探討紅龍果X病毒與仙人掌X病毒及寄主因子之交互作用

Abstract

仙人掌科(Cactaceae)三角柱屬(Hylocereus)的紅龍果為台灣重要的熱帶水果作物，台灣田間的紅龍果病毒有紅龍果X病毒(Pitaya virus X, PiVX)、仙人掌X病毒(Cactus virus X, CVX)與蟹爪蘭X病毒(Zygocactus virus X, ZyVX)三種 Potexvirus屬病毒，其中CVX和PiVX的感染率高於ZyVX。為瞭解CVX和PiVX這兩種病毒在植物中的感染機制，本研究從實驗室前人的紅龍果轉錄組資料庫和交聯免疫共沉澱的研究結果，篩選出可能和病毒具有交互作用之寄主因子，並從中挑選紅龍果的過氧化氫酶 (catalase, CAT)作為研究目標，探討HuCAT2對CVX和PiVX感染過程之影響。過氧化氫酶為植物在H2O2代謝途徑中的重要酵素，且過氧化氫酶家族根據其蛋白表現於不同組織被分成三大類，而親緣關係樹顯示HuCAT2較接近第二類家族，代表在維管束組織有較高之表現。本研究先透過紅龍果轉錄組資料庫和RT-qPCR分析，篩選出ubiquitin-conjugating enzyme E2 variant 1D-like基因為表現量最穩定之參考基因。而後以RT-qPCR分析HuCAT2基因在感染CVX和PiVX間的表現情形，結果顯示於紅龍果植株單獨和共同接種CVX和PiVX病毒，皆會提高HuCAT2 mRNA的表現量。另一方面，為釐清HuCAT2對CVX和PiVX的影響，透過農桿菌注射法 (agroinfiltration)於圓葉菸草中共同表現HuCAT2蛋白和紅龍果病毒株，以西方墨點法分析的結果顯示，過量表現HuCAT2蛋白有助於CVX鞘蛋白之累積，而對於PiVX的影響則較不明顯。另外，透過螢光顯微鏡觀察發現，單獨表現HuCAT2-eGFP蛋白後的二至四天中，其蛋白會從葉肉細胞內移動至表皮細胞之細胞膜附近或維管束中；然而當和紅龍果病毒株共同表現時，並未觀察到HuCAT2-eGFP蛋白的分布有明顯的變化。由於未能透過菸草脆裂病毒 (Tobacco rattle virus, TRV)之病毒載體系統對紅龍果進行基因靜默，因此將基因靜默之目標改為白藜的過氧化氫酶 (CqCAT)，先以RT-qPCR確認目標基因mRNA被靜默，然後接種CVX和PiVX，再分析病毒的累積情形。結果顯示抑制CqCAT基因之表現，對於CVX和PiVX鞘蛋白和基因體RNA的累積量似乎有負面的影響。綜合以上結果，我們認為CVX和PiVX在感染寄主植物過程中，CAT基因產物可能作為寄主因子，扮演影響CVX和PiVX的複製和鞘蛋白累積之角色，其中對CVX鞘蛋白累積的幫助較為明顯。

Pitaya is a member of the genus Hylocereus in the family Cactaceae and also an important tropical fruit crop. The viruses currently reported to infect pitaya are Pitaya virus X (PiVX), Cactus virus X (CVX) and Zygocactus virus X (ZyVX). Among these potexviruses, the infection rate of CVX and PiVX is higher than that of ZyVX. To investigate the infection mechanism of CVX and PiVX, we screened the potential host factors from the previously established pitaya transcriptome database and the LC-MS/MS analysis of co-immunoprecipitation, and HuCAT2, a predicted pitaya catalase, was selected as a candidate host gene. The aim of this study is to investigate the interaction between pitaya viruses and HuCAT2. Catalase is an important antioxidant enzyme that catalyzes the decomposition of H2O2, and plant catalases can be classified into three groups based on their expression properties. According to the phylogenetic tree, HuCAT2 corresponds to Class II catalases, which are mainly expressed in vascular tissues. In order to identify an ideal reference gene for analyzing CVX- and PiVX-infected pitaya by quantitative RT-PCR (RT-qPCR), several candidate reference genes with relatively stable expression level were selected from the pitaya transcriptome database, and finally ubiquitin-conjugation E2 variant 1D-like gene was recognized as the pitaya reference gene based on the result of RT-qPCR. The expression level of HuCAT2 mRNA was all up-regulated in the single and co-inoculated pitaya plants by CVX and PiVX. On the other hand, the overexpression of HuCAT2 protein by agroinfiltration enhanced the accumulation of CVX coat protein (CP) in Nicotiana benthamiana, but not that of PiVX CP as shown by western blot. In addition, the fluorescence microscopy analysis revealed that overexpressed HuCAT2-eGFP proteins localized to the plasma membrane of epidermal cells and vascular tissues at 2-4 days after agroinfiltration, and did not change its locations when co-expressed with pitaya viruses. Because the gene silencing of pitaya could not be achieved by Tobacco rattle virus (TRV)-based vector, the target gene was changed to the CAT gene (CqCAT) of Chenopodium quinoa. Before inoculation of CVX and PiVX to C. quinoa, RT-qPCR was used to confirm the mRNA of CqCAT was silenced, and then the accumulation of the virus was analyzed for 2-4 days. The results indicated that gene silencing of CqCAT appeared to have a negative effect on the accumulation of CVX and PiVX coat protein and genomic RNA. In summary, we suggested that the CAT gene product may play as a host factor to affect coat protein and genomic RNA accumulation of CVX and PiVX, especially in helping the accumulation of CVX coat protein.