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NDR1, a Pathogen-Induced Component Required for Arabidopsis Disease Resistance

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Science  12 Dec 1997:
Vol. 278, Issue 5345, pp. 1963-1965
DOI: 10.1126/science.278.5345.1963

Abstract

Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific corresponding avirulence genes. In Arabidopsis thaliana, resistance to both bacterial and fungal pathogens, mediated by several Rgene products, requires the NDR1 gene. Positional cloning was used to isolate NDR1, which encodes a 660–base pair open reading frame. The predicted 219–amino acid sequence suggests that NDR1 may be associated with a membrane. NDR1 expression is induced in response to pathogen challenge and may function to integrate various pathogen recognition signals.

Genetic analyses of disease resistance in plants show that resistance to pathogens is often highly specific, requiring single corresponding genetic loci in both the plant and the pathogen (1). Disease resistance genes cloned from diverse plant species such as tomato, rice, and Arabidopsis thaliana encode proteins that share one or more similar motifs (2). These motifs include leucine-rich repeat regions (implicated in protein-protein interactions) (3), nucleotide-binding sites, and kinase domains, all of which predict a role for resistance genes as components in signal transduction pathways. These genes confer resistance to a variety of pathogens, including bacteria, fungi, viruses, and nematodes, which suggests a conserved mechanism of plant disease resistance. Therefore, it is possible that the signal transduction pathways used by the different resistance gene products converge at some point. We report the cloning of NDR1 fromArabidopsis, a gene that functions in common among several resistance responses.

The NDR1 locus is required for resistance to both the bacterial pathogen Pseudomonas syringae pv.tomato (Pst) and the fungal pathogenPeronospora parasitica (4). Mutation ofNDR1 causes susceptibility to numerous strains of these pathogens. Thus, NDR1 represents a strong candidate for a conserved signal transduction element required for avirulence (avr) gene–specific disease resistance. NDR1 is located onArabidopsis chromosome three, in an ∼8.5-centimorgan (cM) interval between restriction fragment length polymorphism (RFLP) markers g6220 and g4711 (4). Fine-structure mapping with RFLP and polymerase chain reaction (PCR)–based markers further delimited the genomic region carrying NDR1 (Fig.1A) (5). An overlapping set of yeast artificial chromosome (YAC) clones spanning ∼1200 kb was constructed (Fig. 1B) (6). Two YAC clones, CIC3D12 and CIC7E1, together spanned NDR1, as determined by recombination analysis. A plant-transformation competent cosmid library from each of these two YAC clones was generated, and a cosmid contig was constructed (Fig. 1C) (7). An approximate 1-kb deletion was uncovered in the ndr1-1 fast-neutron–generated mutant (4) when cosmid FH6 from the CIC3D12 library was used as a32P-labeled probe (Fig. 1D). To determine ifNDR1 was contained in the deleted region, ndr1-1plants were transformed with cosmids spanning the deletion and tested for complementation with a hypersensitive response (HR) assay (7). Wild-type Col-0 plants react with an HR to P. syringae pv. maculicola (Psm) expressingavrRpt2, whereas ndr1-1 mutant plants do not. Cosmids spanning the deleted region (Fig. 1C) restored wild-type HR toPsm (avrRpt2) in ndr1-1 plants. In planta bacterial growth analyses (Fig. 2) and cotyledon sporulation assays with P. parasitica (Table1) demonstrated restored, heritable resistance in the complemented transformed plants.

Figure 1

Genetic mapping and positional cloning ofNDR1. (A) Fine-structure RFLP map. Recombinant analysis delimited the genomic region containing NDR1 to a section flanked by RFLP marker pCIT1240 and ARMS (ArabidopsisRFLP mapping set) marker 560B1 (5). (B) YAC contig. Genetic mapping of the insert ends from the YAC clones demonstrated that the contig spanned the NDR1 locus (6). An RFLP marker (14E8LE) derived from one end of yUP14E8 further narrowed the physical genomic region containingNDR1 to a 0.68-cM interval. (C) Cosmid contig. Cosmids derived from CIC3D12 were organized into an overlapping set that spanned NDR1. The ndr1-1 mutant was genetically transformed with these cosmids and tested for complementation by HR analysis (+, HR restored; –, no HR) (7). (D) DNA gel blot demonstrates an ∼1-kb deletion in the mutant ndr1-1. A 14-kb Eco RIArabidopsis DNA fragment from cosmid FH6 was radiolabeled and used as a hybridization probe against Hind III–digested Col-0, La-er, or ndr1-1 genomic DNA. Thendr1-1 lane shows the deletion of an ∼1-kb fragment containing a Hind III site resulting in the larger 1.9-kb single fragment.

Figure 2

Growth of avirulent Pststrain DC3000 within cosmid-complemented ndr1-1 Arabidopsis. (A) Pst DC3000 (avrRpt2). (B) Pst DC3000 (avrRpm1). •, ndr1-1 mutant; ▪,ndr1-1 transformed with noncomplementing CB17 cosmid; ○, wild-type Col-0; and □, ndr1-1 transformed with complementing FH6 cosmid. T3 homozygous plant lines were derived from selfing progeny of a single Col-0ndr11/ndr1-1 transformant heterozygous for kanamycin resistance. Plants were inoculated by vacuum infiltration, and bacterial growth in leaves was monitored as described (27). CB17 cosmid does not contain NDR1 ORF (Fig.1C). Sample means and standard deviations are shown from a representative experiment.

Table 1

Asexual sporulation (measured as the mean number of sporangiophores per cotyledon; maximum of 20 sporangiophores counted per cotyledon) by three incompatible isolates of Peronospora parasitica on Col-0, the mutant ndr1-1, and two transformed lines of ndr1-1. RPP resistance specificities for each isolate are indicated. The cotyledon assay used has been described previously (31). SEM, standard error of the mean; n, number of seedlings inoculated.

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Sequencing of wild-type Col-0 genomic DNA revealed a single 660-base pair open reading frame (ORF) in the region spanned by the deletion in the ndr1-1 mutant (8). Sequencing of the additional mutant alleles ndr1-2 and ndr1-3(9) also revealed alterations in this ORF (Fig.3). The ORF predicts a 219–amino acid gene product (Fig. 3), which shows identity to oneArabidopsis expressed sequence tag (GenBank accession numberT21313). This cDNA clone was obtained from the ArabidopsisBiological Resource Center and was used to probe RNA gel blots (Fig.4). The size of the hybridizing RNA from wild-type Col-0 plants is in agreement with the size of the ORF, indicating that the full-length gene is contained in the single ORF without introns. The message is absent in ndr1-1 mutant plants (10). Accumulation of NDR1 mRNA is up-regulated by both virulent and avirulent bacteria relative to the MgCl2 control treatment in wild-type Col-0 plants (Fig. 4A) and in mutants ndr1-2 and ndr1-3 (10). Over a period of 48 hours, up-regulation of NDR1 was seen as early as 4 hours after inoculation, with maximal expression at 8 hours (Fig. 4B). Therefore, NDR1 is a classically induced defense response gene which is genetically required for resistance.

Figure 3

Primary structure of NDR1-predicted protein (28). Putative transmembrane domains are underlined. The entire NH2-terminal portion of the protein through amino acid Asn179 (indicated by the arrow) is deleted inndr1-1, as well as a portion of the upstream DNA (GenBank accession number AF021346). Molecular alterations in ndr1-2and ndr1-3 alleles convert Trp124 and Phe31, respectively, to premature stop codons and are indicated by asterisks. See the GenBank entry (http://www.mcbi.nlm.nih.gov/Entrer/nucleotide.html) for nucleotide sequence and base pairs altered in the mutant alleles.

Figure 4

(A) RNA gel blot showing expression of NDR1 message in WT Col-0Arabidopsis plants. Five-week-old plants grown under short-day conditions in a growth chamber were vacuum infiltrated with either a 10 mM MgCl2 control or 1 × 107CFU/ml of Pst DC3000, Pst DC3000 (avrB), or Pst DC3000 (avrRpt2). Plants were frozen in liquid nitrogen after an 8-hour induction period, and total RNA was extracted (Tri-Reagent, Sigma). Lanes: C, Col-0 uninduced; M, MgCl2 induction control; V, virulentPst DC3000 induction; avrB, avirulent PstDC3000 (avrB) induction; avrRpt2, avirulentPst DC3000 (avrRpt2). Gel blot analysis was done according to standard protocol (29) using Hybond-NX transfer membrane (Amersham) according to manufacturer's directions. The blot was stripped and reprobed with pea 18S ribosomal DNA (30) as a control for loading. The blot shown is representative of three experiments. (B) NDR1mRNA accumulation after 8 hours in uninoculated tissue (a), leaves infiltrated with MgCl2 control (b), virulent DC3000 (c), or avirulent DC3000 (avrRpt2) (d). Data was generated by combining the results from three separate RNA gel blots and was standardized for loading by comparing with control probes for total RNA. A similar trend of RNA accumulation was seen in plants inoculated with DC3000 (avrB) in two separate experiments (10).

SBASE library (11) analysis of the NDR1-predicted amino acid sequence identified two putative transmembrane domains similar (up to 85%) to membrane-spanning regions in proteins such as the 6K protein of Ockelbo virus (a Sindbis virus) and the inositol 1,4,5-trisphosphate receptor protein from various species. In NDR1, the putative transmembrane domains span amino acids 19 through 36 and 202 through 218 (Fig. 3). These similarities suggest that NDR1 may be a membrane-associated protein; however, the subcellular location of NDR1 is not known.

BLAST searches (12) revealed limited similarity with two tobacco genes, hin1 (13) and clone NG2 (14), which are correlated with the resistance response (15). Both hin1 (13) and NDR1 are induced by avirulent pathogens (Fig. 4). The significance of the similarities between ndr1 and these two tobacco genes remains to be determined.

NDR1 is required for resistance to the bacterial pathogenPst expressing avrB, avrRpt2,avrRpm1, or avrPph3, as well as resistance to numerous isolates of the fungal pathogen Peronospora parasitica (4), but not for expression of the resistance gene RPS2 (16). Therefore, we propose that NDR1 may encode a component in the signal transduction pathway downstream of initial pathogen recognition. Mutation ofNDR1 results in loss of resistance governed by several resistance genes. Because of their specificity, it has been speculated that resistance gene products act as receptors for avirulence signals. NDR1 may interact directly with many specific receptors to transduce the elicitor signal, or it may serve as a transporter or receptor for an elicitor signal or secondary messenger.

Several genes have been identified that are required for the activity of individual resistance genes in tomato and barley (17). However, NDR1 and another Arabidopsis gene,EDS1 (18), have been shown to be necessary for plant defense mediated by numerous resistance genes. That mutation ofNDR1 causes susceptibility to both bacterial and fungal pathogens supports a central role for NDR1 in disease resistance. Further analysis of NDR1, such as identification of important domains, interacting proteins, and cellular localization, will help make significant progress toward the goal of characterizing a complete signal transduction pathway for plant disease resistance.

  • * Present address: Biology Department, San Francisco State University, San Francisco, CA 94132, USA.

  • Present address: Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19717, USA.

  • To whom correspondence should be addressed. E-mail: stask{at}nature.berkeley.edu

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