定點突變矮南瓜黃化嵌紋病毒 HC-Pro 協同性基因導致病毒在單斑寄主奎藜上不引起過敏性反應並且不被局部化

Abstract

In the previous study, the three conserved amino acids, Arg180, Phe205, and Glu396 of helper component-proteinase (HC-Pro) of a severe Taiwan strain of Zucchini yellow mosaic virus (ZYMV), TW-TN3, were substituted with Ile180, Leu205, and Asn396, respectively, by mutating an infectious full-length cDNA clone harboring green fluorescent protein (GFP) gene as a reporter. The three single-mutated viruses GA, GB, GC and two double-mutated viruses GAC and GBC caused various levels of attenuated infection on the systemic host zucchini squash. While both of the double mutants GAC and GAB lost their ability to cause local lesions on the local lesion host Chenopodium quinoa, GAC could induce transient mottling in squash, but GAB was not able to be mechanically transferred from C. quinoa to squash. Infection on the plants of C. quinoa and zucchini squash by the triple-mutated virus GABC was not observed. In this investigation, to examine the infection of the mutants on C. quinoa and zucchini squash, the infection of GAB, GAC, GBC and GABC mutants was monitored by GFP expression in leaf tissue of C. quinoa and their infectivity was directly tested on plants of the systemic host zucchini squash by particle bombardment. The GAB caused slightly more severe mottling than that induced by GAC on plants of zucchini squash. Although the mutant GABC was able to infect plants of C. quinoa without local-lesion formation, symptoms were not observed and the mutant was not recovered from the bombarded zucchini squash plants. GAB, GAC and GABC did not induce local lesions on C. quinoa, but their infection was verified by GFP expression in leaf tissue. The fluorescence generated by GAB, GAC, GBC and GABC was found unlocalized in the leaf tissue. In particular, the spread of the mutant GAC was able to move continuously to the edge of the leaf of C. quinoa at 16 days post-inoculation (dpi). To further verify infection, GFP expression by the mutants on C. quinoa plants was monitored by leaf-tissue image recording at different time courses after inoculation and detected by tissue print immunoblotting and RNA-RNA hybridization. The results showed that GFP expression and the distribution pattern of CP were detected in the corresponding positions to the infected areas on the inoculated leaves. Furthermore, when the leaves inoculated by GAB, GAC, GBC and GABC were monitored by laser-scanning confocal microscopy at both tissue and cellular levels, the expression levels of fluorescence were found only 25.02%, 25.52%, 79.6% and 13.78% as that expressed by the wild type ZGFP, respectively, and the fluorescence in the central regions of expanding spots on the leaves of C. quinoa plants inoculated with GAB, GAC and GABC gradually disappeared. The spread distance of ZYMV mutants on the leaves of C. quinoa plants inoculated with GAB, GAC, GBC and GABC, respectively, at 18 dpi were 1.76 mm (139%), 2.28 mm (180%), 1.55 mm (123%) and 1.19 mm (94%), as compared to that induced by ZGFP of 1.26 mm (100%). Our results indicated that the mutants GAB, GAC and GBC in mesophyll cells are able to spread in leaf tissue of C. quinoa plants in an unlocalized manner. Although the unlocalized spread of GABC was also observed, the fluorescence gradually diminished and the observation became difficult. The mild recombinants GAB-NcMP and GAC-NcMP carrying the reading frame of CMV MP in the C-terminal region of NIb were able to restore the hypersensitive reaction (HR) and cause tiny and necrotic lesions on C. quinoa plants, as confirmed by GFP tagged in the N-terminal region of HC-Pro. When the severe strain ZGFP was used to challenge the C. quinoa plants that were protected with the attenuated mutants GAB or GAC for eight days, the cross-protection effectiveness against ZGFP was observed. Taken all together, our results indicate that mutated HC-Pros of the mutants abolish the ability of ZYMV to induce HR on the local lesion host C. quinoa, leading to unlocalized spread of virus in the leaf tissue of the local lesion host, and reduce the virulence on systemic host squash.