Identification Of Subgenomic Rna From Chinese Wheat Mosaic Virus RNA2 And Functional Analysis Of Its Encoded CRP Protein

Chinese wheat mosaic virus(CWMV)is one of the important pathogens causing wheat mosaic disease,which often result in decrease by 10-30% in wheat production.CWMV is transmitted by Polymyxa graminis and belongs to the genus Furovirus in the family Virgaviridae;its genome contains two positive single-stranded RNAs,encoding seven viral proteins,including replicase,movement protein(MP),coat protein(CP),cysteine-rich protein(CRP),et cetera.Among them,the CRP protein has been considered to be a weaker silencing suppressor,however,further functional analysis has not yet reported.Thus,in this study,we identified its expression and functional activity in its host plant by using methods,including Northern-blot,5’-RACE and genetic transformation.The CRP coding region was first used as probe in Northern-blot assay,and a subgenomic RNA(sgRNA)encoding CRP protein together with the full-length RNA2 was found in CWMV-infected plants.The transcription start site of the sgRNA was localized at nt2533 in 5’-Rapid Amplification of cDNA End(5’-RACE)experiments.Then the CRP protein was successfully expressed in prokaryotic expression system and purified for preparation of its specific antibodies;Western-blot analysis showed that the CRP protein expressed at high level in CWMV-infected plants.In order to further analyze its function,the CRP gene was introduced into Nicotiana benthamianae by agrobacterium-mediated genetic transformation method,and five transgenic lines were identified to overexpress CRP.Phenotype observation showed that these transgenic plants were obviously yellowing and stunting.Measurements of chlorophyll content and photosynthetic rate showed that both chlorophyll synthesis and photosynthesis were significantly inhibited in transgenic plants overexpressing CRP protein.Then total RNAs and proteins were respectively extracted for transcriptome and proteome analysis from transgenic plants,the GO/ KEGG function enrichment analysis consistently indicated that the photosynthetic pathway was significantly affected in transgenic plants overexpressing CRP protein.Further qPCR analysis also confirmed that the overexpression of CRP protein could inhibit photosynthesis pathway in transgenic plants.Taken together,the above results would be helpful for understanding the gene expression regulation strategies of CWMV and lay a foundation for elucidating the pathogenic mechanism of CWMV.