Current progress on pathogenicity‐related transcription factors in Fusarium oxysporum

Abstract Fusarium oxysporum is a well‐known soilborne plant pathogen that causes severe vascular wilt in economically important crops worldwide. During the infection process, F. oxysporum not only secretes various virulence factors, such as cell wall‐degrading enzymes (CWDEs), effectors, and mycotoxins, that potentially play important roles in fungal pathogenicity but it must also respond to extrinsic abiotic stresses from the environment and the host. Over 700 transcription factors (TFs) have been predicted in the genome of F. oxysporum, but only 26 TFs have been functionally characterized in various formae speciales of F. oxysporum. Among these TFs, a total of 23 belonging to 10 families are required for pathogenesis through various mechanisms and pathways, and the zinc finger TF family is the largest family among these 10 families, which consists of 15 TFs that have been functionally characterized in F. oxysporum. In this review, we report current research progress on the 26 functionally analysed TFs in F. oxysporum and sort them into four groups based on their roles in F. oxysporum pathogenicity. Furthermore, we summarize and compare the biofunctions, involved pathways, putative targets, and homologs of these TFs and analyse the relationships among them. This review provides a systematic analysis of the regulation of virulence‐related genes and facilitates further mechanistic analysis of TFs important in F. oxysporum virulence.

grading waxes, cuticles, and cell walls to induce tissue invasion and pathogen dispersal . Additionally, F. oxysporum can produce mycotoxins, including beauvericin (Li et al., 2013), fusaric acid (López-Díaz et al., 2018), and fumonisins (Rheeder et al., 2002), which contribute to pathogenicity in hosts. Beauvericin and fusaric acid have been associated with Foc, causing banana plantlets to wilt, decay, and die in vitro (Li et al., 2013). In Foc tropical race 4 (TR4), fusaric acid plays a key role as a positive virulence factor, particularly at the early stage of disease development (Liu et al., 2020). Beauvericin is a mycotoxin produced by many Fusarium species and by Beauveria bassiana that induces DNA fragmentation and apoptosis by disrupting mitochondrial pathways (Mallebrera et al., 2018). In Fol, beauvericin reduces the level of ascorbic acid in tomato cells, leading to collapse of the ascorbate system and protoplast death (Paciolla et al., 2004).
In addition to CWDEs and mycotoxins, F. oxysporum produces effectors known as Six (secreted in xylem) proteins, a group of cysteine-rich effectors identified from the xylem sap of tomato after inoculation with pathogens. To date, 14 Six effectors have been identified. In Fol, some SIX genes have been found to play significant roles in determining host specificity. For instance, SIX4 (AVR1), SIX3 (AVR2), and SIX1 (AVR3) act as avirulence genes that interact with the tomato resistance genes I-1 (Immunity-1), I-2, and I-3, respectively (Taken & Rep, 2010). SIX genes have been identified in a range of F. oxysporum formae speciales, but each forma specialis of the fungus has a specific combination of these SIX genes that can be used to predict F. oxysporum host specificity (van Dam et al., 2016).
In Fol, the SIX genes are located on lineage-specific chromosomes known as accessory chromosomes that are enriched in transposable elements (TEs) and play a role in host specialization (Ma et al., 2010).
Moreover, accessory chromosomes can be horizontally transferred to another strain, which is associated with the host-specific pathogenicity of F. oxysporum (Ma et al., 2010;van Dam et al., 2017).
Transcription factors (TFs) are the main players in transcriptional regulation of pathways implicated in F. oxysporum virulence. During infection, F. oxysporum connects sensing of host cues to transcriptional reprogramming by activating a number of TFs that govern the physiological adaptation, pathogenesis, and growth of the fungus to adapt to the host environment . Previously, the genome of Fol was sequenced, and approximately 5% (genome size) of the genes were found to encode TFs, making them the largest protein family in F. oxysporum (Ma et al., 2010). Genomic analysis of both Foc race 1 and Foc race 4 showed that the genome structures are highly syntenic with that of Fol and that Foc race 1 encodes 729 TFs compared to 793 for Foc race 4 . To date, only 26 TFs have been functionally analysed in F. oxysporum, but comparison and summarization of the biological functions and mechanisms of these TFs are limited.
Basic leucine zipper (bZIP) transcription factors are highly conserved in eukaryotes and form one of the largest families of dimerizing TFs (Ellenberger, 1994). bZIP family proteins are characterized as having a basic region for DNA binding and a leucine zipper region for protein dimerization consisting of up to nine heptad repeats with a leucine every seven amino acids, thereby allowing two monomers to "zip up" when forming the protein dimer (Ellenberger, 1994;Ellenberger et al., 1992). In F. oxysporum, a total of three bZIP TFs have been functionally analysed: FoAtf1, MeaB, and HapX (López-Berges et al., 2010Qi et al., 2013).
Velvet complex proteins are highly conserved in filamentous fungi and include VeA (VelA), VelB, VelC, and LaeA proteins (Bayram et al., 2008). These proteins have a 150-amino acid region known as the velvet domain, which is thought to be a key regulator of diverse cellular processes, such as secondary metabolism and asexual or sexual sporulation (Bayram & Braus, 2012 (Zhao et al., 2015). In F. oxysporum, two transcription factors belonging to this family, StuA and Swi6, have been functionally analysed (Ding et al., 2018;. Only one transcription factor each from the MADS-box, Pac2/ Gti1, and HSF (heat shock factor) families has been analysed in F. oxysporum. MADS is an acronym of the initials of MINICHROMOSOME MAINTENANCE 1 (MCM1) from S. cerevisiae, AGAMOUS (AG) from Arabidopsis, DEFICIENS (DEF) from Antirrhinum majus, and S for serum response factor (SRF) from Homo sapiens (Yanofsky et al., 1990). In F. oxysporum, MADS-box Rlm1 is the only TF that has been functionally tested (Ding et al., 2020). Gti1/Pac2 is a conserved fungal protein family that regulates morphogenic transition and controls toxin production and pathogenesis in fungi . Pac2 and Gti1 were the first members characterized in this family of TFs.
Sge1 is the only member of this family that has been functionally analysed in F. oxysporum (Michielse, van Wijk, Reijnen, Manders, et al., 2009). Heat shock factor-type transcriptional regulators bind specifically to heat shock sequence elements (HSEs) throughout the genome (Guertin & Lis, 2010). Their activators have a role in maintaining cell wall integrity and regulating the osmotic/oxidative stress response (OSR) in S. cerevisiae, where it is part of a two-component signal transduction system. FoSkn7 is the only member of this family that has been functionally analysed in F. oxysporum (Qi et al., 2019). in F. oxysporum, Sge1 and Ftf1 regulate the expression of effector genes. Xln1, Ctf1, and Ctf2 are responsible for regulation of CWDE activities, while FolCzf1 and the velvet complex act as regulators of mycotoxin production. Research progress on the above TFs will be introduced below.
In Fol, the SGE1 deletion mutant (ΔSGE1) almost loses virulence against tomato plants, and deletion of SGE1 affects parasitic growth within xylem tissue but not colonization of the root surface or root penetration (Michielse, van Wijk, Reijnen, Manders, et al., 2009). but absent in the genome of weakly virulent strains (Michielse, van Wijk, Reijnen, Manders, et al., 2009;Nino-Sanchez et al., 2016 In M. oryzae, XlnR was found to regulate the expression of genes involved in the pentose catabolic pathway but not genes encoding hemicellulolytic enzymes (Battaglia et al., 2013). In F. graminearum, Xyr1, the homolog of XlnR, regulates xylanase but not cellulase secretion (Brunner et al., 2007).

| FolCzf1 and velvet complex regulate mycotoxin production in F. oxysporum
The C2H2 TF FolCzf1 plays an important role in early infection in F. oxysporum (Yun et al., 2019). Deletion of FolCZF1 resulted in a loss of virulence and decreased fusaric acid production (Yun et al., 2019).
A transcriptional profiling study further showed that FolCzf1 is involved in regulating fusaric acid biosynthesis genes (Yun et al., 2019).
The homologs of FolCzf1, F. graminearum GzC2H003, and M. oryzae GCF3 are highly expressed in the early infection stage and are required for the pathogenicity of F. graminearum and M. oryzae (Cao et al., 2016;Son et al., 2011). Furthermore, deletion of GzC2H003 does not affect the biosynthesis of the mycotoxin deoxynivalenol but leads to increased production of zearalenone (Son et al., 2011).
The heterotrimeric velvet complex is a conserved regulator of fungal development and secondary metabolism previously examined in other Fusarium pathogenic fungi, such as F. graminearum, F. verticillioides, and F. fujikuroi (Bayram et al., 2008;Jiang et al., 2012;Wiemann et al., 2010).  tissue. Extracellular alkalization is thought to contribute to the fungal pathogenesis of F. oxysporum (Masachis et al., 2016). PacC is an important pH-responsive TF in F. oxysporum. Another factor affecting the virulence of Fusarium pathogens is light. Light is a signal from the environment that regulates many fungal development processes.

| TR ANSCRIP TIONAL REG UL ATION OF PATHOG ENI CIT Y UNDER AB I OTI C S TRE SS I N F. OX YSP ORUM
Photoreceptors sense light and generate signals that stimulate cellular responses, such as carotenoid biosynthesis, spore formation, and phototropism (Yu & Fischer, 2019). Recent studies have demonstrated that the presence of a photosensor component is needed not only for ecological adaptation of the pathogen but also for regulation of virulence factor expression (Tang et al., 2020). In F. oxysporum, the TF WC-1 is the key element involved in light signal transduction.
The oxidative burst, a defence strategy observed in most host plants during fungal invasion, consists of rapid production of large amounts of reactive oxygen species to control fungal penetration during root colonization (Zeilinger et al., 2016). Therefore, F. oxysporum has evolved different mechanisms to detoxify reactive oxygen species

| Fnr1 and MeaB are involved in the nitrogen catabolic process and the pathogenicity of F. oxysporum
GATA-ZF TF AreA homologs have been identified as regulators of nitrogen nutrition genes in filamentous fungi, and the homolog of AreA in Fol was named Fnr1 (Fusarium nitrogen regulator 1) (Divon et al., 2006). Fnr1 disruption mutants showed a reduced ability to utilize several secondary nitrogen sources, whereas growth on favourable nitrogen sources was not affected (Divon et al., 2006). Deletion of FNR1 led to an obvious reduction in the pathogenicity of Fol on tomato and abolished the in vitro expression of nutrition genes during the early phase of infection, suggesting that Fnr1 mediates adaptation to nitrogen-poor conditions in planta through regulation of secondary nitrogen acquisition and acts as a determinant for fungal fitness during infection (Divon et al., 2006
In Fol, ZAFA expression is induced under zinc-limiting conditions and repressed by zinc at early stages of plant infection, which regulates the expression of many genes in response to zinc deficiency, including high-affinity membrane zinc transporters, such as Zrt2, Zrt3, Fet4, and Zrc1 (López-Berges, 2020). ZafA is also required for full virulence of Fol in plant and animal hosts (López-Berges, 2020). In the opportunistic fungal pathogen A. fumigatus, the homolog of ZafA, AoZafA, also plays an essential role in maintaining zinc homeostasis, and AoZafA was found to be regulated by pH and influenced by the PacC transcriptional regulator (Amich et al., 2009(Amich et al., , 2010.

| PacC regulates the pH response but is a negative regulator of F. oxysporum pathogenicity
The expression level of PACC is elevated in Fol grown under alkaline conditions and is almost undetectable under extreme acidic growth conditions. Gene deletion of PACC led to an acidity-mimicking phenotype, resulting in poor growth at alkaline pH and increased acid protease activity, while overexpression of PACC led to the opposite phenotype. However, loss of PACC resulted in increased virulence in tomato roots, suggesting that Fol PacC acts as a negative regulator of virulence in plants, possibly by preventing transcription of acidexpressed genes important for infection (Caracuel et al., 2003). The homologs of PacC in other filamentous fungi, including A. nidulans, M. oryzae, and Fusarium species, showed a conserved role in regulation of pH-response genes (Bignell et al., 2005;Landraud et al., 2013). In addition, in F. graminearum and F. verticillioides, PacC homologs are also involved in secondary metabolites by regulating the production of trichothecene and fumonisins (Flaherty et al., 2003;Merhej et al., 2011).

| Wc-1 is involved in light signal transduction in F. oxysporum
White collar 1 (WC-1) and WC-2 are two key elements involved in light signal transduction that have been previously studied in N. crassa (Ballario et al., 1996;Liu et al., 2003).

| FoAtf1 and FoSkn7 regulate the oxidative stress response and F. oxysporum pathogenicity
The bZIP TF FoAtf1 has been characterized in Foc race 4. The FoATF1 deletion mutant was highly sensitive to hydrogen peroxide compared with the wild-type strain (Qi et al., 2013). In addition, FoAtf1 is thought to be involved in the reduction of extracellular enzyme activity and the transcription level of catalase. Furthermore, FoAtf1 has been shown to be involved in virulence by regulating the oxidative stress responses of Cavendish banana (Musa spp.) (Qi et al., 2013). In M. oryzae, MoAtf1 is involved in the oxidative stress response by regulating host-derived reactive oxygen species levels and facilitating successful pathogen invasion of host tissues (Guo et al., 2010).
In Foc race 4, FoSkn7 is thought to be involved in growth and conidiation as well as in the reduction of Foc race 4 virulence (Qi et al., 2019). The FoSKN7 deletion mutant is highly sensitive to oxidative stress (Qi et al., 2019). Previous work in A. flavus has revealed that Skn7 induces the expression of several antioxidant genes that provide resistance to hydrogen peroxide, including genes encoding peroxidase, catalase, and thioredoxin Vargas-Perez et al., 2007). The functions of FoSkn7 are similar to those of Skn7 orthologs in Alternaria alternata and in other fungi that participate in the oxidative stress response, such as Penicillium marnefei, C. glabrata, and S. cerevisiae (Cao et al., 2009;Chen et al., 2012;Mulford & Fassler, 2011;Saijo et al., 2010).

| Rlm1, Swi6, and Ste12 act as downstream TFs involved in MAPK pathways in F. oxysporum
Mpk1 is the key kinase in the conserved cell wall integrity (CWI) MAPK pathway, repairing the cell wall and maintaining cellular integrity, which is mediated by Swi4-Swi6 cell cycle box-binding factor (SBF) and the MADS-box transcription factor Rlm1 in the budding yeast S. cerevisiae (Levin, 2011). In Foc, the homologs of Swi6 and Rlm1 have been functionally analysed (Ding et al., 2018(Ding et al., , 2020. Swi6 is crucial, but Rlm1 is dispensable for vegetative growth of Foc; however, deletion of SWI6 or RLM1 results in both reduced virulence in banana plantlets and increased sensitivity to hydrogen peroxide. Interestingly, both Swi6 and Rlm1 are involved in regulation of fusaric acid biosynthesis genes, and Rlm1 is also required for transcription of beauvericin biosynthesis genes, suggesting that Swi6 and Rlm1 may also take part in regulation of secondary metabolism in Foc. In F. graminearum, FgRlm1 and FgSwi6 are required for fungal growth, development, pathogenicity, and secondary metabolism processes, suggesting a conserved role of Rlm1 and Swi6 homologs among Fusarium fungi (Liu et al., 2013;Yun et al., 2014).
The Fmk1 MAPK pathway is highly conserved in fungi and is essential for infection in most plant pathogens (Turra et al., 2014). In Fol, the Fmk1 MAPK cascade promotes expression of the TF Ste12 to activate transcription of genes involved in pathogenicity (Rispail & Di Pietro, 2009). Targeted deletion mutants lacking Ste12 showed dramatic defects in invasive growth, vegetative hyphal fusion, and secretion of pectinolytic enzymes as well as reduced virulence in tomato plants (Rispail & Di Pietro, 2009). Additionally, it has been shown that Ste12 positively regulates extracellular amylase and cellulase activities but is not impaired in the activation of pectinase genes in Fol (Rispail & Di Pietro, 2009 (Deng et al., 2007) and B. cinerea (Schamber et al., 2010), the human pathogens C. albicans (Liu et al., 1994) and C. neoformans (Yue et al., 1999), and the saprophytes N. crassa (Li et al., 2005) and A. nidulans (Vallim et al., 2000). The sexual spores (ascospores) produced by some Fusarium species function as infectious propagules; however, sexual reproduction in F. oxysporum is still unknown.

| TR ANSCRIP TIONAL REG UL ATION OF A S E XUAL CONID IATI ON IN F. OX YSP ORUM
F. oxysporum is unique in its asexual reproduction, producing large quantities of three types of conidia (microconidia, macroconidia, and chlamydospores) . Observation of host colonization suggests that this process is expedited by the accumulation of conidia, which move freely within the xylem, leading to blockade of the vessels by forming a plate at the end. Then, microconidia will germinate and form germ tubes that are able to produce more conidia (Beckman et al., 1961). The germination, penetration, and sporulation series accelerates the speed of host colonization. StuA and Ren1 regulate the development of asexual conidiation in F. oxysporum. In F. oxysporum f. sp. melonis (Fom) REN1 deletion mutants, the production of microconidia and macroconidia are affected, but normal vegetative growth and chlamydospore formation are observed. In Fol, deletion of STUA showed normal microconidium formation and reduced macroconidium formation but increased chlamydospore formation under culture conditions, but the mutants produced markedly fewer macroconidia and microconidia in infected plants than the wild-type strain. However, loss of Ren1 or StuA did not affect pathogenesis, suggesting that microconidia and macroconidia may not be required for F. oxysporum pathogenicity.

| TR ANSCRIP TIONAL REG UL ATION OF G ENER AL ME TABOLIS M AND VIRULEN CE IN F. OX YSP ORUM
Some TFs are specific for virulence, but some TFs affect many developmental processes, acting as general regulators in fungal biological processes. The three TFs, Ebr1, Con7-1, and Snt2, affect general metabolism, which leads to weak fitness of fungi. The basic phenotypes, including virulence, were affected after loss of these three factors.
However, whether these three TFs also have direct roles in virulence regulation is still unknown. In contrast to these three TFs, Fow2 and Cti6 are specific regulators of the pathogenicity of F. oxysporum, and their loss affects virulence, but the mechanism is also unknown.
Ebr1 (enhanced branching 1) has been found to impair growth, reduce pathogenicity, and slightly reduce biocontrol capacities in F. graminearum (Thatcher et al., 2012;Zhao et al., 2011). Interestingly, EBR1 is present as multiple copies in Fol strains, while only a single copy is found in F. graminearum (Thatcher et al., 2012;Zhao et al., 2011). In the Fol 4287 strain, nine paralogs of Ebr1 were retrieved, among which Ebr1 (FOXG_05408) showed the highest similarity to FgEbr1 (89%). Deletion of EBR1 in different Fol strains containing different numbers of EBR genes resulted in similar impaired growth and reduced pathogenicity, suggesting that loss of EBR1 causes a growth and pathogenicity phenotype in Fol strains independent of the other EBR copies (Thatcher et al., 2012).

Con7 has been described as a central regulator of infection-
related morphogenesis in the rice blast fungus M. oryzae (Odenbach et al., 2007). However, three copies of Con7 homologs have been identified in Fol, and Con7-1 (FOXG_11503) shares the closest (69%) identity with Con7 of M. oryzae (Ruiz-Roldan et al., 2015). Gene deletion and comparative microarray-based gene expression analysis demonstrated that Con7-1 acts as a general regulator in polar growth, hyphal branching, conidiation, and pathogenicity (Ruiz-Roldan et al., 2015). In contrast, simultaneous inactivation of both Con7-2 copies caused no detectable defects in the deletion mutants, suggesting that the other two Con7-2 copies are not important for the growth, development, and pathogenicity of F. oxysporum.
Snt2 is involved in regulation of hyphal growth, hyphal septation, conidiation, and fungal respiration in F. oxysporum f. sp. melonis (Fom) (Denisov et al., 2011). SNT2 deletion mutants showed obviously reduced virulence in muskmelon plants and exhibited significantly lower colonization of upper plant stems, suggesting that Snt2 is required for the early stage and host colonization of Fom pathogenesis (Denisov et al., 2011). Furthermore, Snt2 has been found to regulate the expression of four genes (IDI4, PDC, MSF1, eEF1G) in the TOR pathway, but Snt2 is not involved in nitrogen metabolism, suggesting that the Snt2 and TOR pathways share common components to support an overlapping regulatory mechanism in maintaining cell biological processes in F. oxysporum (Denisov et al., 2011).
Fow2, a Zn(II)2Cys6-type TF, is required for full virulence but not hyphal growth and conidiation in Fom (Imazaki et al., 2007). Fow2 also controls the ability of Fom to invade roots and colonize plant tissue (Imazaki et al., 2007). FOW2 is highly conserved in formae speciales of F. oxysporum. Homologs of Fow2 were identified in six other formae speciales of F. oxysporum, and the deletion of FOW2 in Fol also resulted in near nonvirulence in tomato plants (Imazaki et al., 2007).
Cti6, a PHD finger-containing TF, is also required for full pathogenicity of Fol toward tomato plants and participates in chromatin modifications (Michielse, van Wijk, Reijnen, Cornelissen, et al., 2009

| CON CLUS ION
The lack of effective control methods for Fusarium wilt disease threatens the production of many economic crops, especially for F I G U R E 1 Schematic representation of the potential relationship of the characterized transcription factors with the different signalling pathways and cellular processes associated with Fusarium oxysporum virulence cultivation in a greenhouse. Thus, an improved understanding of the sensing, pH response, iron starvation, zinc homeostasis, and oxidative stress, which play an important role in the infection process. As shown in Figure 1, the known TFs involved in regulation of the above process are summarized in this review. However, our understanding of this process is still limited. To further understand the stressresponsive gene regulatory networks in F. oxysporum, components of the regulatory systems should be identified, including genes encoding TFs and genes encoding downstream component products.
The availability of the complete genomes of some formae speciales of F. oxysporum may help in improving understanding of the TFs involved in pathogenesis and has already provided evidence of several potential pathogenic mechanisms Ma et al., 2010). Some of these TFs have been predicted to have essential roles in coordinating gene expression and might coordinate the exchange between core chromosomes and accessory chromosomes. Among the 26 functionally analysed TFs in F. oxysporum, FTF1, PACC, EBR1, and FOW2 are expanded in the genome and contribute to pathogenicity (Caracuel et al., 2003;Jonkers et al., 2014;Ramos et al., 2007).
Interestingly, FTF1 has the largest number of copies (11 genes predicted in Fol and four genes predicted in Fop), while EBR1 and FOW2 have nine and three gene copies in the Fol genome, respectively (Jonkers et al., 2014;Ramos et al., 2007). A recent study investigating the genome of the opportunistic fungal pathogen F. oxysporum NRRL 32931 identified four paralogs of PACC (PACC_0, PACC_a, PACC_b, and PACC_c) . These four TFs comprise a singlecopy gene found on core chromosomes, with several copies distributed on accessory chromosomes. TFs encoded in the core genome have been demonstrated to potentially regulate the expression of other accessory chromosome paralogs, such as EBR1-like paralogs (EBR2, EBR3, and EBR4), which are regulated by core chromosome EBR1 (Jonkers et al., 2014). Similar to EBR, core chromosome PACC plays a predominant function and has been found to regulate alkaline pH . In contrast, the deletion accessory chromosome FTF1 copy displayed a role in virulence, while core chromosome FTF1, up-regulated during infection, showed less effect on virulence (de Vega- Bartol et al., 2011;Niño-Sánchez et al., 2015). The study also revealed that FTF1 on either accessory chromosomes or the core chromosome can be induced to regulate many effectors in Fol, suggesting that accessory chromosome and core chromosome TFs may play various functions (van der Does et al., 2016). Although paralogous TFs share the same DNA-binding motifs, they perform distinct regulatory functions. Further genomic analysis and functional studies of TF expansion can give rise to new TF families and their function in the origin and evolution of pathogenesis.
In this review, we have summarized the pathogenicity-related TFs that have been characterized in F. oxysporum ( Figure 1 and Table 2). However, our understanding of the role of TFs that modulate F. oxysporum virulence is far from complete, and particularly lacking are those involved in the early stages of host-pathogen interactions and the regulatory network of virulence-related factors, thus further exploration of the mechanism and target networks of the known TFs is still needed.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.