AvrPtoBB728a 誘發番茄防禦反應功能區塊分析

Abstract

Pseudomonas syringae pv. tomato (Pst) DC3000是引起番茄細菌性斑點病的病原菌，亦為研究病原細菌與植物宿主間交互作用的模式生物之一。與其他植物病原細菌相似，Pst DC3000 會藉由第三型分泌系統將效應蛋白傳送至宿主細胞中，干擾植物基礎防禦反應或正常生理代謝途徑，造成宿主植物產生病徵。AvrPtoB 為 Pst DC3000 的第三型分泌系統效應蛋白，除了具有前述的致病力活性外，在抗性番茄品種中，AvrPtoB 會被 Pto 抗性蛋白辨認，進而引發過敏性反應 (hypersensitive response, HR)。在先前研究中發現，AvrPtoB 的序列廣泛存在於 Pseudomonas syringae 中，其中，豆科病原菌 P. s. pv. syringae (Psy) B728a 的 AvrPtoB 同源蛋白 (即 AvrPtoBB728a)，除了能誘發 Pto 所介導的抗性反應外，在沒有 Pto 基因的栽培番茄品種中也能引發 HR，此外透過基因靜默分析法發現此抗性與栽培品種中的 Pto 基因家族成員有關。本研究的目的在確認栽培番茄品種中能辨認 AvrPtoBB728a 而誘發抗性反應的抗性蛋白及找出能被成功辨認並誘發 HR 等抗性反應的 AvrPtoBB728a 最小片段為何。分別將 AvrPtoBB728a 之N 端或 C 端切除作出不同長度的片段後進行酵母菌雙雜交分析並結合病原性或 HR 測試，確認僅辨認 AvrPtoBB728a 而不會辨認AvrPtoB728a或 AvrPtoB 的 SlPtoB 應是栽培番茄品種中誘發 AvrPtoBB728a 介導抗性反應的抗性蛋白，此外更成功地找出 AvrPtoBB728a 被 SlPtoB 辨認所需要的最小片段為第 111–330 個胺基酸。在以抗性番茄 Rio-Grande PtoR 作為材料進行病原性測試時發現，AvrPtoBB728a 誘發 Pto 介導的抗性反應所需要的最小片段可能為第 111–215 個胺基酸。將 AvrPtoBB728a 與 AvrPtoB 的胺基酸序列進行比對後發現，兩者的 Pto-interacting domain (PID) 是相似的，但在 Fen (PtoB)-interacting domain (FID) 上，AvrPtoBB728a 需要涵蓋 AvrPtoB 的 PID 和 FID 之較長片段才得以被 SlPtoB 辨認。

Pseudomonas syringae pv. tomato (Pst) DC3000 is the causing agent of tomato bacterial speck disease, and also one of the model organisms for studying the interactions between phytopathogenic bacteria and their hosts. Similar to the other phytopathogenic bacteria, Pst DC3000 can also translocate effector proteins into its host cells via the type III secretion system, to interfere with defense responses and/or normal physiological/-metabolic pathways, leading to symptoms development on host plants. AvrPtoB is a type III secretion effector identified in Pst DC3000. In addition to the virulence activities mentioned above, AvrPtoB can also be recognized by the resistant protein Pto and triggers a hypersensitive response (HR). Previous studies have shown that the sequence of avrPtoB is widely present in Pseudomonas syringae, in which a homolog of AvrPtoB from a bean pathogen P. s. pv. syringae (Psy) B728a (hereafter AvrPtoBB728a), can also induced HR on cultivate tomato lacking Pto. This response is related to the Pto gene family members as determined by gene silencing analysis. The purpose of this study was to confirm the resistant (R) protein(s) responsible for recognizing AvrPtoBB728a to induce resistance responses in cultivate tomato. In addition, the region of AvrPtoBB728a required for interaction with the cognate resistant protein was also determined. The N- or C-terminal truncations of AvrPtoBB728a were constructed for yeast two-hybrid analysis as well as pathogenicity or HR test. The results demonstrated that SlPtoB, which can recognize only AvrPtoBB728a, but not AvrPtoB728a or AvrPtoB, is indeed the corresponding R protein for AvrPtoBB728a-induced resistance in cultivate tomato. The smallest region of AvrPtoBB728a required for SlPtoB recognition is amino acids 111–330. When tomato cultivar Rio-Grande PtoR was substituted for the pathogenicity assay, the fragment of AvrPtoBB728a sufficient for triggering Pto-mediated resistance could be amino acids 111–215. Amino acid sequence alignment of AvrPtoBB728a and AvrPtoB revealed that Pto-interacting domains (PID) of these two effectors are similar. However, AvrPtoBB728a interaction with SlPtoB requires longer fragment equivalent to a region covering the Fen-interacting domain (FID) and PID of AvrPtoB.