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.

Secreted in xylem 6 (SIX6) mediates Fusarium oxysporum f. sp. fragariae race 1 avirulence on FW1-resistant strawberry cultivars.

Fusarium oxysporum f.sp. fragariae (Fof) race 1 is avirulent on cultivars with the dominant resistance gene, FW1, while Fof race 2 is virulent on FW1-resistant cultivars. We hypothesized there was a gene-for-gene interaction between a gene at the FW1 locus and an avirulence gene (AvrFW1) in Fof race 1. To identify a candidate AvrFW1, we compared genomes of twenty-four Fof race 1 and three Fof race 2 isolates. We found one candidate gene that was present in race 1, absent in race 2, highly expressed in planta, and homologous to a known effector, secreted in xylem 6 (SIX6). We knocked out SIX6 in two Fof race 1 isolates by homologous recombination. All SIX6 knockout transformants (ΔSIX6) gained virulence on FW1/fw1 cultivars, whereas ectopic transformants and the wildtype isolates remained avirulent. ΔSIX6 isolates were quantitatively less virulent on FW1/fw1 cultivars Fronteras and San Andreas than fw1/fw1 cultivars. Seedlings from an FW1/fw1 × fw1/fw1 population were genotyped for FW1 and tested for susceptibility to a SIX6 knockout isolate. Results suggested that additional minor-effect quantitative resistance genes could be present at the FW1 locus. This work demonstrates that SIX6 acts as an avirulence factor interacting with a resistance gene at the FW1 locus. The identification of AvrFW1 enables surveillance for Fof race 2 and provides insight into the mechanisms of FW1-mediated resistance.

Kss1 of Verticillium dahliae regulates virulence, microsclerotia formation, and nitrogen metabolism.

Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia (MS) enable V. dahliae to survive for years in soil, thus the fungus is especially difficult to control once it has become established. Previously, we found that the mitogen activated protein kinase VdSte11 (MAPKKK) plays key roles in MS formation, penetration, and virulence in V. dahliae. In this study, two MAPK homologs of the yeast Ste7p and Kss1p were identified and characterized in V. dahliae. Deletion of VdSte7 or VdKss1 reuslted in severe defects in melaninized MS formation and virulence. Furthermore, phosphorylation assays demonstrated that VdSte11 and VdSte7 can phosphorylate VdKss1 in V. dahliae. Proteomic analysis revealed a significant change in sterol biosynthesis with a fold change of ≥ 1.2 after the deletion of VdKss1. In addition, phosphoproteomic analysis showed that VdKss1 was involved in the regulation of nitrogen metabolism. Finally, we identified VdRlm1 as a potentially downstream target of VdKss1, which is involved in regulating ammonium nitrogen utilization. This study sheds light on the network of regulatory proteins in V. dahliae that affect MS formation and nitrogen metabolism.

Transmission of Spinach Downy Mildew via Seed and Infested Leaf Debris.

Spinach downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is a worldwide constraint on spinach production. The role of airborne sporangia in the disease cycle of P. effusa is well established, but the role of the sexual oospores in the epidemiology of P. effusa is less clear and has been a major challenge to examine experimentally. To evaluate seed transmission of spinach downy mildew via oospores in this study, isolated glass chambers were employed in two independent experiments to grow out oospore-infested spinach seed and noninfested seeds mixed with oospore-infested crop debris. Downy mildew diseased spinach plants were observed 37 and 34 days after planting in the two isolator experiments, respectively, in the chambers that contained one of two oospore-infested seed lots or seeds coated with oospore-infested leaves. Spinach plants in isolated glass chambers initiated from seeds without oospores did not show downy mildew symptoms. Similar findings were obtained using the same seed lot samples in a third experiment conducted in a growth chamber. In direct grow out tests to examine oospore infection on seedlings performed in a containment greenhouse with oospore-infested seed of two different cultivars, characteristic Peronospora sporangiophores were observed growing from a seedling of each cultivar. The frequency of seedlings developing symptoms from 82 of these oospore-infested seed indicated that approximately 2.4% of seedlings from infested seed developed symptoms, and 0.55% of seedlings from total seeds assayed developed symptoms. The results provide evidence that oospores can serve as a source of inoculum for downy mildew and provide further evidence of direct seed transmission of the downy mildew pathogen to seedlings in spinach via seedborne oospores.

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.

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.

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.

VdTps2 Modulates Plant Colonization and Symptom Development in Verticillium dahliae.

The trehalose biosynthesis pathway is a potential target for antifungal drugs development. Trehalose phosphate synthase (TPS) and phosphatase are widely conserved components of trehalose biosynthesis in fungi. However, the role of trehalose biosynthesis in the vascular plant-pathogenic fungus Verticillium dahliae remains unclear. Here, we investigated the functions of the TPS complex, including VdTps1, VdTps2, and VdTps3 in V. dahliae. Unlike VdTps2, deletion of VdTps1 or VdTps3 did not alter any phenotypes compared with the wild-type strain. In contrast, the ΔVdTps2 strain showed severely depressed radial growth due to the abnormal swelling of the hyphal tips. Further, deletion of VdTps2 increased microsclerotia formation, melanin biosynthesis, and resistance to cell-wall perturbation and high-temperature stress. Virulence assays and quantification of fungal biomass revealed that deletion of VdTps2 delayed disease symptom development, as evident by the reduced virulence and decreased biomass of the ΔVdTps2 strain in plant stem tissue following inoculation. Additionally, increases in penetration peg formation observed in the ΔVdTps2 strain in the presence of H2O2 suggested that VdTps2 suppresses initial colonization. Our results also revealed the role of VdTps2 as a regulator of autophagy. Together, these results indicate that VdTps2 contributes to plant colonization and disease development. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

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.

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.

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 bZip Transcription Factor VdMRTF1 is a Negative Regulator of Melanin Biosynthesis and Virulence in Verticillium dahliae.

The ascomycete fungus Verticillium dahliae infects over 400 plant species and causes serious losses of economically important crops, such as cotton and tomato, and also of woody plants, such as smoke tree, maple, and olive. Melanized long-term survival structures known as microsclerotia play crucial roles in the disease cycle of V. dahliae, enabling this soilborne fungus to survive for years in the soil in the absence of a host. Previously, we identified VdMRTF1 (microsclerotia-related transcription factor) encoding a bZip transcription factor which is downregulated during microsclerotial development in V. dahliae. In the present study, we showed that VdMRTF1 negatively controls melanin production and virulence by genetic, biological, and transcriptomic analyses. The mutant strain lacking VdMRTF1 (ΔVdMRTF1) exhibited increased melanin biosynthesis and the defect also promoted microsclerotial development and sensitivity to Ca2+. In comparison with the wild-type strain, the ΔVdMRTF1 strain showed a significant enhancement in virulence and displayed an increased capacity to eliminate reactive oxygen species in planta. Furthermore, analyses of transcriptomic profiles between the ΔVdMRTF1 and wild-type strains indicated that VdMRTF1 regulates the differential expression of genes associated with melanin biosynthesis, tyrosine metabolism, hydrogen peroxide catabolic processes, and oxidoreductase activity in V. dahliae. Taken together, these data show that VdMRTF1 is a negative transcriptional regulator of melanin biosynthesis, microsclerotia formation, and virulence in V. dahliae. IMPORTANCE Verticillium wilt is difficult to manage because the pathogen colonizes the plant xylem tissue and produces melanized microsclerotia which survive for more than 10 years in soil without a host. The molecular mechanisms underlying microsclerotia formation are of great importance to control the disease. Here, we provide evidence that a bZip transcription factor, VdMRTF1, plays important roles in melanin biosynthesis, microsclerotial development, resistance to elevated Ca2+ levels, and fungal virulence of V. dahliae. The findings extend and deepen our understanding of the complexities of melanin biosynthesis, microsclerotia formation, and virulence that are regulated by bZip transcription factors in V. dahliae.

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.

Early Detection of the Spinach Downy Mildew Pathogen in Leaves by Recombinase Polymerase Amplification.

Downy mildew of spinach, caused by Peronospora effusa, is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for P. effusa could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the P. effusa specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, P. schachtii. TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect P. effusa in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.

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.