Effect Of TYLCCNB βC1Phosphorylation On Its Suppression Of Transcriptional Gene Silencing

Tomato yellow leaf curl China virus (TYLCCNV) belongs to monopartite geminivirus, and its associated betasatellite DNA (Tomato yellow leaf curl China Betasatellite, TYLCCNB) encodes a βPC1protein which functions as a pathogenicity factor and suppressor of RNA silencing to suppress methylation-directed transcriptional gene silencing (TGS). To defend geminivirus infection, a tomato S1SnRK1[Snf (sucrose non-fermenting)-l-related protein kinasesl] interacted with and phosphorylated βto attenuate the symptoms induced by TYLCCNV. We predict that the functions of βC1may be affected once it is phosphorylated, but the underlying mechanism has not been elucidated. Therefore, the effect of βphosphorylation on its ability to reverse TGS was studied in this article.Previous study indicated that individual or simultaneous substitution of the Ser-33or Thr-78of βC1by aspartate residues reduced the efficiency of virus infection and induced milder symptoms; while substitution of these sites of βC1by alanine, resembled wild type βC1in efficiency of virus infection or symptom severity.To understand whether the phosphorylation of βC1can affect the expression of βC1protein, PVX vectors carrying six βC1point mutants were used to inoculate Nicotiana benthamiana plants. Western blot assay using monoclonal antibody raised against βC1indicated that no significant differences of protein expression level were observed between wild type βC1and its mutants. Subsequentely, the relevant PVX vectors mentioned above were used to inoculate N. benthamiana plants containing a transcriptionally-silenced GFP transgene (line16-TGS). We found that pGR106-βC1S33A、pGR106-βC1T78A and pGR106-βC1S33A/T78A can reverse green fluorescent protein (GFP) as wild type pGR106-βC1observed under UV light, while pGR106-βC1S33D、PGR106-βC1T78D and pGR106-βC1S33D/T78D can not reverse GFP. These reults showed that the constitutive phosphorylation mutants of βC1can not reverse TGS.Chop-PCR assay indicated that infectious clones of βC1phosphorylation mutants, TYLCCNBS33A, TYLCCNBT78A and TYLCCNBS33A/T78A, can reduce the methylation level of TYLCCNV geome as wild type TYLCCNB. However, TYLCCNBS33D, TYLCCNBT78D and TYLCCNBS33D T78D can not reduce the methylation level of TYLCCNV geome, indicating that the constitutive phosphorylation mutants of βC1can not reduce the methylation level of TYLCCNV geome.The above results showed that the constitutive phosphorylation mutants of βC1lose the ability of reversing methylation-mediated transcriptional gene silencing by attenuating the ability reducing the geome methylation of TYLCCNV.