Molecular Identification Of Tomato Chlorosis Virus Of Four Provinces Isolates And Screening Of Host Proteins Interacting With P22

Tomato chlorosis virus(ToCV)is an newly emerging virus in China recently.In 2013,it was first discovered and reported in greenhouses in Beijing area.Subsequently,ToCV spread rapidly and occurred in 13 provinces in China because of its wide host range and unconrolled transmitting vector.Plants infected ToCV have weaker growth,smaller firuts with decreased commercial value than that of healthy plants,posing huge financial loss on tomato production industry.In order to detect ToCV and take control of measures in time in Shanxi,Inner Mongolia,Liaoning and Jilin provinces,molecular biological method was applied to detect and identify ToCV in tomato samples in this study.Totally four positive samples were detected in 123 collected from the four provinces,and this is the first time that ToCV was detected and reported in the above four provinces.The similarity and phylogenetic analysis of nucleotide and amino acid sequences of CP and Hsp70 h showed that the four isolates shared higher nucleotide similarity and closer relationship with other isolates of China.Due to the low dection rates,we speculated that ToCV in these four provinces was in the initial stage of disease development and had not caused significnt loss yet.However,more attention should be paid on this disease and be warned of epidemic and outbreaks of ToCV because of its rapid spread speed and wide infection range.Many studies about ToCV were limited to fundamental researches including diagnosis and identification,optimization of detection methods and evolution analysis.There are very few researches about interaction mechanisms between host plants and ToCV.To partially dissect the pathogenictiy of ToCV,in this study,the RNA silencing suppressor p22 encoded by ToCV RNA1 was used as a bait protein to screen interacting proteins form a tomato cDNA library by yeast two hybrid.In total,we obtained 19 candidate proteins form 193 clones.Among these proteins,as S-phase kinase-associated protein 1(SKP1)was obtained in many clones,and the interacting region coverd the whole open reading frame and shared 100% amino acid similarity,it was selected for further confirmation.Finally,the interaction between p22 and NbSKP1.1 was confirmed by assays of yeast two hybrid and bimolecular fluroscence complementation,but NbSKP1.2,NbSKP1.3 and NbSKP1L1 cannot interact with p22.To find the key interacting domain between p22 and NbSKP1.1 and to further investigate the influence of interaction on the pathogenicity of virus and effects of physiological metabolism of host plants,further anaysis on structure of SKP1 were conducted.The results showed it has 155 amino acids and can be divided into N-terminal domain(aa 1-98)and C-terminal domain(aa 99-155).The N-terminal and C-terminal domains were verified with p22 by yeast two hybrid respectively.The result showed that p22 interacted specifically with C-terminal domain rather than N-terminal domain of NbSKP1.1.