探討鞘蛋白與 TGB1 於紅龍果 X 病毒與仙人掌 X 病毒感染過程所扮演之角色

Abstract

紅龍果為台灣重要經濟果樹之一，實驗室前人研究發現台灣的紅龍果病毒為 Potexvirus 屬之仙人掌 X 病毒 (Cactus virus X, CVX)、紅龍果 X 病毒 (Pitaya virus X, PiVX) 與蟹爪蘭 X 病毒 (Zygocactus virus X, ZyVX)；田間調查發現台灣紅龍果受到 CVX 及 PiVX 複合感染的情況十分常見，且在紅龍果原生質體中可觀察到 CVX 與 PiVX 有協力作用的現象，因而推測兩病毒可能存在交互作用。本研究目的為探討 CVX 及 PiVX 之鞘蛋白 (coat protein, CP) 與 TGB1 (triple gene block 1) 蛋白於病毒感染過程中的角色，主要聚焦於 CP 對病毒累積之影響，以及複合感染對於 TGB1 缺失株其移動能力之影響。前人研究顯示 CP 缺失突變會使 CVX 和 PiVX 於圓葉菸草原生質體內之累積量大幅下降；本研究以野生型和 CP 缺失型之病毒轉錄體共同接種圓葉菸草原生質體，結果發現 CP 缺失株之 RNA 累積量無法藉由對方野生型病毒而提升。而為後續研究建構 35S 啟動子驅動之 CP 缺失病毒株，並以農桿菌短暫表現系統接種至圓葉菸草葉片，透過西方墨點法與北方雜合法進行分析；結果顯示缺失 CP 之 CVX 和 PiVX 無法表現病毒 CP，且其病毒 RNA 累積量顯著低於野生型病毒。當 CP 缺失病毒株與 CP 蛋白同時大量表現於圓葉菸草葉片，北方雜合分析結果顯示此種額外補充 CP 之方式，無法於圓葉菸草中回復 CP 缺失株所減少的RNA 累積量。且當野生型或 CP 缺失型病毒株同時與 CP ORF 轉錄體接種於圓葉菸草原生質體，結果顯示額外添加 CP ORF 轉錄體對病毒 RNA 累積量亦無顯著影響。然而於紅龍果原生質體系統，則是發現當 CP 缺失株與對方野生型共同接種，或額外添加 CP ORF 轉錄體時，野生型和CP 缺失株的 RNA 累積量皆能被略為提升。前人研究顯示缺失 TGB1 會使 CVX 和 PiVX 喪失移動能力，為探討透過複合感染以額外補充 TGB1，能否回復 TGB1 缺失病毒株受損之移動能力，因此於白藜葉片先接種野生型病毒，三日後再接種缺失 TGB1 且帶有 eGFP 的病毒轉錄體；螢光觀察的結果顯示 TGB1 缺失株於接種後第三天，其感染點可擴散至相鄰細胞，第六天則可觀察到感染點擴散至周圍 2 到 3 個細胞，顯示 TGB1 缺失所造成的移動能力損害可藉由自身或對方病毒的複合感染而部分回復。綜合以上結果，在紅龍果原生質體中，CVX 和 PiVX 能利用野生型病毒株或 CP ORF 轉錄體，可略為提升 CP 缺失株之基因體 RNA 累積量，顯示額外補充 CP 可以部分回復其功能，且此現象應具有寄主專一性。另外，透過預先接種野生型病毒株的實驗，驗證了 TGB1 協助病毒於白藜葉片細胞間移動的能力，能以額外補充 TGB1 的方式加以回復。

Pitaya is one of the important fruit crops in Taiwan. There are three species of potexviruses, Cactus virus X (CVX), Pitaya virus X (PiVX) and Zygocactus virus X (ZyVX), infecting pitayas in Taiwan. Referring to the field surveys, CVX and PiVX often co-infected pitaya plants, and these two viruses displayed the synergistic effect in pitaya protoplasts. Accordingly, it seems that there is interaction between CVX and PiVX. The aim of this study is to investigate the roles of coat protein (CP) and triple gene block 1 (TGB1) protein of CVX and PiVX during viral infection; mainly focusing on the effect of CP on viral accumulation, and the influence of mixed infection on the movement ability of TGB1 deletion mutant. Our lab showed that RNA accumulation of CVX and PiVX greatly decreased in Nicotiana benthamiana protoplasts due to CP deletion mutation. In this study, wild type (WT) and CP deletion mutant viral transcripts were co-inoculated to N. benthamiana protoplasts, and the results revealed that viral RNA accumulation of CP deletion mutant could not be increased by the other WT virus. For further study, 35S promoter-driven binary vectors of CVX and PiVX CP deletion mutants were constructed for agroinfiltration in N. benthamiana. According to the results of Western blot and Northern blot, both viral CP deletion mutants could not express coat proteins, and their viral RNA accumulations were significantly lower than those of WT. Northern analysis indicated that co-expression of CP deletion mutants and CP proteins in N. benthamiana leaves could not recover the decreased RNA accumulation of CP deletion mutants. When WT or CP deletion mutant co-inoculated with CP ORF transcripts to N. benthamiana protoplasts, viral RNA accumulations were not significantly affected. Never the less, the experiments of WT viruses and CP deletion mutants of CVX and PiVX in pitaya protoplasts showed that co-inoculation of WT and mutant transcripts or additon of CP ORF transcripts could slightly increase the viral RNA accumulations of WT as well as CP deletion mutants. Previous data of our lab indicated that deletion of TGB1 resulted in the loss of movement ability of CVX and PiVX. To investigate the feasibility of trans-complement of TGB1, leaves of Chenopodium quinoa were first inoculated with CVX or PiVX WT and then inoculated with TGB1-eGFP deletion mutant transcripts after three days. Based on fluorescence observation, the infection foci of TGB1 deletion mutant could expand to neighboring cells at 3-6 days post inoculation, which means the TGB1 deletion mutants could be rescued by trans-complementation of WT viruses. In summary, WT virus and CP ORF transcripts could partially rescue the viral RNA accumulation of CP deletion mutants in pitaya protoplasts. Besides, the trans-complementation of CVX and PiVX CP is host specific. Additionally, the defective movement ability of TGB1 deletion mutants could be recovered by the trans-complementation of TGB1 supplied by the CVX or PiVX-infected C. quinoa leaves.