Construction Of Yeast System For BaMMV Virus Protein And Mechanism Of WRKY55 Gene Involved In BaMMV Infection In Barley

Barley is the fourth-largest cereal crop in terms of cultivated area and total yield in the world.It has a long history of planting in China.Barley is mainly used as livestock feed,food processing raw materials and brewing beer and planted around the Yangtze River in China.Barley yellow mosaic disease is a soil-borne virus disease,caused by Barley yellow mosaic virus(BaYMV)and Barley mild mosaic virus(BaMMV),that inhabit in the spores of fungus Polymyxa graminis for long even ten years.Barley yellow mosaic disease has seriously affected the production of barley.The traditional chemical methods to prevent and treat the disease has failed,breeding for disease-resistant varieties of barley is a promising strategy to prevent the disease.In this project,there are two parts of work.Firstly,we amplified 13 coding genes of BaMMV,including P3?7K1?C1?7K2?VPg?NIa?NIb?CP?NIa-VPg?7K2-NIa-VPg?7K2-VPg in RNA1 and P1 or P2 in RNA2 chain,then were construced to the yeast two-hybrid system,and there are no direct physical interaction between virus structural proteins and either of susceptibility factors eIF4 E and PDIL5-1.Secondly,by uncovering the transcriptome sequencing(RNA-seq),we identified a differentially expressed gene HORVU1Hr1G069640(WRKY55)that was significantly up-regulated after infection with BaMMV.The fluorescence quantitative qPCR showed that the expression level of WRKY55 in susceptible barley was significantly higher than that of resistant varieties,indicating that WRKY55 played a vital role in the process of barley yellow mosaic disease ocurring.To further investigate the mechanism of WRKY55 in virus infection of barley,WRKY55 was cloned from a barley cultivar Morex.This gene contains an 870 bp of the coding sequence that encodes a conserved WRKY domain between nucleotides 415'-594'.Phylogenetic analysis revealed an independent branch,differed to the possible homolog genes from relative species.This gene was highly expressed in roots and young tissues,but rarely expressed in remained tissues.By Agrobacterium-mediated transformation into Nicotiana benthamiana,the WRKY55-GFP fusion protein was detected throughout the cell,including nucleus,cytosol and plasma membrane,which was different from the nucleus location of the general transcription factor,indicating that WRKY55 have a new function.By transforming WRKY55 into yeast,no transcription activity was detected and no physical interaction between WRKY55 and either of susceptibility factors eIF4 E and PDIL5-1 or virus structural proteins were observed.Therefore,this work identified a BaMMV-infected up-regulated gene WRKY55,which was possibly involved in infection of BaMMV,despite that the functional mechanism of action remained to be further illustrated.WRKY55 involved in the process of virus infection pointed out that the direction for exploring the disease resistance mechanism of barley,and provided the guidance for breeding barley resistant varieties in the future.