探討胞外泌體在感染柑橘鱗砧類病毒番茄上系統性傳播的角色

Abstract

類病毒是一類僅由裸露、環狀單股RNA組成且無蛋白編碼能力的植物病原體。柑橘鱗砧類病毒 (Citrus exocortis viroid, CEVd) 不僅可感染柑橘，也能感染多種草本作物，包含茄科植物，但其致病機制及系統性症狀的生成尚不明確。為探討CEVd是否可藉由番茄內源性的胞外泌體 （tomato-derived extracellular vesicles, tEVs）在植物體內進行傳播，我們從接種CEVd後六週的番茄莖及葉中分離tEVs，並分析顆粒大小及濃度、蛋白質及RNA的種類和含量，並使用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 進行 tEVs的型態觀察。我們發現CEVd 的核酸序列可在受CEVd感染的tEVs中被檢測到，顯示CEVd可能通過tEV進行宿主體內傳播。小片段RNA定序進一步顯示，CEVd感染的tEVs中包含來自CEVd的類病毒衍生之小RNA片段（viroid-derived small RNAs, vd-sRNAs），這些vd-sRNAs主要分布於類病毒基因體的變異區域，顯示具有專一性。標靶基因預測分析結果顯示vd-sRNAs主要針對參與植物免疫反應和植物賀爾蒙信號的mRNAs。除此之外，來自CEVd感染葉片的tEV中攜帶多種與分解代謝及防禦路徑相關的蛋白（如過氧化氫酶、類胰蛋白酶及病程相關蛋白），其表限量高於未接種組。為評估CEVd感染的tEVs的致病性，我們將受感染的tEVs重新接種至健康番茄，結果顯示，接種後4週的植株生物量及葉面積顯著低於對照組，且系統葉中的賀爾蒙相關基因表達量亦顯著下調。研究結果首次揭示CEVd可通過胞外泌體在番茄內進行傳播，並可能促進其系統性症狀的發展。

Viroids are a type of plant pathogens solely consisted of naked, circular single-stranded RNAs that do not encode any protein. Citrus exocortis viroid (CEVd) enables infecting not only citrus but also many Solanaceae spp. Nevertheless, the mechanistic nature of pathogenesis and systemic symptoms of CEVd remains largely unknown. First of all, to clarify whether CEVd enables systematic transmission through tomato-derived extracellular vesicles (tEVs) inside of plants, we isolated tEVs from upper stems or young leaves of tomato plants at six wpi of CEVd or mock. Despite of not much differences in the tEV particle concentration, protein, RNA amounts as well as morphology of tEV examined by transmission electron microscopy (TEM) between the CEVd and mock tomato plants, the CEVd RNA genome was detected in the CEVd-infected tEVs but not in the mock, implicating that CEVd might be able to transmit in the host plant via tEVs. Small RNA sequencing of CEVd-infected tEVs revealed viroid-derived small RNAs (vd-sRNAs) enriched in the variable region of the viroid RNA genome, those of which exhibit predicted targets predominantly involved in plant immune responses and hormone signaling. Additionally, tEVs derived from CEVd-infected leaves bear several catabolic and defense pathway-associated proteins by the proteomics analysis, including catalase, subtilisin-like protease, and pathogenesis-related (PR) proteins at a relatively high expression level compared to the mock counterpart. Furthermore, to speculate the pathogenicity of the CEVd-contained tEVs, we re-inoculated the CEVd-infected tEVs on tomatoes and subsequently found significant decreases in the biomass, leaf area, and downregulation of hormone-related genes in systemic leaves at 4 wpi compared to the mock control. Our study provides first and novel insight for the EV-mediated transmission of CEVd within infected tomatoes attributed to the systemic symptom development during CEVd pathogenesis.