Characterization Of Begomovirues DNA-A Component Associated With Of TYLCD Occurred In 2013 And Screening Of Tomato Factors Interacted With C4 Encoded By TYLCV

Tomato yellow leaf curl disease (TYLCD), which is a serious tomato virus disease has causes devastating losses to tomato production in China. In Jiangsu province, TYLCD caused by TYLCV, with typical symptoms of leaf curling, and yellowing, was first reported in 2007.In 2013, a new TYLCD was found in Nanjing, Jiangsu province and the symptoms were quite different from previous reports of TYLCD caused by tomato yellow leaf curl virus (TYLCV) in Jiangsu. The infected plants typically exhibited leaf crumpling, vein swelling and enations on the undersides of leaves, and a whitefly-transmitted geminivirus was detected in the samples collected from the infected plants. So the new TYLCD might be associated with a new begomovirus. The DNA-A component of the associated begomovirus was cloned and sequenced and the results showed that the virus isolates formed a circular ssDNA and contained 2,781 nts, which contained six potential ORFs encoding V1, V2, C1, C2, C3 and C4. BLAST and phylogenetic analysis showed that the virus was an isolate of TYLCV, which shared high (>99%) sequence identities with TYLCV isolates previously reported in Jiangsu and no obvious variation/recombination sites were found between them. But the symptoms of the disease caused by TYLCV tomato yellow leaf different from that previous reported, so we wonder there are other components of the virus.In order to study the function of C4 encoded by TYLCV during the process of TYLCV infection and pathogenicity, C4 gene was cloned and yeast two hybrid vector pGBK-C4 was constructed. A tomato cDNA libraries was screened by yeast two hybrid system using C4 as a bait.541 candidate colonies were idetified and 210 colonies were finally sequenced. Based on the public tomato genome in GenBank database, the sequences were annotated, revealing that there might correspond to 10 kind of proteins of tomato.There were two kind of heat shock protein, two kind of ribosomal protein, two kind of rubisco small subunit, a kind of catalase, a kind of metallothionein, a kind of biotin carboxyl carrier protein of acetyl CoA carboxylase and a F-box protein.Based on the results of yeast two hybrid screen using C4 as a bait, a tomato chloroplast protein, rubisco small subunit (slRbCS), was chosen to study the interaction with C4 using yeast two hybrid. slRbCS was cloned and sequenced and sequence analysis results showed that there was a signal peptide in N-terminal of slRbCS, so pGAD-slRbCS and pGAD-ΔslRbCS (without peptide) were constructed respectively. The two constructs were transformed AH 109 competent cells together with the pGBK-C4 and selected on the defect type of medium. The results confirmed that slRbCS and ΔslRbCS both interacted with C4. slRbCS is the key protein in the process of CO2 fixation in chloroplast. There is no evidence to confirm that it is involved in the synthesis of chlorophyll directly, but silencing of RbCS on some plants including tomato can cause development of yellow symptoms on plant. Therefore, the interaction of C4 and slRbCS might involve in the development of yellow symptoms on tomato after TYLCV infection.