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|標題:||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|
Abolishment of hypersensitive reaction and unlocalized spread on local lesion host Chenopodium quinoa by attenuated strains of Zucchini yellow mosaic virus with point mutations in helper component-proteinase gene
|關鍵字:||Zucchini yellow mosaic virus|
|引用:||Alderz, W. C., Purcifull, D. E., Simone, G. W., and Hiebert, E. 1983. Zucchini yellow mosaic virus: a pathogen of squash and other cucurbits in Florida. Proc. Flor. Sta. Hort. Soc. 96:72-74. Allison, R., Johnston, R. E., and Dougherty, W. G. 1986. The nucleotide sequence of the coding region of Tobacco etch virus genomic RNA evidence for the synthesis of a single polyprotein. Virology 154:9-20. Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., and Vance, V. B. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95:13079-13084. Andrejeva, J., Puurand, U., Merits, A., Rabenstein, F., Jarvekulg, L., and Valkonen, J. P. 1999. Potyvirus helper component-proteinase and coat protein (CP) have coordinated functions in virus-host interactions and the same CP motif affects virus transmission and accumulation. J. Gen. Virol. 80:1133-1139. Arazi, T., Slutsky, S. G., Shiboleth, Y. M., Wang, Y., Rubinstein, M., Barak, S., Yang, J., and Gal-On, A. 2001. Engineering Zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits. J. Biotechnol. 87:67-82. Beauchemin, C., Bougie, V., and Laliberte, J. F. 2005. Simultaneous production of two foreign proteins from a potyvirus-based vector. Virus Res. 112:1-8. Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W., and Baulcombe, D. C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739-6746. Canto, T., and Palukaitis, P. 1999. The hypersensitive response to Cucumber mosaic virus in Chenopodium amaranticolor requires virus movement outside the initially infected cell. Virology 265:74-82. Chen, C. C., Chao, C. H., Chen, C. C., Yeh, S. D., Tsai, H. T., and Chang, C. A. 2003. Identification of Turnip mosaic virus isolates causing yellow stripe and spot on calla lily. Plant Dis. 87:901-905. Chiang, C. H., Lee, C. Y., Wang, C. H., Jan, F. J., Lin, S. S., Chen, T. C., Raja, J. A. J., and Yeh, S. D. 2007. Genetic analysis of an attenuated Papaya ringspot virus strain applied for cross-protection. Eur. J. Plant Pathol. 118:333-348. Choi, I. R., Stenger, D. C., Morris, T. J., and French, R. 2000. A plant virus vector for systemic expression of foreign genes in cereals. Plant J. 23:547-555. Clark, M. F., and Adams, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34:475-483. Cronin, S., Verchot, J., Haldeman, C. R., Schaad, M. C., and Carrington, J. C. 1995. Long-distance movement factor: A transport function of the potyvirus helper component proteinase. Plant Cell 7:549-559. Desbiez, C., and Lecoq, H. 1997. Zucchini yellow mosaic virus. Plant Pathol. 46:809-829. Dietrich, C., and Maiss, E. 2003. Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants. J. Gen. Virol. 84:2871-2876. Dolja, V. V., Haldeman-Cahill, R., Montgomery, A. E., Vandenbosch, K. A., and Carrington, J. C. 1995. Capsid protein determinants involved in cell-to-cell and long distance movement of Tobacco etch potyvirus. Virology 206:1007-1016. Dolja, V. V., McBride, H. J., and Carrington, J. C. 1992. Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proc. Natl. Acad. Sci. USA 89:10208-10212. Dombrovsky, A., Huet, H., Chejanovsky, N., and Raccah, B. 2005. Aphid transmission of a potyvirus depends on suitability of the helper component and the N terminus of the coat protein. Arch. Virol. 150:287-298. Gal-On, A., Antignus, Y., Rosner, A., and Raccah, B. 1991. Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, Zucchini yellow mosaic virus. J. Gen. Virol. 72:2639-2643. German-Retana, S., Candresse, T., Alias, E., Delbos, R. P., and Le Gall, O. 2000. Effects of green fluorescent protein or beta-glucuronidase tagging on the accumulation and pathogenicity of a resistance-breaking Lettuce mosaic virus isolate in susceptible and resistant lettuce cultivars. Mol. Plant-Microbe Interact. 13:316-324. Gleba, Y., Marillonnet, S., and Klimyuk, V. 2004. Engineering viral expression vectors for plants: the ''full virus'' and the ''deconstructed virus'' strategies. Curr. Opin. Plant Biol. 7:182-188. Gopinath, K., Wellink, J., Porta, C., Taylor, K. M., Lomonossoff, G. P., and van Kammen, A. 2000. Engineering Cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants. Virology 267:159-173. Guo, H. S., Lopez-Moya, J. J., and Garcia, J. A. 1998. Susceptibility to recombination rearrangements of a chimeric Plum pox potyvirus genome after insertion of a foreign gene. Virus Res. 57:183-195. Hamamoto, H., Sugiyama, Y., Nakagawa, N., Hashida, E., Matsunaga, Y., Takemoto, S., Watanabe, Y., and Okada, Y. 1993. A new Tobacco mosaic virus vector and its use for the systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco and tomato. Biotechnology 11:930-932. Hammond-Kosack, K. E., Staskawicz, B. J., Jones, J. D. G., and Baulcombe, D. C. 1995. Functional expression of a fungal avirulence gene from a modified Potato virus X genome. Mol. Plant-Microbe Interact. 8:181-185. Hendy, S., Chen, Z. C., Barker, H., Cruz, S. S., Chapman, S., Torrance, L., Cockburn, W., and Whitelam, G. C. 1999. Rapid production of single-chain Fv fragments in plants using a Potato virus X episomal vector. J. Immunol. Methods 231:137-146. Hjulsager, C. K., Lund, O. S., and Johansen, I. E. 2002. A new pathotype of Pea seedborne mosaic virus explained by properties of the P3-6k1- and viral genome-linked protein (VPg)-coding regions. Mol. Plant-Microbe Interact. 15:169-171. Hong, Y., and Hunt, A. G. 1996. RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase. Virology 226:146-151. Hseu, S. H., Huang, C. H., Chang, C. A., Yang, W. Z., Chang, Y. M., and Hsiao, C. H. 1987. The occurrence of five viruses in six cucurbits in Taiwan. Plant Prot. Bull. 29:233-244. Hseu, S. H., Wang, H. L., and Huang, C. H. 1985. Identification of a Zucchini yellow mosaic virus from Cucumis sativus. J. Agric. China. 34:87-95. Hsu, C. H., Lin, S. S., Liu, F. L., Su, W. C., and Yeh, S. D. 2004. Oral administration of a mite allergen expressed by Zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis. J. Allergy Clin. Immunol. 113:1079-1085. Ivanov, K. I., Puustinen, P., Gabrenaite, R., Vihinen, H., Ronnstrand, L., Valmu, L., Kalkkinen, N., and Makinen, K. 2003. Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection. Plant Cell 15:2124-2139. Jenner, C. E., Tomimura, K., Ohshima, K., Hughes, S. L., and Walsh, J. A. 2002. Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes. Virology 300:50-59. Jenner, C. E., Wang, X., Tomimura, K., Ohshima, K., Ponz, F., and Walsh, J. A. 2003. The dual role of the potyvirus P3 protein of Turnip mosaic virus as a symptom and avirulence determinant in brassicas. Mol. Plant-Microbe Interact. 16:777-784. Johansen, I. E., Lund, O. S., Hjulsager, C. K., and Laursen, J. 2001. Recessive resistance in Pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus. J. Virol. 75:6609-6614. Kasschau, K. D., and Carrington, J. C. 1995. Requirement for HC-Pro processing during genome amplification of Tobacco etch polyvirus. Virology 209:268-273. Kasschau, K. D., and Carrington, J. C. 1998. A counterdefensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell 95:461-470. Kasschau, K. D., Cronin, S., and Carrington, J. C. 1997. Genome amplification and long-distance movement functions associated with the central domain of Tobacco etch potyvirus helper component-proteinase. Virology 228:251-262. Kumagai, M. H., Donson, J., della-Cioppa, G., and Grill, L. K. 2000. Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector. Gene 245:169-174. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227:680-685. Lecoq, H., Lisa, V., and Dellavalle, G. 1983. Serological identity of Muskmelon yellow stunt and Zucchini yellow mosaic viruses. Plant Dis. 67:824-825. Leonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M. G., and Laliberte, J. F. 2000. Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J. Virol. 74:7730-7737. Lesemann, D. E., Makkouk, K. M., Koenig, R., and Natafji Samman, E. 1983. Natural infection of cucumbers by Zucchini yellow mosaic virus in Lebanon. Phytopathol. Z. 108:304-313. Lin, S. S., Hou, R. F., Huang, C. H., and Yeh, S. D. 1998. Characterization of Zucchini yellow mosaic virus (ZYMV) isolates collected from Taiwan by host reactions, serology, and RT-PCR. Plant Prot. Bull. 40:163-176. Lin, S. S., Hou, R. F., and Yeh, S. D. 2001. Complete genome sequence and genetic organization of a Taiwan isolate of Zucchini yellow mosaic virus. Bot. Bull. Acad. Sin. 42:243-250. Lin, S. S., Hou, R. F., and Yeh, S. D. 2002. Construction of in vitro and in vivo infectious transcripts of a Taiwan strain of Zucchini yellow mosaic virus. Bot. Bull. Acad. Sin. 43:261-269. Lin, S. S., Wu, H. W., Jan, F. J., Hou, R. F., and Yeh, S. D. 2007. Modifications of the HC-Pro of Zucchini yellow mosaic potyvirus for generation of attenuated mutants for cross protection against severe infection. Phytopathology 97:287-296. Lin, Y. H. 2003. The MP and 2b genes of Cucumber mosaic virus complement the mutated potyviral HC-Pro gene defective in hypersensitive reaction and virulence. Master Thesis. National Chung Hsing University, Department of Plant Pathology. 52 pp. Lisa, V., Boccardo, G., D''Agostino, G., Dellavalle, G., and D''Aquilio, M. 1981. Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathology 71:667-672. Lisa, V., and Lecoq, H. 1984. Zucchini yellow mosaic virus. CMI/AAB Descriptions of Plant Viruses, No. 282. Kew, Surrey. Mahajan, S., Dolja, V. V., and Carrington, J. C. 1996. Roles of the sequence encoding Tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element. J. Virol. 70:4370-4379. Maia, I. G., Haenni, A., and Bernardi, F. 1996. Potyviral HC-Pro: a multifunctional protein. J. Gen. Virol. 77:1335-1341. Maiss, E., Timpe, U., Birsske, A., Jelkman, W., Casper, R., Himmler, G., Mattanovich, D., and Katinger, H. W. D. 1989. The complete nucleotide sequence of Plum pox virus RNA. J. Gen. Virol. 70:513-524. Mas, P., and Pallas, V. 1996. Long-distance movement of Cherry leaf roll virus in infected tobacco plants. J. Gen. Virol. 77:531-540. Masuta, C., Yamana, T., Tacahashi, Y., Uyeda, I., Sato, M., Ueda, S., and Matsumura, T. 2000. Development of Clover yellow vein virus as an efficient, stable gene-expression system for legume species. Plant J. 23:539-546. McCormick, A. A., Kumagai, M. H., Hanley, K., Turpen, T. H., Hakim, I., Grill, L. K., Tuse, D., Levy, S., and Levy, R. 1999. Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc. Natl. Acad. Sci. USA 96:703-708. Nameth, S. T., Dodds, J. A., Paulus, A. O., and Kishaba, A. 1985. Zucchini yellow mosaic virus associated with severe diseases of melon and watermelon in southeastern California desert valleys. Plant Dis. 69:785-788. Pirone, T. P., and Blanc, S. 1996. Helper-dependent vector transmission of plant viruses. Annu. Rev. Phytopathol. 34:227-247. Poch, O., Sauvaget, I., Delarue, M., and Tordo, N. 1989. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 8:3867-3874. Pogue, G. P., Lindbo, J. A., Garger, S. J., and Fitzmaurice, W. P. 2002. Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40:45-74. Provvidenti, R., Gonsalves, D., and Humaydan, H. S. 1984. Occurrence of Zucchini yellow mosaic virus in cucurbits from Connecticut, New-York, Florida, and California. Plant Dis. 68:443-446. Pruss, G., Ge, X., Shi, X. M., Carrington, J. C., and Bowman Vance, V. 1997. Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9:859-868. Rajamaki, M. L., Kelloniemi, J., Alminaite, A., Kekarainen, T., Rabenstein, F., and Valkonen, J. P. 2005. A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions. Virology 342:88-101. Revers, F., Gall, O. L., Candresse, T., and Maule, A. J. 1999. New advances in understanding the molecular biology of plant potyvirus interactions. Mol. Plant-Microbe Interact. 12:367-376. Riechmann, J. L., Cervera, M. T., and Garcia, J. A. 1995. Processing of the Plum pox virus polyprotein at the P3-6K1 junction is not required for virus viability. J. Gen. Virol. 76:951-956. Riechmann, J. L., Lain, S., and Garcia, J. A. 1992. Highlights and prospects of potyvirus molecular biology. J. Gen. Virol. 73:1-16. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L., and Lucas, W. J. 1997. Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237:283-295. Sablowski, R. W., Baulcombe, D. C., and Bevan, M. 1995. Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter. Proc. Natl. Acad. Sci. USA 92:6901-6905. Saenz, P., Cervera, M. T., Dallot, S., Quiot, L., Quiot, J. B., Riechmann, J. L., and Garcia, J. A. 2000. Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K1. J. Gen. Virol. 81:557-566. Saenz, P., Salvador, B., Simon-Mateo, C., Kasschau, K. D., Carrington, J. C., and Garcia, J. A. 2002. Host-specific involvement of the HC protein in the long-distance movement of potyviruses. J. Virol. 76:1922-1931. Schaad, M. C., Anderberg, R. J., and Carrington, J. C. 2000. Strain-specific interaction of the Tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system. Virology 273:300-306. Scholthof, H. B., Scholthof, K. B. G., and Jackson, A. O. 1996. Plant virus gene vectors for transient expression of foreign proteins in plants. Annu. Rev. Phytopathol. 34:299-323. Shi, X. M., Miller, H., Verchot, J., Carrington, J. C., and Vance, V. B. 1997. Mutations in the region encoding the central domain of helper component-proteinase (HC-Pro) eliminate Potato virus X/potyviral synergism. Virology 231:35-42. Shukla, D. D., Ward, C. W., and Brunt, A. A. 1994. The Potyviridae. CAB International, Wallingford, Oxon, UK. 516 pp. Spall, V. E., Shanks, M., and Lomonossoff, G. P. 1997. Polyprotein processing as a strategy for gene expression in RNA viruses. Semin. Virol. 8:15-23. Valli, A., Martin-Hernandez, A. M., Lopez-Moya, J. J., and Garcia, J. A. 2006. RNA silencing suppression by a second copy of the P1 serine protease of Cucumber vein yellowing ipomovirus, a member of the family Potyviridae that lacks the cysteine protease HC-Pro. J. Virol. 80:10055-10063. Ward, C. Q., and Shukla, D. D. 1991. Taxonomy of potyviruses current problems and some solutions. Intervirology 32:269-296. Wittmann, S., Chatel, H., Fortin, M. G., and Laliberte, J. F. 1997. Interaction of the viral protein genome linked of Turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology 234:84-92. Yang, S., and Ravelonandro, M. 2002. Molecular studies of the synergistic interactions between Plum pox virus HC-Pro protein and Potato virus X. Arch. Virol. 147:2301-2312. Yeh, S. D., and Gonsalves, D. 1984. Evaluation of induced mutants of Papaya ringspot virus for control by cross protection. Phytopathology 74:1086-1091.|
|摘要:||矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV) 屬於馬鈴薯 Y 屬病毒，為瓜類作物生長期間最大的危害因子。根據前人研究比對輕症型木瓜輪點病毒 (PRSV HA5-1) 與輕症型 ZYMV (ZYMV-WK) 之協同性蛋白 (HC-Pro) 之胺基酸序列顯示，Arg180、Phe205 與 Glu396 三個胺基酸扮演由強系病毒轉變為弱系病毒之關鍵位置。因此，將這三個胺基酸 Arg180、Phe205 與 Glu396 分別置換成 Ile、Leu 與 Asn，產生單一、雙重與三重不同組合之突變病毒株，將之分別以機械接種之方式直接接種於單斑寄主奎藜或系統性寄主矮南瓜上，分析其致病性。然而，在前人的實驗中並無法證實雙重突變株 GAB 與三重突變株 GABC 是否對系統系寄主具有感染的能力。本實驗乃藉由粒子槍之方式將含有綠螢光蛋白 (green fluorescent protein, GFP) 基因的突變病毒株接種於矮南瓜上，此外也以機械接種之方式將突變病毒株直接接種於奎藜上，於柯達影像系統 (Kodak 4000MM image station) 下觀察綠螢光蛋白產生之情形，以確定不形成系統性病徵的突變株是否會感染奎藜。本實驗結果顯示雙重突變株 GAB 可以感染矮南瓜並且造成比另一雙重突變株 GAC 稍微嚴重之斑駁病徵，然而三重突變株 GABC 仍未觀察到具有感染矮南瓜的能力。另一方面，由在影像系統下觀察突變病毒株在奎藜上綠螢光蛋白產生之結果顯示，雙重與三重突變株都具有感染單班寄主奎藜的能力，而且雙重突變株 GAB、GAC 與三重突變株 GABC 則皆不產生任何局部病斑。此外，持續觀察之後發現，雙重突變株 GAB、GAC、GBC 與三重突變株 GABC 都可以在奎藜葉肉細胞內移動而不被侷限住，GAC 在持續觀察 16 天後，發現甚至可以移動到葉緣的部分。利用組織印痕技術，我們證實在葉片上螢光表現的位置與病毒鞘蛋白 (CP) 表現的位置是吻合的。進一步利用共軛焦顯微鏡觀察病毒感染之奎藜葉片，發現在葉片上只有弱系病毒 GAB、GAC、GBC 與 GABC移動之後的區域，仍可以觀察到螢光，其表現量與強系病毒 ZGFP 相較之下分別為 25.02%、25.52%、13.78% 與 79.6%。此外，計算病毒誘導產生之斑點的擴散距離，在接種後 18 天，GAB、GAC、GBC 與 GABC 之距離分別為 1.76 mm (139%)、2.28 mm (180%)、1.55 mm (123%)、1.19 mm (94%)，而 ZGFP 此時的擴散距離為 1.26mm (100%)。此實驗結果顯示 GAB、GAC、GBC 在奎藜葉片組織內的確具有移動的能力。雖然 GABC 在奎藜上也不會被侷限，但是 GABC 螢光的強度逐漸降低，因此不易觀察。另外，以攜帶胡瓜嵌紋病毒之轉移相關的移動蛋白 (CMV MP) 之弱系重組病毒 ZGAB-NcMP 與 ZGAC-NcMP，發現具有回復原本弱系病毒 GAB 與 GAC 在奎藜上不產生單斑的能力。GAC 與 GAB 接種於奎藜植物8天後，在螢光的觀察之下，則發現具有保護寄主不被強系病毒 ZGFP 感染的能力。本實驗結果得知，在協同蛋白基因上之突變具有破壞強系病毒 ZYMV 在奎藜上誘導產生過敏性反應 (hypersensitive reaction, HR) 的能力，導致突變病毒株可以在奎藜葉肉細胞內移動，且在系統性寄主矮南瓜上的毒力 (virulence) 也大幅減低。|
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.
|Appears in Collections:||植物病理學系|
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