Molecular Mechanism Of Papain-Like Cysteine Protease CysP6 In Nicotiana Benthamiana Against Chinese Wheat Mosaic Virus (CWMV)

Chinese wheat mosaic virus disease is a severe threat to the production of Chinese winter wheat(Triticum aestivum),and it is caused by Chinese wheat mosaic virus(CWMV).In the last century,CWMV was firstly discovered and isolated in a wheat field from Shandong Province in China and it was transmitted by P.graminis.CWMV particles were short rod-shaped,and it was identified as a member of the fungal genus Furovirus.CWMV is an RNA virus that infects plants,and its genome contains two single-stranded sense RNAs(+ ss RNA).It is extremely difficult for us to prevent and control CWMV,therefore the mechanisms of interactions between CWMV and plant hosts are poorly understood until now.Artificial full-length CWMV c DNA clones have been constructed which can successfully infect N.benthamiana plants,currently the most widely used experimental model in plant virology.In this study,label-free quantitative proteomics analysis based on nanoliquid chromatography(LC)-mass spectrometry(MS)/MS technique was conducted to analyze the proteomic changes of plants under CWMV infection.A total of 2,751 host proteins were identified,1,496 of which were quantified.390 proteins were characterized as differentially expressed proteins(DEPs).The DEPs were made up of 146 up-regulated and 244 down-regulated proteins.Subsequently,we identified a cysteine protease Cys P6 which obtained from comparative proteomic analysis and found that Nb Cys P6 belongs to the RD21 As subfamily of the cysteine protease(C1)family-papain.By VIGS technology,we found that silencing of Nb Cys P6 gene enhanced CWMV infection.Meanwhile,over-expression of Nb Cys P6 weakened CWMV infection.In order to further explore the mechanism of Nb Cys P6 resistance against CWMV,we performed an RNA-seq analysis of Nb Cys P6 over-expression transgenic tobacco plants.A total 2,206 differentially expressed genes(DEGs),which were made up of 1,629 up-regulated genes and 577 down-regulated genes,were identified.Through analysis,annotation and verification of these DEGs,we found that the expression levels of a variety of defense signal molecule genes were significantly increased.In particular,the expression levels of several ABA pathway-related genes had changed significantly.We speculated that overexpression of Nb Cys P6 in N.benthamiana regulated ABA signaling pathway to prevent CWMV invasion.More than half(55.33%)of DEPs were predicted to be localized in the chloroplast,an organelle indispensable for abscisic acid(ABA)synthesis.Our results suggested that CWMV infection probably interrupts normal chloroplast functions and affects the contents of ABA in N.benthamiana plants.CWMV infection inhibited the expression of ABA1 and ABA2 in N.benthamiana.Further analysis showed that the ABA pathway was suppressed under CWMV infection and exogenous ABA treatment did induce plant hosts defense against CWMV.Based on these results,corresponding ABA pathway genes(Nb ABA1,Nb ABA2 and Nb PP2C16)involved in plant response to CWMV infection were characterized through virus-induced gene silencing.We found that silencing of Nb ABA1 or Nb ABA2,involved in the ABA synthesis pathway,resulted in reduced ABA contents and inhibited the expression of ABA-responsive genes(Nb ABI5 and Nb RAB18)to significantly enhance CWMV accumulation.Type A PP2 Cs are negative regulators of ABA signaling pathways in plants,we found that silencing PP2C16 in N.benthamiana plants had significantly reduced CWMV accumulation.In summary,these results lay a good foundation for the molecular basis of plant hosts resistance against CWMV.