Functional Analysis Of CFEM Domain-Containing Small Secreted Cysteine-Rich Proteins In Verticillium Dahliae

Vertivillium dahliae is a soil-borne vascular fungus that can invade more than 600 dicotyledonous plants species,over 180 species palnts are the important agricultural crops,which casuses enormous economic loss to agricultural production.V.dahliae has the characteristics of broad host range,abundant genetic variation,various sources of primary infection,complex pathogenic mechanism and strong survivability during evolution,which make the situation very difficult to control once the Vertivillium wilt occurs.Small secreted cysteine-rich proteins?SCRPs?are a common class of effectors that have ability to interact with the host to manipulate the host immune responses.V.dahliae genome encodes 127hypothetical SCRPs by predictive analytics,only a few SCRPs function,however,has been reported.Therefore we would analyze the function of SCRPs family and there is currently a lack of a systematic study on the function of this family of SCRPs.We would analyze the function of 127 SCRPs encoded by Vd991 genome during the interaction of V.dahliae-host systematically.Bioinformatics analysis shows 127 hypothetical proteins encoding SCRPs were predicted as effectors among which four SCRPs containing four cysteine residues were excluded from the set of predicted SCRPs due to the presence of one cysteine residue within the signal peptide,and resulted in mature proteins with only three cysteine residues.123 SCRPs genes distribute on different chromosomes,most SCRPs are between 100 and 300 amino acids and contain 2%-4%cysteine residues.The SCRPs are divided into 7 classes basing on the the distribution pattern of cysteine residues,and nine SCRPs have similar distribution patterns of cysteine residues described as CFEM domain.The results of agro-mediated transient expression on Nicotiana benthamiana leaves showed that three SCRPs could induce cell death and nine could inhibit cell necrosis induced by VdEG1.The nine suppression acitivity SCRPs showed different inhibitory effects on eight cell-death proteins?VdEG1,VdEG3,NEP1,NEP2,CUT11,SCR27,SCR113,SCR126?.Together,these results indicated that the SCRPs family during the infection on the host plant.SCR76 and SCR77 from suppressive SCRPs could inhibit the eight cell-death proteins from V.dahliae effectively and the corresponding encoding genes are coupled at genomic locations which having the CFEM domain.The analysis for the broad-spectrum inhibition of the CFEM SCRPs family proteins showed that the suppression activity of SCR76 and SCR77 were the best,following was SCR99,with only partial inhibition function was SCR43,SCR72,SCR116 and SCR120,and almost no inhibition activity was SCR33 and SCR41.To sum up,these results showed that the SCRPs involved CFEM domains exist functional differentiation in manipulating host immunity.In order to clarify the suppression function of SCRPs family,we select the classical effectors SCR76and SCR77 for the deep research.The signal peptides of SCR76 and SCR77 had the secretory activity by yeast signal trap system and were the necessary for protein inhibition activity.The analysis of subcellular location on Nicotiana benthamiana and onion epidermal cells showed that SCR76 and SCR77 located mainly on the plant cell membrane.To investigate the key domains or sites related in suppression function,genes involved sequence truncation of SCR76 and SCR77 were expressed transiently with VdEG1.The results showed that the suppression activity depended on the integrity of protein structure,and the suppression function of SCR76^N43D,SCR77^N47D,SCR76^?GPI and SCR76^N208A weakened relation to the wild-type genes.The PTI responses of ROS accumulation,electrolyte leakage and resistance-related genes expression induced by VdEG1 were suppressed completely by SCR76 and SCR77,the functions of site-mutagenesis genes were weakened or lost,which indicated that the several sites were important to the suppression function of SCR76 and SCR77.The transient and stable expression of SCR76 and SCR77in N.benthamiana accelerate the infection of Botrytis cinerea.Together,these results demonstrate that SCR76 and SCR77 could inhibit plants resiatance and promote pathogen infection.qPCR analysis showed that the transcripts of SCR33?SCR72?SCR76?SCR77?SCR99?SCR116?SCR120 were significantly increased during cotton and N.benthamiana infection,SCR41 and SCR43,however,had no significant expression during cotton infection,which indicated that the expression of different genes in the same family were induced in different levels to do with the different hosts.The pathogenicity of knockout mutants?SCR76,?SCR77 and?SCR99 decreased on cotton and N.benthamiana,and?SCR120 was also decreased during N.benthamiana infection.Above results showed the virulence function of the SCRPs family genes involved CFEM domain is differentiated in the process of evolution.The complement strains of wild-type genes SCR76 and SCR77 could restore virulence on cotton,but the pathogenicity of complement transformants related with sites-muntanted genes SCR76^N43D,SCR77^N47D,SCR76^?GPI do not get well.These results confirmed that the asparagine residue between the third and fourth cysteine residues in CFEM domain of SCR76 and SCR77,and the GPI site in SCR76 are required for virulence of V.dahliae on cotton.In this study,SCRPs family proteins manipulate immunity by inducing or inhibiting host immunity reponses during V.dahliae infection.Among the family,SCR76 and SCR77 have a broad-spectrum suppression activity and act as the virulence factors that inhibit host immunity responses and promote pathogen infection.