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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
作者: 王南益
Wang, Nan-Yi
關鍵字: Zucchini yellow mosaic virus
Mild strains
Helper component-proteinase
Hypersensitive reaction
Viral movement
出版社: 植物病理學系所
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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.
其他識別: U0005-2708200711003900
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