| Summary: | 碩士 === 國立中興大學 === 植物病理學系所 === 101 === Papaya ringspot virus (PRSV) is the major limiting factor for papaya production worldwide. Current strategy for control relies on generation of coat protein (CP)-transgenic papaya lines through the mechanism of post-transcriptional gene silencing (PTGS). However, during field trials, we encountered a highly virulent strain of PRSV, designated PRSV 5-19, which can overcome the resistance of PRSV YK (a common mosaic strain of Taiwan) CP-3''UTR transgenic papaya lines. The CP gene and the contiguous 3''UTR of the super-strain PRSV 5-19 share high levels of homology with those of PRSV YK, suggesting that PRSV YK CP-transgenic resistance is overcome in a sequence homology-independent manner. In this study, we investigated the possible roles of PRSV 5-19 P1 and HC-Pro responsible for the homology-independent breakdown of the transgenic resistance. The antiserum to the 5-19 HC-Pro protein purified from leaf tissue of infected Cucumis metuliferus plants and P1 protein expressed from bacteria were used to prepare antisera from rabbits for detecting the corresponding antigens. The optimal reactions of HC-Pro antiserum are recommended at 1/5000 dilution, and P1 antiserum at 1/2000 dilution. The involvement and relative strength of P1 and HC-Pro proteins of 5-19 and YK in PTGS suppression were analyzed by their transient expression along with a GFP expresser and a hairpin silencing inducer, by agro-infiltration into leaves of Nicotiana benthamiana plants. Our results indicated that (i) P1 protein was not functional for gene silencing suppression; (ii) in cis or in trans P1 protein reduce the silencing suppression activity of HC-Pro; and (iii) gene silencing suppressor activity of PRSV 5-19 HC-Pro is stronger than that of PRSV YK. The stability analysis of PRSV 5-19 P1 showed that P1 can not be detected by western blotting. When in the presence of HC-Pro in cis, the mRNA of P1 accumulated in higher a levels, and the accumulation of P1 siRNA was lower, suggesting that P1 was not stable, both at protein and mRNA levels. In addition, we found other new PRSV super virulent isolates from transgenic papaya test field. These isolates all broke down the resistance of transgenic papaya after inoculation. Sequencing analysis of their HC-Pros indicated that few amino acid changes in the central region of HC-Pro may enhance the PTGS suppression ability of HC-Pro. Here, we have illustrated the molecular mechanism for the emergence of super virulent virus stain that contain a stronger silencing suppressor, which is selected out by CP-transgenic resistance in a sequence homology independent manner under field conditions. These super virulent virus strains represent a severe threat to CP-transgenic crops. When they are extensively cultivated, they may cause severe damage to other non-transgenic crops.
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