Generation of useful mild virus strains by modification of pathogenicity factor HC-Pro of Papaya ringspot virus for solving the problem of strain-specific cross protection

• Main Authors: Tzu-Tung Lin, 林子彤
• Other Authors: 葉錫東
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/04742009725236488005

碩士 === 國立中興大學 === 植物病理學系所 === 104 === Aphid-borne Papaya ringspot virus seriously limits papaya production in tropical and subtropical areas. A nitrous-acid induced mild strain HA 5-1, derived from Hawaii severe strain HA, has been widely used in Taiwan and Hawaii for control of PRSV by cross protection since 1985. However, cross protection is highly strain- specific, thus rendered this control measure drawn back in Taiwan and difficult to be applied in different geographic regions. In this study, through the manipulation of the in vivo infectious clone of a Taiwan dominant severe strain PRSV YK, four conserved residues of essential motifs of HC-Pro gene responsible for potyviral pathogenicity, including the N-terminal FWKG , highly conserved FRNK and two essential motifs at the central and C-terminal regions, were modified by site directed mutagenesis, singly or in combinations, to generate possible attenuated mutants. Seven mutants were infectious on papaya plants, and among them single mutants of R181I, F206L and D397N induced attenuated symptoms but still prominent on infected papaya and horn melon plants. The other single mutant F7I, and double mutants F7I+R181I, F7I+F206L and F7I+D397N displayed mild symptoms followed with recovery in inoculated plants of papaya and did not display conspicuous symptoms on systemic leaves of horn melon plants. Accumulation of the mutants F7I and F7I+F206L in papaya and horn melon plants showed a typical zigzag trend, with an crease in the initial stage similar to the wild-type following by a sharp drop in titer and an low level equilibrium thereafter, indicating that they can trigge RNA silencing from the host and are able to maintain effective antagonistic suppession against the host defesens. Whereas, the titers of the mutants F7I+R181I and F7I+D397N were at low levels without a zigzag pattern. Transient analysis of individual mutated HC-Pros by agroinfiltration in N. benthamiana plants revealed that F7I and F7I+F206L mutated HC-Pros have lower RNA silencing suppression capabilities, 35% and 55% as that of wild-type HC-Pro, respectively, whereas the mutated F7I+R181I and F7I+D397N HC-Pros did not have the silencing ability. Our results of northern blotting indicated that the GFP mRNA expression levels in F7I and F7I+F206L infiltrated tissue were lower than that of the wild-type infiltrated tissue , but GFP siRNA accumulation level are higher than that of the wild-type. Cross protection tests under greenhouse conditions showed that these the two mutants F7I and F7I+F206L provided complete cross-protection (100%), from three independent trials with a total of 30 papaya plants and 30 horn melon plants challenged with YK 3 wk after the protective inoculation, as compared to that of 10% in papaya plants and 16.7% in horn melon plants provided by HA 5-1. Aphid transmission assay showed that F7I and F7I+F206L do not have the ability to be spread by Myzus persicae. Our resulted indicate that the two mutants F7I and F7I+F206L can solve the problem of strain-specific protection and have a great potential for control of PRSV in Taiwan. Their long-term stability and feasibility for cross protection will be investigated under field conditions.