Cloning And Preliminary Function Analysis Of HSP20 Gene

Small heat shock proteins (sHSPs) exist widely in all kinds of creatures and play an important role in both development and stress responses. Generally, sHSPs function as molecular chaperones, protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. In this study, genes encoding sHSP (OsHSP20、NbHSP20) was cloned from Oryza sativa and Nicotiana benthamiana and sequence analysis showed that such kind of sHSPs were conserved in different plants and could be divided into the cytosolic class Ⅰ (CI). OsHSP20 was sub-cloned into a prokaryotic expression vector and transformed into Eschrichia coli for protein expression and purification. The expression patterns of OsHSP20 and its homologue (NbHSP20) were shown to be affected by heat shock and viral infection with real-time quantitative RT-PCR. In yeast two hybrid (YTH) assays, the homo-and heterologous interactions between the two sHSPs were investigated and their conserved α-crystallin domains (ACDs) were crucial for their interactions. Interestingly, both OsHSP20 and NbHSP20 were found to be localized in the cytoplasm and form numerous moving granules of various sizes in vivo when expressed alone, however, the subcellular localization and distribution patterns of both sHSPs were affected by rice stripe virus (RSV) infection, suggesting that there might be interaction between HSP20s and viral protein.To investigate the interaction between the HSP20s and viral proteins, both sHSPs were used as bait for screening a prey library of RSV cDNA in YTH assays and a strong interaction between HSP20s and the viral RNA-dependent RNA polymerase (RdRp). The interaction was further confirmed by pull-down and BiFC assays. A series of mutation assays revealed that both N-termini of RdRp and HSP20s were crucial for their interaction and only N-terminus (residues 1-296) of the RdRp was responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs. Taken together, these findings could contribute to understanding the function of sHSPs and provide new insight into the interactions between RSV and its host plant.