Screening Of The Host Factors Interacting With CI Of Sugarcane Mosaic Virus By Yeast Two-Hybrid System

Sugarcane(Saccharum spp.hybrid)is a widely planted economic crop.It is not only the main raw material for sugar production in China,but also an important raw material for light industry,chemical industry and energy.About 90%of sugar is produced from sugarcane in China.Sugarcane production play an important role for the national economy development in China.Sugarcane mosaic disease(SMD)seriously harms sugarcane production and is one of the most serious virus diseases.It has a significant impact on the yield and quality of sugarcane.There are three major pathogens of sugarcane mosaic disease:Sugarcane mosaic virus(SCMV),Sorghum mosaic virus(SrMV)and Sugarcane streak mosaic virus(SCSMV).All three viruses belong to the family Potyviridae.Among them,SCMV and SrMV belong to the genus Potyvirus,while SCSMV belongs to the genus Poacevirus.At present,most studies focus on the detection and evolution of these pathogens.In-depth reports on the pathogenic mechanism of the virus are rare.Among them,the mechanism of SCMV infecting sugarcane host molecules has been the focus of the past two years.In this study,the SCMV-FZ-C1 strain CI protein was used as bait to screen the cDNA yeast library constructed from the SCMV infected sugarcane leaves.Three host genes were obtained:ferredoxinl(Fd1),Chloroplast translational elongation factor Tu(EF-Tu),and plastocyanin(Pc).They were designated as ScFd1,ScEF-Tu,and ScPc,respectively.ScFd1 was selected to be explore its role in the process of SCMV infecting sugarcane.The results of the study were as follows:1.The CI gene of SCMV-FZ-C1 strain was cloned,and the yeast twohybrid bait vector pGBKT7-SCMV-CI was successfully constructed using In-Fusion technology.Self-activation and toxicity were tested for pGBKT7-SCMV-CI.The results showed that the bait vector pGBKT7SCMV-CI has no self-activation and toxicity for Y2Hgold yeast and is suitable for the screening of yeast cDNA libraries constructed from sugarcane leaves infected by SCMV.Using SCMV-CI as bait,the yeast two-hybrid system(Y2HS)was used to screen the cDNA library.Three positive clones were obtained.Based on the bioinformatics analysis,three proteins were predicted to be involved in transcriptional regulation of plant chloroplast,stress defense responses,and photosynthesis activities,respectively.The ORFs of these three genes were cloned and subcellular localization experiments showed that all these three proteins were localized on the chloroplasts.Their interactions with CI were complemented by yeast two-hybrid(Y2H)and Bimolecular fluorescence complementation(BiFC).Only ScFd1 directly interacted with the bait protein SCMV-CI.2.The tissue-specific expression profile of the ScFd1 showed that ScFd1 was expressed in the root,the first internode,the fourth internode,the eighth inthernode,leaf roll,the first fully developed leaf and the fourth leaf of sugarcane plant.However,almost no expression of ScFd1 was found in the roots.The expression was abundant in leaves and the highest expression in the leaf roll.Under the infection of SCMV,the expression of ScFd1 was down-regulated at 0-6 h,and then returned to normal level with time going.NbFd1 was virus-induced gene silencing(VIGS)on the model plant Nicotiana benthamiana.The NbFd1 silenced mutated plants showed severely chlorotic leaves and impaired plant growth.We speculated that ScFd1 not only participated in the process of SCMV infecting sugarcane,but also had a certain correlation with the symptoms of sugarcane mosaic disease.