以蛋白體學分析菸草中參與Elicitin作用之SlSOBIR1交互作用蛋白

Abstract

植物藉由細胞膜上之pattern recognition receptor (PRRs)辨識病原保守性構造pathogen/microbe-associated molecular patterns (PAMPs/MAMPs)以啟動PAMP-triggered immunity (PTI)。受到PAMPs活化之PRRs通常會與其他leucine-rich repeat (LRR)-receptor-like kinases (RLKs)交互作用，如BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1)及SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1)，進而啟動PTI下游防禦反應。Elicitins為疫病菌及腐黴菌特有的外泌性elicitor蛋白，能誘發部分雙子葉植物產生過敏性反應 (hypersensitive response, HR)，引起細胞壞疽性死亡。本實驗室近期發現番茄SlSOBIR1與SlSOBIR1-like參與Phytophthora parasitica elicitin ParA1引發壞疽的反應途徑，為能進一步瞭解SlSOBIR1在植物接收ParA1後相關的反應途徑，本研究以Nicotiana benthamiana為研究系統，透過共免疫沉澱與質譜儀分析，分析處理與未處理ParA1時之SlSOBIR1交互作用蛋白體，總共發現157個SlSOBIR1免疫共沉澱蛋白，其中25個蛋白只專一性的出現於ParA1處理組。基因註解分析發現三個biological process Gene ontology (GO) terms在ParA1處理時顯著提升，包含cell communication, response to endogenous stimulus以及 response to stress。為驗證這些基因是否參與ParA1誘導之過敏性反應，進一步以TRV-mediated gene silencing靜默其中11個基因，並於菸草葉片短暫表現ParA1，結果發現降低NbArcA2及NbGBLP (兩者皆為guanosine nucleotide-binding proteins, G protein)，NbPDR1 [ATP-binding cassette tranporter (ABC) transporter]、NbPR10 [pathogenesis-related (PR) protein]，NbOligoA (Oligopeptidase A)與NbrbohB (NADPH oxidase)的表現量皆延遲及減少ParA1引發的細胞壞疽現象，其中尤以NbPDR1的作用效果最為顯著。

Recognition of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) by plasma membrane-localized pattern recognition receptors (PRRs) is the initial step to activate PAMP-triggered immunity (PTI) in plants. In addition to PRRs, other leucine-rich repeat receptor-like kinases are known to play important roles in PTI, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1), and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR), which interact with the receptors to regulate downstream defense responses. Recently, tomato homologs of SOBIR1 (known as SlSOBIR1 and SlSOBIR1-like) are known to mediate plant basal defense against Phytophthora parasitica. Furthermore, they are involved in the signaling of ParA1, an elicitin of P. parasitica. In this study, coimmunoprecipitation (co-IP) and mass spectrometry were performed to identify SlSOBIR1-interacting proteins to find out how SlSOBIR1 mediates ParA1 signaling in Nicotiana benthamiana. A total of 157 SlSOBIR1-interacting proteins were identified, of which 25 of them are associated with SlSOBIR1 only in the presence of ParA1. GO term analysis indicated that three biological process terms were specifically enriched in response to ParA1 treatment, including cell communication, response to endogenous stimulus, and response to stress. To uncover their roles, we silenced 11 of the 25 genes by TRV-mediated gene silencing, and then expressed ParA1 by agroinfiltration. ParA1-induced necrosis is compromised upon silencing of NbArcA2 and NbGBLP (guanosine nucleotide-binding proteins, G protein), NbPDR1 [ATP-binding cassette tranporter (ABC) transporter], NbPR10 [pathogenesis-related (PR) protein], NbOligoA (Oligopeptidase A), and NbrbohB (NADPH oxidase), with silencing of NbPDR1 showing the most prominent effect.