Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways.

John M Smith,Michelle E Leslie,Tong Zhang,Antje Heese,Steven K Backues,David A Korasick,Samuel J Robinson,Abraham J Koo,Peter V Cornish,Sebastian Y Bednarek,Xin Li

doi:10.1371/journal.ppat.1004578

John M Smith, Michelle E Leslie + Show 9 more

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https://doi.org/10.1371/journal.ppat.1004578

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Abstract

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC −, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC−. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Highlights

Eukaryotes have developed highly effective immune mechanisms for protection against microbial pathogens
The plant receptor FLAGELLIN SENSING 2 (FLS2) recognizes the bacterial protein flagellin to initiate host defense responses contributing to immunity
We identify DynaminRelated Protein 2B (DRP2B), previously implicated in membrane trafficking in plants, as a novel component of defense responses against flagellin and bacterial Pseudomonas syringae strains in Arabidopsis thaliana

Summary

Introduction

Eukaryotes have developed highly effective immune mechanisms for protection against microbial pathogens. As the first line of defense, Pattern-Triggered Immunity (PTI) relies on the perception of conserved microbial features called Pathogen- (or Microbe-) Associated Molecular Patterns (PAMPs or MAMPs) by host receptors referred to as Pattern Recognition Receptors (PRRs) [1,2,3]. The bacterial PAMP flagellin is the main proteinaceous component of flagellum filaments essential for mobility of pathogenic bacteria such as Pseudomonades to infect hosts [4,5]. FLS2 undergoes ligand-induced endocytosis and trafficking through the Trans-Golgi Network/Early Endosomes (TGN/EE) and Multi Vesicular Bodies/Late Endosomes (MVB/ LE) for subsequent degradation [9,10,11,12,13,14].

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