Functional analysis of the mating type genes in Verticillium dahliae.

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.

Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

Verticillium wilt, caused by Verticillium dahliae, is one of the most devastating soilborne diseases of lettuce (Lactuca sativa L.). There are three races of V. dahliae and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e, respectively. While the presence of race 1 in coastal California was well-established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non-Verticillium fungal species. Since the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and one endive (Cichorium endivia L.) line, comprised of commercial cultivars (cv.) and breeding lines, including the race 1-resistant heirloom cv. La Brillante and the susceptible cv. Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2 while all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available. Keywords: Lactuca sativa, avirulence, effectors, host resistance, disease severity.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Genetic determinants of lettuce resistance to drop caused by Sclerotinia minor identified through genome-wide association mapping frequently co-locate with loci regulating anthocyanin content.

KEY MESSAGE: GWAS identified 19 QTLs for resistance to Sclerotinia minor, 11 of them co-locating with red leaf color. Lower disease incidence was observed in red and dark red accessions. Lettuce (Lactuca sativa L.), one of the most economically important vegetables grown primarily in moderate climates around the world, is susceptible to many diseases including lettuce drop caused by the soilborne fungus Sclerotinia minor. Complete resistance to S. minor has not been identified in cultivated lettuce or its wild relatives. We conducted five experiments over 4 years with the diversity panel of almost 500 lettuce accessions to evaluate their response to the pathogen in an artificially infested field. The lowest disease incidence (DI) was observed in cultivars Eruption, Infantry, and Annapolis (median DI of 12.1-17.5%), while the highest DI was recorded for cultivars Reine des Glaces, Wayahead, and line FL. 43007 (median DI of 81.0-95.2%). Overall, significantly lower DI was observed in red and dark red accessions compared to those with a lower anthocyanin content. Genome-wide association mapping identified 19 QTLs for resistance to S. minor, 21 for the presence of red leaf color or its variations caused by the anthocyanin content, and one for the green color intensity. Eleven of the QTLs for disease resistance were located within 10 Mb of the loci associated with red color or anthocyanin content identified in this diversity panel. The frequent, non-random co-location of QTLs, together with the lower DI observed in red and dark red accessions suggests that lettuce interaction with S. minor may be partly influenced by anthocyanins. We have identified RLL2 and ANS, the genes of the anthocyanin biosynthesis pathway that co-locate with resistance QTLs, as candidates for functional studies to ascertain the involvement of anthocyanins in lettuce resistance against S. minor. Resistance QTLs closely linked with QTLs for anthocyanin content could be used to develop lettuce with a relatively high partial resistance and red color, while those not associated with anthocyanins could be used to develop partially resistant cultivars of green color.

Genome-wide identification and analysis of a cotton secretome reveals its role in resistance against Verticillium dahliae.

BACKGROUND: The extracellular space between the cell wall and plasma membrane is a battlefield in plant-pathogen interactions. Within this space, the pathogen employs its secretome to attack the host in a variety of ways, including immunity manipulation. However, the role of the plant secretome is rarely studied for its role in disease resistance. RESULTS: Here, we examined the secretome of Verticillium wilt-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2, encoding 95,327 predicted coding sequences) to determine its role in disease resistance against the wilt causal agent, Verticillium dahliae. Bioinformatics-driven analyses showed that the ZZM2 genome encodes 2085 secreted proteins and that these display disequilibrium in their distribution among the chromosomes. The cotton secretome displayed differences in the abundance of certain amino acid residues as compared to the remaining encoded proteins due to the localization of these putative proteins in the extracellular space. The secretome analysis revealed conservation for an allotetraploid genome, which nevertheless exhibited variation among orthologs and comparable unique genes between the two sub-genomes. Secretome annotation strongly suggested its involvement in extracellular stress responses (hydrolase activity, oxidoreductase activity, and extracellular region, etc.), thus contributing to resistance against the V. dahliae infection. Furthermore, the defense response genes (immunity marker NbHIN1, salicylic acid marker NbPR1, and jasmonic acid marker NbLOX4) were activated to varying degrees when Nicotina benthamiana leaves were agro-infiltrated with 28 randomly selected members, suggesting that the secretome plays an important role in the immunity response. Finally, gene silencing assays of 11 members from 13 selected candidates in ZZM2 displayed higher susceptibility to V. dahliae, suggesting that the secretome members confer the Verticillium wilt resistance in cotton. CONCLUSIONS: Our data demonstrate that the cotton secretome plays an important role in Verticillium wilt resistance, facilitating the development of the resistance gene markers and increasing the understanding of the mechanisms regulating disease resistance.

Functional Characterization of Verticillium dahliae Race 3-Specific Gene VdR3e in Virulence and Elicitation of Plant Immune Responses.

Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

The Verticillium dahliae Small Cysteine-Rich Protein VdSCP23 Manipulates Host Immunity.

Verticillium wilt caused by Verticillium dahliae is a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection, V. dahliae secretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs from V. dahliae are unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis in Nicotiana benthamiana leaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on the N-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion of VdSCP23 did not affect the growth and development of mycelia or conidial production in V. dahliae. Unexpectedly, VdSCP23 deletion strains still maintained their virulence for N. benthamiana, Gossypium hirsutum and Arabidopsis thaliana seedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence in V. dahliae.

Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae.

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H2O2, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

Verticillium dahliae Effector VdCE11 Contributes to Virulence by Promoting Accumulation and Activity of the Aspartic Protease GhAP1 from Cotton.

Verticillium dahliae is a soilborne plant fungal pathogen that causes Verticillium wilt, a disease that reduces the yields of many economically important crops. Despite its worldwide distribution and harmful impacts, much remains unknown regarding how the numerous effectors of V. dahliae modulate plant immunity. Here, we identified the intracellular effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana to counter leaf pathogens such as Sclerotinia sclerotiorum and Botrytis cinerea. VdCE11 also contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity, since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further, VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. Taken together, these results indicate a novel mechanism regulating virulence whereby the secreted effector VdCE11 increases cotton susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1. IMPORTANCE Verticclium dahliae is a plant fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, we identified a V. dahliae effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana. Meanwhile, VdCE11 contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further research showed that VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. These results suggested that a novel mechanism regulating virulence whereby VdCE11 increases susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1 in the host.

Genome Resource for the Verticillium Wilt Resistant Gossypium hirsutum Cultivar Zhongzhimian No. 2.

Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the major cause of disease-related yield losses in cotton (Gossypium hirsutum). Despite these losses, the major cultivars of G. hirsutum remain highly susceptible to Verticillium wilt. The lack of understanding on the genetic basis for Verticillium wilt resistance may further hinder progress in deploying elite cultivars with proven resistance, such as the wilt resistant G. hirsutum cultivar Zhongzhimian No. 2. To help remedy this knowledge gap, we sequenced the whole genome of Zhongzhimian No. 2 and assembled it from a combination of PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture technologies. The final assembly of the genome was 2.33 Gb, encoding 95,327 predicted coding sequences. The GC content was 34.39% with 99.2% of the bases anchored to 26 pseudo-chromosomes that ranged from 53.8 to 127.7 Mb. This resource will help gain a detailed understanding of the genomic features governing high yield and Verticillium wilt resistance in this cultivar. Comparative genomics will be particularly helpful, since there are several published genomes of other Gossypium species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Phosphatase VdPtc3 Regulates Virulence in Verticillium dahliae by Interacting with VdAtg1.

Type 2C protein phosphatases regulate various biological processes in eukaryotes. However, their functions in Verticillium dahliae have not been characterized. In this study, homologs VdPtc1, VdPtc3, VdPtc5, VdPtc6, and VdPtc7 were identified in V. dahliae on the basis of homologous comparison with those in Saccharomyces cerevisiae. VdPtc2 and VdPtc4 are missing in the genome of the V. dahliae XJ592 strain. VdPtc3 is the homolog of Ptc2, Ptc3, and Ptc4 proteins in S. cerevisiae, implying that VdPtc3 may play versatile functions in V. dahliae. VdPtc3 promoted conidium development, melanin, and microsclerotium formation in V. dahliae. The ΔVdPtc3 strains showed increased sensitivity to NaCl and sorbitol and augmented the phosphorylation of p38 mitogen-activated protein kinase homolog Hog1 induced by osmotic stress. Besides, the ΔVdPtc3 strains also showed milder Verticillium wilt symptom on cotton. Furthermore, VdPtc3 interacts with VdAtg1, which modulates melanin and microsclerotium formation, as well as pathogenicity.

Transcription Factor VdCf2 Regulates Growth, Pathogenicity, and the Expression of a Putative Secondary Metabolism Gene Cluster in Verticillium dahliae.

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence.

BACKGROUND: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. RESULTS: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. CONCLUSIONS: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

The secretome of Verticillium dahliae in collusion with plant defence responses modulates Verticillium wilt symptoms.

Verticillium dahliae is a notorious soil-borne pathogen that enters hosts through the roots and proliferates in the plant water-conducting elements to cause Verticillium wilt. Historically, Verticillium wilt symptoms have been explained by vascular occlusion, due to the accumulation of mycelia and plant biomacromolecule aggregation, and also by phytotoxicity caused by pathogen-secreted toxins. Beyond the direct cytotoxicity of some members of the secretome, this review systematically discusses the roles of the V. dahliae secretome in vascular occlusion, including the deposition of polysaccharides as an outcome of plant cell wall destruction, the accumulation of fungal mycelia, and modulation of plant defence responses. By modulating plant defences and hormone levels, the secretome manipulates the vascular environment to induce Verticillium wilt. Thus, the secretome of V. dahliae colludes with plant defence responses to modulate Verticillium wilt symptoms, and thereby bridges the historical concepts of both toxin production by the pathogen and vascular occlusion as the cause of wilting symptoms.

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity.

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)-related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site-directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand-binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR-inducing effector in V. dahliae that contributes to the activation of plant immunity.

The Verticillium dahliae Spt-Ada-Gcn5 Acetyltransferase Complex Subunit Ada1 Is Essential for Conidia and Microsclerotia Production and Contributes to Virulence.

Verticillium dahliae is a destructive soil-borne pathogen of many economically important dicots. The genetics of pathogenesis in V. dahliae has been extensively studied. Spt-Ada-Gcn5 acetyltransferase complex (SAGA) is an ATP-independent multifunctional chromatin remodeling complex that contributes to diverse transcriptional regulatory functions. As members of the core module in the SAGA complex in Saccharomyces cerevisiae, Ada1, together with Spt7 and Spt20, play an important role in maintaining the integrity of the complex. In this study, we identified homologs of the SAGA complex in V. dahliae and found that deletion of the Ada1 subunit (VdAda1) causes severe defects in the formation of conidia and microsclerotia, and in melanin biosynthesis and virulence. The effect of VdAda1 on histone acetylation in V. dahliae was confirmed by western blot analysis. The deletion of VdAda1 resulted in genome-wide alteration of the V. dahliae transcriptome, including genes encoding transcription factors and secreted proteins, suggesting its prominent role in the regulation of transcription and virulence. Overall, we demonstrated that VdAda1, a member of the SAGA complex, modulates multiple physiological processes by regulating global gene expression that impinge on virulence and survival in V. dahliae.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence.

BACKGROUND: Verticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown. RESULTS: Nine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type. CONCLUSIONS: In the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Identification of long non-coding RNAs in Verticillium dahliae following inoculation of cotton.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, these functions have not been assessed in Verticillium dahliae, a soil-borne fungal pathogen that causes devastating wilt diseases in many crops. The discovery and identity of novel lncRNAs and their association with virulence may contribute to an increased understanding of the regulation of virulence in V. dahliae. Here, we identified a total of 352 lncRNAs in V. dahliae. The lncRNAs were transcribed from all V. dahliae chromosomes, typically with shorter open reading frames, lower GC content, and fewer exons than protein-coding genes. In addition, 308 protein-coding genes located within 10 kb upstream and 10 kb downstream of lncRNAs were identified as neighboring genes, and which were considered as potential targets of lncRNA. These neighboring genes encode products involved in development, stress responses, and pathogenicity of V. dahliae, such as transcription factors (TF), kinase, and members of the secretome. Furthermore, 47 lncRNAs were significantly differentially expressed in V. dahliae following inoculation of susceptible cotton (Gossyoiumhisutum) cultivar Junmian No.1, suggesting that lncRNAs may be involved in the regulation of virulence in V. dahliae. Moreover, correlations in expression patterns between lncRNA and their neighboring genes were detected. Expression of lncRNA012077 and its neighboring gene was up-regulated 6 h following inoculation of cotton, while the expression of lncRNA007722 was down-regulated at 6 h but up-regulated at 24 h, in a pattern opposite to that of its neighboring gene. Overexpression of lncRNA012077 in wild-type strain (Vd991) enhanced its virulence on cotton while overexpression of lncRNA009491 reduced virulence. Identification of novel lncRNAs and their association with virulence may provide new targets for disease control.

Functional Genomics and Comparative Lineage-Specific Region Analyses Reveal Novel Insights into Race Divergence in Verticillium dahliae.

Verticillium dahliae is a widespread soilborne fungus that causes Verticillium wilt on numerous economically important plant species. In tomato, until now, three races have been characterized based on the response of differential cultivars to V. dahliae, but the genetic basis of race divergence in V. dahliae remains undetermined. To investigate the genetic basis of race divergence, we sequenced the genomes of two race 2 strains and four race 3 strains for comparative analyses with two known race 1 genomes. The genetic basis of race divergence was described by the pathogenicity-related genes among the three races, orthologue analyses, and genomic structural variations. Global comparative genomics showed that chromosomal rearrangements are not the only source of race divergence and that race 3 should be split into two genotypes based on orthologue clustering. Lineage-specific regions (LSRs), frequently observed between genomes of the three races, encode several predicted secreted proteins that potentially function as suppressors of immunity triggered by known effectors. These likely contribute to the virulence of the three races. Two genes in particular that can act as markers for race 2 and race 3 (VdR2e and VdR3e, respectively) contribute to virulence on tomato, and the latter acts as an avirulence factor of race 3. We elucidated the genetic basis of race divergence through global comparative genomics and identified secreted proteins in LSRs that could potentially play critical roles in the differential virulence among the races in V. dahliae. IMPORTANCE Deciphering the gene-for-gene relationships during host-pathogen interactions is the basis of modern plant resistance breeding. In the Verticillium dahliae-tomato pathosystem, two races (races 1 and 2) and their corresponding avirulence (Avr) genes have been identified, but strains that lack these two Avr genes exist in nature. In this system, race 3 has been described, but the corresponding Avr gene has not been identified. We de novo-sequenced genomes of six strains and identified secreted proteins within the lineage-specific regions (LSRs) distributed among the genomes of the three races that could potentially function as manipulators of host immunity. One of the LSR genes, VdR3e, was confirmed as the Avr gene for race 3. The results indicate that differences in transcriptional regulation may contribute to race differentiation. This is the first study to describe these differences and elucidate roles of secreted proteins in LSRs that play roles in race differentiation.