gene deletion and genetic complementation
To generate the VdNop12 disruption construct, a 1.2 kb
upstream fragment (UP) and a 1.2 kb downstream fragment
(DOWN) of VdNop12 were individually amplified from wil-
dtype Vd991 genomic DNA using primer pairs Y0065_F/
Y0065_R and Y0064_F/Y0064_R. HPH-F and HPH-R were
used to amplify the hygromycin cassette (HPH)fromthe
pMD18T-HPH vector. The three fragments of UP, HPH and
DOWN were fused to one fragment by overlap PCR. Sub-
sequently, a nested PCR was performed using the fused
fragment as the template and a nested primer pair KO_F/
KO_R to amplify the knockout fragment. The schematic dia-
gram of generation of ΔVdNop12 mutant by Agrobacterium
tumefaciens-mediated transformation (ATMT) and comple-
mentation has been shown as the knockout fragment was
sequentially cloned into the pDHt2 vector to obtain
pDHt2-VdNop12. The VdNop12 complementation construct
(pCOM-VdNop12) was generated by cloning a 4.3 Kb
fragment containing a 1.2 Kb upstream sequence, the cod-
ing region of VdNop12 (1.9 Kb) and a 1.2 Kb downstream
sequence into the pCOM vector that contained the geneticin
resistance cassette (Zhou et al., 2013).
ATMT was used to transfer the resulting plasmids to fun-
gal strains, as described by Maruthachalam et al. (2011),
with some modifications. Briefly, A. tumefaciens strain
AGL1 containing pDHt2-VdNop12 was transferred into wil-
dtype Vd991 to obtain VdNop12 deletion mutant
(ΔVdNop12). The resulting plasmid pCOM-VdNop12 was
introduced into the M11H01 and ΔVdNop12 strains to
obtain the EC-M11H01 (ectopic complementation of
M11H01) and EC-ΔVdNop12 (ectopic complementation of
ΔVdNop12) strains respectively. Transformants were
selected on PDA medium supplemented with 50 μg/ml
hygromycin B or G418 sulphate as needed. Each drug-
resistant colony was screened by PCR and further verified
by Southern hybridization analysis. RT-PCR was used to
confirm the recovery of the VdNop12 transcript in the com-
plementation transformants. The primer sequences are
shown in Table S1.
Pathogenicity, fungal recovery assays and penetration
assays
Verticillium wilt susceptible cotton plants (G. hirsutum
cv. ‘Junmian No.1’) were used to evaluate the virulence
effect of the Vd991 (wildtype), M11H01, EC-M11H01,
ΔVdNop12 and EC-ΔVdNop12 strains. Each pathogen
was cultured in Czapek liquid medium for 7 days, and the
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 11
inoculum concentration adjusted to 5 ×10
6
spores/ml
before inoculation. Sixteen cotton seedlings were inocu-
lated with a 20 ml conidial suspension from each strain
using the root-dipping method as described by Xu
et al. (2014). Cotton seedlings immersed in sterile water
were used as the control. The experiment was repeated
three times. Disease progress of the inoculated cotton
seedling was recorded from 1 to 25 dpi. The DI was cal-
culated based on one of five levels of severity of Ver-
ticillium wilt disease during the fungal invasion, as
previously described (Zhu et al., 2013). Stem sections
above cotyledons were taken from cotton seedlings at
25 days after inoculation as described by Xu et al. (2014).
The stem sections were surface sterilized, cut into
5–8 mm slices and incubated at 25C on PDA plates.
For fungal biomass studies, genomic DNA was
extracted from roots of three inoculated plants at 25 dpi,
and the fungal biomass was determined by qPCR as pre-
viously described (Gui et al., 2017). Verticillium elonga-
tion factor 1αgene was used to quantify fungal
colonization, whereas the 18S rDNA gene of cotton
plants was served as endogenous plant control.
For penetration assays, sterilized cellophane mem-
brane (Solarbio, Beijing, China) was overlaid on the MM
plates. The cultures were inoculated on the cellophane
membrane and incubated for 3 days at 25C. On the third
day, the membrane was removed and the plates were
incubated for an additional 3 days to observe the hyphal
growth. The experiments were independently repeated at
least three times (Zhao et al., 2016).
Phenotypic analysis of each strain for sensitivity to
different temperature levels and stress agents
To determine sensitivity to abiotic stress agents 10 mm
mycelial plugs of each strain were taken from 7 days old
colony edges were inoculated on CM plates and then
incubated at various temperatures (4C, 10C, 15C,
20C, and 25C). The growth media was also sup-
plemented with various chemical stress agents including
SDS (0.01%), Congo Red (75 μg/ml), Calcofluor white
(30 μg/ml), NaCl (0.7 M), sorbitol (1 M) and H
2
O
2
(15 mM). After incubation for 7 days, the colony diameter
of each plate was measured in two perpendicular direc-
tions with the original mycelial plug diameter (10 mm)
subtracted from each measurement. For each plate, the
average of the colony diameters was used to calculate
RGI values by the following equation: RGI = {(R1−R2)/
R1} * 100% (Liu et al., 2017). R1 and R2 are the diame-
ters of radial growth from the control and treated groups
respectively. The mycelial growth of each strain at 25C
without any stress agents was the control. This assay
was conducted using a randomized design and per-
formed in triplicate.
Yeast complementation assays
The cDNA of VdNop12 was amplified, digested by BamH
I and EcoR I and cloned into pYES2 vector (Invitrogen,
CA, USA). The primer sequences are shown in Table S1.
The resulting plasmid pYES2-VdNop12 was transformed
into the Nop12p mutant of yeast BY4741. Synthetic
medium lacking uracil was used to select yeast trans-
formants. The wildtype strain BY4741 and the strain
transformed with an empty pYES2 vector were used as
controls. For complementation assays, the yeast trans-
formants were grown on medium containing 1% yeast
extract, 2% bactopeptone and 2% agar supplemented
with 2% galactose (YPgal), or 2% glucose (YPG) at all
four temperature levels (15C, 25C, 30C and 37C).
Construction of VdNop12-mCherry plasmid and
microscopic analysis
For VdNop12-mCherry fusion constructs, VdNop12 ORF
and mCherry coding sequences were amplified from
cDNA of Vd991 and pmCherry vector, respectively, using
the specific primers (Table S1). The fusion construct was
then cloned into pCOM vector under the control of Asper-
gillus nidulans TrpC promoter (Zhou et al., 2013). Subse-
quently, the generated construct, together with the
geneticin-resistant cassette, was transferred into the
ΔVdNop12 mutant using ATMT method. Transformants
harbouring VdNop12-mCherry were confirmed by PCR
using primers in Table S1. Later, 10 mm mycelial plugs
of the transformants taken from 5 days old colony edges
were inoculated onto the glass slide and then incubated
at 25C for 2 days. The mycelia were stained with 5 μg/ml
DAPI for 10 15 min to observe the nucleus. After wash-
ing 5 times with PBS, the hyphae were observed using a
Zeiss laser confocal microscope (LSM880; Carl Zeiss,
Jena, Germany). For DAPI, excitation was set at 405 nm
and emission at 440 to 475 nm. For mCherry, excitation
was set at 543 nm and emission at 570 to 620 nm.
RNA extraction and RT-qPCR
Fresh mycelium of the ΔVdNop12 mutant and the wil-
dtype Vd991 were cultured on CM plates at 25C for
7 days. Mycelia were subjected to RNA extraction using
TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Wal-
tham, USA). RNA integrity was confirmed by agarose gel
electrophoresis. First-strand cDNA was synthesized from
3μg of total RNA in a 10 μl reaction mixture by using
PrimeScript® 1st strand cDNA Synthesis Kit (TaKaRa,
Dalian, China). RT-qPCR was performed in triplicate on a
Quant Studio™6 Flex System cycler apparatus with
SYBR Green (NovoStart® SYBR qPCR SuperMix Plus).
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
12 J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
The β-tubulin of V. dahliae was used as an internal con-
trol. All primers used in this study are listed in Table S1.
Statistical analysis
All the experiments were conducted three times and in
triplicate. Data are represented as mean ± standard error.
GraphPad Prsim 7.0 (GraphPad Software, San Diego,
CA) was used to perform the statistical analysis via Stu-
dent’st-test.
Acknowledgements
This research work was partially supported by National Natu-
ral Science Foundation of China (no. 31670143), Beijing
Natural Science Foundation (no. 6192023), National Key
R&D Program of China (2017YFC1600903) and Elite Youth
Program of Chinese Academy of Agricultural Sciences for
WG. We thank Professor Xiaofeng Dai and Dr. Jieyin Chen
at Institute of Food Science and Technology, Chinese Acad-
emy of Agricultural Sciences, for providing experimental
materials and valuable comments.
Authors contributions
WG conceived and designed the experiments. JZ and
WYC performed the experiments. JZ, HAH and WG
analysed the data and wrote the manuscript.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Table S1. Primers used in this study.
Fig. S1. The ΔVdNop12 mutant exhibited no response to
osmotic and oxidative stress.
A. Colony morphology of the wildtype (Vd991), M11H01,
EC-M11H01, ΔVdNop12 and EC-ΔVdNop12 on CM plates
supplemented with 0.7 M NaCl, 1 M Sorbitol or 15 mM H
2
O
2
at 7 dpi.
B. Relative growth inhibition (RGI) of fungal colony growth in
response to different stress agents.
Fig. S2. Generation of the VdNop12 deletion mutant by
Agrobacterium tumefaciens-mediated transformation and
complementation.
A. VdNop12 disruption strategy. Vertical bar, Pst I site; HPH,
hygromycin B phosphotransferase marker gene cassette.
B. Genomic DNA from wildtype Vd991 (lane 1), ΔVdNop12
(lane 2) and the complementation transformant EC-
ΔVdNop12 (lane 3) was digested with Pst Ιfor southern
hybridization using the probe harbouring partial HPH and
VdNop12.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
14 J. Zhang, W. Cui, H. Abdul Haseeb and W. Guo
C. The loss and recovery of VdNop12 transcripts were deter-
mined by RT-PCR. Lane 1, the wildtype; lane 2, ΔVdNop12;
lane 3, EC-ΔVdNop12.
Fig. S3. Penetration assay on the cellophane membrane.
Colonies of wildtype (Vd991), M11H01, EC-M11H01,
ΔVdNop12 and EC-ΔVdNop12 were grown on MM medium
overlaid with a cellophane layer (above) and after removal of
the cellophane membrane (below). Photographs in the first
row were taken at 3 dpi at 25C. The second row shows
growth of all of stains including Vd991, M11H01, EC-
M11H01, ΔVdNop12 and EC-ΔVdNop12 on MM medium
after penetration from the cellophane membrane.
© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Identification and characterization of VdNop12 15
