The glycoside hydrolase 28 member VdEPG1 is a virulence factor of Verticillium dahliae and interacts with the jasmonic acid pathway-related gene GhOPR9

Glycoside hydrolase (GH) family members act as virulence factors and regulate plant immune responses during pathogen infection. Here, we characterized the GH28 family member endopolygalacturonase VdEPG1 in Verticillium dahliae. VdEPG1 acts as a virulence factor during V. dahliae infection. The expression level of VdEPG1 was greatly increased in V. dahliae inoculated on cotton roots. VdEPG1 suppressed VdNLP1-mediated cell death by modulating pathogenesis-related genes in Nicotiana benthamiana. Knocking out VdEPG1 led to a significant decrease in the pathogenicity of V. dahliae in cotton. The deletion strains were more susceptible to osmotic stress and the ability of V. dahliae to utilize carbon sources was deficient. In addition, the deletion strains lost the ability to penetrate cellophane membrane, with mycelia showing a disordered arrangement on the membrane, and spore development was affected. A jasmonic acid (JA) pathway-related gene, GhOPR9, was identified as interacting with VdEPG1 in the yeast two-hybrid system. The interaction was further confirmed by bimolecular fluorescence complementation and luciferase complementation imaging assays in N. benthamiana leaves. GhOPR9 plays a positive role in the resistance of cotton to V. dahliae by regulating JA biosynthesis. These results indicate that VdEPG1 may be able to regulate host immune responses as a virulence factor through modulating the GhOPR9-mediated JA biosynthesis.     

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Chitin‐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.     

VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae

The vascular wilt fungus Verticillium dahliae infects the roots of cotton plants and can seriously diminish the yield and quality of this and other dicotyledons. However, the key genes involved in V. dahliae infection and pathogenesis in cotton remain unclear. Msb encodes a transmembrane mucin that is highly conserved in the MAPK signal pathway. Msb has been implicated previously in pathogenicity in various aerial plant fungi. In this study, V. dahliae Msb (VdMsb) was found to be required for fungal virulence and microsclerotia production. Strains lacking VdMsb exhibited reduced conidiation and microsclerotia formation. Compared with wild-type and gene-complemented strains, the invasive growth and adhesive capacity of VdMsb deletion mutants were significantly decreased. These results suggest that VdMsb plays a role in development and virulence in V. dahliae.     

Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more and less H₂O₂ than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.     

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.     

VdSho1 Regulates Growth,Oxidant Adaptation and Virulence in Verticillium dahliae

Verticillium dahliae infection leads to Verticillium wilt in cotton and other dicotyledon crops. To reduce the loss of economic crops, more attention has been focused on the key genes involved in pathogenicity of this soil‐borne plant fungal pathogen. Sho1 encodes a conserved tetraspan transmembrane protein which is a key element of the two upstream branches of the HOG‐MAPK pathway in fungi. Sho1 is required for full virulence in a wide variety of pathogenic fungi. In this study, sho1 mutant in V. dahliae (designated ΔVdsho1) was generated by Agrobacterium tumefaciens‐mediated transformation. ΔVdsho1 strain was highly sensitive to menadione (at concentration of 120 μm ) and hydrogen peroxide (at concentration of 250 μm ), displayed delayed spore germination and reduced spore production compared with the wild type and the complemented strains. During infection of host cotton plants, ΔVdsho1 exhibited impaired ability of root attachment and invasive growth. Results from the present work suggest that VdSho1 controls external sensing, virulence and multiple growth‐related traits in V. dahliae and might serve as a potential target for control of Verticillium wilt.     

Verticillium dahliae secreted protein Vd424Y is required for full virulence,targets the nucleus of plant cells,and induces cell death

Fungal pathogens secrete effector proteins that regulate host immunity and can suppress basal defence mechanisms against colonization in plants. Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and reduces plant yields. However, little is currently known about how the effectors secreted by V. dahliae function. In this study, we analysed and identified 34 candidate effectors in the V. dahliae secretome and found that Vd424Y, a glycoside hydrolase family 11 protein, was highly upregulated during the early stages of V. dahliae infection in cotton plants. This protein was located in the nucleus and its deletion compromised the virulence of the fungus. The transient expression of Vd424Y in Nicotiana benthamiana induced BAK1- and SOBIR1-dependent cell death and activated both salicylic acid and jasmonic acid signalling. This enhanced its resistance to the oomycetes Phytophthora capsici in a way that depended on its nuclear localization signal and signal peptides. Our results demonstrate that Vd424Y is an important effector protein targeting the host nucleus to regulate and activate effector-triggered immunity in plants.     

Verticillium dahliae VdBre1 is required for cotton infection by modulating lipid metabolism and secondary metabolites

The soil-borne ascomycete Verticillium dahliae causes wilt disease in more than two hundred dicotyledonous plants including the economically important crop cotton, and results in a severe reduction in cotton fiber yield and quality. During infection, V. dahliae secretes numerous secondary metabolites, which act as toxic factors to promote the infection process. However, the mechanism underlying how V. dahliae secondary metabolites regulate cotton infection remains largely unexplored. In this study, we report that VdBre1, an ubiquitin ligase (E3) enzyme to modify H2B, regulates radial growth and conidia production of V. dahliae. The VdBre1 deletion strains show nonpathogenic symptoms on cotton, and microscopic inspection and penetration assay indicated that penetration ability of the ∆VdBre1 strain was dramatically reduced. RNA-seq revealed that a total of 1643 differentially expressed genes between the ∆VdBre1 strain and the wild type strain V592, among which genes related to lipid metabolism were significantly overrepresented. Remarkably, the volume of lipid droplets in the ∆VdBre1 conidia was shown to be smaller than that of wild-type strains. Further metabolomics analysis revealed that the pathways of lipid metabolism and secondary metabolites, such as steroid biosynthesis and metabolism of terpenoids and polyketides, have dramatically changed in the ∆VdBre1 metabolome. Taken together, these results indicate that VdBre1 plays crucial roles in cotton infection and pathogenecity, by globally regulating lipid metabolism and secondary metabolism of V. dahliae.     

Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii

Cyclic diguanylate (c‐di‐GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c‐di‐GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c‐di‐GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c‐di‐GMP, RpoN and T3SS and the potential role of c‐di‐GMP in T3SS regulation among a wide range of bacterial pathogens.