Annotation of the 12th Chromosome of the Forest Pathogen Fusarium circinatum

Abstract

The genus Fusarium comprises more than 300 species, and many of them are pathogens that cause severe diseases in agricultural, horticultural and forestry plants in both antropogenic and natural ecosystems. Because of their importance as plant pathogens, the genomes of several Fusarium spp. have been sequenced. Within this genus, Fusarium circinatum is one of the most harmful pathogens of pine trees attacking up to 60 Pinus species. Till now, the genomes of 13 strains of F.
circinatum have been sequenced. The strain GL1327 we studied lacks a twelfth chromosome, which allows the study of virulence genes on this chromosome. Although the genome of several strains of F. circinatum has been sequenced, it is still almost completely unannotated, which severely limits the possibilities to further investigate the molecular mechanisms of virulence of Fusarium. Therefore, this study aimed to annotate the 12th chromosome of F. circinatum and integrate currently available resources. In silico annotation of the 12th chromosome of F. circinatum revealed the presence of 118 open reading frames (ORFs) encoding 141 proteins which were predicted using an ab initio gene prediction tool. The InterProScan and SMART analyses identified known domains in 30 proteins and
eggNOG additionally in 12 of them. Among them, four groups can be distinguished: genes possibly related to heterokaryon incompatibility (4 genes), regulation of transcription (5 genes), plant cell wall degrading enzymes (7 genes) and
richothecene synthesis (3 genes). This study also integrated data of F.circinatum reference strain CMWF1803 assembled to chromosome level but not annotated with currently best annotated but assembled only to scaffold level strain NRRL 25331.

Full text

Citation: Malewski, T; Mati´c, S;
Okorski, A; Borowik, P.; Oszako, T
Annotation of the 12th Chromosome
of the Forest Pathogen Fusarium
circinatum.Agronomy 2023,13, 773.
https://doi.org/10.3390/
agronomy13030773
Academic Editor: Pedro Talhinhas
Received: 10 February 2023
Revised: 1 March 2023
Accepted: 6 March 2023
Published: 7 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
agronomy
Article
Annotation of the 12th Chromosome of the Forest
Pathogen Fusarium circinatum
Tadeusz Malewski 1, Slavica Mati´c 2, Adam Okorski 3, Piotr Borowik 4and Tomasz Oszako 5,*
1Museum and Institute of Zoology, Polish Academy of Science, ul. Wilcza 64, 00-679 Warszawa, Poland
2
Institute for Sustainable Plant Protection (IPSP), National Research Council of Italy (CNR), Strada delle Cacce
73, 10135 Torino, Italy
3
Department of Entomology, Phytopathology and Molecular Diagnostics, Faculty of Agriculture and Forestry,
University of Warmia and Mazury in Olsztyn, Pl. Łódzki 5, 10-727 Olsztyn, Poland
4Faculty of Physics, Warsaw University of Technology, ul. Koszykowa 75, 00-662 Warszawa, Poland
5Department of Forest Protection, Forest Research Institute, ul. Braci Le´snej 3, 05-090 S˛ekocin Stary, Poland
*Correspondence: T.oszako@ibles.waw.pl
Abstract:
The genus Fusarium comprises more than 300 species, and many of them are pathogens
that cause severe diseases in agricultural, horticultural and forestry plants in both antropogenic
and natural ecosystems. Because of their importance as plant pathogens, the genomes of several
Fusarium spp. have been sequenced. Within this genus, Fusarium circinatum is one of the most harmful
pathogens of pine trees attacking up to 60 Pinus species. Till now, the genomes of 13 strains of F.
circinatum have been sequenced. The strain GL1327 we studied lacks a twelfth chromosome, which
allows the study of virulence genes on this chromosome. Although the genome of several strains
of F. circinatum has been sequenced, it is still almost completely unannotated, which severely limits
the possibilities to further investigate the molecular mechanisms of virulence of Fusarium. Therefore,
this study aimed to annotate the 12th chromosome of F. circinatum and integrate currently available
resources. In silico annotation of the 12th chromosome of F. circinatum revealed the presence of 118
open reading frames (ORFs) encoding 141 proteins which were predicted using an ab initio gene
prediction tool. The InterProScan and SMART analyses identified known domains in 30 proteins and
eggNOG additionally in 12 of them. Among them, four groups can be distinguished: genes possibly
related to heterokaryon incompatibility (4 genes), regulation of transcription (5 genes), plant cell wall
degrading enzymes (7 genes) and trichothecene synthesis (3 genes). This study also integrated data
of F.circinatum reference strain CMWF1803 assembled to chromosome level but not annotated with
currently best annotated but assembled only to scaffold level strain NRRL 25331.
Keywords: Pinus; pathogen; pitch canker; genomics
1. Introduction
Fusarium is a cosmopolitan genus of filamentous ascomycetes (Sordariomycetes:
Hypocreales: Nectriaceae) that includes many toxin-producing plant pathogens of agri-
cultural importance. The genus Fusarium includes over 300 phylogenetically distinct
species [
1
]. Many of these species are plant pathogens that cause serious diseases on
agricultural, horticultural and forestry plants in antropogenic and natural ecosystems [2].
Pine Pitch Canker Disease (PPC), a serious threat that attacks many pine species, is caused by
the pathogenicfungus Fusarium circinatum Nirenberg &O’Donnell
(teleomorph = Gibberella circinata)
.
F. circinatum belongs to the EPPO A2 quarantine pathogen and causes one of the most devastating
diseases in pine forests, afforestations and nurseries, not only in Europe but throughout the world [
3
].
The host range of F. circinatum is very broad and includes up to 60 Pinus species [4,5].
Although the whole genome sequence of about 44 Mb of F. circinatum has been de-
termined [
6
], our knowledge of the fungal genes involved in its pathogenic behaviour is
limited. Seven putative quantitative trait loci associated with mycelial growth and colony
Agronomy 2023,13, 773. https://doi.org/10.3390/agronomy13030773 https://www.mdpi.com/journal/agronomy

Agronomy 2023,13, 773 2 of 19
margins have been described [
7
]. Van Wyk et al. [
8
] discovered a locus that possibly de-
termines the growth rate near the telomere of chromosome 3. The sequence of this locus
is highly conserved in F. circinatum and its close relatives, except for a 12,000 bp insertion
encoding five genes. An in silico analysis of the F. circinatum genome identified five candi-
date genes related to the growth (Fcfga1,Fcfgb1,Fcac,Fcrho1, and FcpacC) [
9
]. Functional
studies of Fcrho1 deletion mutants, a Rho-type GTPase, showed significantly reduced
growth in vitro than the corresponding ectopic and wild-type strains. The knockout mutant
of Ras2, another gene encoding the GTPase, also produced significantly smaller lesions
compared to the complementation mutants and wild-type strains. Growth studies showed
also significantly smaller colonies and delayed germination of conidia in the knockout
mutant strain [10].
Currently, the genomes of 13 strains of F. circinatum have been sequenced [
11
], pro-
viding a solid basis for comparative genomics. One of them (GL 1327) lacks a 12th
chromosome [12]
, which provides favourable conditions for the identification and study of
genes determining the virulence of F. circinatum.
The aim of this study was to identify ORFs localised on the 12th chromosome and
subsequently characterise the encoded proteins.
2. Materials and Methods
Analysis In Silico
The sequence of the 12th chromosome (Assembly ASM2404739v1, Acc. No. CM043929.1)
from the representative genome of F. circinatum Mexican strain CMWF1803 from
Pinus patula [11]
was retrieved from NCBI. Gene prediction was performed with the programme AUGUSTUS
version 3.3.1 trained for F. graminearum with the ab initio gene prediction method [13,14].
The predicted protein sequences were analysed against the protein database NCBI-NR
using BLASTp (default identity
≥
40%, coverage
≥
40%). Functional analysis of predicted
protein sequences was performed using InterProScan 91.0 [
15
,
16
] against the integrated InterPro
database consisting of PRINTS, SMART, Pfam, SUPERFAMILY, CATH -Gene3D, PANTHER
and CDD databases [
17
], Simple Modular Architecture Research Tool
(SMART v.9)
[
18
] and
against the unsupervised orthologous group database EggNOG v6.0 [19].
3. Results
In Silico Characterisation of Putative F. circinatum Genes
The sequence of the 12th chromosome was processed for ab initio gene prediction
using AUGUSTUS. A total of 118 putative genes were predicted, of which 56 are located on
the plus strand and 62 on the minus strand (Table A1 in Appendix A). These genes can be
transcribed into 141 transcripts. Thirty-six transcripts were intronless, while 41, 28, 10, 8, 7,
3, 2 and 1 transcripts have one, two, three, four, five, seven, either six or eight, and nine
introns, respectively.
Six genes (g1,g19,g20,g31,g56 and g76) had two alternative transcription start sites,
and the transcripts of 21 genes had no alternative splicing. The putative genes correspond to five
scaffolds of F. circinatum strain NRRL 25331 (PRJNA565749): JAAQPE010000042.1-142267 bp;
JAAQPE010000057.1-51020 bp; JAAQPE010000172.1-29236 bp; and JAAQPE010000262.1-90105
bp (Figure 1). The identity of the four scaffolds to the CM043929 sequence ranged from 99.09%
to 99.81%, with only scaffold JAAQPE010000057.1 having 93.44%.

Agronomy 2023,13, 773 3 of 19
Figure 1.
Localization of the scaffolds of F.circinatum strain NRRL 25331 on the 12th chromosome of
the representative genome of Fusarium circinatum strain CMWF1803. Black bar - reference sequence
of F.circinatum 12th chromosome CMWF1803 strain with coordinates of the sequence. Blue letters -
names of F.circinatum strain NRRL 25331 scaffolds. Grey/red boxes - scaffolds of F.circinatum strain
NRRL 25331. Insertions are marked with blue two hourglass-like triangles.
The predicted protein sequences were compared with sequences deposited in GenBank
using BLASTp. Out of 141 queries, 130 sequences were highly identical (>90%) to F. circinatum
strain NRRL 25331, seven proteins had lower identity (g99t2, 76.34%; g115t1, 81.17%; g16t1,
81.33%; G99t1, 84.22%; G92t2, 86.52%; G87t2, 87.94%; g78t1, 87.97%) and four (g48t1; g59t1;
g74t1; G98t1) were not identical to F. circinatum but identical to other Fusarium species.
With the help of InterProScan and SMART, domains and protein architectures could
be identified for 30 proteins (Table A3 in Appendix A). Among them, four groups can
be distinguished: Genes possibly related to heterokaryon incompatibility, regulation of
transcription, plant cell wall degrading enzymes and trichothecene synthesis.
Among the genes predicted by Augustus, four genes were found to be related to het-
erokaryon incompatibility (Figure 2). The protein g57t1 has two domains: Heterokaryon incom-
patibility (HET) and protein kinases (S-TKc). This protein sequence is identical to KAF5666823.1
(100% search coverage, 100% identity) of F. circinatum strain NRRL 25331, but is only referred to
as serine-threonine kinase in GenBank. Four proteins (g26t1, g52t1 and g58t1 and g58t2) contain
a NACHT nucleoside triphosphatase domain (named after the NAIP, CIITA, HET-E and TP-1
proteins) flanked by a varying number of ankyrin repeat domains. The alternative splicing
of the g58 transcript has no effect on the protein architecture. In the g26t1 and g52t1 proteins,
the nucleoside phosphorylase domain (PNP-UDP-1) is located proximal to NACHT; in addition,
g52 contains the domain oxoglutarate/iron-dependent dioxygenase (2OG-FeII-Oxy).
Figure 2.
Architecture of proteins potentially involved in heterokaryon incompatibility. Black
rectangles - protein domains related to heterokaryon incompatibility. Green rectangles—ankyrin
domain repeats. Blue polygon - protein kinases domain.

Agronomy 2023,13, 773 4 of 19
The incompatibility reaction is associated with massive transcriptional reprogram-
ming. Four genes (g20,g55,g82 and g83) encoding putative transcription factors were
found on the 12th chromosome. The protein g20t1 contains a Jumonji domain (JmjC).
G82 contains a transcription factor domain specific for fungi (Fungal-trans). This protein
sequence is identical to KAF5673552.1 (100% search coverage, 100% identity) of F. circinatum
strain NRRL 25331, where it is designated cutinase transcription factor 1 alpha. The G55
transcript is subject to alternative splicing, but this does not affect the protein architecture.
Both proteins (g55t1 and g55t2) have two domains: Fungal-trans and GAL4. The G83
protein (Table A4 in Appendix A)
has a fungal binuclear Zn(2)-Cys(6) domain. In addition
to the putative transcription factors, the g103t1 protein contains a SET domain typical of
proteins involved in epigenetic regulation of gene expression.
During infection, Fusarium secretes various virulence factors, including effector pro-
teins and plant cell wall degrading enzymes (CDWEs). The proteins encoded by g7 and g102
contain a lipase GDSL-2 domain. This domain is typical of SGNH hydrolase-type esterases
that act as esterases and lipases. The carboxylesterase domain (COesterase) contains the pro-
teins g81t1 and g82t2. Another type of hydrolases-peptidases-encode the genes g18 and g89.
The protein g18t1 contains peptidase C1A, g98t1 and g89t2 peptidase C14 or caspase do-
main. In addition to hydrolases, there are two genes
(g66 and g105)
on the 12th chromosome
that encode proteins containing a domain of the Major Facilitator Superfamily (MSF1).
One of the features of Fusarium is toxin synthesis. Three putative genes for tri-
chothecene synthesis have been detected. G6t1 and g6t2 contain tyrosinase, g95t1 - p450
and g70t1 - three acyl-CoA domains. In addition to groups of putative genes related to
heterokaryon incompatibility, transcriptional regulation, plant cell wall degrading enzymes
and trichothecene synthesis, 13 proteins were found whose products may be involved
in many metabolic processes of Fusarium metabolic processes. Another 12 proteins were
annotated with eggNOG (Table A4 in Appendix A).
4. Discussion
4.1. Distribution of F. circinatum, the Causative Agent of PPC Disease
As mentioned in the introduction, the fungal pathogen F. circinatum is the causative
agent of PPC disease [
20
]. Does the presence of the 12th chromosome cause the pathogen
to severely attack a variety of pine species in forests and nurseries worldwide? The
fungus can damage seedlings in nurseries and mature trees in forests. Symptoms in
seedlings include wilting and in mature trees bleeding, resinous cankers on trunks or thick
branches and tree death [
20
]. As F. circinatum has already been detected in Europe, it is
considered a serious, potentially invasive forest pathogen that spreads via infected seeds,
seedlings, wood, soil, wind, insect vectors and human activities. In Europe, the fungus has
affected pine trees in northern Spain and Portugal and has also been detected in France
and Italy. Research on the fungus (including its chromosomes) can therefore contribute
to the understanding of its pathogenesis and thus to the development of an appropriate
protection strategy. This should apply to young seedlings as well as to adult trees. Despite
the economic importance of PPC disease, the worldwide distribution of the pathogen
F. circinatum is poorly documented and the pathogenicity of its strains is even less known.
It is likely that the genetic diversity and population structure of the pathogen influence the
spread of PPC, including in Europe (models for the likely spread of the disease), and the
susceptibility of hosts. Chromosome number could be important for the virulence of
F. circinatum
, which also depends on host species, tree age and environmental characteristics.
Knowledge of the above factors is crucial for disease management, containment and
mitigation strategies. The in silico analyses carried out should help countries that are
currently free of F. circinatum to put in place effective procedures and restrictions and
prevent the invasion of the pathogen.

Agronomy 2023,13, 773 5 of 19
4.2. Development of New Diagnostic Methods to Ensure Reduction of PPC
Fusarium circinatum is on the list of species recommended for regulation as a quar-
antine pest in Europe. More than 60 species of Pinus are susceptible to this pathogen,
and it also attacks Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species from
genera such as Picea and Larix. The European Food Safety Authority (EFSA) estimates the
probability of reintroduction into the EU as very high [
21
]. Thanks to the possibility of early
detection, continuous surveillance and inspections by quarantine services, outbreaks of F.
circinatum in Italy and France have been officially eradicated. However, the global spread
of
F. circinatum
suggests that the pathogen will continue to be found in new areas in the
future. Rapid identification of the most virulent strains of the pathogen (using knowledge
of their chromosomes) will be important in Europe and elsewhere to limit the spread of the
disease. Currently, morphological identification methods are being replaced by molecular
methods, which include conventional PCR with a specific target region in the intergenic
interval and various real-time PCR protocols with varying specificity and sensitivity [
22
].
Perhaps the search for chromosome 12 will also be useful for pest risk assessment.
4.3. Pathways of Transmission and Potential Host Risk of the Pathogen
As F. circinatum is the causal agent of one of the most devastating forest diseases
worldwide, its spread over long distances should be controlled, especially by monitoring
infected seeds. On the other hand, at the regional level, seedlings, substrates and containers
play an important role in the spread of the fungus [
21
]. The pathogen enters nurseries
via infected seeds and is further spread by planting infected plants, especially since in-
fected plants (asymptomatic) may appear without disease symptoms. Once established,
F. circinatum
is spread by rain, wind and insects. Natural spread of the pathogen is limited
due to the short spore dispersal distances and the relatively short flight distances of the
spreading insects. To understand how best to intervene in the development of the disease
in nurseries and forests, we conducted annotation of twelfth chromosome.
4.4. Risk of Establishing the Pathogen in New Regions in Europe
Pine trees as potential host plants are important components of native forests and
plantations in Europe, where they play an important role both economically and ecologically.
Pine diseases are mainly caused by fungal pathogens and can significantly affect the
survival, vigour and yield of both individual trees and entire stands or plantations. PPC
caused by F. circinatum, one of the most devastating pine diseases in the world, is an
example of a new invasive disease in Europe.
The susceptibility of Scots pines in Poland (Pinus sylvestris L.) to infection by
F. circinatum
was tested in a greenhouse trial by [
23
]. Sixteen Polish pine cultivars were artificially inoc-
ulated with the 12th chromosome of F. circinatum and six other Fusarium species known to
infect pine seedlings in nurseries. All pines were found to be highly susceptible to PPC and
showed varying degrees of susceptibility to the other Fusarium species tested. The results
suggest that the risk of establishment of the invasive pathogen F. circinatum may be high as a
result of its accidental introduction in Poland.
In the future, the fungus is more likely to spread in the pine forests of southern Europe,
but there is also the possibility of spread in central and northern Europe. In Lithuania,
no occurrence of F. circinatum has been reported so far. In 2018, the susceptibility of three
different native Lithuanian Pinus sylvestris provenances to this pathogen [
24
] was tested.
For each origin, 38 pines were used and the soil was inoculated with a suspension of
F. circinatum
, and DNA was extracted from several plants that appeared unhealthy four
weeks after soil inoculation. Using the real-time PCR method, F. circinatum could not be
detected in these samples. However, the reason could be that the fungal biomass was too
low in relation to the host biomass or that the strain was less pathogenic than others.

Agronomy 2023,13, 773 6 of 19
4.5. Possible Interactions between F. circinatum and Other Fungal Species
The impact of microbiome interactions on plant health and the possible role of the
plant microbiome in disease expression have been the subject of several recent studies [
24
].
In Lithuania, the interaction between 12th chromosome strain of F. circinatum and several
pine-inhabiting fungi such as Dothistroma septosporum,F. oxysporum and Lecanosticta acicola
was also verified [
24
]. It was found that F. oxysporum grows slightly faster than F. circinatum
and inhibits the growth rate of F. circinatum.D. septosporum produced dothistromin, which
also appeared to slow the growth of the F. circinatum culture. In the meantime, L. acicola
was displaced by F. circinatum.
Co-infection of trees with indigenous pathogenic fungi or alien oomycetes and
F. circinatum
is possible. Biotic interactions could play an important role in the establishment of the PPC
pathogen in European nurseries and forests [
25
]. Available information on pine pathogens
that may co-occur with F. circinatum in Europe will have an impact on pine survival and
growth. Early and accurate identification of F. circinatum, a recently introduced pathogen
currently being regulated in Europe, is crucial to prevent its introduction and spread in forests.
Chromosome studies could provide valuable information in this regard if it is confirmed that
the high pathogenicity of some strains of the fungus depends on them and others do not.
4.6. In Silico Approach to the Identification and Characterisation of Genes
In this study, we used a genome-based in silico approach to identify and characterise
genes located on the 12th chromosome of F. circinatum. Chromosome 12 has been shown to
be the smallest of the chromosomes found in species of the F. fujikuroi complex. The size of
these chromosomes varies considerably intra- and interspecifically and shows polymor-
phism in chromosome length compared to the other chromosomes [26].
Fungal cells can interact with each other either vegetatively or sexually. In ascomycete
fungi, sexual interactions are controlled by the alleles at the mating type locus (MAT)
and asexual interactions by the alleles at the loci vic (vegetative incompatibility) or het
(heterokaryon incompatibility) [
27
]. In members of the F. fujikuroi species complex, 8 to
10 vic loci have been identified [
28
]. Vegetative incompatibility leads to programmed cell
death. For programmed cell death associated with vegetative incompatibility, there are
important proteins containing HET [
29
] and NACHT [
30
] domains. In F. circinatum we
have found four putative proteins that contain a central NACHT domain. Two of them
have an N-terminal PNP-UDP effector domain and all three have a C-terminal ANK repeat
domain (Figure 2). This organisation is typical of Ascomycota, where 20% of proteins with
NACH domains have N-terminal PNP-UDP and 42% have C-terminal ANK repeats [
30
].
Proteins containing the NACHT domain are involved in a process of non-self- recognition
and programmed cell death of fungi called heterokaryon incompatibility [31,32].
The incompatibility response has been found to be associated with massive transcrip-
tional reprogramming [
33
]. Transcription factors (TFs) play a key role in regulating gene
expression by binding to DNA in a sequence-specific manner. TFs are usually classified
according to their DNA-binding motif. Representatives of 80 TF families are typically
found in fungal genomes. The largest of these is the zinc cluster (C6 zinc finger) family [
34
].
They play an important role in growth, development and pathogenicity [
35
–
37
]. Fusarium
transcription factor 1 (FTF1) has been described as a potential regulator of effector expres-
sion in F. oxysporum f. sp. phaseoli and F. oxysporum f. sp. lycopersici [
38
]. Mahanty et al. [
39
]
described that specialised C6-type TFs may act as major regulators of F. oxysporum f. sp.
cepae pathogenicity during the development of Fusarium basal rot in onions.
Proteins with a zinc finger domain were found in the g24t1, g55t1 and g55t2 proteins.
SMART identified a transcription factor specific to fungi in the g82t1 domain, while BLAST
found sequence identity with KAF5673552.1, which was annotated as cutinase transcription
factor 1 (CTF1). CTF1 belongs to the C6 zinc TFs. CTF regulates the expression of cutinases
and fatty acid metabolism genes in F. solani f. sp. pisi [
40
] and Aspergillus nidulans [
41
].
Disruption of Ctf1
α
eliminated the phytopathogenicity of F. solani [
40
]. F. oxysporum strains

Agronomy 2023,13, 773 7 of 19
lacking a functional copy of the CTF1 gene are impaired in the induction of cutinase activity
and in the expression of genes encoding cutinase and lipase [42].
Gene expression also depends on the methylation of histones. Acetylation of lysine (K)
residues in histone 3 (H3) is associated with active transcription, while methylation of lysine
or arginine (R) residues leads to a more complex outcome that depends on associated reader
proteins [
43
]. H3K4 and H3K36 are considered to be hallmarks of euchromatin in yeast and
higher eukaryotes [
44
]. In filamentous fungi, the picture appears to be more diverse, as data
showed the ubiquitous presence of the H3K36 trimethylation mark (me3) in F. fujikuroi and
F. graminearum [
45
,
46
]. Methylation of H3K4 has been shown to depend on the conserved
SET domain-containing methyltransferase Set1 [
47
,
48
]. While Set1 is responsible for H3K4
methylation in the fungus, jumonji C is responsible for demethylation [
49
]. On Chr12 of
F. circinatum we have discovered putative genes encoding both of these proteins: G103t1
contains the SET domain and g20t1 jumonji.
During the infection process, Fusarium uses a number of secretion systems and releases
a variety of virulence factors such as mycotoxins, effector proteins and CWDEs to over-
come the target host cells. CWDEs such as polygalacturonases, pectate lyases, xylanases,
peptidases, peptide hydrolases, ribonucleases and cutinases may contribute to pathogene-
sis by degrading waxes, cuticles and cell walls to promote tissue invasion and pathogen
spread [
50
,
51
]. Cutinases and lipases that catalyse the hydrolysis of ester bonds from fatty
acid polymers, facilitating fungal invasion through the cuticle. Disruption of the lipase
gene FGL1 in F. graminearum resulted in reduced extracellular lipolytic activity in culture
and reduced virulence in both wheat and maize [
52
]. Disruption of another lipase gene,
FgATG15, also greatly attenuated wheat head infection [
53
]. An active role of lipases in
establishing full virulence has also recently been suggested for the plant pathogenF. oxyspo-
rum f. sp. lycopersici, where reduced lipolytic activity due to deletion of lipase regulatory
genes resulted in reduced colonisation of tomato plants [54].
The secreted metalloprotease FoMep1 and the serine protease FoSep1 of F. oxysporum
are involved in full virulence against tomato because they can reduce the antifungal activity
of their host plant chitinases [
55
]. The FoAYP1 gene also encodes protease. Surprisingly, this
protease is secreted by F. oxysporum but is localised in the nucleus in plant cells. The knock-
out strain of the FoAYP1 gene showed reduced virulence against tomato plants, but its
mycelial growth and conidiation were unchanged [56]. The Major Facilitator Superfamily
(MFS) is one of the largest known membrane transporter families. MFS transporters are cur-
rently the best characterised superfamily of secondary transmembrane transport proteins
responsible for nutrient uptake, extrusion of metabolites and resistance to various toxic
compounds, including not only secondary metabolites but also fungicides and antibiotics.
On the other hand, MFS transporters play a role in the availability of nutrients for survival,
including the transport of lipids, ions and small metabolites [
57
]. The transcript abundance
of the MFS multidrug transporter was five times higher in pathogenic F. oxysporum than in
non-pathogenic F. oxysporum. This transporter family regulates the movement of sugars,
Krebs cycle metabolites, phosphorylated glycolytic intermediates, amino acids, peptides, os-
moliths, iron siderophores, nucleosides, and organic and inorganic anions and cations [
58
].
In addition, MFS transporters have been linked to fungal pathogenicity by avoiding toxic
compounds produced by the pathogen or protecting against plant defences [
59
]. On Chr12,
not only the putative CTF gene but also putative genes encoding proteins containing lipase
(g71,g102), COesterase (g81), peptidase (g18 and g89) and domains of MFS (g66 and g105)
are localised.
Species of the genus Fusarium produce a wide variety of agriculturally important
trichothecene toxins, which differ from each other in their pattern of oxygenation and
esterification. Trichothecenes are a structurally diverse family of fungal sesquiterpene
epoxides that cause mycotoxicosis in humans and animals and increase the virulence of
some Fusarium species on crops. In F. sporotrichioides and F. graminearum, trichothecene
biosynthetic genes are localised in a 40-kb gene cluster [
60
,
61
]. Genes in this cluster
include trichodiene synthetase, P450 oxygenase, acetyltransferase, a toxin efflux pump

Agronomy 2023,13, 773 8 of 19
and transcription factors containing a Cys2His2 zinc finger motif [
62
]. On Chr12, genes
involved in trichothecene synthesis were found - a putative tyrosinase gene (g6), acyl-CoA
dehydrogenase (g70) and p450 cytochrome oxidase (g95), but not organised in a cluster.
The gene g2 encodes a protein containing a GPI-anchored domain found at the N-
terminus of a group of cell wall synthesis proteins involved in the synthesis of
beta-1,6-glucan
in the cell wall [
63
]. The cell wall shapes and protects the fungal cell. The 1,3-beta-glucan
synthase is responsible for the synthesis of one of the main components of the fungal wall.
This enzyme has been described in F. solani and many other Fusarium species [
64
]. Many
attempts to delete the gene encoding this enzyme have been unsuccessful, suggesting that it
may be a gene essential for cell life [65].
A comparison of the expression of serine/threonine protein kinase genes (ste12) in
pathogenic and non-pathogenic strains of F. oxysporum f. sp. cubense showed a significant
increase in the expression of ste12 in pathogenic strains [
66
]. Deletion of FgPTC1, a ser-
ine/threonine phosphatase, also attenuated the virulence of F. graminearum on wheat [67].
The mutant of F. verticillioides in which the fpk1 gene encoding the cAMP-dependent protein
kinase was disrupted showed reduced vegetative growth, fewer and shorter aerial mycelia,
severely impaired conidiation and reduced spore germination rate. After germination,
the fresh hyphae were stout and unbranched. When inoculated into susceptible maize vari-
eties, infection of the delta fpk1 mutant was delayed and infection efficiency was reduced
compared to the wild-type strain [
68
]. Family of serine/threonine protein kinases and
plays an important role in yeasts and other filamentous fungi. Deletion of FoIme2, which
belongs to this family, in F. oxysporum reduced mycelial growth and conidia production.
The mutants were hypersensitive to the osmotic stressor NaCl but less sensitive to the
membrane stressor SDS. Deletion of FoIme2 also reduced pathogenicity [
69
]. The gene
encoding the protein kinase (g56) is located on Chr 12.
In filamentous fungi, gene silencing by RNA interference (RNAi) affects many bi-
ological processes, including pathogenicity. Deletion of qde3, which encodes helicase,
impaired conidiation and ascosporogenesis in F. graminaceum and contributes to sexual
reproduction [
70
]. Chr. 12 contains the gene g74, which encodes a protein containing a
helicase domain.
5. Conclusions
•
Overall, the knowledge gained in this study about the annotations of genes, ORFs
and domains in the 12th chromosome of F. circinatum could make an important
contribution to the management of PPC disease and to strategies for containment and
mitigation strategies.
•
Our study can serve to clarify the phylogeny of the species and furthermore to develop
new molecular detection tools.
•
The genomic organisation of virulence genes can be used to clarify the relationship
between F. ciricantum and hosts.
• We concluded that at least 14 genes are associated with pathogenesis/virulence.
Author Contributions:
Conceptualization, T.M., S.M., A.O.; methodology, T.M., S.M., A.O.; software,
T.M., P.B.; validation, T.M., P.B.; formal analysis, T.M., P.B.; investigation, T.M.; resources, A.O., T.M.;
data curation, P.B.; writing—Original draft preparation, T.M., T.O.; writing—Review and editing, T.O.,
P.B., S.M.; visualization, P.B., T.M.; supervision, T.M., T.O.; project administration, T.M., T.O.; funding
acquisition, A.O.; All authors have read and agreed to the published version of the manuscript.
Funding:
The publication was written as a part of result of the author’s (AO) internship in Slovak Uni-
versity of Agriculture in Nitra, co-financed by the European Union under the European Social Fund
(Operational Program Knowledge Education Development), carried out in the project Development
Program at the University of Warmia and Mazury in Olsztyn (POWR.03.05. 00-00-Z310/17).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Agronomy 2023,13, 773 9 of 19
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A. Localization and Annotation of Putative F. circinatum Proteins
Table A1.
Localization of putative Open Reading Frames and transcripts, protein sizes, and number
of introns derived from the F. circinatum 12th chromosome annotation.
Gene Strand Localization No. Transcript Localization Predicted
Introns Protein Size
1 + 31217–31960 2 1 31217–31219 196
+ 2 2 31229–31960 192
2 + 33294–34089 1 33294–34089 238
3−36737–36989 1 36737–36989 68
4 + 38485–39264 0 38485–39264 259
5 + 40947–41501 0 40947–41501 184
6−42288–44793 7 1 42288–44793 687
7 2 42288–44793 687
7 + 47832–50343 3 47832–50343 712
8 + 50528–51772 0 50528–51772 414
9−53269–53616 0 53269–53616 115
10 −53692–54501 0 53692–54501 269
11 −58288–59517 1 58288–59517 393
12 −67276–67674 0 67276–67674 132
13 −69092–69550 0 69092–69550 152
14 −71147–71430 0 71147–71430 75
15 + 71985–72521 1 71985–72521 136
16 −76693–77699 1 76693–77699 300
17 + 81870–83876 0 81870–83876 668
18 −83977–85827 0 83977–85827 618
19 −87569–88259 2 87569–88259 194
20 + 89834–92287 0 89834–92287 817
21 + 95681–96392 1 1 95681–96392 219
+ 1 2 95777–96392 187
22 −101909–103049 4 1 101973–103049 148
5 2 101909–103049 195
23 + 104774–105565 2 104774–105565 223
24 + 108785–110371 0 108785–110371 528
25 + 115888–116535 2 115888–116535 146
26 −117728–122084 2 117728–122084 1379
27 −130442–131614 4 130442–131614 323
28 + 133748–134953 3 1 133982–134953 271
6 2 133748–134953 297
29 −138883–139227 0 138883–139227 114
30 −143831–145637 5 143831–145637 441
31 + 145727–148851 11 145727–148851 475
32 + 156385–157122 1 156385–157122 228
33 −157739–158284 0 157739–158284 181
34 −165252–166541 0 165252–166541 429
35 −167760–169128 2 167760–169128 311
36 + 171554–172271 1 1 171554–172271 178
2 171578–172271 170
37 + 173661–174852 2 173661–174852 364
38 + 177631–179403 3 177631–179403 368
39 −179756–180727 2 1 179756–180727 285
2 2 179756–180727 288
40 + 180965–181919 1 180965–181919 209
41 −181947–182603 2 181947–182603 185
42 −184433–186403 3 1 184433–186403 436
4 2 184433–186403 461
43 −197699–198520 1 197699–198520 131
44 + 201599–202213 0 201599–202213 204
45 −206352–206792 1 206352–206792 114
46 −212670–213117 1 212670–213117 117
47 −217694–218296 1 217694–218296 134
48 −219886–220412 1 219886–220412 153

Agronomy 2023,13, 773 10 of 19
Table A1. Cont.
Gene Strand Localization No. Transcript Localization Predicted
Introns Protein Size
49 + 241829–243064 4 241829–243064 292
50 + 249462–249912 1 249462–249912 132
51 −255019–255430 1 255019–255430 117
52 −260511–267687 4 260511–267687 1871
53 + 267833–269506 0 267833–269506 557
54 + 269924–270610 0 269924–270610 228
55 −270814–272503 4 1 270814–272503 442
4 2 270814–272503 433
56 −279820–281924 2 279820–281924 636
57 + 284917–288900 5 284917–288900 1103
58 + 290740–294512 8 1 290740–294512 993
8 2 290740–290742 1008
59 −296603–298423 1 296603–298423 465
60 −299021–300337 0 299021–300337 436
61 + 301149–301535 0 301149–301535 128
62 + 306378–309789 3 306378–309789 987
63 + 312081–313133 0 312081–313133 350
64 −314421–314718 1 314421–314718 82
65 −316363–317384 2 316363–317384 304
66 + 319615–321355 1 1 319636–321355 550
1 2 319615–321355 557
67 −321897–322763 0 321897–322763 288
68 + 324749–325970 3 324749–325970 339
69 + 327367–328119 0 327367–328119 250
70 −328511–329701 0 328511–329701 396
71 −333730–334527 0 333730–334527 165
72 −335535–336444 1 335535–336444 141
73 + 339435–340126 1 1 339435–340042 186
2 2 339435–340126 196
74 −340698–341147 2 340698–341147 116
75 −344219–347477 2 344219–347477 1042
76 + 349423–350519 2 1 349423–350519 331
1 2 349423–350519 347
77 −352248–352879 1 352248–352879 118
78 + 353875–354707 1 353875–354707 235
79 + 358678–362423 5 358678–362423 1046
80 −364509–365599 1 364509–365599 346
81 −368775–372232 5 1 368775–372232 660
5 2 368775–372232 634
82 −373134–374775 1 373134–374775 527
83 + 375893–377234 1 375893–377234 428
84 −377746–379869 2 377746–379869 638
85 + 381383–382024 0 381383–382024 213
86 + 384422–387495 3 1 386043–387495 418
0 2 384422–385309 295
5 3 384422–387495 754
87 + 389607–393031 6 1 389607–393031 550
7 2 389607–393031 531
88 + 393067–393411 0 393067–393411 114
89 + 396581–397378 1 1 396650–397378 167
1 2 396581–397378 190
90 −399885–401207 2 399885–401207 504
91 −403346–403807 0 403346–403807 153
92 −405413–405787 2 1 405413–405787 92
1 2 405413–405738 91
93 −412668–413939 1 412668–413939 407
94 + 418703–419415 2 418703–419415 201
95 −419654–420268 0 419654–420268 204
96 −423279–424095 2 423279–424095 240
97 −432389–433183 0 432389–433183 264
98 −437147–438263 1 437147–438263 327
99 −441782–442974 1 1 441782–442974 379
2 2 441782–442974 349
100 −444928–446536 2 444928–446536 502
101 + 447456–448802 3 447456–448802 392
102 + 451345–452097 0 451345–452097 250
103 + 459258–461038 2 459258–461038 552

Agronomy 2023,13, 773 11 of 19
Table A1. Cont.
Gene Strand Localization No. Transcript Localization Predicted
Introns Protein Size
104 + 462490–462897 1 462490–462897 102
105 −465130–468729 9 465130–468729 888
106 + 476676–478090 1 476676–478090 448
107 −481803–482006 0 481803–482006 67
108 −486871–487349 1 486871–487349 141
109 −490202–492772 4 490202–492772 528
110 + 490202–492772 1 490202–492772 156
111 + 497012–498151 0 497012–498151 379
112 + 499716–500198 0 499716–500198 160
113 −502582–504360 1 502582–504360 508
114 + 506239–507444 3 1 506239–507444 174
2 2 506239–507444 161
115 + 508757–509677 2 1 508757–509677 198
3 2 508757–509677 239
116 + 511677–512903 0 511677–512903 408
117 −513379–514311 2 513379–514311 137
118 + 515695–516607 1 515695–516607 249

Agronomy 2023,13, 773 12 of 19
Table A2. GenBank best matches of putative F. circinatum proteins.
Protein Length
(AA)
Best Match ID Accession No.
(Best Match)
E Value Identity
(%)
g1t1 196 Hypothetical protein FCIRC_1200 F. circinatum KAF5689737.1 5 ×10−129 100.00
g1t2 192 Hypothetical protein FCIRC_1200 F. circinatum
XP_049150203.1
4×10−129 100.00
g2t1 238
Cell wall beta-glucan synthesis, FCIRC_1201, F. circina-
tum
KAF5689738.1 1 ×10−170 100.00
g3t1 68 Hypothetical protein FCIRC_1202, F. circinatum KAF5689739.1 3 ×10−40 100.00
g4t1 258 Hypothetical protein, FCIRC_1203 F. circinatum KAF5689740.1 0.0 100.00
g5t1 184 Hypothetical protein FCIRC_1204 F. circinatum KAF5689741.1 9 ×10−135 100.00
g6t1 687 Tyrosinase precursor, FCIRC_1205, F. circinatum KAF5689742.1 0.0 100.00
g6t2 687 Tyrosinase precursor, FCIRC_1205, F. circinatum KAF5689742.1 0.0 98.84
g7t1 712
Extracellular gdsl-like lipase, FCIRC_1206, F. circinatum
KAF5689743.1 0.0 100.00
g8t1 414 Hypothetical protein FCIRC_1207 F. circinatum KAF5689744.1 0.0 100.00
g9t1 115 Hypothetical protein FCIRC_1208, F. circinatum KAF5689745.1 1 ×10−75 100.00
g10t1 269 Hypothetical protein FCIRC_1209, F. circinatum KAF5689746.1 0.0 100.00
g11t1 393 Hypothetical protein FCIRC_1210, F. circinatum KAF5689747.1 0.0 100.00
g12t1 132 Hypothetical protein FCIRC_1211, F. circinatum KAF5689748.1 2 ×10−90 100.00
g13t1 152 Hypothetical protein FCIRC_1212, F. circinatum KAF5689749.1 2 ×10−107 100.00
g14t1 75 Hypothetical protein FCIRC_1213, F. circinatum KAF5689750.1 3 ×10−47 100.00
g15t1 136 Hypothetical protein FCIRC_1214, F. circinatum KAF5689751.1 1 ×10−94 100.00
g16t1 300 Serine threonine kinase, FCIRC_1215, F. circinatum KAF5689752.1 7 ×10−167 81.33
g17t1 668 Hypothetical protein CIRC_1216, F. circinatum KAF5689753.1 0.0 100.00
g18t1 616 Hypothetical protein CIRC_1217, F. circinatum KAF5689754.1 0.0 100.00
g19t1 194 Hypothetical protein CIRC_1218, F. circinatum KAF5689755.1 2 ×10−136 100.00
g20t1 817
Transcription factor jumonji, FCIRC_1219, F. circinatum
KAF5689756.1 0.0 100.00
g21t1 219 Hypothetical protein CIRC_1220, F. circinatum KAF5689757.1 6 ×10−154 92.80
g21t2 187 Hypothetical protein FCIRC_1220, F. circinatum KAF5689757.1 5 ×10−130 91.67
g22t1 148 Hypothetical protein FCIRC_1222, F. circinatum KAF5689759.1 2 ×10−88 99.23
g22t2 195 Hypothetical protein FCIRC_1222, F. circinatum KAF5228515.1 1 ×10−129 93.33
g23t1 223 Hypothetical protein CIRC_1223, F. circinatum KAF5689760.1 1 ×10−165 100.00
g24t1 528 Hypothetical protein CIRC_1224, F. circinatum KAF5689761.1 0.0 100.00
g25t1 146 Hypothetical protein CIRC_1225, F. circinatum KAF5689762.1 5 ×10−102 100.00
g26t1 1379 Ankyrin repeat protein, FCIRC_1226, F. circinatum KAF5689763.1 0.0 100.00
g27t1 323 Hypothetical protein CIRC_1227, F. circinatum KAF5689764.1 0.0 100.00
g28t1 271 Hypothetical protein FCIRC_1228, F. circinatum KAF5689765.1 0.0 100.00
g28t2 297 Hypothetical protein FCIRC_1228, F. circinatum KAF5689765.1 0.0 100.00
g29t1 114 Hypothetical protein FCIRC_1229, F. circinatum KAF5689766.1 3 ×10−77 100.00
g30t1 441 Hypothetical protein FCIRC_1230, F. circinatum KAF5689767.1 0.0 100.00
g31t1 475
Translation initiation factor IF-2, FCIRC_1231, F. circi-
natum
KAF5689768.1 4 ×10−179 96.51
g32t1 228 Hypothetical protein FCIRC_1232, F. circinatum KAF5689769.1 1 ×10−158 100.00
g33t1 181 Hypothetical protein FCIRC_1233,F. circinatum KAF5689770.1 2 ×10−131 100.00
g34t1 429 Hypothetical protein FCIRC_1234, F. circinatum KAF5689771.1 0.0 100.00
g35t1 311 C2H2 transcription factor, FCIRC_1235, F. circinatum KAF5689772.1 0.0 100.00
g36t1 178 Hypothetical protein FCIRC_1237, F. circinatum KAF5689774.1 1 ×10−126 100.00
g36t2 170 Hypothetical protein FCIRC_1237, F. circinatum KAF5689774.1 9 ×10−120 100.00
g37t1 364 Hypothetical protein FCIRC_1729, F. circinatum KAF5688719.1 0.0 100.00
g38t1 368 Hypothetical protein FCIRC_1731, F. circinatum KAF5688721.1 0.0 100.00
g39t1 285 Hypothetical protein FCIRC_1732, F. circinatum KAF5688722.1 0.0 100.00
g39t2 288 Hypothetical protein FCIRC_1732, F. circinatum KAF5688722.1 0.0 98.61
g40t1 209 Hypothetical protein FCIRC_1733, F. circinatum KAF5688723.1 1 ×10−150 100.00
g41t1 185 Hypothetical protein FCIRC_1734, F. circinatum KAF5688724.1 2 ×10−135 100.00
g42t1 436 Hypothetical protein FCIRC_1735, F. circinatum KAF5688725.1 0.0 99.74
g42t2 461 Hypothetical protein FCIRC_1735, F. circinatum KAF5688725.1 0.0 99.74
g43t1 131 FK506-binding protein, FCIRC_1236, F. circinatum KAF5688726.1 8 ×10−93 100.00
g44t1 204 Hypothetical protein FCIRC_1737, F. circinatum KAF5688727.1 2 ×10−144 100.00
g45t1 114 Hypothetical protein FCIRC_1738 F. circinatum KAF5688728.1 5 ×10−78 100.00
g46t1 117 Hypothetical protein FCIRC_1739 F. circinatum KAF5688729.1 7 ×10−77 100.00
g47t1 134 Hypothetical protein FCIRC_1740, F. circinatum KAF5688730.1 1 ×10−92 100.00
g48t1 153 Arginine deiminase type-3, F. mexicanum KAF5555127.1 1 ×10−95 95.83

Agronomy 2023,13, 773 13 of 19
Table A2. Cont.
Protein Length
(AA)
Best Match ID Accession No.
(Best Match)
E Value Identity
(%)
g49t1 292 Hypothetical protein FCIRC_10050, F. circinatum KAF5666814.1 0.0 100.00
g50t1 132 Hypothetical protein FCIRC_10051, F. circinatum KAF5666815.1 1 ×10−92 100.00
g51t1 117
Sterol 3beta-glucosyltransferase, FCIRC_10052, F. circina-
tum
KAF5666816.1 2 ×10−60 100.00
g52t1 1871
NACHT ankyrin domain-containing protein,
FCIRC_10053, F. circinatum
KAF5666817.1 0.0 100.00
g53t1 557
NCS1 nucleoside transporter, FCIRC_10054, F. circina-
tum
KAF5666818.1 0.0 100.00
g54t1 228
Asp glu hydantoin racemase, FCIRC_10055, F. circina-
tum
KAF5666819.1 6 ×10−163 100.00
g55t1 442 C6 transcription factor, FCIRC_10056, F. circinatum KAF5666820.1 0.0 97.96
g55t2 433 C6 transcription factor, FCIRC_10056, F. circinatum KAF5666820.1 0.0 100.00
g56t1 638 CMGC DYRK kinase, FCIRC_10058, F. circinatum KAF5666822.1 0.0 100.00
g57t1 1103 Serine threonine kinase, FCIRC_10059, F. circinatum KAF5666823.1 0.0 100.00
g58t1 993
NACHT domain-containing protein, FCIRC_5226, F. circi-
natum
KAF5682015.1 0.0 99.59
g58t2 1008
NACHT domain-containing protein, FCIRC_5226, F. circi-
natum
KAF5682015.1 0.0 92.73
g59t1 465 TPR domain-containing protein, F. denticulatum KAF5674688.1 0.0 55.26
g60t1 438
TPR domain-containing protein, FCIRC_5228, F. circi-
natum
KAF5682016.1 0.0 100.00
g61t1 128 Hypothetical protein FCIRC_5229, F. circinatum KAF5682017.1 2 ×10−88 100.00
g62t1 987 Hypothetical protein FCIRC_5230, F. circinatum KAF5682018.1 0.0 92.40
g63t1 350 Hypothetical protein FCIRC_5231, F. circinatum KAF5682019.1 0.0 98.86
g64t1 82 Hypothetical protein FCIRC_5232, F. circinatum KAF5682020.1 5 ×10−53 100.00
g65t1 304
Aspartate aminotransferase, FCIRC_5233, F. circinatum
KAF5682021.1 1 ×10−136 100.00
g66t1 550
Multidrug resistance protein fnx1, FCIRC_8030, F. circina-
tum
KAF5673567.1 0.0 100.00
g66t2 557
Multidrug resistance protein fnx1, FCIRC_8030, F. circina-
tum
KAF5673567.1 0.0 100.00
g67t1 288
D-isomer specific 2-hydroxyacid dehydrogenase,
FCIRC_8029, F. circinatum
KAF5673566.1 0.0 100.00
g68t1 339 Macrophomate synthase, FCIRC_8028, F. circinatum KAF5673565.1 0.0 100.00
g69t1 250
Demethylmenaquinone methyltransferase family,
FCIRC_8027, F. circinatum
KAF5673564.1 0.0 100.00
g70t1 396 Hypothetical protein FCIRC_8026 F. circinatum KAF5673563.1 0.0 100.00
g71t1 165 Hypothetical protein FCIRC_8025, F. circinatum KAF5673562.1 1 ×10−119 100.00
g72t1 141 Hypothetical protein FCIRC_8024, F. circinatum KAF5673561.1 2 ×10−98 100.00
g73t1 186 Hypothetical protein FCIRC_8023, F. circinatum KAF5673560.1 3 ×10−133 100.00
g73t2 196 Hypothetical protein FCIRC_8023, F. circinatum KAF5673560.1 4 ×10−124 100.00
g74t1 116 SNF2 family domain containing protein, F. agapanthi KAF4497424.1 6 ×10−65 92.24
g75t1 1042 Hypothetical protein FCIRC_8022, F. circinatum KAF5673559.1 0.0 100.00
g76t1 331 Hypothetical protein FCIRC_8021, F. circinatum KAF5673558.1 0.0 100.00
g76t2 347 Hypothetical protein FCIRC_8021, F. circinatum KAF5673558.1 0.0 95.39
g77t1 118 Hypothetical protein FCIRC_8020, F. circinatum KAF5673557.1 5 ×10−82 100.00

Agronomy 2023,13, 773 14 of 19
Table A2. Cont.
Protein Length
(AA)
Best Match ID Accession No.
(Best Match)
E Value Identity
(%)
g78t1 235 Kinase-like domain-containing protein, FCIRC_8019, F. circinatum KAF5673556.1 9 ×10−167 87.97
g79t1 1046 Hypothetical protein FCIRC_8018, F. circinatum KAF5673555.1 0.0 100.00
g80t1 346 Hypothetical protein FCIRC_8017, F. circinatum KAF5673554.1 0.0 100.00
g81t1 660 Para-nitrobenzyl esterase, FCIRC_8016, F. circinatum KAF5673553.1 0.0 100.00
g81t2 634 Para-nitrobenzyl esterase, FCIRC_8016, F. circinatum KAF5673553.1 0.0 96.06
g82t1 527 Cutinase transcription factor 1 alpha, FCIRC_8015, F. circinatum KAF5673552.1 0.0 100.00
g83t1 428 Hypothetical protein FCIRC_8014, F. circinatum KAF5673551.1 0.0 100.00
g84t1 638 Hypothetical protein FCIRC_8013, F. circinatum KAF5673550.1 0.0 100.00
g85t1 213 Hypothetical protein FCIRC_8012, F. circinatum KAF5673549.1 0.0 100.00
g86t1 418 Hypothetical protein FCIRC_8010, F. circinatum KAF5673547.1 0.0 100.00
g86t2 295 Hypothetical protein FCIRC_8011, F. circinatum KAF5673548.1 0.0 97.97
g86t3 754 SGL domain-containing protein, Fusarium sp. LHS14.1 KAI8724150.1 2 ×10−95 52.38
g87t1 550 ATP synthase F1, FCIRC_8009, F. circinatum KAF5673546.1 0.0 91.65
g87t2 531 ATP synthase F1, FCIRC_8009, F. circinatum KAF5673546.1 0.0 87.94
g88t1 114 Hypothetical protein FCIRC_8008, F. circinatum KAF5673545.1 8 ×10−77 100.00
g89t1 167 Caspase, FCIRC_8007, F. circinatum KAF5673544.1 1 ×10−121 100.00
g89t2 190 Caspase, FCIRC_8007, F. circinatum KAF5673544.1 2 ×10−121 100.00
g90t1 405 Hypothetical protein FCIRC_8006, F. circinatum KAF5673543.1 0.0 100.00
g91t1 153 Hypothetical protein FCIRC_8005, F. circinatum KAF5673542.1 1 ×10−105 100.00
g92t1 92 Hypothetical protein FCIRC_8004, F. circinatum KAF5673541.1 8 ×10−60 100.00
g92t2 91 Hypothetical protein FCIRC_8004, F. circinatum KAF5673541.1 3 ×10−46 86.52
g93t1 407 Transaldolase, FCIRC_7317, F. circinatum KAF5675701.1 0.0 100.00
g94t1 201 Aromatic prenyltransferase, FCIRC_7316, F. circinatum KAF5675700.1 2 ×10−142 100.00
g95t1 204 Cytochrome P450 monooxygenase, FCIRC_7315, F. circinatum KAF5675699.1 2 ×10−150 100.00
g96t1 240 Nonribosomal peptide synthase, FCIRC_7314, F. circinatum KAF5675698.1 8 ×10−176 100.00
g97t1 264 Hypothetical protein FCIRC_7313, F. circinatum KAF5675697.1 0.0 100.00
g98t1 327 Uncharacterized protein FSUBG_13770, F. subglutinans XP_036530762.1 7 ×10−158 64.23
G99t1 379 Hypothetical protein FCIRC_7311, F. circinatum KAF5675696.1 0.0 84.22
g99t2 349 Hypothetical protein FCIRC_7311, F. circinatum KAF5675696.1 0.0 76.34
g100t1 502
Rhs repeat-associated core domain-containing protein,
FCIRC_7310, F. circinatum
KAF5675695.1 0.0 100.00
g101t1 392 Hypothetical protein FCIRC_7309, F. circinatum KAF5675694.1 0.0 100.00
g102t1 250
Esterase SGNH hydrolase-type subgroup, FCIRC_7308,
F. circinatum
KAF5675693.1 0.0 100.00
g103t1 552 SET domain-containing protein, FCIRC_7305, F. circinatum KAF5675692.1 0.0 100.00
g104t1 102 Hypothetical protein FCIRC_7306, F. circinatum KAF5675691.1 3 ×10−67 100.00
g105t1 888
Major facilitator superfamily transporter, FCIRC_7305
F. circinatum
KAF5675690.1 0.0 100.00
g106t1 448 Hypothetical protein FCIRC_7304, F. circinatum KAF5675689.1 0.0 95.12
g107t1 67 Hypothetical protein FCIRC_7303, F. circinatum KAF5675688.1 4 ×10−39 100.00
g108t1 141 Hypothetical protein FCIRC_7302, F. circinatum KAF5675687.1 2 ×10−95 100.00
g109t1 528 Polyketide synthase FCIRC_7301, F. circinatum KAF5675686.1 0.0 100.00
g110t1 156 Taurine dioxygenase family FCIRC_7300, F. circinatum KAF5675685.1 2 ×10−110 100.00
g111t1 379 Hypothetical protein FCIRC_7299, F. circinatum KAF5675684.1 0.0 100.00
g112t1 160 Hypothetical protein FCIRC_7298, F. circinatum KAF5675683.1 6 ×10−116 100.00
g113t1 508 Hypothetical protein FCIRC_7297, F. circinatum KAF5675682.1 0.0 100.00
g114t1 174 Kinase-like (PK-like), FCIRC_7296 F. circinatum KAF5675681.1 8 ×10−88 100.00
g114t2 161 Kinase-like (PK-like), FCIRC_7296 F. circinatum KAF5675681.1 7 ×10−88 100.00
g115t1 198 Hypothetical protein FCIRC_7295, F. circinatum KAF5675680.1 4 ×10−132 81.17
g115t2 239 Hypothetical protein FCIRC_7295, F. circinatum KAF5675680.1 5 ×10−178 100.00
g116t1 408 Alpha beta-hydrolase, FCIRC_7294, F. circinatum KAF5675679.1 0.0 100.00
g117t1 137 Hypothetical protein FCIRC_7293, F. circinatum KAF5675678.1 8 ×10−96 100.00
g118t1 249 Telomere-associated recQ-like helicase, FCIRC_7292, F. circinatum KAF5675677.1 0.0 100.00

Agronomy 2023,13, 773 15 of 19
Table A3.
Protein domains identified by InterPro scan and SMART in putative F. circinatum proteins.
Gene Pfam Acc.
No.
InterPro
Acc. No.
Domain Name Domain Name
Abbreviation
Localization
(AA)
E Value
g2t1 PF10342
IPR018466
Kre9/KNH-like N-terminal Ig-like do-
main GPI-anchored 29–123 2.6 ×10−17
g6t1 PF00264
IPR002227
Tyrosinase copper-binding Tyrosinase 60–358 5.9 ×10−39
g6t2 PF00264
IPR002227
Tyrosinase copper-binding Tyrosinase 60–359 5.9 ×10−39
g7t1 PF13472
IPR013830
SGNH hydrolase-type esterase Lipase_GDSL_2 174–343 2.8 ×10−12
g18t1 PF00112
IPR000668
Peptidase C1A, papain C-terminal Peptidase_C1 434–607 4.9 ×10−7
g20t1 -
IPR003347
Jumonji JmjC 339–498 2.04 ×10−5
g26t1 PF01048
IPR000845
Nucleoside phosphorylase PNP_UDP _1 42–358 7.5 ×10−12
g26t1 PF05729
IPR000845
NACHT nucleoside triphosphatase NACHT 407–589 7.4 ×10−7
g26t1 -
IPR002110
Ankyrin repeat ANK
15 rpt. from
854 to 1374
g52t1 PF03171
IPR005123
Oxoglutarate/iron-dependent dioxyge-
nase 2OG-FeII_Oxy 49–158 2.8 ×10−17
g52t1 PF01048
IPR000845
Nucleoside phosphorylase PNP_UDP _1 175–472 3.3 ×10−7
g52t1 PF05729
IPR007111
NACHT nucleoside triphosphatase NACHT 575–742 1 ×10−6
g53t1 PF02133
IPR001248
Purine-cytosine permease Transp_cyt_pur 30–487 2.3 ×10−86
g54t1 PF01177
IPR015942
Asp/Glu/hydantoin racemase Asp_Glu_race 42–219 4 ×10−10
g55t1 PF04082
IPR007219
Transcription factor, fungi Fungal_trans 131–318 6×10−6
g55t1 -
IPR001138
Zn(2)-C6 fungal-type DNA-binding GAL4 6–54 4.19 ×10−4
g55t2 PF04082
IPR007219
Transcription factor, fungi Fungal_trans 130–318 5.7×10−6
g55t2 -
IPR001138
Zn(2)-C6 fungal-type DNA-binding GAL4 6–54 4.19 ×10−4
g56t1 -
IPR000719
Protein kinase S_TKc 29–359 9.33 ×10−9
g57t1 - PR000719 Protein kinase S_TKc 167–466 0.142
g57t1 PF06985
IPR010730
Heterokaryon incompatibility HET 693–821 5.4 ×10−7
g58t1 PF05729
IPR007111
NACHT nucleoside triphosphatase NACHT 291–469 9.7 ×10−8
g58t1 -
IPR002110
Ankyrin repeats ANK
6 rpt. from
711 to 963
g58t2 PF05729
IPR007111
NACHT nucleoside triphosphatase NACHT 291–469 9.9 ×10−8
g58t2 -
IPR002110
Ankyrin repeats ANK
6 rpt. from
726 to 978
p65t1 PF00155
IPR004839
Aminotransferase, class I/class II Aminotran_1_2 27–208 1.8 ×10−25
p66t1 PF07690
IPR011701
Major facilitator superfamily MSF1 61–459 2 ×10−45
p66t2 PF07690
IPR011701
Major facilitator superfamily MSF1 86–466 2.1 ×10−45
p67t1 PF02826
IPR006140
D-isomer specific 2-hydroxyacid dehy-
drogenase 2-Hacid_dh_C 60–256 1.1 ×10−46
p68t1 PF03328
IPR005000
HpcH/HpaI aldolase/citrate lyase HpcH_HpaI 37–257 5.9 ×10−25
g70t1 PF02771
IPR013786
Acyl-CoA dehydrogenase/oxidase, N-
terminal Acyl-CoA_dh_N 5–117 9.2 ×10−18
g70t1 PF02770
IPR006091
Acyl-CoA dehydrogenase/oxidase,
middle Acyl-CoA_dh_M 121–222 6.9 ×10−18
g70t1 PF00441
IPR009075
Acyl-CoA dehydrogenase/oxidase, C-
terminal Acyl-CoA 234–390 2.7 ×10−32
g74t1 PF00271
IPR001650
Helicase, C-terminal helicase_C 14–116 1.8 ×10−8
g79t1 -
IPR002110
Ankyrin repeats ANK
9 rpt. from
275 to 275
g81t1 PF00135 IPR00201 Carboxylesterase, type B COesterase 33–488 1.1 ×10−93
g81t2 PF00135 IPR00201 Carboxylesterase, type B COesterase 33–364 4.9 ×10−83
g82t1 -
IPR007219
Transcription factor, fungi Fungal_trans 242–314 1.16×10−6
g89t1 PF00656
IPR011600
Peptidase C14, caspase Caspase 40–158 7 ×10−9
g89t2 PF00656
IPR011600
Peptidase C14, caspase Caspase 63-181 1 ×10−8
g93t1 PF03702
IPR005338
Anhydro-N-acetylmuramic acid kinase AnmK 2–382 1.9 ×10−60
g94t1 PF11991
IPR017795
Aromatic prenyltransferase, DMATS-
type Trp_DMAT 1–193 1.1 ×10−27
g95t1 PF00067
IPR001128
Cytochrome P450 p450 24–182 6.1 ×10−15
g96t1 PF00501
IPR000873
AMP-dependent synthetase/ligase AMP-binding 4–83 9.5 ×10−8

Agronomy 2023,13, 773 16 of 19
Table A3. Cont.
Gene Pfam Acc.
No.
InterPro
Acc. No.
Domain Name Domain Name
Abbreviation
Localization
(AA)
E Value
g100t1 PF03534
IPR003284
Salmonella virulence plasmid protein SpvB 47–240 1.1 ×10−51
g102t1 PF13472
IPR013830
SGNH hydrolase-type esterase Lipase_GDSL_2 22–239 1.5 ×10−11
g103t1 -
IPR001214
SET domain SET 7 –155
8.47
×
10
−12
g105t1 PF07690
IPR011701
Major facilitator superfamily MSF1 38–416 3.5 ×10−38
g109t1 PF00109
IPR014030
Beta-ketoacyl synthase, N-terminal Ketoacyl-synt 1–76 3.2 ×10−10
g109t1 PF00109
IPR014030
Beta-ketoacyl synthase, N-terminal Ketoacyl-synt 72–166 4.2 ×10−23
g109t1 PF02801
IPR014031
Beta-ketoacyl synthase, C-terminal Ketoacyl-synt_C 174–274 5.3 ×10−22
Table A4. Putative proteins of F. circinatum annotated by eggNOG.
Gene eggNOG Description
g9t1 arCOG00379 trimeric autotransporter adhesin
g10t1 7KF05 fibrous sheath CABYR-binding protein
g24t1 BKZCK ZnF_C2H2
g27t1 5K2KN sjoegren syndrome nuclear autoantigen 1
g47t1 5J4GB anthrone oxygenases
g48t1 KOG1724 S-phase kinase-associated protein 1
g59t1 KOG4626 protein O-GlcNAc transferase
g60t1 KOG4626 protein O-GlcNAc transferase
g75t1 KOG1546 nicotinamide-nucleotide amidase
g81t1 7NBP8 abhydrolase_1 alpha/beta hydrolase
g81t2 7NBP8 abhydrolase_1 alpha/beta hydrolase
g101t1 7K74H cupin domain
References
1.
Aoki, T.; O’Donnell, K.; Geiser, D.M. Systematics of key phytopathogenic Fusarium species: Current status and future challenges.
J. Gen. Plant Pathol. 2014,80, 189–201. https://doi.org/10.1007/s10327-014-0509-3.
2.
Dean, R.; Van Kan, J.A.; Pretorius, Z.A.; Hammond-Kosack, K.E.; Di Pietro, A.; Spanu, P.D.; Rudd, J.J.; Dickman, M.; Kahmann,
R.; Ellis, J.; et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol.
2012
,13, 414–430. https:
//doi.org/10.1111/j.1364-3703.2011.00783.x.
3.
Martín-García, J.; Paraschiv, M.; Flores-Pacheco, J.A.; Chira, D.; Diez, J.J.; Fernández, M. Susceptibility of several northeastern
conifers to Fusarium circinatum and strategies for biocontrol. Forests 2017,8, 318.
4.
Wingfield, M.; Hammerbacher, A.; Ganley, R.; Steenkamp, E.; Gordon, T.; Wingfield, B.; Coutinho, T. Pitch canker caused by
Fusarium circinatum — A growing threat to pine plantations and forests worldwide. Australas. Plant Pathol. 2008,37, 319–334.
5.
Bezos, D.; Martinez-Alvarez, P.; Fernandez, M.; Diez, J.J. Epidemiology and management of pine pitch canker disease in
Europe—A review. Balt. For. 2017,23, 279–293.
6.
Wingfield, B.D.; Steenkamp, E.T.; Santana, Q.C.; Coetzee, M.P.; Bam, S.; Barnes, I.; Beukes, C.W.; Chan, W.Y.; Vos, L.D.; Fourie, G.;
et al. First fungal genome sequence from Africa: A preliminary analysis. S. Afr. J. Sci. 2012,108, 1–9.
7.
Vos, L.D.; van der Nest, M.A.; van der Merwe, N.A.; Myburg, A.A.; Wingfield, M.J.; Wingfield, B.D. Genetic analysis of growth,
morphology and pathogenicity in the F1 progeny of an interspecific cross between Fusarium circinatum and Fusarium subglutinans.
Fungal Biol. 2011,115, 902–908. https://doi.org/10.1016/j.funbio.2011.07.003.
8.
Van Wyk, S.; Wingfield, B.D.; De Vos, L.; Santana, Q.C.; Van der Merwe, N.A.; Steenkamp, E.T. Multiple independent origins for a
subtelomeric locus associated with growth rate in Fusarium circinatum.IMA Fungus 2018,9, 27–36.
9.
Muñoz-Adalia, E.J.; Fernández, M.; Wingfield, B.D.; Diez, J.J. In silico annotation of five candidate genes associated with
pathogenicity in Fusarium circinatum.For. Pathol. 2018,48, e12417.
10.
Phasha, M.; Wingfield, M.; Wingfield, B.; Coetzee, M.; Hallen-Adams, H.; Fru, F.; Swalarsk-Parry, B.; Yilmaz, N.; Duong, T.;
Steenkamp, E. Ras2 is important for growth and pathogenicity in Fusarium circinatum.Fungal Genet. Biol.
2021
,150, 103541.
https://doi.org/10.1016/j.fgb.2021.103541.
11.
Maphosa, M.N.; Steenkamp, E.T.; Kanzi, A.M.; Van Wyk, S.; De Vos, L.; Santana, Q.C.; Duong, T.A.; Wingfield, B.D. Intra-species
genomic variation in the pine pathogen Fusarium circinatum.J. Fungi 2022,8, 657.
12. der Nest, M.V.; Olson, Å.; Lind, M.; Vélëz, H.; Dalman, K.; Durling, M.B.; Karlsson, M.; Stenlid, J. Distribution and evolution of
het gene homologs in the basidiomycota. Fungal Genet. Biol. 2014,64, 45–57. https://doi.org/10.1016/j.fgb.2013.12.007.

Agronomy 2023,13, 773 17 of 19
13.
Stanke, M.; Morgenstern, B. AUGUSTUS: A web server for gene prediction in eukaryotes that allows user-defined constraints.
Nucleic Acids Res. 2005,33, W465–W467.
14.
Hoff, K.J.; Stanke, M. WebAUGUSTUS—A web service for training AUGUSTUS and predicting genes in eukaryotes. Nucleic
Acids Res. 2013,41, W123–W128.
15. InterProScan. Available online: https://www.ebi.ac.uk/interpro/search/sequence/ (accessed on 15 January 2023).
16.
Jones, P.; Binns, D.; Chang, H.Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.; Nuka, G.; et al.
InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014,30, 1236–1240.
17.
Blum, M.; Chang, H.Y.; Chuguransky, S.; Grego, T.; Kandasaamy, S.; Mitchell, A.; Nuka, G.; Paysan-Lafosse, T.; Qureshi, M.;
Raj, S.; et al. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res.
2020
,49, D344–D354.
https://doi.org/10.1093/nar/gkaa977.
18.
Letunic, I.; Khedkar, S.; Bork, P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Res.
2020
,
49, D458–D460. https://doi.org/10.1093/nar/gkaa937.
19.
Hernández-Plaza, A.; Szklarczyk, D.; Botas, J.; Cantalapiedra, C.P.; Giner-Lamia, J.; Mende, D.R.; Kirsch, R.; Rattei, T.; Letunic, I.;
Jensen, L.J.; et al. eggNOG 6.0: Enabling comparative genomics across 12 535 organisms. Nucleic Acids Res.
2022
,51, D389–D394.
https://doi.org/10.1093/nar/gkac1022.
20.
Drenkhan, R.; Ganley, B.; Martín-García, J.; Vahalík, P.; Adamson, K.; Adamˇcíková, K.; Ahumada, R.; Blank, L.; Bragança, H.;
Capretti, P.; et al. Global geographic distribution and host range of Fusarium circinatum, the causal agent of pine pitch canker.
Forests 2020,11, 724.
21.
Zamora-Ballesteros, C.; Diez, J.J.; Martín-García, J.; Witzell, J.; Solla, A.; Ahumada, R.; Capretti, P.; Cleary, M.; Drenkhan, R.;
Dvoˇrák, M.; et al. Pine pitch canker (PPC): Pathways of pathogen spread and preventive measures. Forests 2019,10, 1158.
22.
Vainio, E.J.; Bezos, D.; Bragança, H.; Cleary, M.; Fourie, G.; Georgieva, M.; Ghelardini, L.; Hannunen, S.; Ioos, R.; Martín-Garcí, J.;
et al. Sampling and detection strategies for the pine pitch canker (PPC) disease pathogen Fusarium circinatum in Europe. Forests
2019,10, 723.
23.
Davydenko, K.; Nowakowska, J.A.; Kaluski, T.; Gawlak, M.; Sadowska, K.; García, J.M.; Diez, J.J.; Okorski, A.; Oszako, T. A
Comparative Study of the Pathogenicity of Fusarium circinatum and other Fusarium Species in Polish Provenances of P. sylvestris L.
Forests 2018,9, 560.
24.
Raitelaityt
˙
e, K.; Oszako, T.; Markovskaja, S.; Radzijevskaja, J.; Paulauskas, A. Fusarium circinatum research on Pinus sylvestris of
different provenances and interaction with other pine-inhabiting fungi. In Proceedings of the Smart Bio: ICSB 2nd International
Conference, Kaunas, Lithuania, 3–5 May 2018; Vytautas Magnus University: Kaunas, Lithuania, 2018.
25.
Elvira-Recuenco, M.; Cacciola, S.O.; Sanz-Ros, A.V.; Garbelotto, M.; Aguayo, J.; Solla, A.; Mullett, M.; Drenkhan, T.; Oskay, F.;
Kaya, A.G.A.; et al. Potential interactions between invasive Fusarium circinatum and other pine pathogens in Europe. Forests
2019
,
11, 7.
26.
Xu, J.R.; Yan, K.; Dickman, M.B.; Leslie, J.F. Electrophoretic karyotypes distinguish the biological species of Gibberella fujikuroi
(Fusarium section Liseola). MPMI-Mol. Plant Microbe Interact. 1995,8, 74–84.
27.
Saupe, S.J. Molecular Genetics of Heterokaryon Incompatibility in Filamentous Ascomycetes. Microbiol. Mol. Biol. Rev.
2000
,
64, 489–502. https://doi.org/10.1128/mmbr.64.3.489-502.2000.
28.
Puhalla, J.E.; Spieth, P.T. A comparison of heterokaryosis and vegetative incompatibility among varieties of Gibberella fujikuroi
(Fusarium moniliforme). Exp. Mycol. 1985,9, 39–47.
29.
Paoletti, M.; Clavé, C. The Fungus-Specific HET Domain Mediates Programmed Cell Death in Podospora anserina.Eukaryot. Cell
2007,6, 2001–2008. https://doi.org/10.1128/ec.00129-07.
30.
Dyrka, W.; Lamacchia, M.; Durrens, P.; Kobe, B.; Daskalov, A.; Paoletti, M.; Sherman, D.J.; Saupe, S.J. Diversity and Variability of
NOD-Like Receptors in Fungi. Genome Biol. Evol. 2014,6, 3137–3158. https://doi.org/10.1093/gbe/evu251.
31.
Saupe, S.; Turcq, B.; Bégueret, J. A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein
with a GTP-binding motif and Gβhomologous domain. Gene 1995,162, 135–139. https://doi.org/10.1016/0378-1119(95)00272-8.
32.
Koonin, E.V.; Aravind, L. The NACHT family–a new group of predicted NTPases implicated in apoptosis and MHC transcription
activation. Trends Biochem. Sci. 2000,25, 223–224.
33.
Bidard, F.; Clavé, C.; Saupe, S.J. The Transcriptional Response to Nonself in the Fungus Podospora anserina.G3 Genes Genomes
Genet. 2013,3, 1015–1030. https://doi.org/10.1534/g3.113.006262.
34.
Shelest, E. Transcription Factors in Fungi: TFome Dynamics, Three Major Families, and Dual-Specificity TFs. Front. Genet.
2017
,
8, 53.https://doi.org/10.3389/fgene.2017.00053.
35.
Chang, P.K.; Ehrlich, K.C. Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus.Appl.
Microbiol. Biotechnol. 2013,97, 4289–4300. https://doi.org/10.1007/s00253-013-4865-2.
36.
Galhano, R.; Illana, A.; Ryder, L.S.; Rodríguez-Romero, J.; Demuez, M.; Badaruddin, M.; Martinez-Rocha, A.L.; Soanes, D.M.;
Studholme, D.J.; Talbot, N.J.; et al. Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and
virulence in the rice blast fungus. PLoS Pathog. 2017,13, e1006516. https://doi.org/10.1371/journal.ppat.1006516.
37.
Hou, Z.; Chen, Q.; Zhao, M.; Huang, C.; Wu, X. Genome-wide characterization of the Zn(II)
2
Cys
6
zinc cluster-encoding gene
family in Pleurotus ostreatus and expression analyses of this family during developmental stages and under heat stress. PeerJ
2020
,
8, e9336. https://doi.org/10.7717/peerj.9336.

Agronomy 2023,13, 773 18 of 19
38.
Niño-Sánchez, J.; Castillo, V.C.D.; Tello, V.; Vega-Bartol, J.J.D.; Ramos, B.; Sukno, S.A.; Mínguez, J.M.D. The FTF gene family
regulates virulence and expression of SIX effectors in Fusarium oxysporum.Mol. Plant Pathol.
2016
,17, 1124–1139. https:
//doi.org/10.1111/mpp.12373.
39.
Mahanty, B.; Mishra, R.; Joshi, R.K. Molecular characterization of Zn(II)2Cys6 cluster gene family and their association with
pathogenicity of the onion basal rot pathogen, Fusarium oxysporum f. sp. cepae.Physiol. Mol. Plant Pathol.
2022
,117, 101782.
https://doi.org/10.1016/j.pmpp.2021.101782.
40.
Li, D.; Sirakova, T.; Rogers, L.; Ettinger, W.F.; Kolattukudy, P. Regulation of constitutively expressed and induced cutinase genes
by different zinc finger transcription factors in Fusarium solani f. sp. pisi (nectria haematococca). J. Biol. Chem.
2002
,277, 7905–7912.
41.
Hynes, M.J.; Murray, S.L.; Duncan, A.; Khew, G.S.; Davis, M.A. Regulatory genes controlling fatty acid catabolism and peroxisomal
functions in the filamentous fungus Aspergillus nidulans.Eukaryot. Cell 2006,5, 794–805.
42.
Rocha, A.L.M.; Pietro, A.D.; Ruiz-Roldán, C.; Roncero, M.I.G. Ctf1, a transcriptional activator of cutinase and lipase genes in Fusarium
oxysporum is dispensable for virulence. Mol. Plant Pathol. 2008,9, 293–304. https://doi.org/10.1111/j.1364-3703.2007.00463.x.
43.
Gacek, A.; Strauss, J. The chromatin code of fungal secondary metabolite gene clusters. Appl. Microbiol. Biotechnol.
2012
,
95, 1389–1404.
44.
Wagner, E.J.; Carpenter, P.B. Understanding the language of Lys36 methylation at histone H3. Nat. Rev. Mol. Cell Biol.
2012
,
13, 115–126.
45.
Connolly, L.R.; Smith, K.M.; Freitag, M. The Fusarium graminearum histone H3 K27 methyltransferase KMT6 regulates development
and expression of secondary metabolite gene clusters. PLoS Genet. 2013,9, e1003916.
46. Janevska, S.; Baumann, L.; Sieber, C.M.; Münsterkötter, M.; Ulrich, J.; Kämper, J.; Güldener, U.; Tudzynski, B. Elucidation of the
two H3K36me3 histone methyltransferases Set2 and Ash1 in Fusarium fujikuroi unravels their different chromosomal targets and
a major impact of Ash1 on genome stability. Genetics 2018,208, 153–171.
47.
Lee, J.H.; Skalnik, D.G. CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4
methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. J. Biol. Chem. 2005,280, 41725–41731.
48. Freitag, M. Histone methylation by SET domain proteins in fungi. Annu. Rev. Microbiol. 2017,71, 413–439.
49.
Gacek-Matthews, A.; Berger, H.; Sasaki, T.; Wittstein, K.; Gruber, C.; Lewis, Z.A.; Strauss, J. KdmB, a Jumonji histone H3
demethylase, regulates genome-wide H3K4 trimethylation and is required for normal induction of secondary metabolism in
Aspergillus nidulans.PLoS Genet. 2016,12, e1006222.
50.
Ma, L.J.; Geiser, D.M.; Proctor, R.H.; Rooney, A.P.; O’Donnell, K.; Trail, F.; Gardiner, D.M.; Manners, J.M.; Kazan, K. Fusarium
pathogenomics.Annu. Rev. Microbiol. 2013,67, 399–416.
51.
van Dam, P.; Fokkens, L.; Ayukawa, Y.; van der Gragt, M.; Ter Horst, A.; Brankovics, B.; Houterman, P.M.; Arie, T.; Rep, M. A
mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species. Sci. Rep. 2017,7, 9042.
52.
Voigt, C.A.; Schäfer, W.; Salomon, S. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of
cereals. Plant J. 2005,42, 364–375.
53.
Nguyen, L.N.; Bormann, J.; Le, G.T.T.; Stärkel, C.; Olsson, S.; Nosanchuk, J.D.; Giese, H.; Schäfer, W. Autophagy-related lipase
FgATG15 of Fusarium graminearum is important for lipid turnover and plant infection. Fungal Genet. Biol.
2011
,48, 217–224.
https://doi.org/10.1016/j.fgb.2010.11.004.
54.
Bravo-Ruiz, G.; Ruiz-Roldán, C.; Roncero, M.I.G. Lipolytic system of the tomato pathogen Fusarium oxysporum f. sp. lycopersici.
Mol.-Plant-Microbe Interact. 2013,26, 1054–1067.
55.
Jashni, M.K.; Dols, I.H.M.; Iida, Y.; Boeren, S.; Beenen, H.G.; Mehrabi, R.; Collemare, J.; de Wit, P.J.G.M. Synergistic action
of a metalloprotease and a serine protease from Fusarium oxysporum f. sp. lycopersici cleaves chitin-binding tomato chitinases,
reduces their antifungal activity, and enhances fungal virulence. Mol.-Plant-Microbe Interact.
2015
,28, 996–1008. https:
//doi.org/10.1094/mpmi-04-15-0074-r.
56.
Qian, H.; Song, L.; Wang, L.; Wang, B.; Liang, W. The secreted FoAPY1 peptidase promotes Fusarium oxysporum invasion. Front.
Microbiol. 2022,13, 1040302. https://doi.org/10.3389/fmicb.2022.1040302.
57.
Yan, N. Structural advances for the major facilitator superfamily (MFS) transporters. Trends Biochem. Sci.
2013
,38, 151–159.
https://doi.org/10.1016/j.tibs.2013.01.003.
58.
Stergiopoulos, I.; Zwiers, L.H.; Waard, M.A.D. Secretion of Natural and Synthetic Toxic Compounds from Filamentous Fungi by
Membrane Transporters of the ATP-binding Cassette and Major Facilitator Superfamily. Eur. J. Plant Pathol.
2002
,108, 719–734.
https://doi.org/10.1023/a:1020604716500.
59.
Sorbo, G.D.; jan Schoonbeek, H.; Waard, M.A.D. Fungal Transporters Involved in Efflux of Natural Toxic Compounds and
Fungicides. Fungal Genet. Biol. 2000,30, 1–15. https://doi.org/10.1006/fgbi.2000.1206.
60.
Brown, D.W.; McCormick, S.P.; Alexander, N.J.; Proctor, R.H.; Desjardins, A.E. A Genetic and Biochemical Approach to
Study Trichothecene Diversity in Fusarium sporotrichioides and Fusarium graminearum.Fungal Genet. Biol.
2001
,32, 121–133.
https://doi.org/10.1006/fgbi.2001.1256.
61.
Kimura, M.; Tokai, T.; O’Donnell, K.; Ward, T.J.; Fujimura, M.; Hamamoto, H.; Shibata, T.; Yamaguchi, I. The trichothecene
biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed
non-essential genes. FEBS Lett. 2003,539, 105–110. https://doi.org/10.1016/s0014-5793(03)00208-4.
62.
Hohn, T.M.; Krishna, R.; Proctor, R.H. Characterization of a Transcriptional Activator Controlling Trichothecene Toxin Biosynthesis.
Fungal Genet. Biol. 1999,26, 224–235. https://doi.org/10.1006/fgbi.1999.1122.

Agronomy 2023,13, 773 19 of 19
63.
Costachel, C.; Coddeville, B.; Latgé, J.P.; Fontaine, T. Glycosylphosphatidylinositol-anchored fungal polysaccharide in Aspergillus
fumigatus.J. Biol. Chem. 2005,280, 39835–39842.
64.
Ruiz-Herrera, J.; Ortiz-Castellanos, L. Cell wall glucans of fungi. A review. Cell Surf.
2019
,5, 100022. https://doi.org/10.1016/j.
tcsw.2019.100022.
65.
Ha, Y.s.; Covert, S.F.; Momany, M. FsFKS1, the 1, 3-
β
-Glucan Synthase from the Caspofungin-Resistant Fungus Fusarium solani.
Eukaryot. Cell 2006,5, 1036–1042. https://doi.org/10.1128/ec.00030-06.
66.
Sutherland, R.; Viljoen, A.; Myburg, A.A.; den Berg, N.V. Pathogenicity associated genes in Fusarium oxysporum f. sp. cubense race
4. S. Afr. J. Sci. 2013,109, 10. https://doi.org/10.1590/sajs.2013/20120023.
67.
Jiang, L.; Yang, J.; Fan, F.; Zhang, D.; Wang, X. The Type 2C protein phosphatase FgPtc1p of the plant fungal pathogen Fusarium
graminearum is involved in lithium toxicity and virulence. Mol. Plant Pathol.
2010
,11, 277–282. https://doi.org/10.1111/j.1364-3
703.2009.00598.x.
68.
Pei-Bao, Z.; Ren, A.Z.; Xu, H.J.; Li, D.C. The gene fpk1, encoding a cAMP-dependent protein kinase catalytic subunit homolog,
is required for hyphal growth, spore germination, and plant infection in Fusarium verticillioides.J. Microbiol. Biotechnol.
2010
,
20, 208–216.
69.
Xiao, J.; Zhang, Y.; Yang, K.; Tang, Y.; Wei, L.; Liu, E.; Liang, Z. Protein kinase Ime2 is associated with mycelial growth, conidiation,
osmoregulation, and pathogenicity in Fusarium oxysporum.Arch. Microbiol.
2022
,204.https://doi.org/10.1007/s00203-022-02964-0.
70.
Gaffar, F.Y.; Imani, J.; Karlovsky, P.; Koch, A.; Kogel, K.H. Different Components of the RNA Interference Machinery Are Required
for Conidiation, Ascosporogenesis, Virulence, Deoxynivalenol Production, and Fungal Inhibition by Exogenous Double-Stranded
RNA in the Head Blight Pathogen Fusarium graminearum. Front. Microbiol.
2019
,10, 1662.https://doi.org/10.3389/fmicb.2019
.01662.
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