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標題: 利用矮南瓜黃化嵌紋病毒載體及農桿菌暫時性表現系統對西瓜銀斑病毒之非結構性NSs蛋白上重要區域對基因靜默及病原性之功能性分析
Analysis of essential regions of NSs protein of Watermelon silver mottle virus for gene silencing suppression and pathogenicity by Zucchini yellow mosaic virus vector and Agrobacterium-mediated expression system
作者: 蕭文榮
Hsiao, Wen-Rong
關鍵字: WSMoV;西瓜銀斑病毒;NSs;WNSscon;ZYMV;Gateway system;gene silencing suppressor;pathogenicity;非結構性蛋白;矮南瓜黃化嵌紋病毒;基因靜默抑制子;病原性
出版社: 植物病理學系所
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西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 為Bunyaviridae 科,蕃茄斑萎病毒屬 (Tospovirus) 之病毒,是亞洲瓜類栽培重要限制因子之一。 其中由病毒所表現出之非結構性 NSs (nonstructural, NSs) 蛋白,在前人研究中已指出此蛋白與病徵的嚴重度有關,且為一基因靜默抑制子 (gene silencing suppressor)。 在本實驗室先前研究中,製備了對抗西瓜銀斑病毒NSs 蛋白的單株抗體,此單株抗體可辨識同為西瓜銀斑病毒血清群的他種蕃茄斑萎病毒屬病毒的NSs蛋白,並且證實此單株抗體可辨識NSs蛋白的N 端第89至125個胺基酸的區域 (WNSscon),且此區域屬於西瓜銀斑病毒血清群屬的病毒中NSs 蛋白上的高保留區,並在該研究結果中指出Cys108及Lys109胺基酸為 N 端高保留區的關鍵胺基酸。 因此,本實驗之目的,在於分析 NSs 蛋白的 N 端 WNSscon 區域及C 端區域在NSs蛋白中,對病原性及基因靜默抑制作用上所扮演的角色。 利用本實驗室已構築,生物體內具感染力之矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV) 輕症型 (ZYMVAC) 作為載體來表現不同長度的NSs片段,並將重組病毒以機械接種方式接種於產生局部病斑的寄主奎藜或系統性寄主矮南瓜上,藉由寄主植物病徵之變化,以分析其對毒力的影響。 此外,在本研究中,進一步將原來的病毒載體改造成Gateway® 系統,可利用該系統特異點重組之特性,將想要表現之蛋白基因利用此重組技術,以重組的方式放到載體上進行表現,並且附帶一個GFP螢光蛋白以利追蹤及觀察各表現不同片段及定點突變NSs蛋白的重組病毒之病毒活性之用。 另一方面,同時利用農桿菌注射法 (agro-infiltration) 以及表現GFP螢光蛋白的菸草 (Nicotiana benthamiana line 16C),做為實驗的平台進行基因靜默的研究。 結果顯示利用輕症型載體 (ZYMVAC) 表現1/3、2/3及N端高度保留區 (WNSscon) 已移除的NSs蛋白後,經接種後在單斑寄主奎藜上不引發單斑反應,在系統性寄主矮南瓜上僅造成輕微病徵。 另一方面利用p35SZAC-DC-nGFP 輕症型具Gateway® 系統之載體表現各長度片段及突變的NSs蛋白,只有ZAC-DC-nGFP-WNSsF可表現完整NSs蛋白及ZAC-DC-nGFP-WNSsC108A其表現的 NSs 蛋白的Cys108突變成Ala108之重組病毒可在單斑寄主奎藜上形成典型單斑。 利用農桿菌注射法進行基因靜默功能的分析,也只有 WNSsF 及 WNSsC108A 此二蛋白在農桿菌共同注射法 (co-infiltration) 的分析下,在五天之後可觀察到具有基因靜默抑制子的活性。 以西方轉漬法 (western blotting) 對此二表現系統進行產物偵測時,實驗結果顯示, NSs 蛋白的Lys109胺基酸遭突變後 (WNSsK109A), NSs 蛋白的專一性單株抗體則無法偵測此一定點突變 NSs 蛋白。 同時 WNSsK109A定點突變蛋白,即失去對 ZYMV突變後受損的HC-Pro 功能上有所互補,基因靜默抑制子的活性也受到影響。 另一方面, C 端第352到第388 胺基酸遭移除後 (WNSsΔ352-388) 的 NSs 蛋白,也不具有對因突變而功能受損的 HC-Pro 在功能上有所互補,也觀察不到基因靜默抑制子的活性。 根據以上結果,我們認為 NSs 蛋白的 N 端WNSscon 高度保留區和 C 端的第352到第388 胺基酸區域對NSs 蛋白的基因靜默抑制能力及病原性扮演重要角色。 更進一步, N 端高保留區中的 Lys109胺基酸在 NSs 蛋白的專一性單株抗體辨識上、基因靜默抑制子活性及毒力上都具有相當重要具不可或缺的角色。

Watermelon silver mottle virus (WSMoV), a member of the genus Tospovirus, is a major limiting factor for growing watermelon, melon, wax gourd, and other cucurbits in Asia. A highly conserved WNSscon region (aa 89 ~ 125) in NSs protein specifically targeted by a mouse monoclonal antibody (MAb) was previously identified. In this investigation, the WNSscon and C-terminal region NSs protein was analyzed for their roles in pathogenicity and gene silencing suppression. The engineered mild strain ZYMVGAC, which contained two amino acid changes in the HC-Pro gene, induced mild symptoms on zucchini squash plants and infected plants of Chenopodium quinoa without local lesions, was used to express the nonstructural (NSs) protein of WSMoV in both hosts. In addition, the mild strain ZYMVGAC was further modified to generate the destination vector ZAC-DC-nGFP with a Gateway® system in between P1 and HC-Pro regions and with a GFP coding sequence inserted between NIb and CP regions. Different deletion forms and point-mutated forms of NSs protein were constructed and expressed by ZYMVGAC and ZAC-DC-nGFP vector separately to analyze the essential part of the NSs protein for its functions in complementation of the HC-Pro mutations. The Agrobacterium-mediated transient expression system and GFP transgeneic Nicotiana benthanamiana line 16C were used to analyze the activity of silencing suppression of the deleted or mutated NSs proteins by co-infiltration with a partial GFP gene for inducing GFP silencing. The mild recombinants derived from strain p35SZYMVAC carrying one-third, two-third or full-length NSs ORF without the WNSscon common epitope induced no local lesions on plants of C. quinoa and caused mild mottling on squash plants without virulence enhancement. Furthermore, the modified infectious clone p35SZAC-DC-nGFP was used to express deleted or mutated NSs proteins. Only the recombinants ZAC-DC-nGFP-WNSsF carrying the full length of the NSs protein and ZAC-DC-nGFP-WNSsC108A with Cys108 replaced by Ala108 induced local lesions on C. quinoa plants. The expression of different deleted or mutated NSs proteins were also analyzed by agro-infiltration system. Only the construct WNSsF carrying full-length NSs protein and WNSsC108A with Cys108 replaced with Ala108 suppressed the GFP silencing 5 days after co-infiltration. In western blotting assay, the mutated NSs protein, WNSsK109A the Lys109 of the NSs protein changed to Ala109, was no longer recognized by the mouse monoclonal antibody. The ability to complement the function of the attenuated ZYMV HC-Pro and gene silencing suppression of the mutated NSs protein, WNSsK109, both was abolished. On the other hand, the deleted NSs protein, WNSsΔ352-388 a deletion from 352-388 aa of NSs protein, did not complement the function of the attenuated ZYMV HC-Pro and did not suppress gene silencing. Our results indicated that both the WNSscon common eiptope and the C-terminal region (352-388 aa) of the NSs protein play important roles in induction of hypersensitive reaction on C. quinoa and virulence enhancement on squash plants. Moreover, the residue Lys109 in the common WNSscon epitope of the NSs protein, which is indispensable for MAb recognization, is crucial for silencing suppression and virulence enhancement.
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