鑑定與研究青枯病菌效應蛋白 RipBJ 之植物交互作用蛋白質

Abstract

青枯病菌 (Ralstonia solanacearum, Rs) 之寄主範圍廣泛，常造成全球大規模農作物經濟損失，然而目前關於青枯病菌毒力之分子機制瞭解仍有限。本研究室先前發現 Rs 效應蛋白基因 ripBJ 僅存於低/中毒力菌株；在中毒力菌株Pss4中將 ripBJ 剔除會提高 Pss4 之致病力，而在原本無 ripBJ 的高毒力菌株Pss190中大量表現 ripBJ 基因則可降低 Pss190 之致病力，故 RipBJ 之存在會導致 Rs 致病力下降；且在植物大量表現 ripBJ 後會提升番茄對多種特性迥異之重要病菌的防禦力。此外，位於番茄細胞膜之 NADPH oxidase SlWfi1 (SlRbohB) 與 RipBJ 有直接交互作用且正向調控 RipBJ 所誘導之植物反應。本研究旨在藉由研究RipBJ之植物交互作用蛋白探討其誘發植物防禦反應之作用機制。雙分子螢光互補實驗結果顯示 RipBJ 會藉由N端1-47氨基酸 (RipBJ 1-47aa) 與 SlWfi1 之N端1-233氨基酸進行交互作用。在圓葉菸草中大量表現 SlWfi1 會促進 RipBJ 誘導之植物細胞凋亡。靜默圓葉菸草之 SlWfi1 同源蛋白質 NbRbohB 基因可能會降低 RipBJ 在植物中之蛋白質累績。經由NADPH Oxidase酵素活性實驗發現 RipBJ 1-47aa 足以穩定SlWfi1 之NADPH oxidase酵素活性。RT-qPCR結果顯示感染青枯病菌會影響番茄根部及莖基部SlWfi1之轉錄表現量。為進一步探討 RipBJ 是否有其他作用途徑，透過共免疫沈澱與奈流液相層析串連二次質譜 (Co-IP-LC-MS/MS) 已篩選出數個可能與 RipBJ 有交互作用之番茄蛋白質，包含SlHCF164、SlCYP1及SlCAT2。雙分子螢光互補實驗結果顯示三個候選蛋白質可能會與RipBJ交互作用，且在圓葉菸草中大量表現SlHCF164會促進RipBJ 誘導之植物細胞凋亡，而將SlCYP1 之圓葉菸草同源基因 NbCysP6 在圓葉菸草中基因靜默後則會抑制RipBJ 誘導之植物細胞凋亡，雖RipBJ 是否確實與之有交互作用尚待後續驗證，這兩個蛋白質在 RipBJ 誘導之植物細胞凋亡上扮演正調控角色。本研究之結果將有助於瞭解青枯病菌致病力與植物防禦之分子機制。

Ralstonia solanacearum (Rs) causes bacterial wilt (BW), a vascular disease leading to severe global crop losses. However, information about how virulence-related factors function is still limited. Our previous studies showed that R. solanacearum effector RipBJ is present in low/medium-virulence strains and it plays a crucial negative role in virulence. RipBJ expression in tomato and Nicotiana benthamiana enhances plant defense against distinct important pathogens by modulating multiple plant defense signaling pathways. Furthermore, tomato NADPH oxidase SlWfi1 interacts with RipBJ, and plays a positive role in RipBJ-triggered effects on plants. This study aimed to elucidate the mechanism underlying the RipBJ-triggered defense by studying its interacting proteins in plants. Bimolecular fluorescence complementation (BiFC) assays showed that the interacting domains of RipBJ and SlWfi1 are 1-47 aa and 1-233 aa, respectively. Overexpression of SlWfi1 in N. benthamiana promotes RipBJ-induced plant cell death. Silencing of the homolog of SlWfi1 in N. benthamiana, NbRbohB, might affect the accumulation of RipBJ in planta. In vitro NADPH oxidase assay showed that RipBJ1-47 is sufficient to prolong the enzymatic activity of SlWfi1. Expression of SlWfi1 was differentially expressed in tomato root and stem-base after R. solanacearum infection. Furthermore, SlHCF164, SlCYP1 and SlCAT2 are candidate interacting proteins of RipBJ identified by Co-IP-LC-MS/MS. BiFC assays revealed these proteins may interact with RipBJ. Overexpression of SlHCF164 in N. benthamiana promoted RipBJ-induced plant cell death, and silencing of the homolog of SlWfi1 in N. benthamiana, NbCysP6, reduced RipBJ-induced plant cell death. Although the authentic interactions between RipBJ and these proteins remains to be determined further, SlHCF164 and NbCysP6 have a positive role in RipBJ-induced plant cell death. These results are expected to shed light on mechanisms involved in R. solanacearum virulence and plant defense.