Functional analysis of cutinase transcription factors in Fusarium verticillioides

Abstract

Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide. F. verticillioides produces highly toxic mycotoxin Fumonisin B1 (FB1) in infested commodities which makes these food and feeds unsafe for humans and animals. For pathogenic fungi to successfully penetrate its plant hosts, the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer. However, there is limited information on how cutinases transcriptionally regulated to impact F. verticillioides pathogenicity. In this study, our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F. verticillioides pathogenicity and FB1 biosynthesis. Gene deletion of cutinase transcription factor FvCTF1α did not affect the growth and morphology of the fungal mycelia on CMII medium, whereas the conidiation, utilization of sodium acetate and sodium oleate, stress tolerance against cell wall interfering agent_, and the cutinase and pectinase activities in the ΔFvctf1α mutant were negatively impacted. FvCtf1α regulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters. In addition, FvCtf1α, containing a novel function in regulating FB1, interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6, resulting in decreased production of FB1 in the ΔFvctf1α strain. ΔFvctf1α exhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F. verticillioides. We also demonstrated that FvCtf1α regulated FvCUT3 and FvCUT4 differently; FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB, a homologous protein of FvCtf1α. Moreover, RNA-seq analysis showed that FvCtf1α was associated with many pathways, such as fatty acid metabolism, carbon source utilization, cell wall integrity, oxidative stress, and fumonisin synthesis in F. verticillioides. Our study demonstrated that FvCtf1α was not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F. verticillioides virulence and mycotoxin biosynthesis.

Background

Cutin is the main structural component of the plant cuticle, a polymer consisting of hydroxy and epoxy fatty acids of n-C16 and n-C18 types (Kolattukudy 2001). Cutinases hydrolyze substrates such as p-nitrophenyl palmitate (pNPP), p-nitrophenyl butyrate (pNPB), and triglycerides (Martinez et al. 1992). The role of cutinases and their transcription factors in fungal pathogenicity against plants remains ambiguous. Some fungal pathogens secrete cutinases that hydrolyzes ester bonds from fatty acid polymers, thus facilitating fungal penetration through the cuticle (Hynes et al.2006). Four promoter elements involved in cutinase gene regulation are a positive-acting G-rich Sp1-like element, a silencer binding a basal transcription factor, and two overlapping palindromic sequences (palindrome 1 and palindrome 2) (Kämper et al. 1994; Li et al. 2002). Palindrome 1 (Pal 1) remains repressed until induced by cutin monomers (Li et al. 2002), while palindrome 2 (Pal 2) is the sequence that confers inducibility by plant cuticular monomers hydroxy fatty acids (Li and Kolattukudy 1997). Based on the binding preference to different cis-elements, especially the two overlapping palindromes in the cutinase gene promoter under varying environmental conditions, cutinase transcription factors (CTFs) are divided into four types, i.e., palindrome-binding protein (PBP), Ctf1α, Ctf1β, and Ctf2, which regulate the expression of two cutinase types (constitutive expression and induction expression). PBP binds to Pal 1, while both Ctf1α and Ctf1β, which contain a Cys6Zn2 binuclear cluster motif, bind to Pal 2. Ctf2 binds to the cis-element located between the TATA box and the transcription initiation sites. In the absence of cutin monomers, PBP binds only to Pal 1 of the induction expression cutinase promoter to keep the gene repressed. However, in the presence of cutin monomers, Ctf1α phosphorylation displaces PBP (the repressor) from Pal 1 binding, thereby leading to the inactivation of dominant inhibitor PBP, and selectively binds to Pal 2, which induces gene expression (Li et al. 2002). Ctf1β usually can only bind to Pal 2 leading to the activation of constitutive expression cutinase genes and the production of low levels of cutin monomers. Ctf1β usually regulates constitutive expression cutinases, but if PBP does not preemptively bind to their sites, CTF1β can also regulate the induction of cutinase expression.

For example, if Pal 1 of the induction expression cutinase gene cannot bind to PBP, such as in the case of nucleotide substitutions in Pal 1, Ctf1β may bind to Pal 2 of the induction expression cutinase (Li et al. 2002). During the infection process in Fusarium solani f. sp. pisi virulent strains, the contact of a fungal spore with cutin, produced by small amounts of constitutively expressed cutinase, triggers the induction of cutinase transcription within minutes (Köller et al. 1982; Woloshuk and Kolattukudy 1986).

In addition to the induction of plant cuticular components, cutinase expression is under catabolite repression by glucose (Lin and Kolattukudy 1978). Glucose is involved in the regulation of cutinase expression by CTF. In a glucose-depleted condition, Ctf1α binds to its target sequence whether or not induced by cutin hydrolysate. Ctf2 binds to the cis-elements located between the TATA box and the transcription initiation sites in F. solani, which may be another gene-specific activating or repressing DNA-binding protein involved in cutinase gene regulation, potentially mediating glucose repression. CUT1 of F. solani f. sp. pisi is also under glucose catabolite repression (Li et al. 2002). Altogether, Ctf1α can compete for PBP on cutin induction or Ctf2 can regulate the induction expression cutinase on the glucose-depleted situation.

Homologs of Ctf1 and Ctf2 have been found in several plant fungal pathogens, including Aspergillus nidulans, Nectria haematococca, Fusarium oxysporum f. sp. lycopersici, and Magnaporthe oryzae (Li et al. 2002; Hynes et al. 2006; Bravo-Ruiz et al. 2013; Bin Yusof et al.2014). FarA, the homolog of Ctf1α in Aspergillus oryzae, has conserved functions in the lipolytic system and fatty acids (Hynes et al. 2006; Garrido et al. 2012). These Ctf1 and Ctf2 orthologs are also present in human pathogenic yeast Candida albicans and alkane-assimilating yeast Yarrowia lipolytica, where they are involved in fatty acid utilization (Rocha et al. 2008; Ramírez et al.2009; Poopanitpan et al. 2010). Ctf1, a Ctf1α homolog, of the stem pathogen F. solani regulates CUT1 and LIP1 genes that are essential for virulence (Bravo-Ruiz et al. 2013), but Ctf1α of the root pathogen F. oxysporum is not essential for its virulence (Rocha et al. 2008). FarA and FarB of A. nidulans, two proteins with homology to Ctf1α, regulate the expression of genes implicated in the metabolism of short-chain and long-chain fatty acids, respectively (Hynes et al. 2006). FarA is implicated in the expression of cutinase protein Cut1 and hydrophobic surface-binding protein HsbA, required for the degradation of biodegradable plastic, butylene succinate-co-adipate (PBSA), as well as in the expression of lipolytic genes such as mono- and di-acylglycerol lipase MDLB and triacylglycerol lipase TGLA for lipid hydrolysis in A. oryzae (Garrido et al. 2012).

DNA motif identification plays a fundamental role in the elucidation of regulatory mechanisms of transcription factors. Among the many reported motifs, the AGGGG motif (G-box) has been observed in the promoters of fungal chitinase, cutinase, and many other genes (Kämper et al. 1994; Chen et al. 2021). The G-box motif in N. haematococca cutinase gene promoter is required for maintaining the basal level of cutinase gene transcription (Kämper et al. 1994). In addition, the C2H2-type zinc finger protein in Y. lipolytica Mhy1 and Trichoderma atroviride Seb1 also bind to the G-box motif in stress-response elements (Hurtado and Rachubinski 1999; Peterbauer et al. 2002). Despite this information, mechanistic insights into how the G-box binding transcription factor governs fungal infection remain unexplored. Gene induction by cutin monomers is regulated by Ctf1α, most likely a dimeric DNA-binding protein with a palindromic recognition site CCGAGG in F. solani (Li et al. 2002). FarA and FarB of A. nidulans, two proteins with homology to Ctf1α, bind in vitro to the same core DNA element that mediates the binding of the F. solani Ctf1α (Li and Kolattukudy 1997, Lin et al. 2022; Hynes et al. 2006).

The fungal pathogen F. verticillioides can cause significantly damaging grain diseases worldwide, particularly in China. The presence of F. verticillioides in grain can cause both substantial yield loss and grain contamination by Fumonisin B1 (FB1), a mycotoxin with significant pathological consequences for livestock and potentially for humans. The role of cutinase in F. verticillioides virulence and FB1 biosynthesis is not clearly defined. Although studies of Ctfs and Cuts had been performed in phytopathogenic fungi, such as F. solani (Li and Kolattukudy 1997, Lin et al. 2022), their complex regulatory mechanism in F. verticillioides on pathogenicity remains unclear. Our previous studies found that FvCtf1α regulates the production of toxin FB1. In this study, our aim was to characterize the role of F. verticillioides CTFs, mainly FvCtf1α, in expression of cutinases, as well as virulence and mycotoxin production.

Results

FvCTF1 and FvCUT genes of F. verticillioides

In the F. verticillioides genome, 12 cutinase genes (including FvCUT1, FvCUT3, FvCUT4), one cutinase palindrome-binding protein (CPBP), four Ctf1α (including FvCTF1α and FvFARA), and seven FvCTF1β (including FvFARB) were found. Except for one CTF1β and three cutinases, all other genes were detected by RNA-seq analysis in infected maize kernels and CMII medium (Table 1). FvCPBP, FvFARA, FvCTF1α, FvCTF1αC, FvFARB, FvCTF1β, and FvCUT3 were constitutively expressed under different conditions according to RNA-sequencing data, and their expressions were significantly induced when F. verticillioides infected maize kernel (Table 1).

Table 1 The expression of cutinase transcription factor and cutinase gene in Fusarium verticillioides on different medium and the effect of ∆Fvctf1α mutant on kernel medium

Identification of FvCtf1α as a transcription factor for cutinases FvCUT1 and FvCUT4 in F. verticillioides

When blasting Ctf1α and Ctf1β protein sequences of the pea stem pathogen F. solani to the F. verticillioides genome database, we found only one homologous protein (FVEG_00228) with similarities of 29.62% and 27.51%, respectively. Functional domain analysis revealed that FVEG_00228 contained two conserved domains: GAL4-like Zn(II)2Cys6 and a fungal-specific transcription factor domain (Fungal trans) (Additional file 1: Figure S1a). Moreover, amino acids 341-373 of the FVEG_00228 protein sequence were predicted to be a bipartite nuclear localization signal (NLS). A phylogenetic tree was generated with FvCtf1α homologs from nine ascomycete fungi cutinase transcription factors. The results revealed that FvCtf1α was most similar to the proteins FoCtf1 and FsCtf1β (Additional file 1: Figure S1b). Therefore, FVEG_00228 was termed as FvCtf1α.

To examine the transcriptional activity of FvCtf1a, a yeast two-hybrid assay was performed. FvCtf1α exhibited self-activation activity in yeast transformants harboring the BD-FvCtf1α/pGADT7 vectors (Fig. 1a). To confirm the subcellular localization of FvCtf1α, the FvCtf1α-GFP vector was transformed into the ΔFvctf1α protoplast. FvCtf1α-GFP was observed to co-localize with the nucleus stained by DAPI (Fig. 1b), indicating that it localized in the nucleus of F. verticillioides. These results suggested that FvCtf1α is a transcription factor.

Fig. 1

Identification of transcription factors FvCtf1α in the regulation of FvCUT1 and FvCUT4 in F. verticillioides. a Self activation activity was verificated by yeast two-hybrid assays. Yeast transformants harboring the BD-FvCtf1α/pGADT7 vectors were assayed for growth on selective plates (SD/-Leu/Trp/His/Ade), and X-α-Gal added to test for β-galactosidase (LacZ) activities. X-α-Gal: 5-Bromo-4-chloro-3-indolyl-α-D-galactoside. The pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T were used as positive and negative controls, respectively. Scale bars: 10 µm. b The localizations of CTF transcription factor FvCtf1α was observed in the nucleus with the nucleus stained by DAPI. c–e The expression patterns of three cutinase genes within 12 h of cutin induction: the expression levels of cutinase genes FvCUT1, FvCUT3, and FvCUT4 in WT and mutants were detected at 1, 2, 3, 4, 5, and 12 h, respectively. f FvCtf1α can bind the promoters of the two cutinase genes FvCUT1 and FvCUT4 by yeast one-hybrid. pAbAi::FvCUT1pro vector, pAbAi::FvCUT4pro vector, and pGADT7-FvCtf1α were constructed and the Y1H-Gold strain was sequentially transferred into two vectors, first transferred into the pAbAi::FvCUT1pro vector (or pAbAi::FvCUT4pro vector), selecting with SD/-Ura medium, and re-introduced into the pGADT7-FvCtf1α vector, selecting with SD/-Ura media with 0, 100, 150, 200 ng/mL Aureobasidin A (AbA).The vector pair pAbAi::p53pro/p53-pGADT7 transformants serves as the positive control and the vector pAbAi::p53pro/pGADT7 transformants serves as the negative control. g FvCtf1α can bind the promoters of the two cutinase genes FvCUT1 and FvCUT4 at GC-rich site by Multiple EM for Motif Elicitation (MEME) software. p < 0.01. h The expression of FvCUT1, FvCUT3, FvCUT4 were assayed by qRT-PCR under low H₂O₂ stress. i The expression level of three genes including β-oxidation (FvPOT1) and peroxisome biogenesis (FvPEX5, FvECL1) were compared by WT and ΔFvctf1α, respectively. The transcription level of the target gene was determined using qRT-PCR assay and calculated using the 2^−ΔΔCt method with TUB2 as reference gene. Analysis of variance is three independent repeated experiments and asterisks represent a significant difference. (t-test, *: p < 0.05, **: p < 0.01)

To characterize the functional roles of FvCtf1α in F. verticillioides, the FvCTF1α gene was deleted using a split-marker approach (Additional file 1: Figure S2a). The ΔFvctf1α mutants were first screened by PCR (Additional file 1: Figure S2b) and further confirmed by Southern blot assay (Additional file 1: Figure S2c). To verify that the phenotypic defects in the mutants were caused by the targeted gene deletion, a gene-complementation strain ΔFvctf1α-C was generated and further confirmed by qRT-PCR (Additional file 1: Figure S2f).

The corresponding homologs of four A. nidulans cutinase genes (AnCUT1-AnCUT4) were identified in F. verticillioides, termed as FvCUT1–FvCUT4. Functional domain analysis demonstrated that FvCut1–FvCut4 contained one cutinase domain and a signal peptide (Additional file 1: Figure S1c). However, FvCUT2 was not expressed in either CMII or kernel medium, according to RNA-sequencing data. Compared with the wild-type strain Fv7600, the expression levels of FvCUT1 and FvCUT4 in ΔFvctf1α were down-regulated, while that of FvCUT3 remained unaffected (Table 1). In addition, the expression levels of the four cutinase genes (FvCUT1–FvCUT4) were further assayed by qRT-PCR with or without cutin induction. The results showed that only three of these (excluding FvCUT2) were found to encode active cutinases (Fig. 1c–e). Therefore, a yeast one-hybrid assay was used to detect the relationships between the transcription factor FvCtf1α and three cutinase genes (FvCUT1, FvCUT3, and FvCUT4). The results showed that FvCtf1α can bind to the promoters of FvCUT1 and FvCUT4 cutinase genes (Fig. 1f). Further analysis using the Multiple EM for Motif Elicitation (MEME) program revealed that FvCtf1α can bind to the GC-box (GCGCCSC) region in the promoters of FvCUT1 and FvCUT4 cutinase genes (Fig. 1g). Thus, the results indicate that FvCtf1α is a transcription factor for cutinases FvCUT1 and FvCUT4.

FvCtf1α regulates F. verticillioides cutinase genes under different conditions

To test whether FvCtf1α regulates the expression of the three cutinase genes (FvCUT1, FvCUT3, and FvCUT4) under cutin induction or H₂O₂ stress, we compared their expression patterns in the ΔFvctf1α mutant and wild-type (WT) strains using qRT-PCR. Firstly, the expression profiles of these cutinase genes were measured at different stages of cutin induction. Although FvCUT1, FvCUT3, and FvCUT4 in the ΔFvctf1α mutant were slightly up-regulated compared to the WT without cutin induction at early stages, after 1 h cutin induction, the expressions of FvCUT3 and FvCUT4 were significantly increased (2 to 8-fold) in the wild-type. However, the expressions of FvCUT1, FvCUT3, and FvCUT4 in ΔFvctf1α did not change. The induced expression peaks of FvCUT1, FvCUT3, and FvCUT4 were reached at 3–5 h of cutin induction and decreased at 12 h in WT, while the expression levels for ΔFvctf1α remained unchanged (Fig. 1c-e) during these stages. However, after 3 days of cutin induction with low H₂O₂ stress, FvCUT1 and FvCUT4 were abundantly induced in WT, especially for the expression of FvCUT1, but the expression levels in the ΔFvctf1α mutant were reduced (Fig. 1h). Therefore, we hypothesize that FvCtf1α positively regulates FvCUT (FvCUT1, FvCUT3, and FvCUT4) gene expression under cutin induction, and more specifically regulates FvCUT1 and FvCUT4 under cutin induction with low oxidative stress. Simultaneously, under cutin induction with low H₂O₂ stress, the expression levels of three genes, including β-oxidation (FvPOT1) and peroxisome biogenesis (FvPEX5 and FvECL1), were reduced in the ΔFvctf1α mutants (Fig. 1i). These observations suggest that the ΔFvctf1α mutant may not respond to low H₂O₂ stress as it does not stimulate the ROS clearance function of peroxisomes.

Investigating the relationship between FvCtf1α and other FvCtfs

To further investigate the regulatory mechanism of constitutively expressed and induced cutinase genes by different cutinase transcription factors (FvCtfs) in F. verticillioides, other FvCtfs were studied. The homologous proteins FvCtfs FarA and FarB of A. nidulans were found through a blast search in the F. verticillioides genome, with gene numbers FVEG_16071 (FvFARA) and FVEG_07971 (FvFARB). Like FvCtf1α, both FvFarA and FvFarB contain two domains: the GAL4 domain and the transcription factor domain (Fungal trans) (Additional file 1: Figure S1a). DAPI staining was used for nuclear localization markers. The green fluorescence of FvFarA-GFP and FvFarB-GFP colocalized with the nuclear blue light signal, respectively, indicating that FvFarA-GFP and FvFarB-GFP of F. verticillioides were also localized in the nucleus (Fig. 2a). Fluorescence could be observed in the hyphae of the transformants containing FvCtf1α-NYFP and FvFarB-CYFP, but not in the hyphae of the transformants containing FvCtf1α-NYFP and FvFarA-CYFP. This indicates that FvCtf1α can interact with FvFarB but not with FvFarA in F. verticillioides (Fig. 2b).

Fig. 2

Identification the relationship of FvCtf1α with other transcription factors CTFs. a The localizations of CTF transcription factor FvFarA-GFP and FvFarB-GFP were observed in the nucleus with the nucleus stained by DAPI, respectively. b The interaction of FvCtf1 with FvFarA or FvFarB analyses by BiFC fluorescence. Five plasmid pairs were co-transformed into wild-type Fv7600 protoplast, with three plasmid pairs FvCtf1α-NYFP and CYFP, NYFP and FvFarA-CYFP, NYFP and FvFarB-CYFP using as negative controls, while the plasmid pairs FvCtf1α-NYFP and FvFarA-CYFP, FvCtf1α- NYFP, and FvFarB-CYFP were used as tests. BiFC fluorescence images were captured on confocal laser scanning microscope. Scale bars: 10 µm

FvCtf1α affects fatty acid metabolism and carbon source utilization

To test whether F. verticillioides FvCtf1α affects the utilization of various carbon nutrients, we cultured WT, ΔFvctf1α mutants, and ΔFvctf1α-C on minimal medium (MM) agar plates with different carbon sources. The results showed that there was no significant difference in the growth rates of the ΔFvctf1α mutants compared with that of the WT and the complementary strain ΔFvctf1α-C when ethanol absolute, glycerol, and sodium butyrate were used as the sole carbon sources (Fig. 3a). However, the vegetative growth rates of ΔFvctf1α were significantly reduced on sodium acetate and sodium oleate medium when compared with those of the WT and ΔFvctf1α-C strains (Fig. 3a–d). Growth inhibition on sodium acetate and sodium oleate medium were more drastic for ΔFvctf1α than other carbon sources (Fig. 3c, d). These results suggest that ΔFvctf1α mutants have defects in fatty acid metabolism and the utilization of certain carbon sources, such as sodium acetate and sodium oleate.

Fig. 3

Defect on carbon metabolic, producing conidia and cell wall degrading enzyme activity of ΔFvctf1α deleted mutants. a To compare the nutrient utilization capacity of the WT and mutant strains, the vegetative growths of the strains were monitored on MM with short chain carbon, respectively. b The vegetative growths of the strains were monitored on MM with long chain carbon, respectively. c, d The colony diameters of the cultures were measured and inhibition of mycelial growth analyzed by t-test on short chain carbon, on long chain carbon. e The expression levels of the carbon metabolic related genes were significantly down-regulated in ΔFvctf1α by qRT-PCR with the reference gene TUB2 using the 2^−ΔΔCt method. f Measurement and statistical comparison of conidia from WT and mutant strains after inoculation on CMII at 28°C for 3 days. Error bars denote standard deviations from three repeated experiments and asterisks represent a significant difference. g The activity of three cell wall degrading enzyme including cutinase, pectatinase, and celluase were assessed in ΔFvctf1α and WT, respectively. The experiment was performed three times with similar results and error bars represent the standard deviation and asterisks represent a significant difference (t-test, *: p < 0.05,**: p < 0.01)

Three carbon metabolism pathway-related genes, FvFBP1 (FVEG_03829), FvICL1 (FVEG_02611), and FvFOX2 (FVEG_04199), were selected for further analysis. The results showed that their expression was significantly reduced in ΔFvctf1α mutants (Fig. 3e). These observations suggest that the ΔFvctf1α mutant may affect carbon metabolism due to significantly reduced expression levels of carbon metabolism-related genes.

Deletion of FvCtf1α resulted in a conidial production defect

To investigate whether the sporulation of ΔFvctf1α mutants is affected, WT, ΔFvctf1α, and ΔFvctf1α-C were inoculated in potato dextrose agar (PDA) medium. The results showed that the sporulation of the ΔFvctf1α mutants decreased significantly compared to the WT and the complementary strain ΔFvctf1α-C (Fig. 3f). In addition, the expression of the conidia-related gene FvCON7 (FVEG_10320) was significantly reduced in ΔFvctf1α mutants (Fig. 4c). These results suggest that the effect of FvCtf1α on conidial production defects may be due to a significant decrease in the expression level of FvCON7.

Fig. 4

FvCtf1α alters tolerance to cell wall and oxidative stress. a The growth of colony were monitored for tolerance to cell wall and oxidative stress on MM by addition with CR, CFW, SDS, or H₂O₂. CR: congo red, CFW: calcofluor white, SDS: sodium dodecyl sulfate. b The inhibition rates of mycelial growth under cell wall and oxidative stresses were analyzed and subjected to statistical analysis, respectively. c The expression level of six cell wall synthase related genes FvCHS1, FvCHS6, FvCHS7, FvFKA, FvPKCA, and one conidia related gene FvCON7 were compared by ΔFvctf1α and WT, respectively. qRT-PCR was used to quantify transcript level of genes to the reference gene TUB2 using the 2^−ΔΔCt method. Error bars represent the standard deviation and asterisks represent a significant difference (t-test, *: p < 0.05, **: p < 0.01)

FvCtf1α affects cell wall-degrading enzyme (CWDE) activity and contributes to cell wall integrity stress and H₂O₂ stress

The activity of three CWDE, including cutinase, pectinase, and cellulase, was assessed in ΔFvctf1α. Compared with the WT, cutinase, and pectinase activities were significantly reduced in ΔFvctf1α, but cellulase activity was not affected in ΔFvctf1α (Fig. 3g).

To explore the roles of FvCtf1α in the regulation of different stress responses, mutant strains were cultured in MM containing SDS, CFW, CR, or H₂O₂. The results showed that ΔFvctf1α mutants were more sensitive to the chemical treatments of SDS and H₂O₂, and the inhibition rate under these two stresses increased significantly compared with the WT and ΔFvctf1α-C strain (Fig. 4a, b). These findings indicate that FvCtf1α is involved in the response to cell wall integrity stresses and oxidative stress.

Six cell wall synthase-related genes, chitin synthases (CHS), FvCHS1 (FVEG_02839), FvCHS6 (FVEG_07280), FvCHS7 (FVEG_07296), FvFKA (FVEG_12144), FvPKCA (FVEG_06268), and FvCON7 (FVEG_10320) were selected for further analysis. Compared to the WT, the expression levels of these six cell wall synthase-related genes were significantly reduced in ΔFvctf1α mutants (Fig. 4c). The results suggest that the effect of Fvctf1α on cell wall integrity may be due to a significant decrease in the expression level of cell wall synthase genes.

FvCtf1α is a regulatory transcription factor for fumonisin synthesis genes and is important for normal FB1 biosynthesis

The effect of FvCTF1α gene deletion on fumonisin B1 (FB1) biosynthesis was investigated. The wild-type and ΔFvctf1α mutant strains were inoculated in solid maize powder medium, and the concentration of FB1 produced in mycelium and medium was detected after culturing at 28°C for 10 days, respectively. The results showed that the vast majority of FB1 was secreted extracellularly and accumulated in the medium (Fig. 5a). The FB1 secreted into the medium by the ΔFvctf1α mutant was significantly lower than that of the wild-type, and FB1 accumulated in the mycelium showed the same trend (Fig. 5a). We further examined the expression levels of four key FUM genes including FvFUM1, FvFUM8, FvFUM19 and FvFUM21- in the wild-type and ΔFvctf1α mutant strains. The results showed that the expression levels of these four genes in the ΔFvctf1α mutant were significantly lower than those of the wild-type strain (Fig. 5b).

Fig. 5

FvCtf1α plays key role in fumonisin B1 (FB1) production. a FB1 levels in the samples were measured using the formula FB1/TUB2 DNA. Surface sterilized B73 corn kernels were inoculated with the WT and mutant conidia suspensions and incubated for 10 d. FB1 levels were quantified using ELISA Kit. F. verticillioides biomass was quantified by measuring F. verticillioides TUB2 DNA in samples. ELISA: enzyme-linked immunosorbent assay. b Relative FUM genes expression were compared by ΔFvctf1α and WT, respectively. qRT-PCR was used to quantify transcript level of FUM genes to the reference gene TUB2 using the 2^−ΔΔCt method. The experiment was performed three times. Error bars represent the standard deviation. (t-test, *: p < 0.05, **: p < 0.01). c FvCtf1α can bind the promoters of the five FUM genes FvFUM1, FvFUM2, FvFUM6, FvFUM14, and FvFUM16 by yeast one-hybrid, respectively. pAbAi::FvFUM1pro, pAbAi::FvFUM2pro, pAbAi::FvFUM6pro, pAbAi::FvFUM14pro, pAbAi::FvFUM16pro vectors, and pGADT7-FvCtf1α were constructed. The Y1H-Gold strain was sequentially transferred into two vectors, first transferred into the pAbAi::FvFUM1pro vector (or pAbAi::FvFUM2pro vector), selecting with SD/-Ura medium, and reintroduced into the pGADT7-FvCtf1α vector, selecting with SD/-Ura media with 0, 100, 150, 200 ng/mL Aureobasidin A (AbA). The p53-AbAi vector transformants were used as the negative control and pAbAi::pro vector transformants as the positive control. d FvCtf1α can bind the promoters of the five FUM genes at CAMCA site by Multiple EM for Motif Elicitation (MEME) software

To determine whether FvCtf1α is a regulatory transcription factor for FB1 synthesis genes, we conducted yeast one-hybrid (Y1H) assays to determine the interaction between FvCtf1α and each FUM gene. The results showed that FvCtf1α can bind to the promoters of 5 FUM genes: FvFUM1, FvFUM2, FvFUM6, FvFUM14, and FvFUM16 (Fig. 5c). Further analysis by Multiple EM for Motif Elicitation (MEME) software revealed that FvCtf1α may bind to the CAMCA DNA element regions of the 5 FUM genes promoters (Fig. 5d). Y1H assays (Fig. 5c) and RNA-sequencing (Table 2) results confirmed that FvCtf1α was a new regulatory transcription factor of the FB1 biosynthesis genes.

Table 2 DEG of FUM genes between ΔFvctf1α mutants and WT

FvCtf1α, FvCut4, and FvCut3 contribute to pathogenicity

To explore the role of FvCtf1α in the infection process of F. verticillioides on different crops, we first inoculated the mycelium blocks of the WT and ΔFvctf1α strains on the stems of susceptible maize (B73) seedlings. The ΔFvctf1α showed a severe decrease in pathogenicity after 7 days post-inoculation (dpi) when compared to the WT (Fig. 6a). Next, we inoculated WT and ΔFvctf1α mutant strains on maize leaves and used real-time quantitative PCR (qRT-PCR) to detect the expression of six resistance genes (PRm3, PRm6, PR-1, PR-5, NPR1, and LOX10) in the leaves 2 dpi. The expression levels of maize resistance genes were significantly up-regulated following ΔFvctf1α mutant infection (Fig. 6b). Additionally, the susceptible sugarcane (badila) stem was inoculated with a toothpick soaked in a spore suspension of WT and ΔFvctf1α. After 7 dpi at 28°C, we used ImageJ software to statistically analyze the disease area. The results showed that the pathogenicity of ΔFvctf1α mutant was significantly reduced compared to the WT and complemented strain ΔFvctf1α-C (Fig. 6c, d). These results indicate that the FvCtf1α is essential for the pathogenicity of F. verticillioides on the stem of maize and sugarcane.

Fig. 6

FvCtf1α plays key role in pathogenicity. a The B73 maize seedling were inoculated with colony disk and then observed after 7 dpi. b The expression of relative six resistance genes of B73 maize by qRT-PCR were compared between ΔFvctf1α and WT, respectively. qRT-PCR was used to quantify transcript level of genes to the reference gene GAPDH (X07156) using the 2^−ΔΔCt method. c Sugarcane was split longitudinally to visually inspect rot symptoms after 7 dpi. Sugarcane (badila) was inoculated with immersed conidia toothtip at the internodal region after 7 dpi. The cane were inoculated with sterile toothtip as a negative control. d The area of discoloration of split longitudinal section of cane was quantified by ImageJ software and subjected to statistical analysis. The experiment was performed three times. Error bars represent the standard deviation and asterisks represent a significant difference. (t-test, *: p < 0.05, **: p < 0.01)

Most fungal pathogens produce cutinases that can hydrolyze host cutin and promote pathogen invasion, especially to leaves. Thus, we further attempted to explore whether the loss of pathogenicity of the ΔFvctf1α mutant was correlated to the function of cutinase genes. We tried to knockout three cutinase genes, FvCUT1, FvCUT3, and FvCUT4, to investigate the role of cutinase in pathogenicity. However, we only obtained ∆Fvcut3 and ∆Fvcut4 mutants, while FvCUT1 knockout mutants were not obtained. No open reading frame (ORF) was detected in the ∆Fvcut3 and ∆Fvcut4 mutants by PCR assay, but the correct linkage UA was detected as an alternative insertion sequence (Additional file 1: Figure S3a, b). Subsequently, qRT-PCR assay confirmed that the genes knocked out in their respective mutants were not expressed (Additional file 1: Figure S3c, d). The deletion of FvCUT3 and FvCUT4 did not affect mycelial growth in each case (Additional file 1: Figure S4a). However, the deletion of FvCUT4 and FvCUT3 did not affect pathogenicity on the stem of sugarcane but on maize leaves (Fig. 7, Additional file 1: Figure S4b, c). On the other hand, to investigate whether the deletion of two other CTFs, FvFARA and FvFARB, had an impact on pathogenicity, we constructed knockout mutants of these two genes (Additional file 1: Figure S2d–h). However, there was no difference in growth and pathogenicity between the wild-type and the two mutant strains (Additional file 1: Figure S5a–c).

Fig. 7

FvCut3 and FvCut4 play a role in pathogenicity. a, b The B73 maize leaves were inoculated with colony disk and the infection leaf with water-logging of wide-type, ΔFvcut3,and ΔFvcut4 were observed after 5 dpi

FvCtf1α is involved in multiple metabolic pathways

To investigate genes whose expression levels are regulated by FvCtf1α, we conducted RNA-sequencing analysis on the ΔFvctf1α mutants at the maize-infested stage. Differential gene expression (DEG) analysis was conducted using cuffdiff v2.1.1 with parameters: -FDR (False Discovery Rate) = 0.05 -library-norm-method classic-fpkm -u/-multi-read-correct-b/-frag-bias-correct. A gene that was considered to be differentially expressed must have at least 1.2-fold|Log₂fold change|variation between WT and ΔFvctf1α mutant. Our results found that 617 genes were down-regulated and 807 genes were up-regulated in the ΔFvctf1α mutant compared with the WT among the DEGs (Additional file 1: Figure S6a).

GO enrichment and KEGG enrichment pathways were further analyzed in DEGs. Using GO annotation and enrichment (Ye et al. 2018), the down- and up-regulated DEGs were enriched to 17 and 19 GO terms (Additional file 1: Figure S6b, c), respectively. The top 6 down-regulated enrichment pathways were mainly associated with carbohydrate metabolic processes, transmembrane transporter activity, transcription, catalytic activity hydrolase, and heme binding (Additional file 1: Figure S6b), while the top 5 up-regulated enrichment pathways were mainly classified into membrane, carbohydrate metabolic processes, transmembrane transporter activity, proteolysis, and hydrolase (Additional file 1: Figure S6c). Moreover, using KEGG annotation and enrichment (Dennis et al. 2003), the down- and up-regulated DEGs were enriched to 20 and 5 pathways (Additional file 1: Figure S6d, e), respectively. The top 5 down-regulated enrichment pathways were associated with biosynthesis of secondary metabolites, biosynthesis of antibiotics, microbial metabolism in diverse environments, carbon metabolism, fatty acid degradation, and arginine and proline metabolism (Additional file 1: Figure S6d). For the secondary metabolite FB1, all genes related to its metabolism are down-regulated (Table 2). However, their FDR were high in 5 up-regulated pathways (Additional file 1: Figure S6e). The analysis of enrichment pathways, combining both GO and KEGG enrichment, showed that the deletion of FvCTF1α affects transcription, membrane, carbon metabolism, and biosynthesis of secondary metabolites.

Discussion

Cutinases are extracellular enzymes that catalyze the hydrolysis of the ester bonds of cutin, suberin, lipids, and waxes. A variable number of genes encoding cutinase enzymes have been found, ranging from three to seventeen within a single organism (Skamnioti et al. 2008). In our study, RNA-seq analysis of F. verticillioides in CM medium and maize kernel identified one cutinase palindrome-binding protein (CPBP), four CTF1α (including FvCTF1α and FARA), six CTF1β (including FARB), and nine cutinase genes (Table 1). The divergent evolution of cutinase-encoding genes could lead to more efficient enzymes and better adaptation to different niches or conditions. In the CMII medium, transcription factors CPBP, FvFARA, and FvFARB, as well as CUT genes FvCUT3, were highly expressed in F. verticillioides. In addition, maize kernel infected by F. verticillioides induced the expression of many other CTFs and CUT genes, such as the transcription factor FvCTF1α, FvCTF1αA, FvCTF1αB, FvCTF1αC, FvCTF1β, FvCTF1βC, as well as CUT genes FvCUT1, FvCUT4, FvCUT6, FvCUT7, FvCUT8, FvCUT9, and FvCUT10. Our results also showed that FvCtf1α affected the fatty acid metabolism and the utilization of certain carbon sources, such as sodium acetate and sodium oleate. The specific mechanism of different cutinase expression in F. verticillioides still needs further research, whether it is consistent with F. solani, where makes a substrate-induced, catabolite-repressed cutinase (Lin and Kolattukudy 1978).

However, the role of cutinase in different pathogen species varies. Pea stem pathogen F. solani cutinase can help to breach the cuticular barrier of the host plant, playing a significant role in pathogenesis (Woloshuk and Kolattukudy 1986). Three F. solani cutinase genes, FsCUT1, FsCUT2, and FsCUT3 share a high degree of identity. While FsCUT2 and FsCUT3 are expressed constitutively at basal levels (Lin et al. 2022), the expression of FsCUT1 is strongly induced by cutin monomers and mediated by the zinc finger transcription factor Ctf1α in F. solani (Li and Kolattukudy 1997, Lin et al. 2022). Deletion of the FsCUT1 resulted in decreased virulence in peas (Kämper et al. 1994; Li et al. 2002), and insertion of FsCUT1 into Mycosphaerella, a pathogen that normally requires wounds on the papaya fruits surface to cause infection, allowed the transgenic strains to penetrate an intact surface (Dickman et al. 1989). Magnaporthe grisea CUT2 was shown to be associated with pathogenicity (Skamnioti and Gurr 2007). Yeast Pseudozyma antarctica on the leaf may be utilizing an cutinase-like enzymes (CLEs) to extract fatty acids as nutrients, and leaf surfaces were heavily damaged by high concentrations of CLEs (Ueda et al. 2015). These CLEs can degrade tomato leaf cutin, enabling plant pathogens to easily invade leaves (Ueda et al. 2018, Ueda et al. 2021). However, numerous gene mutation studies have failed to show an essential role for cutinase in different pathogen species, such as M. grisea CUT1 and Botrytis cinerea cutinase A (Sweigard et al. 1992; Van kan et al 1997). Unlike F. solani and M. grisea where only specific cutinase enzymes were linked to host infection, both FvCUT4, the inducible enzyme, and FvCUT3, the constitutive enzyme, were associated with F. verticillioides infection of maize leaves. Therefore, compared with F. solani and M. grisea, F. verticillioides contains more cutinase genes and more complex mechanisms for regulating pathogenicity.

The presence of different binding sites for different transcription factors (TFs) in CUT genes, e.g., CCTGCC/GGCAGG for FARA and FARB, GGAATTGGGGCATTGG for NAPA/NF-Y1, and GGC(n3)GCC for CTF1, result in their expression under different conditions (Kämper et al. 1994; Lin et al. 2022; Bermúdez-García et al. 2019). Some transcription factors recognize a DNA sequence with two inverted repeats of CGG elements, separated by a characteristic number of bases. It is recorded that some TFs recognize 5’-CGG(n)CCG with a spacer of different nucleotides, e.g., the spacers for GAL4, PUT3, PPR1, and LEU3 are 11, 10, 6, and 4 nucleotides, respectively (Zhang and Guarente 1994). Ctf1α and Ctf1β bind to an oppositely oriented palindrome, 5’-GCC(n2)GGC, in F. solani (Kämper et al. 1994), and FvCtf1α can bind a canonical palindrome 5’-GGC(n3)GCC with FvCUT4 in F. verticillioides. The GC-rich palindrome is essential for cutinase induction by cutin monomers. Induction and enhancement of FvCUT1 occur by binding the GC-rich (GCGCCSC) region at its promoters, resembling a positive-acting G-rich Sp1-like element in an enhancer in numerous viral and mammalian promoters (Jones et al. 1986). They also appear in the promoters of the A. nidulans PGKA and GPDA genes, which encode phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase, respectively (Hoskins et al.1994). There are two binding sites in some cutinase genes: a silencing sequence that keeps basal gene expression low and affects cutinase gene inducibility and a G-rich positive-acting Sp1-like element that restores high expression levels by antagonizing the silencer. However, the G-rich activator from F. solani did not function as a true enhancer (Kämper et al. 1994). FvCUT1 was highly induced under low H₂O₂ stress, suggesting that the GC-rich element plays a role in inducing enhancers for cutin induction with low H₂O₂ stress. FvCUT1 is consistent with the high expression of chitin monomers through FvCtf1α binding to the GC-rich element. Isolation and characterization of other Ctf1α and PBP proteins, as well as the silencer- and activator-binding proteins, are necessary to further elucidate the mechanisms of cutinase gene regulation in F. verticillioides. On the other hand, FvCtf1α can bind to the CAMCA DNA element regions of 5 FvFUM genes promoters, FvFUM1, 2, 6, 14, 16 genes, as detected by Multiple EM for Motif Elicitation (MEME) software. The conditions for CAMCA binding also need further exploration. Therefore, we speculated that FvCtf1α is a non-specific transcription factor with multiple promoter binding sites. In addition to bind to the inducible cutin gene promoter site GCGCCSC, it has also been found for the first time to bind to the promoter site CAMCA of FUM related genes. FvCtf1α regulates the transcription of inducible cutinase to regulate pathogenicity and also regulates the transcription of key FUM genes to regulate FB1 production. Overall, our results provide new insights into the mechanism of FvCtf1α-mediated gene regulation in F. verticillioides pathogenesis and FB1 production. This study will provide a theoretical basis for reducing the toxicity and yield loss due to F. verticillioides.

Interpretations of data on fungal cutinase activity and pathogenicity are contradictory, and range from cutinase having no apparent influence on pathogenicity to enhancing the adhesion of fungal spores to the plant surface (Schäfer 1993). Different cutinase transcription factors CTF and CUT genes were expressed under various conditions. Ctf1β is involved in the constitutive expression of CUT2 in the virulent strain F. solani, and its competitor CPBP cannot bind to palindrome 1 of CUT2, thus CUT2 is not repressed (Li et al. 2002). However, in the saprophytic fungus A. nidulans, either glucose or starch can strongly repress the expression of AnCUT2 (Castro-Ochoa et al. 2012). Later, the researchers discovered that lipid metabolism transcription factors (TFs) FarA regulated AnCUT2 and FarB regulated AnCUT3 (Bermúdez-García et al. 2019). However, CUT2 was not detected virulence in the F. verticillioides strain under the tested conditions. We suggested that FvCUT2 was repressed in CM medium with glucose and in maize kernel with starch. FvCtf1α indirectly regulates constitutive cutinase FvCUT3, and FvCtf1α interacts with FvFarB, suggesting that FvCtf1α may indirectly regulate FvCUT3 through FvFarB. FarA also regulated AnCUT1, while NapA regulated AnCUT4 in A. nidulans (Bermúdez-García et al. 2019).

F. solani f. sp. pisi CUT1 is also under glucose catabolite repression, and its expression is highly induced by cutin monomers and is positively regulated by Ctf1α (Li et al. 2002). F. solani palindrome-binding protein (PBP) contains a zinc finger motif that shares homology with those in mammals, Saccharomyces cerevisiae, Neurospora crassa, and Ustilago maydis (Li and Kolattukudy 1995). F. solani PBP is believed to interfere with the binding of Ctf1α, the transcription factor involved in induction, to the CUT1 promoter, and thus keeping the CUT1 gene repressed until induced by cutin monomers (Lin et al. 2022). That is, Ctf1α competes with PBP for the binding site on the promoter of the inducible chitinase gene, and Ctf1α binding induces expression while PBP binding inhibits expression. For example, the expression of inducible FvCUT1 was inhibited in CMII medium containing glucose, but was induced by cutin of maize based on RNA-seqencing data (Table 1). In our work, we found that FvCtf1α directly regulates cutinase (FvCUT1 and FvCUT4) induction. Cutin monomers, generated by low levels of constitutively expressed cutinase, induce high levels of cutinase that can help pathogenic fungi penetrate into the host through the cuticle, whose major structural polymer is cutin. We suggest that low levels of F. verticillioides FvCUT3 induce Ctf1α regulated high levels of cutinase FvCUT1 and FvCUT4, which collaborate with the degradation of the host cutin to cause disease.

It is also important to recognize that the regulatory mechanisms of CTFs are involved in many biochemical metabolic pathways. In F. oxysporum, the CTF regulates the expression of cutinase and other enzymes involved in fatty acid hydrolysis. In A. oryzae, a Zinc finger TF involved in lipid metabolism affects the expression levels of cutinase and other lipolytic enzymes (Garrido et al. 2012). The lipid metabolism transcription factors FarA for AnCUT1 and AnCUT2, and FarB for AnCUT3, are involved in constitutive expression (Ramírez 2009). The phylogenetic tree showed that FvCtf1α was closer to FoCtf1α. Although both FoCtf1α and FvCtf1α regulate the transcription of cutin and lipase, FvCtf1α regulates pathogenicity while FoCtf1α does not. However, the homologous proteins of F. solani Ctf1α and Ctf1β are the same as FvCtf1α in the genome database of F. verticillioides. Ctf1α regulates β-oxidation and redox metabolism in C. albicans (Ramírez 2009), and the expression of a cutinase from Monilinia fructicola was enhanced using low H₂O₂ stress with cutin induction (Lee et al. 2010). Ctf1α responds to low H₂O₂ stress metabolic pathways and regulates the expression of these two genes (FvCUT1 and FvCUT4). This is different from the transcription regulatory process in A. nidulans, which involves NapA functioning on AnCUT4 under low H₂O₂ stress with cutin (Bermúdez-García et al. 2019). FvCtf1α regulates the expression of cell wall chitin synthase (Chs) in F. verticillioides. Three MoCHSs (CHS1, CHS6, and CHS7) in M. oryzae were found to be important for plant infection (Kong et al. 2012). It is need to further confirm whether the pathogenicity of F. verticillioides is related with FvCtf1α regulating FvChs.

Pathogens use enzymes such as lipases and cutinases to facilitate their penetration through the plant cuticle (Voigt et al. 2005; Hynes et al 2006; Srivastava et al. 2012). Prolonged pathogen adhesion promotes tighter and steadier attachment between the plant cuticle and spores, as they secrete a polysaccharide-based extracellular mucilaginous matrix, including pectinases, cellulases, and cutinases, towards the plant surface during the infection stage of spore germination on the host plant cuticle (Deising et al. 1992;Doss 1999). FvCtf1α may also affect its pathogenicity through a decrease in cell wall degradation enzyme capacity and spore production. The dormant spores of pathogenic fungi contain "constitutive-type" cutinases, previously also termed "sensing" cutinases, which release small amounts of cutin monomers from the host plant cuticle in a spatially localized manner (Köller et al. 1982). These cutin monomers are essential for subsequent stages of infection (Deising et al. 1992; Arya and Cohen 2022). "Constitutive-type" cutinase activity has been detected during the early stages of infection in the dormant spores of fungal species with different infection strategies, such as Botrytis cinerea, Fusarium graminearum, Curvularia lunata, Pyrenopeziza brassicae, M. grisea, and Colletotrichum spp. (Leroch et al. 2013; Liu et al. 2016; Davies et al. 2000; Oliver and Ipcho 2004; Auyong et al. 2015; Skamnioti and Gurr 2007). "Constitutive-type" cutinase FvCut3 has been detected in early infection strategies and affects pathogenicity. FvCtf1α and "constitutive-type" cutinase FvCut3 affect pathogenicity, but FvFarB does not. This suggests that the interaction between FvCtf1α and FvFarB may play a major role in affecting the expression of FvCUT3 and subsequently impact pathogenicity. Further studies are needed to confirm this. In all, FvCtf1α, its induced cutinase FvCut4, and "constitutive-type" cutinase FvCut3 co- regulated cutin recognition in host leaves, the release of cutinase, causing leaf infection and subsequent water-logging.

Conclusion

In summary, our results demonstrate that the F. verticillioides transcription factor FvCtf1α regulates cutinase gene expression under cutin induction with low oxidative stress. It is also involved with fatty acid metabolism, carbon source utilization, cell wall integrity, conidiation, pathogenicity, fumonisin synthesis, and FvFUM genes expression. The ΔFvctf1α mutant grown on inducing substrates failed to activate extracellular cutinolytic activity, nor to transcribe FvCUT1, FvCUT4, and FvFUM1, FvFUM2, FvFUM6, FvFUM14, FvFUM16 genes. Our results suggest that FvCtf1α is a broad regulatory factor, acting not only on cellular degradation enzymes but also on genes related to FB1 toxin synthesis. Our results provide new insights into the mechanism of FvCtf1α-mediated gene regulation in F. verticillioides pathogenesis, which could be used as new effective strategies for controlling corn ear rot.

Methods

Bioinformatics and phylogenetic analyses

The full sequences of cutinase transcription factor genes (FvCTF1α, FvFARA, and FvFARB), cutinase genes, and other FvCtf1α target genes were downloaded from the National Center for Biotechnology Information (NCBI) by using homologous F. solani or A. nidulans protein sequence as queries. Protein domains were predicted using the SMART software (http://smart.emblheidelberg.de/). The nuclear signal was predicted using the NLS_Mapper software (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). A phylogenetic tree was constructed using the MEGA 6.0 software, and the Maximum likelihood algorithm involving 1000 bootstrap replicates was employed.

Targeted gene deletion, complementation, and Southern blot assay

To further investigate the functions of FvCTF1α (FVEG_00228), the gene was replaced with hygromycin by homologous recombination (Lin et al. 2022). For complementation experiments, a fragment containing the FvCTF1α native promoter region and gene without the termination codon was ligated with the pKNTG vector and then transferred into the ΔFvctf1α protoplast. The deletion (ΔFvctf1α) and complementation (ΔFvctf1α-C) strains were confirmed by PCR, qRT-PCR, and Southern hybridization. For Southern analysis, the genomic DNA isolated from the individual strains (F. verticillioides 7600, ΔFvctf1α, and ΔFvctf1α-C) was digested with EcoRI. The specific Southern hybridization probe was amplified from genomic DNA using primers of upstream flanking sequences (Additional file 1: Figure S2a), labeled, and subsequent hybridization and detection were performed according to a previously described protocol (Lin et al. 2022). Subsequently the validated strains were further chosen for functional characterization. Gene deletion and complementation of other target genes, FvFARA, FvFARB, FvCUT3, and FvCUT4, were performed using the same method described. All primers used in this study are listed (Additional file 2: Table S1).

Strains and culture conditions

F. verticillioides wild-type strain Fv7600 and all transformants were cultured on a minimal medium (MM) with 2% sucrose (Lin et al. 2022). To test the ability of ΔFvctf1α strain to utilize various short carbon sources, it was inoculated in MM medium with 40 mM ethanol absolute, 40 mM sodium acetate, 20 mM glycerol, and 10 mM sodium butyrate as the sole carbon source, and the growth was observed after culturing at 28°C for 3 days. In addition, to test the ability of ΔFvctf1α to utilize long carbon sources, it was inoculated in MM medium with sucrose-containing long-chain fatty acids, including 3 mM linoleic acid, 3 mM sodium oleate, and 0.2% olive oil, respectively. The colony diameter was measured after 3 days of incubation. Cell wall integrity was tested by growing the ΔFvctf1α strain on MM with sucrose supplemented with 100 mg/mL calcofluor white (CFW), 100 μg/mL Congo red (CR), or 0.01% sodium dodecyl sulfate (SDS).

The tolerance of the ΔFvctf1α strain to exogenous reactive oxygen species (ROS) was evaluated by measuring growth on MM with sucrose containing 10 mM H₂O₂. After 3 days of stress (CFW, CR, SDS, and H₂O₂) at 28°C, the colony diameter and inhibition were measured. After 3 days in PDA, the conidia of the strains were measured. Moreover, to further assay the relation between cutinase and low H₂O₂ stress, 0.1 mM H₂O₂ was added to 1% cutin medium (Lee et al. 2010). After 3 days of culture, the expression of cutinase, β-oxidation, and peroxisome biogenesis related genes were detected by qRT-PCR.

The expression of cutinase and other target genes detection and cell wall degrading enzyme assay. Crude cutin (1%) was added to MM liquid medium, and then wild-type F. verticillioides and the ΔFvctf1α mutants were inoculated, and cultured at 28°C with agitation (180 rpm). The expression levels of target cutinase genes at different cutin induction periods (cultured for 1, 2, 3, 4, 5, and 12 h) were detected by qRT-PCR. In addition, after 3 days of cultivation in an MM liquid medium, the expression levels of carbon metabolic-related genes and cell wall synthase genes in the strains were detected by qRT-PCR as previously described (Yu et al. 2022).

Wild-type F. verticillioides Fv7600, mutant strains ΔFvctf1α-25 and ΔFvctf1α-103, and complement strain ΔFvctf1α-C were inoculated in MM liquid medium containing 1% cutin, 1% pectin, and 1% carboxymethyl cellulose (CMC), respectively. The activities of cutinase, pectinase, and cellulase secreted by strains were detected after culturing at 28°C and 150 rpm for 10 days. The supernatant of the culture was obtained after 5 min centrifugation at 4°C and used as a crude enzyme preparation in the assay of CWDE cutinase activity, pectinase activity, and cellulase activity assay.

Extracellular cutinase activities were determined by the formation of yellow color p-nitrophenol butyrate (pNPB) after reaction with 5 mM paranitrophenyl butyrate dissolved in 50 mM potassium phosphate (pH 5.0) for 10 min and measured at A405 nm. Crude cutin was prepared from tomato fruit peel. Pectinase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method, determining the amount of reducing sugar released from the substrates (Zhou et al. 2015). D-( +)-galacturonic acid monohydrate (Sigma) was used to generate a standard curve. Cellulase activity was determined by measuring the amounts of reducing sugar glucose released from 0.5% carboxymethyl-cellulose (CMC) with a 50 mmol/L pH 5.0 citrate buffer and reacted with DNS reagent under alkaline conditions (You and Chung 2007). The reducing sugar glucose was calculated using the standard curve.

GFP fusion and Bimolecular fluorescence complementation (BiFC) fluorescent experiment, confocal microscopy

To investigate the localization of FvCtf1α, FvFarA, and FvFarB, the recombinant fluorescent vectors (FvCtf1α-GFP, FvFarA-GFP, FvFarB-GFP) were constructed following our previously described method (Lin et al. 2022). FvCTF1α, FvFARA, and FvFARB genes with their native promoters were amplified from the genomic DNA of Fv7600, and the PCR products were cloned into the pKNT-GFP vector by one-step cloning, respectively. Each recombinant fluorescent vector was then transferred to its corresponding mutant. The positive transformants obtained from the corresponding mutants were stained with nuclear dye DAPI and observed with a confocal microscope (Nikon, Japan).

To investigate the interaction of FvCtf1α with FvFarA or FvFarB, the fluorescent vectors (FvCtf1α-NYFP, FvFarA-CYFP, FvFarB-CYFP) were constructed. FvCTF1α genes with their native promoter were cloned into the pKNT-NYFP vector with G418 resistance through a one-step cloning process. FvFARA and FvFARB genes with their native promoters were separately cloned into a pCX62-CYFP vector with hygromycin resistance via one-step cloning. Pairs of recombinant plasmids, FvCtf1α-NYFP with FvFarA-CYFP and FvCtf1α-NYFP with FvFarB-CYFP, were co-transformed into the wild-type Fv7600, respectively. BiFC fluorescence images were captured using a confocal laser scanning microscope (Nikon, Japan).

Pathogenicity assay and quantification of FB1 mycotoxin

The susceptible maize B73 and sugarcane Badila were used in pathogenicity assays. Toothpicks were soaked in the F. verticillioides spore suspension (1 × 10⁶ conidia/mL). A needle was used to poke a hole in the middle section of the sugarcane internode, then inserted a toothpick soaked in the spore suspension, and finally wrapped the hole with a sealing film. After 7 dpi, the average area of sugarcane stem rot was calculated for statistical analysis. Moreover, 1 piece of mycelium block was inoculated onto the stem of a 2-week-old maize seedling, incubated at 28℃ in a greenhouse for 5 days, and the pathogenicity was observed. In addition, a mycelium block was inoculated onto the leaves of 4-week-old maize seedlings and incubated at 28℃ for 4 days to observe its pathogenicity. Tissue at the inoculation site was collected, and then the expression of disease-resistance genes was measured by qRT-PCR. This experiment was conducted with three independent biological replicates and analyzed statistically.

For the FB1 assay, spore suspension (1 × 10⁶ conidia/mL) was inoculated on surface-sterilized B73 corn kernels for 10 days, and the FB1 content was quantified using an FB1 ELISA Kit following the manufacturer's suggested protocol (Finder Biotech, Shenzhen). DNA from infected maize kernels was extracted to detect the infection amount of F. verticillioides. F. verticillioides biomass quantification was calculated by qRT-PCR based on the β-tubulin2 gene TUB2 (FVEG_04081) standard curve. Meanwhile, the expression of FB1 biosynthesis genes in F. verticillioides by qRT-PCR were detected from infected maize kernels. Each experiment was repeated three times.

RNA sequencing and quantitative real-time PCR (qRT-PCR)

Maize kernels (B73) were processed in the same way as prepared for the FB1 assay. After 10 dpi, total RNA from infected kernels was extracted using the EastepTM Total RNA Extraction Kit (Promega, China) according to the manufacturer's instructions. The reagents were provided by the Illumina NextSeq 500 Kit, and sequencing was performed on an Illumina NextSeq 500 instrument (Illumina, USA). The sequenced reads were then filtered using PRINSEQ to ensure data quality. For subsequent identification of DEGs between the wild type and mutants, gene enrichment and functional annotation methods for DEGs, such as KOG (Clusters of Orthologous Groups of proteins in Eukaryotic), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes), were used as previously reported (Ye et al. 2018; Yu et al. 2022). Three biological replicates were conducted for each treatment.

Fungal total RNA was extracted using an RNA Kit 200 (OMEGA, USA), and cDNA templates were prepared with the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit. The TransStart® Tip Green qPCR SuperMix (TransGen Biotech, China) was used to perform quantitative real-time PCR (qRT-PCR). The qRT-PCR detection of F. verticillioides and maize was standardized based on the expression levels of their respective housekeeping genes, F. verticillioides TUB2 and maize B73 glyceraldehyde-3-phosphate dehydrogenase GAPDH (X07156). Data were obtained from three biological replicates.

Yeast one-hybrid assay and yeast two-hybrid assays

To detect the direct one-to-one regulatory relationship between transcription factor FvCtf1α and its regulatory genes, yeast one-hybrid experiments were conducted. The FvCTF1α cDNA sequence was ligated to pGADT7, and the pGADT7-FvCtf1α vector was constructed as prey. The Promoter 2.0 and BDGP websites (http://www.fruitfly.org/seq_tools/promoter.html) were used to predict the promoters of the tested target genes. The putative promoter of each detected target gene, including 100 bp upstream and downstream regions, was amplified and ligated to the pAbAi vector (Clontech) as bait. First, the pAbAi::pro vectors were transformed into Y1H-Gold (Clontech) cells, and the transformed strains were isolated on SD/-Ura medium and confirmed by PCR. Subsequently, the pGADT7-FvCtf1α vector was transformed into the previously constructed Y1H-Gold cells harboring the pAbAi::pro vector, and the transformants were isolated on SD/-Ura medium containing 0, 100, 150, 200 ng/mL Aureobasidin A (AbA). The transformants with pAbAi-p53 vector and pGADT7-p53 vector were used as positive controls.

To determine whether FvCtf1α exhibits self-activation function, yeast two-hybrid experiments were conducted following our previous protocol (Lin et al. 2022). The FvCTF1α cDNA sequence was cloned into pGBKT7 as the bait vector, and the empty pGADT7 vector was used as the prey vector. A pair of plasmids, pGBKT7-P53 and pGADT7-T, was used as a positive control, while another pair of plasmids, pGBKT7-Lam and pGADT7-T, served as a negative control.

Statistical analyses

Data were subjected to analysis of variance (ANOVA), and means were separated by the Least Significant Difference (LSD) test (p < 0.05).