Dahliae Strain Research Articles

Functional Characterization of Target of Rapamycin Signaling in Verticillium dahliae.

More than 200 plants have been suffering from Verticillium wilt caused by Verticillium dahliae (V. dahliae) across the world. The target of rapamycin (TOR) is a lethal gene and controls cell growth and development in various eukaryotes, but little is known about TOR signaling in V. dahliae. Here, we found that V. dahliae strain is hypersensitive to rapamycin in the presence of rapamycin binding protein VdFKBP12 while the deletion mutant aaavdfkbp12 is insensitive to rapamycin. Heterologous expressing VdFKBP12 in Arabidopsis conferred rapamycin sensitivity, indicating that VdFKBP12 can bridge the interaction between rapamycin and TOR across species. The key across species of TOR complex 1 (TORC1) and TORC2 have been identified in V. dahliae, suggesting that TOR signaling pathway is evolutionarily conserved in eukaryotic species. Furthermore, the RNA-seq analysis showed that ribosomal biogenesis, RNA polymerase II transcription factors and many metabolic processes were significantly suppressed in rapamycin treated cells of V. dahliae. Importantly, transcript levels of genes associated with cell wall degrading enzymes (CWEDs) were dramatically down-regulated in TOR-inhibited cells. Further infection assay showed that the pathogenicity of V. dahliae and occurrence of Verticillium wilt can be blocked in the presence of rapamycin. These observations suggested that VdTOR is a key target of V. dahliae for controlling and preventing Verticillium wilt in plants.

Comparative transcriptome analysis reveals regulatory networks and key genes of microsclerotia formation in the cotton vascular wilt pathogen

Verticillium dahliae is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Verticillium wilt in a broad range of economic crops. The microsclerotia (MS), which act as the main host inoculum, can survive long-term in soil resulting in uncontrollable disease. In order to clarify the mechanism of MS formation, we sequenced the whole genome-wide expression profile of V. dahliae strain V991. Compared with M1 (no MS formation), during the process of MS formation and maturation, 1354, 1571, and 1521 unique tags were significantly regulated in M2, M3, and M4 library, respectively. During MS formation, melanin synthesis-related genes were preferentially upregulated. The process is more likely to regulated by transcription factors (TFs) including C2H2, Zn2Cys6, bZIP, and fungal-specific TF domain-containing proteins; additionally, G-protein coupled receptors, Ca2+, small GTPases, and cAMP were involved in signalling transduction. Protein kinase-encoding (VDAG_06474) and synthase-encoding (VDAG_05314) genes were demonstrated to negatively and positively influence MS production, respectively. The gene expression dynamics revealed during MS formation provide comprehensive theoretical knowledge to further understanding of the metabolism and regulation of MS development in V. dahliae, potentially providing targets to control Verticillium wilt through interfering MS formation.

The Colonization Process of Sunflower by a Green Fluorescent Protein-Tagged Isolate of Verticillium dahliae and its Seed Transmission.

Sunflower Verticillium wilt is a widespread and destructive disease caused by the soilborne pathogen Verticillium dahliae. To better understand the process of infection and seed transmission of the fungus, sunflower roots were inoculated with a V. dahliae strain (VdBM9-6) labeled with green fluorescent protein (GFP) and monitored microscopically. After 24 to 96 h postinoculation (hpi), conidia germinated and developed into mycelium on root hairs, elongation zones, and caps of lateral roots. Mycelium colonized vascular bundles of lateral roots and taproots at 7 days postinoculation (dpi). At 10 weeks postinoculation (wpi), the epidermal cells, cortical tissues, and vascular elements of stem, petiole, and leaf veins were colonized by mycelium. By 12 wpi, strong GFP signals were detected not only on different tissues of inflorescence but also on testa of seed and a small fraction of pollen grains. A GFP signal was not observed on cotyledon tissues in the seed. Additionally, the colonization of V. dahliae on testa was also confirmed with MNP-10 selection medium, indicating that the testa of seed is the main carrier for the long distance transmission of sunflower yellow wilt.

Verticillium Wilt of Okra Caused by Verticillium dahliae Kleb. in China

Okra (Abelmoschus esculentus (L.) Moench) has gained more popularity as an economically significant plant for its nutritional and medicinal value, especially in China. During 2014–2016, the root disease of okra was discovered in four okra commercial fields surveyed in China. A fungul was isolated from the infected tissues, and was identified by Verticillium dahliae based on morphological characteristics. Pathogenicity test demonstrated that the fungus was pathogenic on okra, and fulfilled Koch’s postulates. The analysis of three sequences revealed 99–100% identity with the reported V. dahliae strain in GenBank. Neighbor-joining analysis of the gene sequences revealed that the representative isolates were clustered with V. dahliae. To the best of our knowledge, this is the first report of Verticillium wilt of okra in China.

Host-induced gene silencing of a regulator of G protein signalling gene (VdRGS1) confers resistance to Verticillium wilt in cotton.

Summary Verticillium wilt (VW), caused by soil‐borne fungi of the genus Verticillium, is a serious disease affecting a wide range of plants and leading to a constant and major challenge to agriculture worldwide. Cotton (Gossypium hirsutum) is the world's most important natural textile fibre and oil crop. VW of cotton is a highly devastating vascular disease; however, few resistant germplasms have been reported in cotton. An increasing number of studies have shown that RNA interference (RNAi)‐based host‐induced gene silencing (HIGS) is an effective strategy for improving plant resistance to pathogens by silencing genes essential for the pathogenicity of these pathogens. Here, we have identified and characterized multifunctional regulators of G protein signalling (RGS) in the Verticillium dahliae virulence strain, Vd8. Of eight VdRGS genes, VdRGS1 showed the most significant increase in expression in V. dahliae after treating with the roots of cotton seedlings. Based on the phenotype detection of VdRGS1 deletion and complementation mutants, we found that VdRGS1 played crucial roles in spore production, hyphal development, microsclerotia formation and pathogenicity. Tobacco rattle virus‐mediated HIGS in cotton plants silenced VdRGS1 transcripts in invaded V. dahliae strains and enhanced broad‐spectrum resistance to cotton VW. Our data demonstrate that VdRGS1 is a conserved and essential gene for V. dahliae virulence. HIGS of VdRGS1 provides effective control against V. dahliae infection and could obtain the durable disease resistance in cotton and in other VW‐susceptible host crops by developing the stable transformants.

Natural distribution of Verticillium wilt on invasive Ailanthus altissima in eastern Austria and its potential for biocontrol

SummaryAilanthus altissima is a highly invasive tree species that has become established in warmer climatic zones of Europe. Verticillium wilt shows promise for biocontrol of this invasive tree species. In this study, we conducted a survey on the natural distribution of Verticillium species on A. altissima in eastern Austria. Results of a survey, across the range of A. altissima in Austria, indicated a widespread distribution of V. dahliae (56 of 77 sampled sites) and a rare occurrence of V. nonalfalfae (two of 77 sites). All sites positive for Verticillium spp. were situated at 130 m to 240 m above sea level. In addition, pathogenicity and virulence of an Austrian isolate of V. nonalfalfae, which is considered the most effective Verticillium species as biocontrol agent against A. altissima, were tested in field inoculations studies. Results revealed that disease progression on A. altissima is negatively influenced by high temperatures in summer. Therefore, inoculation in spring turned out to be the most effective to combat A. altissima. However, due to the common occurrence of V. dahliae in Austria, this pathogen might be considered as alternative biocontrol agent of A. altissima on sites in warmer climates. For that purpose, further investigations of the host range and non‐target effects of selected V. dahliae strains should be carried out.

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts.

SummaryChitin‐binding lysin motif (LysM) effectors contribute to the virulence of various plant‐pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil‐borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage‐specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage‐specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin‐induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage‐specific LysM effector of strain VdLs17 contributes to virulence in planta.

Potential of Endophytic Fungi Isolated from Cotton Roots for Biological Control against Verticillium Wilt Disease.

Verticillium wilt is a soil-borne disease, and severely limits the development of cotton production. To investigate the role of endophytic fungi on Verticillium wilt, CEF-818 (Penicillium simplicissimum), CEF-714 (Leptosphaeria sp.), CEF-642 (Talaromyces flavus.) and CEF-193 (Acremonium sp.) isolated from cotton roots were used to assess their effects against cotton wilt disease caused by a defoliating V. dahliae strain Vd080. In the greenhouse, all treatments significantly reduced disease incidence and disease index, with the control efficacy ranging from 26% (CEF-642) to 67% (CEF-818) at 25 days (d) after inoculation. In the disease nursery, compared to controls (with disease incidence of 33.8% and disease index of 31), CEF-818, CEF-193, CEF-714 and CEF-642 provided a protection effect of 69.5%, 69.2%, 54.6% and 45.7%, respectively. Especially, CEF-818 and CEF-714 still provided well protection against Verticillium wilt with 46.9% and 56.6% or 14.3% and 33.7% at the first peak of the disease in heavily infected field, respectively (in early July). These results indicated that these endophytes not only delayed but also reduced wilt symptoms on cotton. In the harvest, the available cotton bolls of plant treated with CEF-818 and CEF-714 increased to 13.1, and 12.2, respectively. And the seed cotton yield significantly increased after seed bacterization with CEF-818 (3442.04 kg/ha) compared to untreated control (3207.51 kg/ha) by 7.3%. Furtherly, CEF-818 and CET-714 treatment increased transcript levels for PAL, PPO, POD, which leads to the increase of cotton defense reactions. Our results indicate that seed treatment of cotton plants with CEF-818 and CET-714 can help in the biocontrol of V. dahliae and improve seed cotton yield in cotton fields. This study provided a better understanding of cotton-endophyte interactions which will aid in developing effective biocontrol agents for Verticillium wilt of cotton in futhre.

Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen.

Plant pathogenic fungi represent the largest group of disease-causing agents on crop plants, and are a constant and major threat to agriculture worldwide. Recent studies have shown that engineered production of RNA interference (RNAi)-inducing dsRNA in host plants can trigger specific fungal gene silencing and confer resistance to fungal pathogens1-7. Although these findings illustrate efficient uptake of host RNAi triggers by pathogenic fungi, it is unknown whether or not such an uptake mechanism has been evolved for a natural biological function in fungus-host interactions. Here, we show that in response to infection with Verticillium dahliae (a vascular fungal pathogen responsible for devastating wilt diseases in many crops) cotton plants increase production of microRNA 166 (miR166) and miR159 and export both to the fungal hyphae for specific silencing. We found that two V. dahliae genes encoding a Ca2+-dependent cysteine protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15), and targeted by miR166 and miR159, respectively, are both essential for fungal virulence. Notably, V. dahliae strains expressing either Clp-1 or HiC-15 rendered resistant to the respective miRNA exhibited drastically enhanced virulence in cotton plants. Together, our findings identify a novel defence strategy of host plants by exporting specific miRNAs to induce cross-kingdom gene silencing in pathogenic fungi and confer disease resistance.

Generation of transcriptome profiling and gene functional analysis in Gossypium hirsutum upon Verticillium dahliae infection

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae, is a devastating disease in cotton. To get more candidate genes related to wilt resistance, a normalized, full-length cDNA library was used to generate the transcriptome profile from a wilt-resistant Gossypium hirsutum variety (Zhongzhimian KV3) upon V. dahliae strain V991 infection. Total 3486 high-quality ESTs were focused from randomly selected 4000 clones, which included 3065 singletons and 421 contigs. To categorize these unigenes, they were compared to functional domain databases. Total 106 unigenes were found to be homologous to known defense-related genes. Among them, PR genes were the major group. Furthermore, knockdown of GhWRKY22, GhWRKY33, GhChitinase, GhCML, and GhDirigent resulted in increased susceptibility of resistant cotton to V. dahliae infection. The results of transcriptome profiles and virus induced gene silencing (VIGS) analysis laid a good foundation for further investigation of cotton resistance to wilt disease.

Isolation and Characterization of Two Types of Xyloglucanases from a Phytopathogenic Fungus, Verticillium dahliae.

Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).

A Ve homologous gene from Gossypium barbadense, Gbvdr3, enhances the defense response against Verticillium dahliae

The tomato Ve1 gene and several Ve1 homologues are involved in the resistance to Verticillium dahliae. Here, we report on another Ve homologous gene, Gbvdr3, from a Verticillium wilt-resistant cotton cultivar, Gossypium barbadense Hai7124, which has a 3207-bp region that encodes a predicted receptor-like protein. Transient expression analyses indicated that Gbvdr3 is localized in the plasma membrane, and virus-induced gene silencing of Gbvdr3 compromised the resistance of Hai7124 cotton to a defoliating strain of V. dahliae, V991, but not to a non-defoliating strain, BP2. This resistance pattern was further confirmed by over-expression of Gbvdr3 in transgenic Arabidopsis, which significantly elevated the expression of the ethylene-regulated gene GST2, the ethylene- and jasmonic acid-regulated defense-related genes PR3 and PDF1.2, and the salicylic acid-regulated genes PR1 and PR5, but not the PR2 gene. It also triggered the accumulation of hydrogen peroxide and callose at early time points during infection by the V991 defoliating strain. In contrast, elevated accumulation of hydrogen peroxide or callose in Gbvdr3-expressed Arabidopsis leaves was not apparent under infection by the non-defoliating strain, BP2. These results suggested that Gbvdr3 is involved in the resistance to a unique spectrum of defoliating V. dahliae strains.

Construction of a Large Mutational Library from a Defoliating Verticillium dahliae Strain and its Evaluation

Construction of a Large Mutational Library from a Defoliating Verticillium dahliae Strain and its Evaluation

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.

Characterization and distribution of two subtypes of Verticillium longisporum isolated from cabbage fields in Japan

Verticillium longisporum is an allodiploid hybrid fungus that consists of at least three groups, A1/D1, A1/D2, and A1/D3. PCR-based analysis of 18S rDNA, 5.8S rDNA-ITS region, mtSSU-rDNA, cytochrome b gene, and mating type gene, as well as RAPD analysis, revealed that Verticillium wilt of cabbage in Gunma Prefecture in Japan is caused by two groups of V. longisporum—A1/D1 and A1/D3—and by V. dahliae. A1/D1 and A1/D3 lineage strains were equally distributed in cabbage fields, whereas V. dahliae strains were most frequently isolated. The proportion of the three types of Verticillium had not changed in 1998 and 2008. Although both V. longisporum strains produced longer conidia than the V. dahliae strains, the A1/D3-type strains produced larger conidia than A1/D1-type strains. In addition, the A1/D3-type strains formed microsclerotia that were distinguishable from those of A1/D1-type of V. longisporum and V. dahliae. The pathogenicity of the A1/D3-type strains on cabbage was similar to that of the A1/D1-type strains. These results will contribute to understanding of genotypic diversity, distribution, and pathogenicity of Verticillium species pathogenic on cabbage.

Horizontal transfer generates genetic variation in an asexual pathogen.

There are major gaps in the understanding of how genetic variation is generated in the asexual pathogen Verticillium dahliae. On the one hand, V. dahliae is a haploid organism that reproduces clonally. On the other hand, single-nucleotide polymorphisms and chromosomal rearrangements were found between V. dahliae strains. Lineage-specific (LS) regions comprising about 5% of the genome are highly variable between V. dahliae strains. Nonetheless, it is unknown whether horizontal gene transfer plays a major role in generating genetic variation in V. dahliae. Here, we analyzed a previously sequenced V. dahliae population of nine strains from various geographical locations and hosts. We found highly homologous elements in LS regions of each strain; LS regions of V. dahliae strain JR2 are much richer in highly homologous elements than the core genome. In addition, we discovered, in LS regions of JR2, several structural forms of nonhomologous recombination, and two or three homologous sequence types of each form, with almost each sequence type present in an LS region of another strain. A large section of one of the forms is known to be horizontally transferred between V. dahliae strains. We unexpectedly found that 350 kilobases of dynamic LS regions were much more conserved than the core genome between V. dahliae and a closely related species (V. albo-atrum), suggesting that these LS regions were horizontally transferred recently. Our results support the view that genetic variation in LS regions is generated by horizontal transfer between strains, and by chromosomal reshuffling reported previously.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-324) contains supplementary material, which is available to authorized users.

Expression Profile Analysis and Target Gene Prediction of Three Conserved MicroRNAs in Resistant <i>Gossypium hirsutum</i> Cv. KV-1 Responding to <i>Verticillium dahliae</i>

Plant microRNAs (miRNAs) play important roles in the post-transcriptional regulation of plant growth, development, flowering, metabolism, and responses to stress. Verticillium wilt is a vascular disease in plants caused by the fungal pathogen Verticillium dahliae. In order to find and investigate miRNAs related to the upland cotton variety Zhongzhimian KV-1 resistant Verticillium wilt, deep sequencing technology was used to construct small RNA libraries of two samples, which from seedlings of KV-1 cotton by different pathogenicity strains Verticillium wilt pathogen infections. The V. dahliae strains D07038 and V991 were used in this study and are moderately virulent and virulent, respectively. miRNAs with differential expression among the samples were obtained through analysis of sequencing data and three miRNAs (miR1423a-5p, miR3444a-5p and miR5562) were chosen to be identified by quantitative real-time RT-PCR (qRT-PCR). At the same time, their target genes were predicted. The results of qRT-PCR were consistent, which indicated 3444a-5p and miR5562 were with the highest expression level in virulent condition, but miR1423a-5p was a low-level expression. The results of experiments agreed with deep sequencing data basically. Analysis of the transcript data for target genes of three conserved miRNAs indicated that they play an important role in plant-pathogen interaction mechanism. The identification and characterisation of miRNAs from upland cotton may help to further the study of miRNA regulatory mechanisms that are involved in resistance to Verticillium wilt.

RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae

BackgroundThe soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae.ResultsTo shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.ConclusionCollectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.