Characterization of cDNAs from Nicotiana benthamiana that encode proteins which interact with tomato golden mosaic virus AL2 protein in the yeast two-hybrid system

Abstract

The AL2 protein of tomato golden mosaic virus (TGMV) is multifunctional. It is required for derepression of the TGMV AR1 gene in phloem tissue, and for trans-activating the AR1 and BR1 genes in mesophyll cells. It also enhances virus susceptibility when expressed in transgenic plants. It is thought that TGMV AL2 protein accomplishes these functions by interactions with unknown host factors. In this study, cDNAs from Nicotiana benthamiana plants that encode proteins which interact with TGMV AL2 were characterized. A yeast two-hybrid assay identified two cDNA clones, Nb#51 and Nb#62, that specifically interacted with TGMV AL2, but not with negative control proteins. Sequences of these two cDNA clones were determined by primer walking, which revealed that Nb#51 appears to be a 3'-coterminal truncated version of Nb#62. Inspection of the amino acid sequences encoded by Nb#62 found the presence of both ankyrin-repeats and tetratricopeptide repeats (TPR). Deletion analysis showed that the TPR motif, together with its flanking regions, was sufficient to confer on Nb#62 the ability to interact with TGMV AL2, whereas the ankyrin-repeats were not required for this interaction. Nb#62-specific mRNAs were detected in N. benthamiana plants by northern hybridization in potato virus X (PVX) infection experiments, but not in heat-shock experiments. Virus-induced gene silencing (VIGS) assays were used to investigate the possible function(s) of the Nb#62-encoded protein in normal plants and in the context of a TGMV infection. When PVX carrying a 618-bp fragment from the 5'-end of the Nb#62 cDNA was used as a silencing trigger in N. benthamiana plants, VIGS effectively targeted the transcripts which contained sequence similarity with the trigger fragment. However, the infected plants didn't have difference in the phenotype when compared to the PVX vector infection. When TGMV carrying a 93-bp fragment from the 5'-end of the Nb#62 cDNA infected plants, viral DNA accumulation in the upper leaves was reduced, when compared to wild-type TGMV or a control TGMV construct containing a 95-bp fragment from the tobacco Sulfur gene.