Whole Genome Analysis Of The Pathogenic Function Of The Small Molecular Weight Cysteine-rich Protein Of Verticillium Dahliae

Verticillium dahliae can cause verticillium wilt and to infect hundreds of dicotyledonous plant species,such as cotton,eggplant,potato,which causes heavy agricultural economic losses worldwide.In China,cotton verticillium wilt has been the primary barrier for cotton highly productive.Previous studies illustrated secreted proteins play important roles in the immune response of the host to V.dahliae infection.Therefore,screening and functional identifying pathogenicities secreted proteinsh has been the research focus of mechanisms discovered in V.dahliae.Previous studies during host-pathogen interactions,have found that the secreted proteins of SCPs(small,cysteine-rich protein,Scp)have been proposed to be likely candidates for effector proteins,and play a critical role during host-pathogen interaction.SCPs are potential fungal effectors designated as small(400 amino acids),cysteine-rich(4 cysteine residues)proteins.One obvious and common feature of SCPs,as well as some effectors that maintain structural stability in oxidizing environments,is the presence of multiple cysteine residues and their disulfide bonds.These SCPs is a major virulence factor during soybean infection when plant have no trigger immunity genes.But when plant have proteins genes,SCPs be recognized by plant trigger genes,which activate immunity.Secreted small cysteine-rich proteins(SCPs)play a critical role in modulating host immunity.Bioinformatic analyses showed that V.dahliae encodes more than one hundred VdSCPs,but their roles in host-pathogen interactions have not been fully characterized.Here,large-scale transient expression of 123 VdSCP-encoding genes were carried out in the heterologous system of Nicotiana benthamiana to identify the candidate genes involved in host-pathogen interactions by cell-death inducing activity.Among these,only VdSCP27,VdSCP113,and VdSCP126 displayed celldeath-inducing activity associated with host immune response.The three VdSCPs trigger immunity in N.benthamiana,including a ROS burst,callose deposition,induction of defense genes,and protection against disease development.Sitedirected mutagenesis showed that cysteine residues that form disulfide bonds are essential for the functioning of VdSCP126,but not VdSCP27 and VdSCP113.Investigation of the transcript levels of VdSCP27,VdSCP113 and VdSCP126 revealed that each of these were significantly up-regulated during infection on N.benthamiana and cotton plants,suggesting that these three VdSCPs may play an important role in host-pathogen interactions.To confirm the subcellular location of VdSCP27,VdSCP113 and VdSCP126 during interaction with host plant,coding regions of these VdSCPs were fused with green fluorescent protein(GFP)and transiently expressed in N.benthamiana leaves by agro-infiltration.The results showed that all three VdSCPs were aggregated along the peripheral regions and were nearly undetectable inside of plant cells.Finally,disruption mutant assays established that VdSCP27,VdSCP113,and VdSCP126 individually are not essential for V.dahliae infection in N.benthamiana and cotton plants.We tested whether VdCBM1 has the ability to suppress the immunity triggered by these three VdSCPs.VdSCP27,VdSCP113,and VdSCP126 were each separately expressed with VdCBM1 in N.benthamiana by co-agro-infiltration.The results suggested that cell death triggered by three VdSCPs is effectively suppressed by VdCBM1 in N.benthamiana.Verticillium dahliae employs the effector from VdCBM1 to suppress immunity that is triggered by VdSCP27,VdSCP113,and VdSCP126 to promote colonization.