Functional Analysis Of Tomato Yellow Leaf Curl China Virus AV2 Gene

Tomato yellow leaf curl China virus (TYLCCNV) isolate Y10 is a member of the genus Begomovirus of the family Geminiviridae. Recently, they cause destructive diseases in many crops, such as tomato, tobacco and so on. The genome of TYLCCNV is about 2.7 kb size and a circular single-stranded DNA. which encodes 6 proteins including a 348 bp gene named AV2. In this study, TYLCCNV Y10 AV2 was identified a RNA silencing suppressor.The TYLCCNV Y10 AV2 protein was expressed in E. coli strain BL21 (DE3) using pET-32a vector, and the recombinant fusion protein was purified with Ni²⁺-NTA affinity column. The purified recombinant protein was used as immunogen to immunize rabbits for producing polyclonal antibodies against TYLCCNV Y10 AV2 protein. GFP green fluorescence in the leaves of 16c transgenic N. benthamiana plants co-infiltrated with the A. tumefaciens harboring GFP gene and the A. tumefaciens harboring AV2 gene could be observed and a narrow red ring around the edge of infiltrated patch could also be found at 6 days post inoculation (dpi), which indicated that TYLCCNV Y10 AV2 protein can suppress local RNA silencing of GFP gene, but can not interfere with the spread of RNA silencing signal of GFP gene by cell-to-cell. In addition, GFP green fluorescence could be observed in the systemic leaves of 16c transgenic N. benthamiana plants co-infiltrated with GFP and AV2 genes at 30 dpi, which suggested that TYLCCNV Y10 AV2 protein can inhibit systemic RNA silencing of GFP gene. Moreover, TYLCCNV Y10 AV2 protein could interfere with the spread of systemic RNA silencing signal of GFP gene by Agrobacterium-mediated infiltration assay. The above results indicated TYLCCNV Y10 AV2 protein is a RNA silencing suppressor. Subcellular localization assay indicated that the AV2 protein was distributed in the cytoplasm of N. benthamiana cell, and accumulated in irregular cytoplasmic bodies at about 48 hours post-agroinfiltration by confocal microscopy. The AV2 gene was cloned into pGR106 vector containing an infectious clone of potato virus X (PVX). Veinal chlorosis was visible as early as at 5 dpi in the N. benthamiana infiltrated with PVX-AV2 and symptoms including chlorotic spots, crimp, necrotic mottling and severe necrosis were observed later. This result indicated that TYLCCNV Y10 AV2 protein is a pathogenicity determinant in the PVX heterogenous system. Northern blot analysis indicated that the concentration of PVX RNA was higher in the N. benthamiana infiltrated with PVX-AV2 than that in the N. benthamiana infiltrated with PVX at 5 dpi and 7 dpi. It could be speculated that the higher concentration of PVX in the N. benthamiana infiltrated with PVX-AV2 was due to the AV2 protein suppressing the antivirus RNA silencing of the plants.