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Different Requirements for EDS1 and NDR1 by Disease Resistance Genes Define at Least Two R Gene-Mediated Signaling Pathways in Arabidopsis

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Different Requirements for EDS1 and NDR1 by Disease Resistance Genes Define at Least Two R Gene-Mediated Signaling Pathways in Arabidopsis Proc. Natl. Acad. Sci. USA Vol. 95, pp. 10306–10311, August 1998 Plant Biology Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis NICOLE AARTS*, MATTHEW METZ†,ERIC HOLUB‡,BRIAN J. STASKAWICZ†,MICHAEL J. DANIELS*, AND JANE E. PARKER*§ *Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom; †Department of Plant Biology, Koshland Hall, University of California, Berkeley, CA 94720-3102; and ‡Horticultural Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom Edited by Frederick M. Ausubel, Harvard Medical School, Boston, MA, and approved June 11, 1998 (received for review April 3, 1998) ABSTRACT The Arabidopsis genes EDS1 and NDR1 were localized either extracellularly or within the cytoplasm are shown previously by mutational analysis to encode essential structurally different to Pto and contain leucine-rich repeats components of race-specific disease resistance. Here, we ex- (LRRs) (5), implicating protein–protein interactions as part of amined the relative requirements for EDS1 and NDR1 by a the recognition process. In the putatively cytoplasmic R pro- broad spectrum of Resistance (R) genes present in three tein members, the LRRs lie adjacent to sequences that con- Arabidopsis accessions (Columbia, Landsberg-erecta, and stitute a nucleotide binding site (NBS) (5). The NBSyLRR Wassilewskija). We show that there is a strong requirement class of R proteins can be subdivided into members that for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and possess an amino-terminal leucine zipper (LZ) motif or those RPS4), conferring resistance to the biotrophic oomycete Per- that have amino-terminal similarity to the cytoplasmic do- onospora parasitica, and to Pseudomonas bacteria expressing mains of the Drosophila Toll and mammalian interleukin 1 the avirulence gene avrRps4. The requirement for NDR1 by transmembrane receptors (referred to as the ‘‘TIR’’ domain) these EDS1-dependent R loci is either weak or not measurable. (1, 6). Conversely, three NDR1-dependent R loci, RPS2, RPM1, and The representation of a limited number of common struc- RPS5, operate independently of EDS1. Another RPP locus, tural motifs in the R proteins identified so far raises the RPP8, exhibits no strong exclusive requirement for EDS1 or possibility that they may function, at least in part, by similar NDR1 in isolate-specific resistance to P. parasitica, although signaling mechanisms. Indeed, the high level of sequence resistance is compromised weakly by eds1. Similarly, resis- conservation between the tobacco N gene conferring resis- tance conditioned by two EDS1-dependent RPP genes, RPP4 tance to a virus (7), the flax L6 gene specifying rust fungus and RPP5, is impaired partially by ndr1, implicating a degree resistance (8), and the Arabidopsis RPP5 gene for resistance to of pathway cross-talk. Our results provide compelling evi- an oomycete pathogen (9) suggests conservation of resistance dence for the preferential utilization of either signaling com- pathways between different plant species and differing patho- ponent by particular R genes and thus define at least two gen types. A major goal now is to understand how early R disease resistance pathways. The data also suggest that strong protein-mediated recognition events lead to effective resis- dependence on EDS1 or NDR1 is governed by R protein tance. structural type rather than pathogen class. The model crucifer Arabidopsis has been exploited as a host for bacterial, viral, fungal, and oomycete pathogens (10), and Disease resistance in plants commonly is specified by geneti- analysis of natural genetic variation between different plant cally paired products that are encoded by plant resistance (R) accessions and pathogen isolates has led to the identification genes and pathogen avirulence (avr) genes (1). Susceptibility of distinct R loci. The cloned RPS2 and RPM1 genes, control- to a particular pathogen race only can occur when either one ling race-specific resistance to the bacterial pathogen Pseudo- y of the genetic components is missing or inactivated. In many monas syringae, encode proteins of the LZ-NBS LRR class plant-pathogen combinations, this type of resistance is asso- (11–13). In contrast, the RPP5 gene mediating isolate-specific ciated with localized cell necrosis at the site of attempted resistance to the biotrophic oomycete Peronospora parasitica y pathogen entry (the hypersensitive response). However, the encodes a protein of the TIR-NBS LRR class (9). precise mechanisms that lead to pathogen containment are not Mutational analyses in Arabidopsis have identified other understood. wild-type genes that are required for R gene-mediated resis- The cloning of avr genes from prokaryotic and eukaryotic tance (14). A mutation in NDR1 (nonrace-specific disease plant pathogens and, more recently, corresponding R genes resistance) abolished resistance conferred by RPS2 and RPS5 from several different plant species has provided some impor- to P. syringae expressing avrRpt2 and avrPph3, respectively, as tant insights to the process of pathogen recognition. For well as a dual specificity resistance encoded by RPM1 to the example, the tomato Pto gene specifying resistance to a bacterial genes avrB and avrRpm1 (15). ndr1 plants also were bacterial pathogen encodes a functional serineythreonine pro- compromised in RPP gene-mediated resistance to several tein kinase that specifically phosphorylates a second protein incompatible P. parasitica isolates, suggesting that the wild- kinase, Pti1 (2). Pto also interacts with the corresponding type NDR1 protein may function at a common point down- bacterial Avr protein AvrPto in a yeast two-hybrid assay (3, 4), stream from the perception of these prokaryotic and eukary- strongly supporting its role as the physiological receptor for the otic pathogens (15). Another resistance signaling component bacterial ligand. Other R proteins that are predicted to be is encoded by EDS1 (enhanced disease susceptibility) (16). A The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: LRR, leucine-rich repeats; NBS, nucleotide binding payment. This article must therefore be hereby marked ‘‘advertisement’’ in site; LZ, leucine zipper; Ler, Landsberg-erecta; Ws, Wassilewskija; accordance with 18 U.S.C. §1734 solely to indicate this fact. Col, Columbia. © 1998 by The National Academy of Sciences 0027-8424y98y9510306-6$2.00y0 §To whom reprint requests should be addressed. e-mail: parkerj@ PNAS is available online at www.pnas.org. bbsrc.ac.uk 10306 Downloaded by guest on September 26, 2021 Plant Biology: Aarts et al. Proc. Natl. Acad. Sci. USA 95 (1998) 10307 mutation in EDS1 abolished resistance conferred by several was distinguished from eds1-2 in F2 plants initially by using the RPP loci but had no effect on RPM1-specified resistance, m249-specific primers, as above. Genotypes were later con- suggesting a function before the convergence of downstream firmed by using EDS1-specific primers to detect a 900-bp pathways (16). deletion in eds1-2 (A. Falk, B. Feys, and J.E.P., unpublished We thought it possible that the ndr1 and eds1 mutations may data). Homozygous NDR1 or ndr1-1 plants from Col ndr1-1 3 reflect the operation of distinct disease resistance signaling Ler F3 families that had been phenotypically selected kindly pathways and that particular R proteins exhibit a preference were provided by K. Century (University of California, Berke- for one signaling mode. To address this possibility, we ex- ley). The NDR1yndr1-1 genotypes were confirmed by using tended our analysis of the relative requirements for EDS1 and NDR1-specific primers that detected a 1-kb deletion in ndr1-1 NDR1 to a wide spectrum of Arabidopsis R genes in three (22). different genetic backgrounds (accessions). Our results show that the requirements for EDS1 or NDR1 appear to be mutually exclusive. Furthermore, the data suggest that pref- RESULTS erential utilization of either EDS1 or NDR1 is determined Identification of Three New eds1 Alleles. The eds1 mutation more by R protein structure than pathogen type. in the Arabidopsis accession Wassilewskija (Ws-0) abolished resistance to several P. parasitica isolates mediated by RPP1, METHODS RPP10, and RPP14 on chromosome 3 and RPP12 on chromo- somes 4 (16). The Ws eds1 allele hereafter is denoted eds1-1. Maintenance of Pathogen Isolates. Pseudomonas syringae Three further defective eds1 alleles were identified in two pv. tomato DC3000 containing the avirulence genes avrRps4 different screens of fast neutron-mutagenized seedlings of (17), avrRpm1 (13), avrRpt2 (11), or avrPph3 (18) in the broad accession Landsberg-erecta (Ler). In the first screen, a loss of host range vector pVSP61 or DC3000 containing empty RPP5-mediated resistance to isolate Noco2 revealed two in- pVSP61 were cultured as described. The Peronospora para- dependent mutant alleles, eds1-2 and eds1-3. In the second sitica isolates from Arabidopsis have been described before (16, screen, a loss of RPS4-specified resistance to the compatible 19–21). These were cultured on their corresponding compat- Pseudomonas syringae pv. tomato strain DC3000 expressing ible hosts as described (21). avrRps4 (17) identified a fourth allele, eds1-4. It was significant Plant Material, Cultivation, and Pathogenicity Tests. Seeds that the requirement for EDS1 in RPS4-specified
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