N-Glycosylation of Effector Proteins by an α-1,3-Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity

Xiao-Lin Chen,Wei Shi,Xiaowen Xu,Deng Chen,Xusheng Gao,Nicholas J Talbot,Jun Yang,Tao Shi,Jin-Rong Xu,You-Liang Peng

doi:10.1105/tpc.114.123588

Abstract

Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.

Highlights

Plants defend themselves from microbial attack using pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) to initiate innate immune responses (Chisholm et al, 2006; Jones and Dangl, 2006)
We demonstrated that ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3) is required for full N-glycosylation of two effector proteins, Secreted LysM Protein1 (Slp1) and Bas4, and the vacuolar protein CYP
We showed that oligosaccharides linked to Slp1 or Carboxypeptidase Y (CPY) in the ALG3 deletion mutant could not be digested by the enzyme endoglycosidase H (Endo H); it can be postulated that they are Man5-oligosaccharides but not Man9-oligosaccharides in the wild type

Summary

Introduction

Plants defend themselves from microbial attack using pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) to initiate innate immune responses (Chisholm et al, 2006; Jones and Dangl, 2006). Immune reactions include the regulated synthesis of reactive oxygen species (ROS), which can be induced by the recognition of cell wall PAMPs, such as oligosaccharides originating from chitin and glucans, released by invading pathogens during infection (Kaku et al, 2006). In response to these defenses, plant pathogens have developed strategies to avoid or overcome PAMP-triggered immunity. Secreted proteins, including cell wall glycoproteins and effector proteins, are synthesized in the endoplasmic reticulum (ER), where they often undergo N-glycosylation and/or O-glycosylation (Helenius and Aebi, 2004). Enzymes involved in protein N-glycosylation and glycoproteins that are N-glycosylated have been widely identified and found to play vital roles in diverse aspects of development and physiology, including salt tolerance and plant immunity (Lerouge et al, 1998; Kang et al, 2008; Qin et al, 2008; Liebminger et al, 2009; Saijo et al, 2009; Häweker et al, 2010; Liu and Howell, 2010; Ruiz-May et al, 2012)

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