Functional Analysis Of Proteins Encoded By RNA2 Of Wheat Yellow Mosaic Virus

A yellow mosaic disease caused by soil-borne viruses is becoming increasingly important, particularly in winter wheat in China. The field surveys conducted from 2008 to 2010 in China indentified that the major pathogen agent was Wheat yellow mosaic virus (WYMV). WYMV belongs to the genus Bymovirus of family Potyviridae. Its genome has two positive sense ssRNAs in which each RNA encodes a single polyprotein that is post translationally cleaved into functional proteins. RNA1 encodes P3,7K, CI,14K, NIa-Vpg, NIa-Pro, NIb and CP, while RNA2 encodes P1 and P2. P1 is similar to HC-Pro, which is encoded by viruses of the genus Potyvirus of the same family Potyviridae and is known to play crucial roles in virus replication, transmission, movement, RNA silencing suppression and viral pathogenicity. As there are currently no effective cultural practices or environmental friendly chemicals for disease control, growing resistant cultivars is the only an available practical control. Although the identification of host plant resistance to WYMV is an important objective in breeding programs, the molecular mechanism by which WYMV induces disease in wheat plants has not been studied yet. In this study, the possible roles of P1 and P2 in WYMV life cycle were investigated using several methods.An immunoassay using PI antiserum indicated that P1 does not associate with the virus particle. Agrobacterium coinfiltration assays in Nicotiana benthamiana was carried out to analyze silencing suppressor activity of proteins encoded by WYMV RNA2. HC-pro showed a strong silencing suppression activity, whereas PI and P2 failed to suppress RNA silencing. To determine the effect of P1 and P2 on viral pathogenicity, P1 and P2 were cloned into PVX vector and inoculated into N. benthamiana. P1 or P2, or P1 plus P2 only had little effect on PVX symptom expressions, whereas HC-pro strongly enhanced the severity of PVX symptom. Bimolecular Fluorescence Complementation (BiFC) assay in the plant and insect cells showed that P1 interacts with its self. Next, BiFC assay was used to screen the PI interaction with other protein encoded by WYMV. The assay revealed that P1 specifically interacts with VPg. Moreover, the analysis sub-cellular localization of P1 and VPg when they were either expressed alone or together suggests that P1 alters VPg sub-cellular distribution. In summary, this study indicates that P1 does not posses the functional characteristics of potyviral HC-Pro, but instead P1 may function to facilitate VPg activity through regulating VPg sub-cellular distribution.