探討類熱休克因子轉錄因子在植物抗病毒免疫中的功能與分子機制

Abstract

內質網（endoplasmic reticulum, ER）在植物細胞中扮演蛋白質的合成、折疊與修飾的角色。然而，當植物遭受病毒入侵等生物性逆境時，大量錯誤或未完成折疊的蛋白質會在內質網中堆積造成內質網壓力（ER stress）。為了維持ER的恆定，植物會啟動未摺疊蛋白反應（unfolded protein response, UPR），以維持蛋白質摺疊恆定性並調控植物免疫反應。先前研究已鑑定出兩條主要的 UPR 訊號分支IRE1/bZIP60 與 bZIP17/bZIP28能正向調控植物的抗病毒反應。除這些既有途徑外，亦有研究指出，類熱休克轉錄因子 TBF1（TL1 binding factor 1）是 UPR 中另一個重要的關鍵調控因子。本研究發現，在菸草鑲嵌病毒（Tobacco mosaic virus, TMV）感染期間，TBF1 的表現受到抑制。病毒累積分析顯示，tbf1 突變株中的 TMV 累積量顯著降低，而 TBF1 過量表現植株中的病毒累積量則顯著增加，顯示 TBF1 為 TMV 病毒累積的正向調控因子。為了進一步釐清 TBF1 影響病毒累積量的機制，我們探討 TBF1 是否會影響病毒複製複合體（viral replication complex, VRCs）的形成，結果顯示tbf1突變株原生質體 TMV-MP 所形成的點狀訊號明顯減少，顯示 TBF1 有助於病毒累積。進一步地，我們利用轉錄體分析（RNA-seq）比較野生型與 tbf1 突變株在病毒感染下的基因表現差異，結果顯示tbf1突變體中RNA干擾（RNA interference, RNAi）相關基因SGS3、RDR6與DCL4表現上升，顯示其可能增強抗病毒RNAi反應。轉錄體分析結果進一步顯示，在 TMV 感染條件下，tbf1 突變株中與水楊酸（salicylic acid, SA）訊息傳遞及 RNA 干擾（RNA interference, RNAi）相關的防禦基因表現量上升，而與生長素（auxin）相關的基因表現量則下降，顯示TBF1在植物中可能扮演連結ER壓力訊號傳遞、病毒複製以及調控RNAi基因的角色。雙螢光素酶報告實驗 （dual-luciferase reporter assay）進一步顯示 TBF1 可降低 CBP60g 啟動子活性，暗示 TBF1 可能參與負向調控 CBP60g 的轉錄以調控SA介導的防禦反應。綜合以上結果，本研究推測 TMV 感染所誘導的 TBF1 下調可能是宿主將轉錄調控重心由生長轉向抗病毒防禦的一種策略。研究結果顯示，TBF1 在病毒感染期間作為調控植物生長及防禦（growth-defense trade-off）的重要節點，並揭示 TBF1 在植物與病毒交互作用中的新功能，以及 UPR 相關轉錄調控與抗病毒防禦之間潛在連結的新見解。

The endoplasmic reticulum (ER) plays a central role in protein synthesis, folding, and modification in plant cells. Under biotic stress such as viral infection, misfolded proteins accumulate in the ER, leading to ER stress and activation of the unfolded protein response (UPR), which restores protein-folding homeostasis and modulates immune responses. Two major UPR signaling branches, IRE1/bZIP60 and bZIP17/bZIP28, have been shown to positively regulate antiviral defense in plants. In addition, the heat shock factor–like transcription factor TBF1 (TL1 binding factor 1) has recently been suggested to function in UPR regulation, although its role in plant antiviral immunity remains unclear. Here, we show that TBF1 expression is induced by salicylic acid (SA) but suppressed during tobacco mosaic virus (TMV) infection. TMV accumulation is reduced in tbf1 mutants but increased in TBF1-overexpressing Arabidopsis, suggesting that TBF1 negatively regulates TMV resistance. Further analysis revealed that TBF1 promotes viral replication complex (VRC) formation, as indicated by reduced TMV-MP:mCherry signals in tbf1 protoplasts. Moreover, the tbf1 mutant exhibits elevated expression of the RNA interference-related gene SGS3, RDR6 and DCL4, potentially enhancing antiviral RNAi activity. Transcriptome analysis further showed upregulation of SA-related genes (CBP60g, NPR1, and SARD1) and downregulation of auxin-related genes in tbf1 mutants during TMV infection. Dual-luciferase assays demonstrated that TBF1 suppresses the promoter activity of CBP60g, suggesting that TBF1 may negatively regulate SA-mediated defense signaling through transcriptional repression of key immune regulators. Collectively, these findings indicate that TBF1 functions as a negative regulator of antiviral immunity by suppressing SA- and RNAi-associated defense pathways while promoting TMV replication. This study reveals a previously unrecognized role of TBF1 in plant–virus interactions and provides new insights into the complex relationship between UPR-associated transcriptional regulation and antiviral defense.