Dianguang Xiong

Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae

BackgroundVerticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a wide range of plant hosts. V. dahliae produces multicelled, melanized resting bodies, also known as microsclerotia (MS) that can survive for years in the soil. The MS are the primary source of infection of the Verticillium disease cycle. Thus, MS formation marks an important event in the disease cycle of V. dahliae.ResultsIn this study, next generation sequencing technology of RNA-Seq was employed to investigate the global transcriptomic dynamics of MS development to identify differential gene expression at several stages of MS formation in strain XS11 of V. dahliae, isolated from smoke tree. We observed large-scale changes in gene expression during MS formation, such as increased expression of genes involved in protein metabolism and carbohydrate metabolism. Genes involved in glycolytic pathway and melanin biosynthesis were dramatically up-regulated in MS. Cluster analyses revealed increased expression of genes encoding products involved in primary metabolism and stress responses throughout MS development. Differential expression of ubiquitin-dependent protein catabolism and cell death-associated genes during MS development were revealed. Homologs of genes located in the lineage-specific (LS) regions of V. dahliae strain VdLs.17, were either not expressed or showed low expression. Furthermore, alternative splicing (AS) events were analyzed, revealing that over 95.0% AS events involve retention of introns (RI).ConclusionsThese data reveal the dynamics of transcriptional regulation during MS formation and were used to construct a comprehensive high-resolution gene expression map. This map provides a key resource for understanding the biology and molecular basis of MS development of V. dahliae.

The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae

The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. V. dahliae produces melanized resting bodies, known as microsclerotia, which can survive for 15 years in the soil, and are thus critically important in its disease cycle. However, the molecular mechanisms that underpin microsclerotia formation, survival, and germination remain poorly understood. In this study, we observed that deletion of VdHog1 (ΔVdHog1), encoding a homolog of a high-osmolarity glycerol (HOG) response mitogen-activated protein kinase, displayed decreased numbers of melanized microsclerotia in culture, heightened sensitivity to hyperosmotic stress, and increased resistance to the fungicide fludioxonil. Through RNA-Seq analysis, we identified 221 genes differentially expressed in the ΔVdHog1 strain. Interestingly, the expression levels of genes involved in melanin biosynthesis, as well as the hydrophobin gene VDH1, involved in the early stage of microsclerotia formation, were significantly decreased in the ΔVdHog1 strains relative to the wild-type expression levels. The ΔVdHog1 strains exhibited decreased virulence relative to the wild type strain on smoke tree seedlings. These results indicate that VdHog1 regulates hyperosmotic stress responses in V. dahliae, and establishes the Hog1-mediated pathway as a target to further probe the up- and downstream processes that regulate asexual development in this fungus.

MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae.

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae.

VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae.

Calcium signaling plays crucial roles in ion stress tolerance, sporulation and pathogenicity in fungi. Although the signaling pathway mediated by calcineurin and the calcineurin-responsive zinc finger transcription factor Crz1 is well characterized in other fungi, this pathway is not well characterized in the phytopathogenic fungus, Verticillium dahliae. To better understand the role of this calcineurin-dependent transcription factor in V. dahliae, an ortholog of CRZ1, VdCrz1, was identified and characterized functionally. Transcriptional analysis of VdCrz1 and GFP expression driven by the VdCrz1 promoter indicated that VdCrz1 was involved in microsclerotia development. After targeted deletion of VdCrz1, microsclerotia formation and melanin accumulation were impaired. Furthermore, the ΔVdCrz1 mutants were hypersensitive to high concentrations of Ca(2+) and cell wall-perturbing agents, such as sodium dodecyl sulfate. The addition of Mg(2+) to the medium restores the microsclerotia formation in ΔVdCrz1 mutants. The ΔVdCrz1 mutants exhibited delayed Verticillium wilt symptoms on smoke tree. These results suggest that VdCrz1 plays important roles in Ca(2+) signaling, cell wall integrity, microsclerotia development and full virulence in V. dahliae.

The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

Phylogenic analysis revealed an expanded C2H2-homeobox subfamily and expression profiles of C2H2 zinc finger gene family in Verticillium dahliae

C2H2 zinc finger (CZF) proteins are a major class of transcription factors that play crucial roles in fungal growth, development, various stress responses, and virulence. Little genome-wide data is available regarding the roles of CZF proteins in Verticillium dahliae, a destructive pathogen that causes vascular wilt disease in more than 200 plant species. We identified a total of 79 typical CZF genes in V. dahliae. Comparative analysis revealed that four plant pathogenic fungi, V. dahliae, Fusarium oxysporum, Magnaporthe oryzae, and Botrytis cinerea, have comparable numbers of predicted CZF genes with similar characteristics. Phylogenetic analysis identified a C2H2-homeobox subfamily in V. dahliae containing seven genes with similar gene structures. V. dahliae and F. oxysporum (Hypocreomycetidae) have more genes of this subfamily than M. oryzae (Sordariomycetidae) and B. cinerea (Leotiomycetes). Furthermore, gene-expression analysis of the smoke tree wilt fungus V. dahliae strain XS11 using digital gene-expression profiling and RT-qPCR revealed that a number of CZF genes were differentially expressed during microsclerotia formation, nutritional starvation, and simulated in planta conditions. Furthermore, the expression profiles revealed that some CZF genes were overrepresented during multiple stages, indicating that they might play diverse roles. Our results provide useful information concerning the functions of CZF genes in microsclerotia formation, nutritional stress responses, and pathogenicity in V. dahliae, and form a basis for future functional studies of these genes.

Transcriptomic profiles of the smoke tree wilt fungus Verticillium dahliae under nutrient starvation stresses

Verticillium dahliae is a notorious plant pathogen that causes vascular wilt on more than 200 plant species. During plant infection, efficient pathogen nutrition during the interaction with the host is a requisite for successful infection. However, little attention has been focused on nutrient uptake and starvation responses in this fungus. Here, we used RNA-Seq to analyze the response of V. dahliae to nutrient starvation, including carbon and nitrogen depletion. Gene expression profile analysis showed that 1854 genes were differentially expressed under carbon starvation (852 upregulated and 539 downregulated genes) and nitrogen starvation (487 upregulated and 291 downregulated genes). Among the differentially expressed genes, genes involved in utilization or production acetyl-CoA, including glycolysis, fatty acid biosynthesis or metabolism, and melanin biosynthesis, were repressed under carbon starvation, whereas melanin biosynthesis genes were strongly induced under nitrogen starvation. These results, combined with VDH1 expression data, suggested that melanin biosynthesis and microsclerotia development were induced under nitrogen starvation, but microsclerotia development was suppressed under carbon starvation. Furthermore, many genes encoding carbohydrate-active enzymes and secreted proteins were induced under carbon starvation. Overall, the results improve our understanding of the response of V. dahliae to nutrient starvation and help to identify potential virulence factors for the development of novel disease control strategies.

Functional characterization of two bZIP transcription factors in Verticillium dahliae

bZIP transcription factors play various biological roles in stress responses, conidiation, and pathogenicity in pathogenic fungi. Here, we report two bZIP transcription factors (VDAG_08640 and VDAG_08676) of Verticillium dahliae, which were differentially expressed during microsclerotia development and induced by hydrogen peroxide as well. We find that deletion of either gene does not affect microsclerotia formation and the sensitivity to hydrogen peroxide; however, the mutants manifest decreased activity of extracellular peroxidase and laccase. Other phenotypic characterization reveals that VDAG_08676 disruption results in significant reduction of conidial production and virulence, while VDAG_08640 disruption does not lead to observable phenotypic variances compared with the wild-type strain. To elucidate whether they exhibit functional redundancy, double deletion mutants were generated. The double deletion mutants show remarkably increased sensitivity to hydrogen peroxide stress, whereas the two genes are not involved in microsclerotia formation. Taken together, our data demonstrate that a bZIP transcription factor gene VDAG_08676 is involved in the conidial production, oxidative stress response and virulence which may lay a foundation for further analysis of other bZIP transcription factors in V. dahliae.

Deletion of VdKu80 enhances targeted gene replacement in Verticillium dahliae

Verticillium dahliae is a notorious phytopathogenic fungus and causes severe Verticillium wilt diseases worldwide. The available genomic sequence of V. dahliae has facilitated genome-wide investigation of its life cycle and disease process. However, inefficient targeted gene replacement hampers gene functional analysis of V. dahliae. Ku heterodimer genes Ku70 and Ku80 have been proved to be involved in the recognition of DNA double-strand breaks, which will reduce the rate of homologous recombination. Here, in order to improve the frequency of the homologous recombination, we identified and deleted the V. dahliae VdKu80 with bioinformatics methods and protoplast-mediated transformation, respectively. The phenotypes of VdKu80 deletion mutants, such as fungal growth, microsclerotia formation and virulence, were similar to those of the wild type strain. Remarkably, the gene replacement frequencies of two genes (VDAG_00736 and VDAG_07169 which encode the cell division control protein) were increased by about 20% with VdKu80 deletion mutant as a recipient strain compared with that of the wild type strain. The results suggest that deletion of VdKu80 triggered the enhancement of the gene replacement frequency, indicating that the ∆VdKu80 strain can be used as an efficient recipient for the targeted gene manipulation in V. dahliae.

The two-component response regulator VdSkn7 plays key roles in microsclerotial development, stress resistance and virulence of Verticillium dahliae

The fungus Verticillium dahliae causes vascular wilt disease on various plant species resulting in devastating yield losses worldwide. The capacity of V. dahliae to colonize in host plant xylem and disseminate by microsclerotia has led to studies to evaluate genes associated with pathogenesis and microsclerotia formation. Here, we identified and characterized a V. dahliae homolog to Skn7, a two-component stress response regulator of Saccharomyces cerevisiae. Results showed that melanized microsclerotia formation and conidiation were significantly inhibited in the VdSkn7 deletion mutants. VdSkn7-deficient mutants displayed severe growth defect under heat shock, cell wall perturbing agents and H2O2, and were significantly less virulent but were not sensitive to osmotic stresses compared to the wild-type strain. Finally, we demonstrated that VdSkn7 is required for the plant penetration. Taken together, our study thus provides new evidence on the functional conservation and divergence of Skn7 orthologs among fungal organisms and indicates that VdSkn7 contributes to microsclerotial development, virulence and stress response of V. dahliae.