The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 (also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7’s ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.
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We thank the members of the Alfano laboratory for many fruitful discussions, Y. Zhou and C. Elowsky for technical assistance with confocal microscopy, T. Clemente and S. Sato for constructing transgenic plants, G. Li and C. Bryan for assistance in the identification of the A. thaliana grp7 mutants, A. Collmer for reviewing the manuscript, P. Seitz for assistance in plasmid constructions, and J. T. Barbieri for help initiating the ADP-RT assays in our laboratory. We are grateful to the Ohio State University Arabidopsis Biological Resource Center, the Salk Institute Genomic Analysis Laboratory, and the Arabidopsis research community for providing the Arabidopsis SALK lines used in this study. This research was supported by grants from the National Science Foundation and the National Institutes of Health, and funds from the Plant Science Initiative at the University of Nebraska to J.R.A, and a grant from the German Research Council to D.S.
Author Contributions Z.Q.F. constructed the DC3000 ΔhopU1 mutant, made the transgenic HopU1–HA-expressing A. thaliana plants, and performed the experiments in Figs 1a, b; 2a, b; 3; 4a, d and Supplementary Figs 1, 2, 4, 5b–d and 6d; M.G. identified the homozygous A. thaliana grp7 mutant plants, cloned the HopU1–His substrate complementary DNAs, and performed the experiments in Figs 2c, d and 5 and Supplementary Figs 3, 4, 6a–c and 7; B.-r.J. performed the experiments in Figs 1c and 4c and Supplementary Fig. 5a, d; and F.T. provided technical support for several experiments. T.E.E. helped direct the identification of the HopU1–His substrates; R.L.C. performed the mass spectrometry and peptide database searches; D.S. provided the anti-GRP antibody, plasmids pGRP7-Gly and pGRP7-RRM, and insights on RNA-binding proteins; J.R.A. helped design the experimental plan, designed Supplementary Fig. 8, and was the primary writer of the paper. All of the authors discussed the results and commented on the paper.
This file contains Supplementary Methods, Supplementary Tables 1-3, Supplementary Figures 1-8 with Legends, Supplementary Notes and additional references. Supplementary Methods section describes materials and procedures. The Supplementary Tables contain a list of the identified HopU1 substrates (Supplementary Table 1), bacterial strains and plasmids used in this study (Supplementary Table 2), and the nucleotide sequences of the primers used in this study. The Supplementary Figures show that HopU1 is injected into plant cells by the type III secretion system of P. syringae and that a hopU1 mutant is reduced in virulence (Supplementary Fig. 1); that HopU1 suppresses ion leakage in plants (Supplementary Fig. 2); that HopU1-expressing plants are altered in the AvrRpt2-dependent HR (Supplementary Fig. 3); representative two-dimensional PAGE gels that led to the identification of HopU1 substrates (Supplementary Fig. 4); localization of HopU1 and HopU1 substrates in plant cells (Supplementary Fig. 5); data showing that an A. thaliana Col-0 knock-out mutant is homozygous for the T-DNA insert in the AtGRP7 locus (Supplementary Fig. 6); Similar data for an independent Atgrp7 mutant and pathogenicity-related phenotypes (Supplementary Fig. 7); and a proposed model of suppression of plant innate immunity by HopU1.