Resistance to tamarillo mosiac potyvirus: targeting and mechanism

Abstract

The present study continues the research by a previous PhD student, Robin MacDiarmid (formerly Eagles), who engineered resistance and recovery from tamarillo mosaic potyvirus (TaMV) infection by introducing different forms of the TaMV coat protein (CP) gene into Nicotiana benthamiana plants. My results showed that in TaMV-CP transgenic plants, resistance to and recovery from TaMV infection was due to post-transcriptional gene silencing (PTGS). In RAT (recovery at top) -lines (where PTGS was induced by TaMV infection) and resistant lines (where PTGS was already in place), PTGS targeted for degradation TaMV-CP transgenic mRNA, TaMV viral RNA, and TaMV-CP sequences delivered by a WClMV (white clover mosaic virus) -based chimeric viral vector. Differences in the targeted sequences and the efficiency of the targeting were observed between both types of transgenic lines. The targeting of TaMV-CP sequences delivered by a chimeric viral vector was more consistent and stronger in resistant plants than in the RAT tissue of recovered plants. Mapping experiments carried out, in UT-II-R plants showed that the chimeric viruses W-TaMV#1, W-TaMV#2, W-TaMV#3, and W-TaMV#4 accumulated in 44 to 94% of the II-R inoculated leaves. However, the W-TaMV#5 chimeric virus containing the 3’-most fragment of the TaMV-CP gene accumulated, in only 5.5% of the inoculated II-R plants. In the upper leaves, W-TaMV#1 progeny were detected in 78% of the II-R plants, while progeny from W-TaMV#2, W-TaMV#3, and W-TaMV#4 were detected systemically in 17, 17, and 5.5% of II-R plants, respectively. Differences of targeting in inoculated and upper leaves suggested that the efficacy of PTGS might be stronger in the vascular tissue of plants. Overall, the targeted sequence was mapped to a 134-nucleotide 3’-region, but other regions were also targeted by PTGS. Several WClMV-based viral vectors were built to deliver foreign sequences/genes into mechanically inoculated plants. Efficient delivery was achieved with chimeric constructs (7K*, W-TaMVfull, W-TaMV #1-#6), which contained foreign sequences but did not express foreign proteins. However, constructs designed to express reporter genes in plants (W-XGUS [beta-D-glucuronidase gene from Escherichia coli], W-GFPo/c, and W2X-GFP [green fluorescent protein]) were unable to move efficiently throughout the plant. These viral constructs, efficiently expressed the reporter genes in the inoculated leaves. However, they either were unable to move systemically (W-GFPo/c) or showed a very high degree of instability of the insert (w-XGUS and W2X-GFP). Data in two areas showed unexpected results. Firstly, plants from one transgenic line, UT-IV4, showed, different phenotypes when inoculated with TaMV and B11, two very similar strains of PVA. TaMV and B11 infection of plants from all other transgenic lines tested induced similar PTGS phenotypes. Secondly, TaMV-CP RNA sequences that were targeted for degradation when delivered in a WClMV-based viral vector were not targeted for degradation when delivered by particle bombardment. These results suggest that high sequence identity is not always enough for efficient PTGS.