Secretome Analysis And Pathogenicity Study Of Verticillium Dahliae In Cotton-extract Induction System

To study the growth, generation of conidium and pathogenicity for secretome of V. dahliae andwhcich are influenced with natural sources, this fungus was cultured in the presence of cotton tissueextracts to mimic in vivo cotton infection system. The results showed that V. dahliae growth wassignificantly favored in the presence of host extracts, exhibiting the production of more spores andlarger amount of secretome compared with basic medium cultivation. Especially, the secretome inducedby cotton tissue caused more serious wilt phenotype in cotton leaves. To further identify theup-regulated secreted proteins induced by cotton tissue extract, we conducted an itraq-labeledcomparative proteomics of V. dahliae secretomes between the cotton tissue induction or non-inductionsamples. In total,502differentially expressed proteins were identified (Fold change>1.5or <0.6,p-value<0.05). Of which, a large number of potential pathogenicity factors were identified, including221secreted proteins,107plant cell wall degradation enzyme (PCWDE),86pathogen-host interaction(PHI) candidates,17small cysteine-rich proteins,2necrosis and ethylene-inducing-like proteins (NLPs)and a LysM effector candidate. Meanwhlie, we found that86secreted proteins were strongly induced(fold change>5.0, p-value<0.05), and76of which were PCWDEs which are suggested to involve inplant cell wall degradation and contribute to virulence in many fungal pathogens. To verify the inducedproteins exactly involved in cotton-V. dahliae interaction, we randomly selected some PCWDE genesand examined their transcripts by real-time quantitative PCR. The results revealed that most of selectedPCWDEs were activated in V. dahliae-cotton interaction, especially at the early of infection process. Inaddition, two of pectin lyses (PL) genes (VdPL3.1and VdPL3.3) belonging to PL3subfamily (there are11PL3genes in the V. dahliae genome) were further selected for knock-out assays. The resultsindicated that deletion of VdPL3.1, which separated from other PL3proteins in phylogenetically, causedover50%virulence reduction. However, loss-of-function of VdPL3.3did not affect virulence. Thatwould be caused by functional redundance among VdPL3.3and other PL3proteins in the samephylogenetic clade. This study provides a comprehensive resource for pathogenicity factor discovery inV. dahliae, and the highly induced PCWDEs are suggested to play significant roles in pathogenicity atthe early of cotton infection process. Further functional characterization of these PCWDEs may greatlyenhance the research on molecular mechanisms of pathogenicity in V. dahliae.