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Functional analysis of the mating type genes in Verticillium dahliae

Abstract

Background

Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood.

Results

In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence.

Conclusions

These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Background

Sexual reproduction is a ubiquitous characteristic of eukaryotes, which can combine elite alleles from different individuals and repair random epigenetic or conventional genetic damage through the processes of crossover and recombination during meiosis [21, 22, 48]. In contrast to sexual reproduction, strictly asexual reproduction is often considered as an evolutionary dead-end, mainly because there is no meiotic recombination, leading to an increase in the accumulation of harmful mutations [41, 56]. These effects are known as Muller’s ratchet [24, 43]. Approximately, 20% of all species of fungi are considered strictly asexual, without a recognized sexual cycle, and in the Ascomycota, the rate is higher, at up to 40% [56]. However, the sexual cycle of some species may be not absent, but rather cryptic since the hallmarks of a sexual cycle have not been directly observed [25, 35, 47].

In the fungal kingdom, sexual reproduction has evolved into two sexual breeding systems, and these systems are homothallism or heterothallism. Sexual reproduction in homothallic ascomycetes is being self-fertile; however, in heterothallic ascomycetes, mating occurs only between two different strains belonging to opposite mating types [1, 39]. In heterothallic fungi, the determination of mating type is governed by the presence of idiomorphic alleles MAT1-1 and MAT1-2. Conversely, homothallic species possess genes for both MAT loci, which may be situated either at a singular MAT locus or on distinct chromosomes [45]. The mating type locus encodes transcription factors that determine mating type identity and serve as master regulators of sexual reproduction [55]. The MAT1-1 locus encodes an α-domain transcription factor named as MAT1-1-1, while its idiomorph, the MAT1-2 locus, encodes a high mobility group (HMG) transcription factor referred to as MAT1-2-1 [38, 57]. MAT genes are critical for regulating cell differentiation, development, and mating of compatible cells. In addition, MAT genes also play important roles in fungal vegetative growth, conidial morphology, conidiospore germination, mycelial development, amino acid, secondary, and iron metabolisms [5, 8, 36].

A vital step in the early stages of sexual reproduction among heterothallic fungi is mate recognition, which depends on the pheromone systems of strains with opposing MAT loci. One fungal strain secretes pheromones that bind to heptahelical pheromone receptors expressed by the strain with opposite MAT type. The interaction between pheromone and its receptor triggers the G-protein-regulated signal transduction and induces the expression of mating-related genes, causing chemotactic growth of strains towards cells of the opposite mating type [3]. Pheromone precursor genes have been identified in several heterothallic filamentous ascomycetes, including Gibberella zeae, Sordaria macrospora, and Neurospora crassa [7, 37, 42]. In these ascomycetes, one of the genes encodes a pheromone polypeptide with a C-terminal carboxy methyl isoprenylated cysteine, which is derived from pheromone precursor with C-terminal CaaX (C, cysteine; a, aliphatic; and X, any amino acid residue) motifs. The other precursor genes encode a polypeptide containing multiple repeats of a putative pheromone sequence bordered by protease processing sites [51]. Deletion of pheromone receptor pre1 in N. crassa causes female sterility [30], while deletion of either pheromone precursor gene causes male sterility, as spermatia could no longer attract female trichogynes [31]. In Gibberella zeae, Δppg1 reduced male fertility and Δpre2 reduces female fertility in outcrossing tests [37]. Similarly, in S. macrospora, the absence of any compatible pheromone receptor pair (Δpre2ppg2, Δpre1ppg1) and the double-pheromone mutant (Δppg1ppg2), results in a significant reduction in the number of perithecia and sexual spores. Moreover, the deletion of both receptor genes (Δpre1pre2) prevents the formation of fruiting bodies and ascospores [42].

V. dahliae is a soilborne plant pathogen that invades and colonizes the xylem tissue, resulting in Verticillium wilt diseases on over 200 plant species [20, 34]. V. dahliae has a complex population structure, comprising physiological races 1, 2, and 3 [10]; defoliating (D) and nondefoliating phenotypes (ND) [66]; vegetative compatibility groups (VCGs) [6, 13, 14, 29], and clonal lineages [4, 18, 44, 53]. In addition, V. dahliae has two mating types, MAT1-1 and MAT1-2, and has maintained all the machinery required for sexual reproduction [53], but whether it undergoes sexual recombination has been a controversial topic [44, 53, 54]. Some reports suggest that V. dahliae may reproduce strictly asexually as it has a clonal population structure, with little genetic variation between strains of the same clonal groups and there is weak evidence for recombination [2, 52]. In addition, the distribution frequencies of these two mating type strains MAT1-1 and MAT1-2 differ significantly in V. dahliae population (with < 1% of sampled strains carrying the MAT1-1 idiomorph) [54]. Therefore, the probability of sexual reproduction between individuals of opposite mating type in V. dahliae in nature is low. Moreover, the genome of V. dahliae has undergone chromosomal rearrangements that may interfere with meiosis and reduce the probability of successful sexual reproduction between existing lineages [15, 46]. Lastly, no fructifications from sexual reproduction, such as the apothecia, perithecia, or cleistothecia, have been found in V. dahliae, either in nature or in the laboratory. In contrast, the possibility that V. dahliae undergoes cryptic sexual reproduction cannot be discounted as the generation of new clonal lineages is suggestive of sexual recombination [13, 14, 29]. In addition, the evolutionary relationship between different lineages is closer than the relationship between two distinct lineages derived from the same lineage, a pattern that could be explained more parsimoniously by recombination rather than mutation [44]. Also, each isolate of V. dahliae contains either the MAT1-1 or MAT1-2 idiomorph, indicating that V. dahliae is heterothallic [58, 59], and many sex-related genes that are necessary for the sexual cycle in other fungi are conserved in V. dahliae [54]. V. dahliae is one of the parents of V. longisporum, a hybrid species derived from three separate hybridization events [27]. Thus, V. dahliae may have a cryptic sexual cycle or has had an ancestral sexual lifestyle.

MAT genes play a pivotal role in fungal sexual reproduction, as they are essential for regulating cell differentiation, development, and mating of compatible cells. V. dahliae populations possess two mating type strains, MAT1-1 and MAT1-2, but the functions of the mating type genes VdMAT1-1-1 and VdMAT1-2-1 remain largely unexplored. The crucial step in the initial phase of fungal sexual reproduction is mate recognition, which depends on the strain’s pheromone system. Yet, the roles of the pheromone precursor genes and pheromone receptor genes in V. dahliae are still not well understood.

In the present study, we characterized the mating type genes VdMAT1-1-1 and VdMAT1-2-1 in V. dahliae. Our results revealed VdMAT1-1-1 and VdMAT1-2-1 possess the capability to bind directly to the promoter regions of pheromone precursor and receptor genes, thereby exerting regulatory control over their expression. Subsequently, we demonstrated that V. dahliae exhibits chemotactic behavior by sensing and orienting growth in response to pheromone signals. Finally, our findings indicated that VdMAT1-1-1 and VdMAT1-2-1 are integral not only to the pathogen’s ability to adapt to varying environmental conditions but also play a crucial role in its vegetative growth and pathogenicity. These insights contribute significantly to our understanding of the molecular mechanisms that underpin pathogenicity and complex regulatory processes in V. dahliae.

Results

Identification of the MAT loci V. dahliae

The strains DK015 and DK038, with opposite MAT loci, were identified from the Verticilli-Omics project. Whole genome comparative analyses indicated that DK015 and DK038, isolated from spinach seeds, had a similar karyotype with very little chromosome rearrangement, and shared 94.6% (9739 orthologs) orthologous genes (unpublished data). DK015 and DK038 contained the MAT1-1 and MAT1-2 idiomorphs, respectively (Fig. 1A). Both VdMAT1-1 and VdMAT1-2 loci and the corresponding flanking sequences were aligned. The coding genes within the flanking sequences of the two MAT idiomorphs are highly conserved, such as the APN2 (AP endonuclease 2), COX13 (Cytochrome c oxidase, subunit VIa), and APC5 (Anaphase-promoting complex subunit 5). The genes coded by the two MAT loci are markedly different, with the MAT1-1 locus containing VdMAT1-1-1 and VdMAT1-1-3 and the MAT1-2 locus containing VdMAT1-2-1 (Fig. 1A). In addition, MAT idiomorphs of other MAT1-1 and MAT1-2 strains isolated from tomato, potato, cotton, sunflower, and watermelon were also analyzed. The results were similar to those of DK015 and DK038 strains, indicating a high conservation of the MAT idiomorphs in V. dahliae (Additional file 1: Fig. S1).

Fig. 1
figure 1

Characteristics of two MAT loci in the Verticilium dahliae strains DK015 and DK038 of opposite mating type. A Diagram of MAT1-1 and MAT1-2 loci and flanking sequences in the MAT1-1 strain DK015 and the MAT1-2 strain DK038. Rectangles represent the genes within the two loci. The dash line indicates the syntenic relationship between two homologous genes flanking both loci. The text above describes the functional annotation of each gene, with the gene IDs of the first and last gene marked. Red and bright light blue rectangles indicate genes specific to VdMAT1-1 locus, VdMAT1-1-1, and VdMAT1-1-3, respectively, while the green rectangle indicates the gene specific to VdMAT1-2 locus, VdMAT1-2-1. Short vertical lines indicate that the gene function has not been annotated. B Structure schematic of VdMAT1-1-1 and VdMAT1-2-1 proteins. The red column represents the MATα_HMG box in VdMAT1-1-1. The purple column represents the HMG domain in VdMAT1-2-1. Light blue, nuclear localization signal (NLS). Gray boxes represent unknown protein structure. Aa, amino acid. HMG, high mobility group. C Subcellular localization of VdMAT1-1-1 and VdMAT1-2-1 fused with GFP proteins in V. dahliae conidia. The nuclear signal was confirmed with the aid of 4, 6-diamidino-2-phenylindole (DAPI), a nuclear dye. The expressed GFP fusions were excited with a 484 nm wavelength, with the emission captured at 507 nm wavelength. DAPI was excited at 360 nm, captured at an emission wavelength of 460 nm. Bars = 5 μm

Cloning and sequence analysis confirmed that VdMAT1-1-1 encodes a protein with 434 amino acids containing a MATalpha_HMGbox domain, while VdMAT1-2-1 encodes a protein with 232 amino acids containing an HMG domain (Fig. 1B). Both VdMAT1-1-1 and VdMAT1-2-1 contain one nuclear localization signal (NLS) that was consistent with their conventional function of regulating the expression of downstream genes. Correspondingly, subcellular localization results verified that both GFP-fused MAT proteins were located in the nucleus (Fig. 1C).

VdMAT1-1-1 and VdMAT1-2-1 regulate the expression of pheromone and pheromone receptor genes by directly binding to their promoter regions

A key role of MAT gene products in heterothallic species is to regulate the expression of the pheromone signaling system, which is involved in the recognition of mating partners [3]. The expression of pheromone precursor and pheromone receptor genes are directly controlled by MAT transcription factors [17]. To investigate the roles of VdMAT1-1-1 and VdMAT1-2-1 in regulating the pheromone (VdPpg1 and VdPpg2) and pheromone receptor genes (VdPre1 and VdPre2), we first performed the dual-luciferase reporter assay in N. benthamiana. The results indicated that VdMAT1-1-1 and VdMAT1-2-1 could inhibit the transcription activity of the VdPre1 and VdPre2 promoters, while enhancing the transcription activity from the VdPpg1 and VdPpg2 promoters (Fig. 2A, B). Electrophoretic mobility shift assay (EMSA) further demonstrated that both VdMAT1-1-1 and VdMAT1-2-1 could directly bind to the promoter regions of VdPpg1, VdPpg2, VdPre1, and VdPre2 (Fig. 2C). In addition, sequence analysis revealed a conserved binding motif, 5′-AACAAT-3′, within the promoter regions of VdPpg2 and VdPre1, which exhibited interactions with both VdMAT1-1-1 and VdMAT1-2-1 (Fig. 2D). Additionally, direct interactions between VdMAT1-1-1 and VdMAT1-2-1 and the VdPpg1 and VdPre2 promoter regions were substantiated through yeast one-hybrid (Y1H) assays with VdMAT1-1-1 and VdMAT1-2-1 and the conserved sequence motif 5′-ATTGA-3′ found upstream of the VdPpg1 and VdPre2 coding regions (Fig. 2D). These results indicate that VdMAT1-1-1 and VdMAT1-2-1 regulate the expression of VdPpg1, VdPpg2, VdPre1, and VdPre2 genes by directly binding to their promoter regions.

Fig. 2
figure 2

Verticillium dahliae VdMAT1-1-1 and VdMAT1-2-1 regulate the expression of pheromone precursors and receptor genes by binding to promoter motifs. A, B Dual-luciferase reporter assays of VdMAT1-1-1 and VdMAT1-2-1 and their ability to regulate the expression of VdPpg1, VdPpg2, VdPre1, and VdPre2. C Electrophoretic mobility transfer assay (EMSA) for the analysis of VdMAT1-1-1 and VdMAT1-2-1 binding to the promoter regions of the VdPpg1, VdPpg2, VdPre1, and VdPre2 genes. A shifted band was observed when purified glutathione S-transferase (GST)-tagged VdMAT1-1-1/VdMAT1-2-1 protein were co-incubated with promoter fragments of VdPpg1, VdPpg2, VdPre1, and VdPre2, respectively. After the addition of proteinase K, the hysteretic band disappeared, leaving only the free probe visible. When GST protein were co-incubated with promoter fragments of VdPpg1, VdPpg2, VdPre1, and VdPre2, no shifted band occurred. D Yeast one hybridization (Y1H) experiment confirmed that VdMAT1-1-1 and VdMAT1-2-1 could directly bind the [5′-ATTGA-3′] motifs in the promoter region of VdPpg1 and VdPre2 genes, and the [5′AACAAT-3′] motifs in the promoter region of VdPpg2 and VdPre1 genes

V. dahliae strains perceive pheromones and exhibit chemotactic growth

Similar to the pheromone peptides present in most filamentous fungi, VdPpg1 encodes a putative pheromone precursor of 212 amino acids containing 6 short tandem repeats of a dodecapeptide sequence, which are always accompanied by the basic dipeptide KR (Kex2-cleavage site) (Fig. 3A). VdPpg2 encodes a short polypeptide of 66 amino acids with five repeats of a undecapeptide sequence and a CaaX motif at the C-terminus (Fig. 3B). Signal peptide activity assays demonstrated that the signal peptide of VdPpg1 had secretory activity (Fig. 3C). Unlike VdPpg1, VdPpg2 has no predicted signal peptide. Moreover, the pheromone receptor genes VdPre1 and VdPre2 of V. dahliae were predicted to encode proteins with seven transmembrane domains (Additional file 1: Fig. S2).

Fig. 3
figure 3

Functional analyses of peptide pheromones from Verticillium dahliae. A The amino acid sequences of the Ppg1-like peptide pheromones from V. dahliae. Repeated parts are highlighted in green font and underlined. Kex2 processing sites (KR) are indicated in green, with putative STE13 processing sites in brown. The hydrophobic leader sequence is underlined. B The amino acid sequence of the Ppg2-like peptide pheromone from V. dahliae. Repeated parts are highlighted with a black underline. Lysine residues and asparagine residues of the putative processing sites are indicated in blue and green, respectively. The C-terminal CaaX farnesylation motif and internal Caax-like motifs are bolded. C Functional validation of the signal peptide of VdPpg1 by a yeast signal trap assay. The region encoding the 18 aa N-terminal peptide of VdPpg1 was fused in-frame to the invertase sequence in the pSUC2 vector and transformed into yeast strain YTK12. The signal peptide of the oomycete effector Avr1b was used as a positive control. The untransformed YTK12 strain and the YTK12 strain carrying the empty pSUC2 vector were used as negative controls. The yeast strain YTK12 cannot grow on a CMD-W medium without tryptophan, whereas strains containing pSUC2 vector could grow based on the function of the Trp operons. Only yeast strains that secrete invertase can convert 2, 3, 5-triphenyltetrazole chloride (TTC) to red triphenylformazan. If the yeast strain can secrete the invertase, it can convert 2, 3, 5-triphenyltetrazole chloride (TTC) into red triphenylmethylzan. D Test of chemotactic pheromone-responsive growth of V. dahliae. MEOH served as a negative control while FO-a and FO-α are a-like and α-like peptide pheromones from F. oxysporum, respectively. VdPpg1 is a Ppg1-like peptide pheromone from V. dahliae, VdPpg2 is Ppg2-like peptide pheromone from V. dahliae. The dotted circle on the left is a schematic diagram. The pheromone was added to the circular filter paper in the middle, and the strain to be tested was inoculated at the four surrounding sites as illustrated

To elucidate whether V. dahliae can perceive or respond to pheromone, we measured the chemotropic response of the wild-type strains (DK015 and DK038), MAT gene deletion strains (ΔVdMAT1-1-1 and ΔVdMAT1-2-1), and the pheromone receptor gene deletion strains (ΔVdPre1 and ΔVdPre2) to the synthesized single-repeat pheromone peptides VdPpg1 and VdPpg2. Considering that F. oxysporum and V. dahliae have a relatively close evolutionary relationship, the F. oxysporum pheromones a-factor and α-factor [60] were also synthesized as controls for the test. With the exception of the responses to the pheromone VdPpg1, all strains exhibited chemotaxis for each of the other types of pheromones. Deletion of mating type genes ΔVdMAT1-1-1 and ΔVdMAT1-2-1 had no significant influence on the chemotactic growth of mycelia, while pheromone receptor gene deletion strains (ΔVdPre1 and ΔVdPre2) showed weaker chemotaxis than wild-type strains (Fig. 3D). Since chemotactic growth was anticipated in a direction only towards the pheromone of the opposite mating type strains, further experiments are necessary to clarify this result.

VdMAT1-1-1 and VdMAT1-2-1 impact V. dahliae carbon source utilization, stress tolerance, and conidia production

Previous reports indicate that MAT genes in filamentous fungi are not only involved in sexual reproduction, but also involved in asexual development, pellet morphology, polar hyphal growth, conidiospore germination, and secondary metabolism [19]. To detect the influence of VdMAT1-1-1 and VdMAT1-2-1 on V. dahliae vegetative development, the phenotypes of wild-type (DK015 and DK038), knockout (ΔVdMAT1-1-1 and ΔVdMAT1-2-1), and complemented strains (ECΔVdMAT1-1-1 and ECΔVdMAT1-2-1) of two MAT genes were evaluated on media containing different carbon sources and abiotic stressors.

Compared to the wild-type strain DK015, the colony diameter of the ΔVdMAT1-1-1 strain was significantly smaller on three media containing sucrose, pectin, and starch as carbon sources (Fig. 4A, B). However, the ΔVdMAT1-1-1 strain exhibited a larger colony diameter than DK015 on the medium containing cellulose as a carbon source (Fig. 4A, B). The growth phenotype and colony diameter of the complemented strain ECΔVdMAT1-1-1 reverted to the phenotypes similar to those of the wild-type strain on the media examined (Fig. 4A, B). On the other hand, the ΔVdMAT1-2-1 strains grew faster than the wild-type strain DK038 on the media containing sucrose and starch as carbon sources, while there were no evident differences on the media containing pectin or cellulose as carbon sources (Fig. 4A, B). Similarly, there was also no significant difference in growth between ECΔVdMAT1-2-1 and DK038 on these media (Fig. 4A, B). Whether MAT genes regulate tolerance to abiotic stress was examined subsequently. The ∆VdMAT1-1-1 strains was more sensitive to sorbitol, but less sensitive to Congo red and H2O2 compared with wild-type and complemented strains (Fig. 4C, D). The ∆VdMAT1-2-1 strains were more sensitive to Congo red, but less sensitive to sorbitol and H2O2 (Fig. 4C, D). The above results indicate divergent responses of the two MAT genes for carbon utilization and stress tolerance in the two mating type strains.

Fig. 4
figure 4

Functional analyses of VdMAT1-1-1 and VdMAT1-2-1 in regulating carbon source utilization, stress tolerance, and conidia production of Verticillium dahliae. Wild-type strains (DK015 and DK038), MAT gene deletion strains (ΔVdMAT1-1-1 and ΔVdMAT1-2-1), and the corresponding complementary strains (ECΔVdMAT1-1-1 and ECΔVdMAT1-2-1) were used in the experiments. A, B Growth and diameter (mm) of the colonies of different strains on Czapek medium containing 30 g/L sucrose, 10 g/L pectin, 17 g/L starch, and 10 g/L cellulose, respectively at 25 ℃ for 9 days. C, D Growth and diameter (mm) of the colonies of different strains on PDA medium containing 1 M sorbitol, 200 μg/ml Congo red, and 2 mM H2O2, respectively at 25 ℃ for 9 days. E, F SEM observation of the conidia and mycelia morphology of different strains. G, H Variation in conidial size. Photographs were taken after 9 days of incubation. I Conidial production of different strains. Error bars represent the standard deviation calculated from three replicate experiments compared with the wild-type, and the asterisks show significant differences (one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001)

To analyze whether VdMAT1-1-1 and VdMAT1-2-1 affect the growth of conidia and mycelia in V. dahliae, scanning electron microscopy (SEM) was used to observe the morphology of conidia and mycelia. Though deletion of VdMAT1-1-1 and VdMAT1-2-1 did not obviously affect the morphology (Fig. 4E, F), the conidia of the ΔVdMAT1-1-1 and ΔVdMAT1-2-1 strains were longer than those in the corresponding wild-type strains (Fig. 4G, H). The widths of ΔVdMAT1-2-1 conidia were also increased compared to that of DK038, while no significant difference was observed between ΔVdMAT1-1-1 and DK015 in conidial width (Fig. 4G, H). In addition, the conidial yield of ΔVdMAT1-1-1 and ΔVdMAT1-2-1 was significantly increased compared to that of the wild-type strains (Fig. 4I). These results indicate a negative regulatory role of the two MAT genes in conidia production in two different MAT strains.

VdMAT1-1-1 and VdMAT1-2-1 negatively regulate the virulence of V. dahliae

To evaluate the roles of VdMAT1-1-1 and VdMAT1-2-1 in the pathogenicity of V. dahliae, the virulence of the wild-type strain (DK015 and DK038), MAT gene deletion strains (ΔVdMAT1-1-1 and ΔVdMAT1-2-1), and the corresponding complemented strains (ECΔVdMAT1-1-1 and ECΔVdMAT1-2-1) were inoculated on spinach, the host from which they were originally obtained and another host Nicotiana benthamiana. The results revealed that ΔVdMAT1-1-1 and ΔVdMAT1-2-1 strains increased Verticillium wilt symptoms on these two hosts compared with the wild-type strains DK015 and DK038, which only caused mild disease on spinach and N. benthamiana (Fig. 5A, C, E, and G). Correspondingly, the fungal biomass of inoculated spinach and N. benthamiana plants with the ΔVdMAT1-1-1 and ΔVdMAT1-2-1 strains were significantly increased compared to the wild-type strain (Fig. 5B, D, F, and H). Furthermore, the virulence phenotype or fungal biomass was restored to the level of wild-type strains in the complemented ECΔVdMAT1-1-1 and ECΔVdMAT1-2-1 strains (Fig. 5A–H). The same results were also obtained using V. dahliae strains of different MAT loci, such as the MAT1-1 strain S109 and the MAT1-2 strain S12, isolated from sunflower (Additional file 1: Fig. S3). These results indicate that VdMAT1-1-1 and VdMAT1-2-1 negatively regulate the virulence of V. dahliae.

Fig. 5
figure 5

Virulence assays with VdMAT1-1-1 and VdMAT1-2-1 deletion mutants and complemented transformant strains of Verticillium dahliae on spinach and tobacco. A, B, E, F Phenotypes and the fungal biomass of spinach and N. benthamiana seedlings inoculated with wild-type strain DK015, ΔVdMAT1-1-1 mutants, and the corresponding complemented transformants ECΔVdMAT1-1-1. C, D, G, H Phenotypes and the fungal biomass of spinach and N. benthamiana seedlings inoculated with wild-type strain DK038, ΔVdMAT1-2-1 mutants, and the corresponding complemented transformants ECΔVdMAT1-2-1. The fungal biomass was determined by qPCR. V. dahliae elongation factor 1-α (VdEF-1α) was used to quantify fungal colonization, while the spinach actin gene and the N. benthamiana EF-1α gene were used as endogenous plant reference genes, respectively. Error bars represent the standard deviation calculated from three replicate experiments compared with the wild-type. The asterisks show significant differences (one-way ANOVA, **P < 0.01)

Discussion

Mating-type genes play important roles not only in regulating the sexual cycle but also in the vegetative growth and pathogenicity of fungi [32, 64, 67]. V. dahliae, a heterothallic fungus, has two mating type idiomorphs, MAT1-1 and MAT1-2, located in different strains. In this study, we demonstrated that the MAT gene locus of V. dahliae was highly conserved, that the genes flanking MAT1-1 and MAT1-2 loci have high collinearity (Fig. 1A), and that the core transcription factors VdMAT1-1-1 and VdMAT1-2-1 were localized to the nucleus (Fig. 1C). These MAT proteins play a pivotal role in regulating the expression of pheromone and pheromone receptor genes by directly binding to their respective promoter regions (Fig. 2C, D). V. dahliae expresses pheromone precursor genes, with the strains being responsive to pheromones, subsequently exhibiting chemotactic growth (Fig. 3D). In addition, VdMAT1-1-1 and VdMAT1-2-1 also play important roles in regulating vegetative growth, stress resistance, and virulence (Figs. 4 and 5). Investigations into the functions of MAT genes in V. dahliae may facilitate studies to further explore a potential sexual cycle of this destructive pathogen.

One of the most important functions of mating type genes is to regulate the fertility of fungi. Mating-pheromone signaling is required for mate recognition in sexual reproduction of heterothallic fungi [3]. Mating type genes directly control the expression of pheromone precursor and receptor genes, but the regulation mode by which this occurs can differ among fungal species. In S. macrospora, the transcription levels of pheromone precursor genes ppg1 and ppg2 were significantly downregulated in the ΔSmtA-1 mutant compared to the wild-type. The expression of ppg2 was significantly increased in the ΔSmtA-2 mutant, while the expression of ppg1 remained unchanged. Additionally, neither mating type proteins SMTA-1 nor SMTA-2 had a clear effect on the expression of pheromone receptor genes pre1 and pre2 [33]. In FusariumF graminearum, both MAT1-1-1 and MAT1-2-1 promoted the expression of pheromone precursor gene GzPPG1, while the expression of GzPPG2 was inhibited by MAT1-1-1 but promoted by MAT1-2-1 [37]. Our results showed that VdMAT1-1-1 and VdMAT1-2-1 promote the expression of pheromone precursor genes VdPpg1 and VdPpg2 but inhibit the expression of pheromone receptor genes VdPre1 and VdPre2 (Fig. 2A, B). These results indicate that the mechanism by which mating type genes regulate pheromone precursor and receptor genes may be different among fungi and may vary conditionally. Furthermore, pheromones can also induce the expression of mating type genes [12, 16]. This suggests that there is a reciprocal regulation between pheromones and mating type genes to achieve orderly transmission and reception of pheromone signals.

Mate recognition, a critical initial step in sexual reproduction, is mediated in many fungi by the perception of pheromones secreted by partners of the opposite mating type [3]. Our research has demonstrated that the pheromone precursors and receptors in V. dahliae exhibit a high degree of similarity in protein sequence to those found in other fungal species (Fig. 3A, B) [7, 37, 42]. Additionally, we have determined that the signaling peptide of VdPpg1 possesses secretory activity (Fig. 3C). Notably, V. dahliae displays chemotactic growth in response to mating pheromones, including VdPpg2, FO-a, and FO-α, but not VdPpg1 (Fig. 3D). Initial assays show that V. dahliae can undergo chemotactic growth in response to the pheromones of F. oxysporum, likely a result of the significant homology between these species [11]. This chemotactic response, however, does not necessarily lead to cell fusion, which could be hindered by heterokaryon incompatibility, akin to a species barrier [50]. The lack of chemotactic growth in response to the synthetic VdPpg1 pheromone raises questions about the congruence between the predicted and actual secreted molecules, a finding that will prompt further investigation into pheromone secretion in V. dahliae. Our observations also suggest that both mating types can exhibit chemotaxis towards VdPpg2, a finding that diverges from traditional expectations, such as in S. cerevisiae. MATα and MATa secrete α-factor and a-factor, respectively; the α-factor binds to a specific receptor (Ste2) on a MATa cells, whereas the a-factor binds to a specific receptor (Ste3) on MATα cells, which subsequently promotes the chemotactic growth of the two mating strains until fusion [63]. The capacity of V. dahliae to grow chemotactically in response to pheromones indicates a conserved communication pathway in its reproductive process, analogous to that observed in known sexually reproducing fungi such as S. cerevisiae, S. macrospora, and N. crassa [31, 42, 63]. However, there may be differences in the mechanisms that regulate pheromone responses in V. dahliae. This has prompted us to explore in future work whether V. dahliae can secrete pheromones, the specific type of pheromone secreted by MAT1-1 and MAT1-2 mating type strains, the corresponding pheromone receptor, the preference of MAT1-1 and MAT1-2 strains for different types of pheromones, the signal pathway used for pheromone transmission, and the gene network involved in information exchange between MAT1-1 and MAT1-2 strains mediated by pheromones.

Mating type genes are essential for the ascospore formation, mycelial morphology, conidial formation, and stress responses in many fungi [8, 61, 62, 65]. In V. dahliae, VdMAT1-1-1 and VdMAT1-2-1 are involved in carbon utilization and stress tolerance, but the extent of this involvement differentiates the two mating type strains, especially in vegetative growth (Fig. 4A–D). The conidia of the ΔVdMAT1-1-1 and ΔVdMAT1-2-1 mutants became slender (Fig. 4E–H). Similarly, in Ulocladium botrytis, MAT1-1-1 regulates the vegetative growth and the size of the conidia [62]. In Penicillium chrysogenum, MAT1-1-1 controls hyphal morphology and conidia formation [8]. However, in Villosiclava virens, the deletion of MAT1-1-1 resulted in slower growth and abnormal conidial morphology, yet no significant differences were detected in conidial production [65]. These studies, including our work, further suggest that MAT1-1-1 and MAT1-2-1 genes have different functions in regulating vegetative growth, asexual reproduction, and responding to external stress in different fungi.

Deletion of either the VdMAT1-1-1 or VdMAT1-2-1 genes resulted in a significantly increased pathogenicity (Fig. 5 and Additional file 1: S3). Interestingly, in F. graminearum, the virulence of the MAT1-1-1 gene deletion mutant was reduced on cornstalks [67] and the pathogenicity of the MAT-2 gene deletion mutant of Sclerotinia sclerotiorum was also significantly decreased [17]. In contrast, in Magnaporthe oryzae, deletion of MAT1-1-1 or MAT1-2-1 genes did not affect appressorium formation and virulence [61]. The functional differences of mating type genes between closely related fungal species indicate the complexity in the network of MAT1-1-1 or MAT1-2-1 protein interactions. The mechanism by which the VdMAT1-1-1 and VdMAT1-2-1 genes regulate the virulence of strains is still unclear at present. In subsequent studies, it may be possible to screen differentially expressed genes regulated by mating type genes under host-induced conditions and directly identify the target genes. Analysis of the function of these genes may facilitate exploitation of the mechanism by which mating type genes regulate virulence.

Conclusions

Mating type genes hold significant importance in fungi, and gaining insights into their molecular functions can substantially aid in unraveling the life cycle of these organisms. While V. dahliae is traditionally regarded as a strictly asexual fungus, the function of the mating type genes within this species has not been sufficiently examined. This study shows that VdMAT1-1-1 and VdMAT1-2-1 play important roles in asexual reproduction, and perhaps also in sexual reproduction of V. dahliae. The findings from this research will serve as a valuable reference for furthering our understanding of sex-related genes and potential mating in V. dahliae. In the ongoing follow-up studies, we are exploring whether the two mating type genes can mediate nuclear fusion and sexual reproduction in V. dahliae, and the regulatory mechanisms governing sexual reproduction. This work is further expected to illuminate on the mode of cell-cell communication and recognition processes between strains with the opposite mating type idiomorphs, and may offer a novel perspective on the mode of sexual reproduction in Verticillium spp. and potentially other ascomycetes.

Methods

All primers used in this study are listed in Additional file 2: Table S1.

Bioinformatics analysis

The wild-type strains DK015 and DK038 used in this study were collected from spinach plants exhibiting wilt symptoms. The mating type loci of DK015 and DK038 were identified by the BLASTN using the VdMAT1-1-1 and VdMAT1-2-1 from VdLs.17 as the query sequences [58]. The sequences of VdMAT1-1-1, VdMAT1-2-1, VdPpg1, VdPpg2, VdPre1, and VdPre2 were cloned from DK015 and DK038. The genomic sequences of these strains were archived in the Verticilli-Omics database (https://db.cngb.org/Verticilli-Omics/), which served as a vital reference for our cloning process.

Subcellular localization assays

To determine the subcellular localization of VdMAT1-1-1 and VdMAT1-2-1, the fragment of TrpC-promoter region, the coding sequence of VdMAT1-1-1 or VdMAT1-2-1, GFP sequence, and Nos-terminator were fused and cloned into a pCOM vector [68]. The positive recombinant vectors were then introduced into Agrobacterium tumefaciens strain AGL-1 for fungal transformation. The positive transformants were selectively cultured on potato dextrose agar (PDA) medium supplemented with 50 µg/mL geneticin. After 7-day culture, conidia were observed using a Leica TCS SP8 confocal microscope. Fluorescent signals were detected at 532 nm excitation and 588 nm emission wavelengths for RFP, 484 nm excitation and 507 nm emission wavelengths for GFP, and 340 nm excitation and 488 nm emission wavelengths for DAPI, respectively.

V. dahliae transformations for gene deletion and complementation

Gene deletion vectors of VdMAT1-1-1, VdMAT1-2-1, VdPre1, and VdPre2 were generated by homologous recombination. In brief, approximately 1.5-kb sequences of the regions flanking the coding sequence of each gene were amplified by PCR using the appropriate primer sets. The plasmid pGKO2 was linearized by restriction endonuclease EcoRI or HindIII, and the amplified up- and downstream fragments were ligated with the hygromycin resistance gene cassette (hyg). To generate the vector for mutant complementation, the genomic sequence of each gene, including its native promoter, coding region, and terminator were amplified by PCR using the appropriate primer sets, and then fused into the pCOM vector which carries geneticin resistance cassette (G418) [68].

The A. tumefaciens mediated transformation (ATMT) method, as previously described, was employed to generate gene deletion and complementation transformants [40]. The positive gene deletion strains were selected on PDA medium supplemented with 50 μg/mL hygromycin, 200 µg/mL cefotaxime, and 200 μg/mL 5-fluoro-2’-deoxyuridine. The complemented strains were selected on PDA medium supplemented with 50 µg/mL geneticin. All positive strains were further verified by conducting PCR to amplify the corresponding specific sequence, using the appropriate primer sets as listed in Additional file 2: Table S1.

Yeast signal sequence trap system

Functional validation of the signal peptide of VdPpg1 was performed as previously described [28]. The sequence encoding the predicted signal peptide of VdPpg1 was cloned into the pSUC2 vector. The resulting recombinant plasmid, pSUC2::SPVdPpg1, was transformed into the yeast strain YTK12 and screened on CMD-W medium.

The recombinant YTK12 strain carrying the signal peptide sequence of Avr1b (pSUC2::SPAvr1b), and the untransformed YTK12 strain and YTK12 strain with an empty pSUC2 vector were used as a positive control and negative controls, respectively. These strains were incubated in a 10 mM acetic acid-sodium acetate solution (pH = 4.7) and 10% sucrose medium for 10 min at 37 °C. Post-incubation, the supernatant was collected and incubated with 0.1% 2,3,5-triphenyltetrazole ammonium chloride (TTC) for 10 min. Invertase enzymatic activity was confirmed by observing an increase in insoluble red-colored triphenyl formazan, indicating the reduction of TTC and signifying successful signal peptide functionality.

Verification of hyphal chemotropism

To verify hyphal chemotropism, pheromones of VdPpg1 (GKPCPRPGQGCW), VdPpg2 (NGGGHSGCAIM), Fo-α (WCTWRGQPCW), and Fo-a (ANGQTPGYPLSCTVM) were chemically synthesized and dissolved in 50% (v/v) methanol (MeOH) to a concentration of 378 μM [60]. For the assay, 25 μL of the pheromone solution was applied to a 1.2-cm-diameter piece of filter paper, which was then placed at the center of water agar medium plates. A 3 μL conidial suspension, with a concentration of 5 × 106 conidia/mL of the different strains, was cultured on water agar medium plates and positioned 1.2 cm away from the filter paper. The plates were incubated for 7 days at 25 °C to observe chemotropic responses.

Dual-luciferase report assay

The coding regions of VdMAT1-1-1 and VdMAT1-2-1 were cloned and fused into the pCAMBIA1300-Cluc vector [9] to generate the effector constructs. Meanwhile, the promoter regions of VdPpg1, VdPpg2, VdPre1, and VdPre2 were introduced into the pGreenII 0800-LUC vector [26], each generating a distinct reporter construct. Both recombinant vectors were transformed into A. tumefaciens strain GV3101 cells. The firefly luciferase and Renilla luciferase activities were analyzed at 60 hpi using the Dual-Luciferase Reporter Assay System (Promega) and the GloMax96 Microplate Luminometer (Promega). Each experiment was performed with three biological replicates.

Electrophoretic mobility shift assay (EMSA)

The full coding regions of VdMAT1-1-1 and VdMAT1-2-1 were amplified from the cDNA of the DK015 and DK038 strains, respectively. These amplified sequences were inserted into the EcoRI/SalI-digested pGEX-4T-1 vector by homologous recombination of multiple fragments (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing, China), to generate the prokaryotic expression vectors pGEX-VdMAT1-1-1 and pGEX-VdMAT1-2-1. Positive recombinant vectors were transformed into the Escherichia coli BL21(DE3) strain. VdMAT1-1-1 and VdMAT1-2-1 proteins were purified following the instructions of GST-tag protein purification kit (Beyotime, Shanghai, China). For DNA shift assays, the promoter fragments of the target genes were labeled using 6-carboxyfluorescein (FAM). The assays were performed with the EMSA binding buffer kit, following the instructions of the manufacturer (Beyotime, Shanghai, China).

Yeast one-hybrid assays

Yeast one-hybrid assays were performed using the Matchmaker Gold Yeast One-Hybrid System Kit (Takara) according to the manufacturer’s protocol. Briefly, gene (VdPpg1, VdPpg2, VdPre1, and VdPre2) promoter fragments were ligated into the pAbAi vector, while the full-length CDS of VdMAT1-1-1 and VdMAT1-2-1 were cloned into the pGADT7 vector (gene-AD). The gene-AD vectors were then used to transform Y1HGold cells harboring the pAbAi-bait and then screened on SD/-Leu/AbA medium.

Strains growth, stress response, and conidiation assays

The strains were routinely cultured on PDA medium or maintained in a shaking incubator using complete medium (CM) for 5 days at 25 °C in the dark. For phenotype analysis, 3 μL of the conidial suspension with a concentration of 5 × 106 conidia/mL was cultured on Czapek plates (FeSO4, 0.01 g/L; KCl, 0.5 g/L; MgSO4·7H2O, 0.5 g/L; K2HPO4, 1 g/L; NaNO3, 3 g/L; and agar, 18 g/L) prepared with different carbon sources: either sucrose, 30 g/L; pectin 10 g/L, starch, 17 g/L, or cellulose, 10 g/L.

For stress response assays, 3 μL of the conidial suspensions, each with a concentration of 5 × 106 conidia/mL, was cultured on PDA medium, supplemented with either 1 M sorbitol, 200 μg/mL Congo red, or 1.5 mM H2O2, each prepared separately. After 7 days of incubation at 25 °C, the phenotypes including colony diameters were observed.

To measure the sporulation of V. dahliae, 3 μL of each conidial suspension with a concentration of 5 × 106 conidia/mL was cultured on PDA plates for 9 days at 25 °C. Three same areas of the fungus were collected and suspended in 1mL of sterile water by vortex for 1 min. The numbers of conidia were counted using a hemocytometer.

Virulence assays

Six-week-old spinach and 5-week-old tobacco seedlings were inoculated by the root irrigation method [23]. Each pot was inoculated with 50 mL of the conidial suspension with a concentration of 5 × 106 conidia/mL. Plants were maintained on the greenhouse benches at 24 ± 2 °C under a 16-h photoperiod after inoculation. The phenotypes of spinach seedlings were investigated 30 days after inoculation, while those of tobacco seedlings were evaluated on the 21st day. For molecular analysis, the root-stem junctions of spinach and tobacco plants were collected and DNA was extracted. Biomass of V. dahliae was quantified by qPCR following the procedure of Santhanam et al. [49]. The V. dahliae elongation factor 1α (VdEF-1α) was used to quantify fungal colonization and the spinach actin gene and N. benthamiana EF-1α gene were used as endogenous reference genes.

Availability of data and materials

All study data are included in the article and/or supplementary information.

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Funding

This work was supported by the National Key Research and Development Program of China (2022YFE0130800, 2022YFD1400300, 2022YFE0111300), the Fundamental Research Funds for Central Non‑profit Scientific Institution in CAAS (Y2021XK22), the National Natural Science Foundation of China (32370213, 32270212), the Agricultural Sciences Talent Program CAAS (J.Y.C.), and the Agricultural Science and Technology Innovation Program grant (J.Y.C.).

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Contributions

JYC and DDZ designed and supervised this study. JYC and KVS provided the materials. YDZ performed main experiments. XBJ and JZ participated in preparation of plasmids and verifying the virulence of mutants on spinach and tobacco. YDZ analyzed the data. YDZ and DDZ wrote the initial draft. KVS, SJK, JYC, and XFD reviewed and edited the manuscript. JYC and XFD provided the funding for this research. All authors read and approved the final manuscript. The authors declare no competing financial interests.

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Correspondence to Krishna V. Subbarao, Dan-Dan Zhang or Jie-Yin Chen.

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Supplementary Information

Additional file 1: Figure S1. 

MAT1-1 and MAT1-2 loci and flanking sequences in the MAT1-1 and the MAT1-2 strains isolated from different hosts. Figure S2. Schematic diagram of the transmembrane domains in VdPre1 and VdPre2 proteins predicted by TMHMM. Figure S3. Virulence assays with VdMAT1-1-1 and VdMAT1-2-1 deletion mutant of Verticillium dahliae on sunflower.

Additional file 2: Table S1.

Information on the primer pairs used to construct the vector in this study.

Additional file 3.

Raw data of experimental results.

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Zhang, YD., Ji, XB., Zong, J. et al. Functional analysis of the mating type genes in Verticillium dahliae. BMC Biol 22, 108 (2024). https://doi.org/10.1186/s12915-024-01900-6

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