番茄黃化捲葉泰國病毒隱藏開放閱讀框之功能性分析

Abstract

病毒基因組通常相當精簡，並經常透過非典型轉譯 (non-canonical translation) 策略擴增其編碼能力，使得病毒基因體中具功能性的編碼區域難以被完整鑑定。近年研究顯示，許多過去未被發現的隱藏開放閱讀框 (hidden open reading frames, ORFs) 實際上可編碼具有生物學功能之蛋白質，並參與病毒感染過程。在雙分體 (bipartite) begomovirus 番茄黃化捲葉泰國病毒 (tomato yellow leaf curl Thailand virus, TYLCTHV) 基因組中，已鑑定出多個候選隱藏 ORFs，然而其生物學功能大多仍尚未釐清。本研究進一步針對 DNA-A 上九個功能尚未明確的候選 ORFs 進行分析。突變分析結果顯示，多數候選 ORFs 之突變會降低病毒在 Nicotiana benthamiana 與 Solanum lycopersicum cv. Micro-Tom 所造成的病徵嚴重程度與病毒 DNA 累積量，顯示其可能參與病毒感染與致病過程。其中，AV2-135 與 AV2-189 為經由不同轉譯起始點所產生之兩個 AV2 蛋白異構體。雖然 AV2-135 被認為是主要表現之 AV2 異構體，但 AV2-189 突變同樣會顯著降低病徵發展與病毒 DNA 累積量。進一步分析顯示，AV2-189 參與病毒感染早期之 DNA 累積，並影響後續系統性感染。蛋白質交互作用分析顯示，AV2-135 可與多個病毒蛋白產生交互作用並具有自體交互作用能力；相較之下，AV2-189 僅與 AV2-135 產生交互作用。此外，共表達試驗進一步顯示，AV2-189 可調節 AV2-135 之自體交互作用，以及 AV2-135 與 AC4 之交互作用。另一方面，在 GFP 基因靜默抑制試驗中，AV2-135 與 AV2-189 均未呈現明顯且穩定之 RNA silencing suppressor (RSS) 活性。進一步分析植物免疫反應發現，於菸草植株中瞬時表達 AV2-135 或 AV2-189，皆可降低水楊酸 (salicylic acid, SA) 反應標誌基因 PATHOGENESIS-RELATED PROTEIN 1 (PR1) 之表現。此外，表達 AV2-189 之 Micro-Tom 轉殖株於 SA 處理後，其 PR1 與 PR5 表現量皆顯著低於野生型植株，顯示 AV2-189 可能參與調控寄主 SA 相關防禦反應。綜合而言，本研究顯示多個候選 ORFs 參與 TYLCTHV 之感染與致病過程。其中，AV2-189 為具有生物學功能之隱藏 ORF，可參與影響病毒 DNA 累積、蛋白質交互作用及寄主防禦反應，進一步提供對 begomovirus 中 AV2 蛋白異構體功能多樣性之新見解。

Plant viruses possess compact genomes and often employ non-canonical translation strategies to expand their coding capacity, making the identification of all functional coding regions challenging. Increasing evidence indicates that many previously unrecognized hidden open reading frames (ORFs) encode functional proteins involved in viral infection. Recently, multiple candidate hidden ORFs were identified in the genome of tomato yellow leaf curl Thailand virus (TYLCTHV), a bipartite begomovirus; however, the biological roles of most of these ORFs remain largely unknown. In this study, nine candidate ORFs located on the DNA-A component of TYLCTHV were investigated. Mutational analyses revealed that disruption of most candidate ORFs reduced symptom severity and viral DNA accumulation in both Nicotiana benthamiana and Solanum lycopersicum cv. Micro-Tom, suggesting their involvement in viral infection and pathogenicity. Among the identified hidden ORFs, AV2-135 and AV2-189 represent two AV2 protein isoforms generated through alternative translation initiation. While AV2-135 is considered the major AV2 isoform, mutational analyses demonstrated that disruption of AV2-189 significantly reduced symptom development and viral DNA accumulation. Further analyses showed that AV2-189 contributes to early viral DNA accumulation and systemic infection. Protein-protein interaction assays demonstrated that AV2-135 interacted with multiple viral proteins and exhibited self-interaction, whereas AV2-189 interacted only with AV2-135. Co-expression experiments further revealed that AV2-189 modulates AV2-135 self-interaction as well as the interaction between AV2-135 and AC4. In contrast, neither AV2-135 nor AV2-189 exhibited obvious and reproducible RNA silencing suppressor activity in the GFP-based silencing suppression assay. Transient expression assays in N. benthamiana revealed that both AV2-135 and AV2-189 reduced the expression of the salicylic acid (SA)-responsive marker gene PATHOGENESIS-RELATED PROTEIN 1 (PR1). Furthermore, transgenic Micro-Tom plants expressing AV2-189 exhibited reduced expression of PR1 and PR5 following SA treatment, suggesting that AV2-189 may modulate SA-related defense responses. Together, these findings demonstrate that multiple candidate ORFs contribute to TYLCTHV infection and pathogenicity. In particular, AV2-189 functions as a biologically relevant hidden ORF that affects viral DNA accumulation, protein interaction networks, and host defense responses, providing new insights into the functional diversity of AV2 isoforms in begomoviruses.