以病毒基因靜默技術驗證候選轉錄因子基因在日日春對抗日日春葉片黃化病植物菌質體之角色及日日春ARF17於植物抗病反應中的可能角色

Abstract

植物菌質體是一種缺少細胞壁的絕對寄生植物病源細菌，引起的常見病徵有枝條增生、花器綠化及花器葉化，並對作物造成危害進而造成經濟上的損失。釐清植物與植物菌質體之間的交互作用有助於發展控制此類病害的策略。先前的實驗中發現日日春感染日日春葉片黃化病菌質體（Periwinkle leaf yellowing phytoplasma，PLY phytoplasma）後，日日春會產生系統性的抗病反應，在有病徵及無病徵枝條並且都有誘導CrPR1a的表現，本實驗室利用病毒誘導基因靜默技術（virus-induced gene silencing，VIGS）篩選出11個潛在調控CrPR1a的日日春轉錄因子，包含五個ARF、3個bZIP及兩個AP2/ERF和1個WRKY。本研究進一步利用VIGS來驗證這些轉錄因子是否參與日日春抵抗植物菌質體的防禦反應，我們發現靜默CrWRKY31或CrTINY2會造成植物大量累積水楊酸並抑制TRV的累積，然而若基因靜默植物感染日日春葉片黃化病，其病程反而被加速。這顯示在日日春可能有與SA無關的抗植物菌質體防禦途徑而CrTINY2及CrWRKY31可能也參與其中。先前實驗發現靜默CrARF17之後降低了CrPR1a的表現並加速日日春葉片黃化病的發展，為了更了解CrARF17在植物抗病反應上的角色，我們將CrARF17與其同源的AtARF17於阿拉伯芥上過量表現，過量表現CrARF17的阿拉伯芥展現出捲葉及果莢短小的性狀，與文獻中的過量表現AtARF17的阿拉伯芥性狀相似，顯示CrARF17在阿拉伯芥上與AtARF17仍有相似的功能。在接種CMV的試驗中發現植物產生強烈的免疫反應，並且誘導了的AtPR1的表現，但是過量表現CrARF17及AtARF17在病毒的累積量上則沒有明顯的抑制效果。本研究找到兩個可能參與日日春抵抗菌質體的轉錄因子以及發現過量表現CrARF17會引發強烈的免疫反應但對於植物抗病毒的反應沒有明顯的幫助。

Phytoplasma, a wall-less obligate bacterial plant parasite, can cause symptoms of witches’-broom, virenscence, as well as phyllody, and result in economic losses. Understanding plant-phytoplasma interactions should help us to develop new control strategies of this pathogen. We found that when Catharanthus roseus (periwinkle) plants are infected with periwinkle leaf yellowing (PLY) phytoplasma, they show systemic defense responses, which was proven by the finding of CrPR1a induction in non-symptomatic shoots. Previously, potential CrPR1a regulatory transcription factors, including 5 ARFs, 3 bZIP, 2 AP2/ERF and 1 WRKY, have been identified using virus-induced gene silencing (VIGS). In this study, I further confirmed whether changes of their transcript abundance affect plant defense against phytoplasma in periwinkle using VIGS. Silencing of CrWRKY31 and CrTINY2 changed the accumulation of SA and resulted in repression of TRV accumulation in periwinkles; however, symptom progression of PLY was accelerated in these plants when inoculated with PLY phytoplamsa. It is possible that CrTINY2 and CrWRKY31 could be involved in a SA-independent defense pathway that is also important for plant to defend against phytoplasma. Previously, it was found that silencing of CrARF17 repressed CrPR1a indcution and accelerated symptom progression of PLY. To understand more about CrARF17 in plant defense responses, it was ectopically expressed in Arabidopsis thaliana and compared its function with its counterpart, AtARF17. It is found that ectopic expression of CrARF17 resulted in curly leaves and short siliques. The phenotypical changes were resemble the phenotype of transgenic plants harbored 35S::5mARF17. These results suggest that CrARF17 shares similar functions with AtARF17. The transgenic plants showed strong immune responses and induction of AtPR1; however, no significant changes in CMV accumulation in these transgenic plants.