十字花科黑腐病菌XpsF與XpsL或XpsE蛋白交互作用之分析

Abstract

摘要 十字花科黑腐病菌為革蘭氏陰性細菌，會侵染白菜、甘藍、花椰菜等十字花科蔬菜造成黑腐病，其致病性依賴完整的第二型蛋白分泌系統，以將多種蛋白如果膠酵素、蛋白水解酵素等分泌至胞外，藉此感染植物。十字花科黑腐病菌的第二型蛋白分泌系統是由XpsD~O等十二個蛋白所組成，其中除了XpsF、XpsE 與XpsL蛋白外，絕大多數的蛋白區塊分佈於胞質周緣區至外膜上。XpsF為一個穿過內膜三次的蛋白，面向細胞質側由穿膜區可分割成兩個大的蛋白區塊，XpsFN與XpsFC；XpsE為細胞質蛋白，推測具有ATP-binding motif，且可藉由內膜蛋白XpsL附著於內膜上。本實驗利用大腸桿菌表現與GST融合之XpsFC蛋白，以及與數種不同標誌融合之XpsE、XpsEN、XpsEC、XpsFN與XpsLN等蛋白，分別利用親和性管柱層析下拉實驗，證實XpsFC/XpsFN、XpsFC/XpsLN、XpsFC/XpsE、XpsFC/XpsEN、以及XpsFC/XpsEC之間均具有蛋白交互結合關係，其中XpsFC/XpsEC之交互作用的關係於酵母菌雙雜交法中並未偵測到。此外，於黑腐病菌中大量表現XpsFC蛋白區塊，發現會抑制野生型菌株的分泌能力，同時大量表現全長或C端XpsE蛋白時，菌體的分泌能力則有明顯恢復，然而同時大量表現N端XpsL蛋白時並無效果。我們推論，XpsFC蛋白區塊仍部分保有XpsF蛋白與其他Xps蛋白交互作用的能力，因而未來可利用XpsFC之親和性管柱層析蛋白下拉實驗系統更進一步地分析XpsF蛋白之功能。

Abstract Xanthomonas campestris pv. campestris is a Gram-negative bacteria that infect crucifers and cause black-rot. Ability to secrete proteins extracellularly by the type II secretion apparatus is critical for its pathogenicity. Among the 12 members (XpsD-O) in secretion apparatus, most proteins have no major domains located at the cytoplasmic side except for XpsF, XpsE, and XpsL. XpsF is predicted to be a polytopic inner membrane protein with two large cytoplasmic-located domains connected by three trans-membrane regions. XpsE is a cytoplasmic protein with an ATP-binding motif and is associated with inner membrane through interaction with an inner membrane protein, XpsL. By yeast two-hybrid analyses conducted previously, the C-terminal domain of XpsF (XpsFC) was revealed interacting with the N-terminal domains of itself (XpsFN), of XpsE (XpsEN), and of XpsL (XpsLN) as well. To further characterize those physical interactions, we expressed GST-fused XpsFC and various fusion proteins of XpsEN, XpsFN, or XpsLN, in E. coli. In vitro affinity pull-down assay demonstrated that there are associations between XpsFC/XpsE, XpsFC/XpsEN, XpsFC/XpsEC, XpsFC/XpsFN, and XpsFC/XpsLN, although interaction between XpsFC and XpsEC was not detected previously in the yeast two-hybrid experiment. Overexpression of XpsFC fragments in the wild type Xanthomonas trans-dominantly interfere secretion of -amylase. Such inhibitory effect was partially lessened when XpsE or XpsEC, but not XpsLN, was co-expressed. We conclude that XpsFC still preserve part of the interaction ability of XpsF, therefore the in vitro pull-down assay can be used to further dissect functions of XpsF.