影響仙人掌X病毒與紅龍果X病毒間協力作用之因子探討

Investigation of the synergistic interaction between Cactus virus X and Pitaya virus X

吳悅民(Yueh-Min Wu)

指導教授 ： 張雅君

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900422

摘要

仙人掌科之紅龍果因營養與經濟價值甚高，且對於生長環境有較強的耐受力，因此近年來逐漸活躍於臺灣市場，且於臺灣的栽培面積也逐年上升。自田間調查發現，Potexvirus屬之仙人掌X病毒(Cactus virus X, CVX)與紅龍果X病毒(Pitaya virus X, PiVX)的複合感染在臺灣紅龍果果園相當常見。為了解兩種病毒在寄主體內是否會相互影響，實驗室前人以北方雜合分析法證實CVX與PiVX感染圓葉菸草(Nicotiana benthamiana)或紅龍果之原生質體時，複合接種之病毒RNA累積量皆較單獨接種高，因而推論CVX與PiVX間具有協力作用。為進一步瞭解CVX與PiVX間協力作用之影響因子，參考potexviruses與其他屬病毒所共同參與協力作用之機制報告，發現病毒之RNA靜默作用抑制子可能在協力作用中扮演重要角色。因此本研究欲先偵測是否有特定的CVX或PiVX病毒蛋白具有抑制RNA靜默作用的能力，並且觀察這些病毒蛋白是否會影響協力作用的產生及其效果，期望能探討可能參與協力作用之因子為何。透過農桿菌短暫表現蛋白法，發現在所有CVX及PiVX轉譯之病毒蛋白中，僅有CVX TGBp1 (triple gene block protein 1)、PiVX TGBp1、CVX CP (capsid protein)以及PiVX CP可抑制由GFP正股RNA所引起的RNA靜默作用(S-PTGS)而表現出綠色螢光；以上四種病毒蛋白中，又僅有CVX TGBp1及PiVX TGBp1具有些微抑制由GFP雙股RNA所引發之RNA靜默作用(IR-PTGS)的能力。後續實驗進一步分析可抑制RNA靜默作用之CVX和PiVX之TGBp1與CP，在單細胞內對另一種病毒RNA累積之影響。製備CVX和PiVX之TGB1以及CP開放閱讀框(open reading frame)的轉錄體，與CVX或PiVX共同接種至圓葉菸草原生質體。北方雜合分析法結果顯示，無論是哪一種病毒之TGB1及CP，皆可以提升CVX與PiVX於原生質體中的病毒RNA累積量。另一方面，我們構築CVX和PiVX的TGB1及CP之病毒突變株，與另一種野生型病毒之轉錄體共同接種至圓葉菸草原生質體，觀察TGB1及CP基因突變對於協力作用之影響。自北方雜合分析結果發現，缺少CVX或PiVX之TGB1或CP任何一種基因，皆會影響協力作用的發生。除此之外，當CVX CP及PiVX CP病毒突變株分別與PiVX及CVX野生型病毒共同接種時，突變株於原生質體中之複製及累積的缺失有機會受到互補(trans-complement)而部分回復。在本研究中，另外測試並優化了白藜原生質體的製備方法，提高了原生質體之產量，提供未來研究CVX與PiVX另一種材料的選擇。綜合上述研究結果，我們認為CVX與PiVX之TGBp1和CP影響了病毒間協力作用的發生，但尚不能確定此協力作用是否與TGBp1和CP具有抑制RNA靜默作用的能力有關，需要更進一步的研究以釐清其機制。

關鍵字

仙人掌X病毒 ； 紅龍果X病毒 ； 協力作用 ； RNA靜默作用

並列摘要

Pitaya, a kind of popular tropical fruit, is rich in several nutrients. Due to the growing market demand as well as good fitness of strict environments of pitaya, the planting area in Taiwan is expanding year by year. Cactus virus X (CVX) and Pitaya virus X (PiVX), belonging to the genus Potexvirus, often co-infect pitaya plants in Taiwan. Based on our previous study, CVX and PiVX were beneficial to each other’s virus accumulation in Nicotiana benthamiana and pitaya protoplasts analyzed by northern blot. Therefore, we suggested that these two viruses may have synergistic interaction. Referring to previous reports of synergistic effect which potexviruses are involved, it is found that viral silencing suppressor might play an important role in synergistic effect. Therefore, we planned to figure out whether CVX and PiVX proteins are able to suppress RNA silencing and also have positive influences on synergism between CVX and PiVX. Agrobacterium-mediated transient expression assay demonstrated that only TGBp1 (triple gene block protein 1) and CP (capsid protein) of both viruses could suppress sense transgene-mediated post-transcriptional gene silencing (S-PTGS) and thus GFP fluorescence expressed. Among these four viral proteins, only CVX and PiVX TGBp1 were proved to suppress inverted repeat transgene-mediated post-transcriptional gene silencing (IR-PTGS) slightly. Hence, TGBp1 and CP which could suppress RNA silencing were further investigated for their effects on viral RNA accumulation in protoplasts. Co-inoculation of CVX or PiVX along with TGBp1 or CP transcripts indicated that both viruses gained higher RNA accumulation with addition of either TGB1 or CP transcripts. In addition, the influences of TGB1 and CP mutation on synergistic effect were studied by co-inoculating transcripts of mutant and wild type virus to N. benthamiana protoplasts. The results revealed that TGBp1 and CP of both viruses were all important to synergistic interaction. Moreover, the reduced RNA accumulation of CP mutant of CVX or PiVX in protoplasts could be slightly trans-complemented by the other virus. In this study, we also modified and optimized the preparation of Chenopodium quinoa protoplasts, and this method provides a new study system for CVX and PiVX. In summary, TGBp1 and CP of both CVX and PiVX were viral RNA silencing suppressors, and were also involved in synergistic interaction. However, the ability of RNA silencing suppression has not yet been proved to associate with synergism between CVX and PiVX. The mechanisms of synergistic effect are still unclear, and need further experimental evidences to clarify.

並列關鍵字

Cactus virus X ； Pitaya virus X ； synergistic interaction ； RNA silencing

參考文獻

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