Physical Association of Arabidopsis Hypersensitive Induced Reaction Proteins (HIRs) with the Immune Receptor RPS2

Yiping Qi,Kenichi Tsuda,Le V Nguyen,Xia Wang,Jinshan Lin,Angus S Murphy,Jane Glazebrook,Hans Thordal-Christensen,Fumiaki Katagiri

doi:10.1074/jbc.m110.211615

Abstract

Arabidopsis RPS2 is a typical nucleotide-binding leucine-rich repeat resistance protein, which indirectly recognizes the bacterial effector protein AvrRpt2 and thereby activates effector-triggered immunity (ETI). Previously, we identified two hypersensitive induced reaction (AtHIR) proteins, AtHIR1 (At1g09840) and AtHIR2 (At3g01290), as potential RPS2 complex components. AtHIR proteins contain the stomatin/prohibitin/flotillin/HflK/C domain (also known as the prohibitin domain or band 7 domain). In this study, we confirmed that AtHIR1 and AtHIR2 form complexes with RPS2 in Arabidopsis and Nicotiana benthamiana using a pulldown assay and fluorescence resonance energy transfer (FRET) analysis. Arabidopsis has four HIR family genes (AtHIR1-4). All AtHIR proteins could form homo- and hetero-oligomers in vivo and were enriched in membrane microdomains of the plasma membrane. The mRNA levels of all except AtHIR4 were significantly induced by microbe-associated molecular patterns, such as the bacterial flagellin fragment flg22. Athir2-1 and Athir3-1 mutants allowed more growth of Pto DC3000 AvrRpt2, but not Pto DC3000, indicating that these mutations reduce RPS2-mediated ETI but do not affect basal resistance to the virulent strain. Overexpression of AtHIR1 and AtHIR2 reduced growth of Pto DC3000. Taken together, the results show that the AtHIR proteins are physically associated with RPS2, are localized in membrane microdomains, and quantitatively contribute to RPS2-mediated ETI.

Highlights

Doctoral dissertation fellowship from the University of Minnesota. 2 To whom correspondence should be addressed: Dept. of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, 1500 Gortner Ave., St
To test the specificity of the obtained anti-AtHIR1 antibody, all four AtHIR proteins were tagged with a Myc epitope tag at the C terminus and expressed in N. benthamiana leaves through Agrobacterium-mediated transient expression [48]
To examine whether the Arabidopsis AtHIR proteins are induced by infection with P. syringae strains, Col wild-type plants were inoculated with pv. tomato (Pto) DC3000 AvrRpm1, Pto DC3000 AvrRpt2, Pto DC3000, and Pto DC3000 hrcC strains

Summary

Introduction

Doctoral dissertation fellowship from the University of Minnesota. 2 To whom correspondence should be addressed: Dept. of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, 1500 Gortner Ave., St. We present a biochemical and genetic study of the Arabidopsis HIR (AtHIR) gene family, which suggests that at least some AtHIR proteins are physically associated with RPS2 and involved in RPS2-mediated ETI. Anti-Myc antibody detected all four AtHIR-Myc proteins, and no signal was detected in the negative control sample (supplemental Fig. S2).

Results

Conclusion