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Broad-Spectrum Mildew Resistance in Arabidopsis thaliana Mediated by RPW8

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Science  05 Jan 2001:
Vol. 291, Issue 5501, pp. 118-120
DOI: 10.1126/science.291.5501.118

Abstract

Plant disease resistance (R) genes control the recognition of specific pathogens and activate subsequent defense responses. We show that the Arabidopsis thaliana locusRESISTANCE TO POWDERY MILDEW8 (RPW8) contains two naturally polymorphic, dominant R genes, RPW8.1and RPW8.2, which individually control resistance to a broad range of powdery mildew pathogens. Although the predicted RPW8.1 and RPW8.2 proteins are different from the previously characterizedR proteins, they induce localized, salicylic acid–dependent defenses similar to those induced byR genes that control specific resistance. Apparently, broad-spectrum resistance mediated by RPW8 uses the same mechanisms as specific resistance.

The majority of characterizedR genes participate in gene-for-gene interactions, in which the R product appears to act as a receptor that recognizes a product of the corresponding avirulence (Avr) gene from the pathogen, inducing defense responses. The R gene–mediated defenses typically involve a rapid, localized necrosis, or hypersensitive response (HR), at the site of infection, and the localized formation of antimicrobial chemicals and proteins that restrict growth of the pathogen (1, 2). Many crops rely onR genes for resistance to specific pathogens, but resistance fails in the presence of strains of the pathogen that lack the corresponding Avr genes. Broad-spectrum disease resistance is therefore desirable, and has been achieved through the use of recessive mutations (3); a challenge is to develop broad-spectrum resistance with dominant R genes (4). More than 20 of the R genes that confer specific resistance have been characterized and they form five classes of protein with differing combinations of five conserved structural motifs. With the exception of Hm1, a toxin reductase, and Pto, a protein kinase, the characterized R proteins contain a leucine-rich repeat (LRR) motif believed to specify recognition (1, 5, 6). All of the characterized A. thaliana R genes encode proteins with motifs for a nucleotide-binding site (NBS) and an LRR, and similar Rgenes have been isolated from other plant species (1). Probes for conserved sequences in NBS-LRR motifs have detected numerous homologs in the genomes of crop plants (7), and more than 100 in the genome of A. thaliana (8). TheA. thaliana loci RPW7 and RPW8 from accession Ms-0 map to the same interval on chromosome 3, and confer resistance to the powdery mildew pathogens Erysiphe cruciferarum UEA1 and E. cichoracearum UCSC1, respectively (9). During the mapping of RPW8, we discovered that this locus segregated from the mapped NBS-LRRR-gene homologs (8), suggesting the presence of a different type of resistance gene, which we characterize here.

We genetically mapped RPW8 to a fragment of genomic DNA from Ms-0 in cosmid B6 (Fig. 1A) (10), and confirmed that Col-0 plants containing the B6 transgene (T-B6) were resistant to E. cichoracearumUCSC1 (Fig. 2A) (10). The B6 DNA sequence (11) revealed three open reading frames (ORFs) (Fig. 1A) encoding a serine/threonine protein kinase 2 (SPK-2) (GenBank accession number S56718) and two uncharacterized genes, which we named MSC1 andMSC2. Subclones of B6 in a plant transformation vector were introduced into Col-0 plants by Agrobacterium-mediated transformation (11, 12), and those that contained eitherMSC1 or MSC2, or both of these ORFs, conferred resistance to E. cichoracearum UCSC1 (Fig. 1A).This indicated that RPW8 comprises two independently acting genes, MSC1 and MSC2, which we therefore renamedRPW8.1 and RPW8.2, respectively. Ms-0 cDNAs forRPW8.1 and RPW8.2 (13) were introduced into accession Col-0 plants under control of the highly active cauliflower mosaic virus 35S promoter (11, 12), and transgenic plants T-35S::RPW8.1 and T-35S::RPW8.2, homozygous for the corresponding transgene, were resistant to E. cichoracearum UCSC1, whereas T-35S::SKP-2 plants used as controls were susceptible (Fig. 1B) (10).

Figure 1

Isolation of the RPW8 locus. (A) Restriction sites in cosmid B6 used for making the indicated subclones (11). Subclones introduced into Col-0 byAgrobacterium-mediated transformation are marked (+) if they conferred resistance to E. cichoracearum UCSC1, and (–) if they did not. ORFs detected in the B6 sequence (11) are shown as thick lines. Only subclones containing either or bothMSC1 and MSC2 conferred resistance. (B) cDNAs for RPW8.1 and RPW8.2(13), and for SKP-2 were cloned in vector pKMB (26) under control of the highly active viral35S promoter, and introduced into A. thalianaCol-0 by Agrobacterium-mediated transformation (11,12). Plants containing the 35S::RPW8.1 or35S::RPW8.2 transgenes were resistant (10), indicated by (+), and plants containing the35S::SKP-2 transgene were susceptible, indicated by (–), to E. cichoracearum UCSC1. (C) Organization of RPW8.1 and RPW8.2 and homologs in accessions Ms-0 and Col-0. Exons are indicated as thick lines; each gene has a single intron indicated by a gap.

Figure 2

Analysis of RPW8-mediated resistance of A. thaliana to E. cichoracearum. (A) Ten days after plants were inoculated with conidia fromE. cichoracearum UCSC1 (27), (left to right) white mycelium and vegetative spore masses of the pathogen had developed on leaves of accession Col-0, which was susceptible, but not on leaves of Col-0 plants containing the RPW8 transgene, T-B6, which was resistant. T-B6 plants in theeds1-2/eds1-2 background (20) were susceptible, T-B6 plants in thendr1-1/ndr1-1 mutant background (20) were resistant, and T-B6 plants containing the NahG transgene encoding salicylate hydroxylase were susceptible. (B)Germinated conidia (c, blue) on surfaces of leaves 30 hours after inoculation. Hydrogen peroxide formation was detected with diamino benzidine (28) as a brown reaction product (r) in T-B6 (right) epidermal cells penetrated by the pathogen, but could not be detected in Col-0 (left) epidermal cells penetrated (p) by the pathogen. Bar, 30 μm. (C) Northern analysis indicated that defense gene PR-1 was induced in T-B6 (B) leaves 48 hours after inoculation with E. cichoracearumUCSC1, but not in Col-0 (C) leaves.

The characterized R genes normally control resistance to only a narrow range of isolates of a species of pathogen (1). To examine the range of pathogens controlled byRPW8, we inoculated A. thaliana Col-0 plants transgenic for RPW8 with a selection of pathogens virulent on accession Col-0. Transgenic plants T-B6, T-35s::RPW8.1, and T-35s::RPW8.2 were resistant to all of the tested powdery mildew pathogens of Arabidopsis. These included 15 isolates of E. cichoracearum, E. cruciferarumisolate UEA1 [indicating that RPW7 (9) is also determined by RPW8.1 and RPW8.2], E. orontii isolate MGH, representing three distinct species (14), and Oidium lycopersici isolate Oxford. A feature of these powdery mildew pathogens is that they each have a very wide host range. For example, E. cichoracearumUCSC1 also infects members of the Cucurbitaceae (15),E. cruciferarum UEA1 also infects members of the Brassicaceae (9, 15), and O. lycopersici Oxford attacks members of the Cucurbitaceae and the Solanaceae (16). T-B6 plants were susceptible to the other pathogens tested, including Peronospora parasitica Noco2 (10), the cauliflower mosaic virus, and the bacteriumPseudomonas syringae pv tomato DC3000 (17).

The genomic sequence of RPW8.1 contained a single 197-nucleotide (nt) intron and produced a 711-nt transcript with 444 nt of predicted coding sequence, and the genomic sequence ofRPW8.2 contained a 128-nt intron and produced a 798-nt transcript with 522 nt of predicted coding sequence (GenBank accession number AF273059). Northern analysis indicated that RPW8.1and RPW8.2 were expressed constitutively, but increased after infection with E. cichoracearum UCSC1 (10). The predicted proteins RPW8.1 and RPW8.2 had 45.2% sequence identity (10), and were relatively small (molecular weights 17,000 and 19,973, respectively) and basic (isoelectric points of 9.46 and 10.05, respectively). Their sequences predicted an NH2-terminal transmembrane (TM) domain, or possibly a signal peptide, and a coiled-coil (CC) domain (10). Although RPW8.1 and RPW8.2 had no significant homology to any characterized proteins, they had 22 and 33% identity, and 51 and 62% similarity, respectively, to a 130 amino acid sequence forming the NH2-terminus of the predicted product of resistance protein–like gene BAB08633, which has an NBS and an LRR motif at residues 171 to 751.

We compared the DNA sequences of the RPW8.1 andRPW8.2 alleles from seven A. thaliana accessions resistant and susceptible to E. cichoracearum UCSC1 (11, 15). Resistance of accessions Kas-1 and Wa-1 toE. cichoracearum UCSC1 is controlled by lociRPW10 and RPW13, respectively, both of which map to the RPW8 locus (18). The DNA sequences of theRPW8.1 and RPW8.2 alleles from Kas-1 and from Wa-1 were identical to those from Ms-0. Accessions Ler, Nd-0, and Ws-0 were moderately susceptible, and the DNA sequences of theirRPW8.1 and RPW8.2 alleles were predicted to encode proteins with 90.5 to 98.3% amino acid sequence identity to those encoded by the Ms-0 alleles. There is an insertion at the 3′ end of the allele for RPW8.1 in Nd-0, and a premature stop at the 3′ end of the alleles for RPW8.2 in Ws-0 and Sy-0. Other differences from Ms-0 were conserved: for example, in RPW8.2, two (T64S and D116G) of the five amino acid differences occurred in all four susceptible accessions (10). Accession Col-0 is extremely susceptible to mildew and we could not detect alleles of either RPW8.1 or RPW8.2 by Southern analysis (10). However, the corresponding sequence in Col-0 [bacterial artificial chromosome (BAC) T20E23] revealed a single gene, CAB62477, predicted to encode a protein with 52 and 33% amino acid sequence identity to RPW8.1 and RPW8.2, respectively.

Some R-gene loci consist of clusters of homologs that represent sites of rapidly evolving R-gene specificity (19). Analysis of the nucleotide sequence of cosmid J4-2 from Ms-0 (10) revealed that RPW8 was linked to three ORFs with 62.6 to 82.3% DNA sequence identity toRPW8.1 and RPW8.2. These three paralogs were named Homologous to RPW8 1 (HR1), HR2, and HR3 (GenBank accession number AF273059). They are closely related (99.4 to 99.9% DNA sequence identity) to their orthologs CAB 62474, CAB62475, and CAB62476, respectively, in BAC T20E23 from accession Col-0 (10); we named CAB62477 asHR4 (Fig. 1C). A recombination breakpoint betweenRPW8.2 and HR3, detected with marker 3B3-L (10) indicated that HR1, HR2, andHR3 did not confer resistance to the powdery mildews. TheRPW8 locus of Ms-0 therefore contained five RPW8paralogs, and the locus in Col-0 contained four (Fig. 1C).

A majority of the characterized A. thaliana R genes mediate resistance mechanisms that involve an HR, induced H2O2 formation and expression of the pathogenesis-related (PR) genes, and require salicylic acid (1) and the signal pathway genes EDS1 orNDR1 (20). We examined whether these features occur in RPW8-mediated resistance. Defense responses in T-B6 plants could be detected 30 hours after inoculation with E. cichoracearum UCSC1, as the formation of H2O2 in epidermal cells penetrated by the pathogen (Fig. 2B); the penetrated cells subsequently collapsed, forming microscopic lesions characteristic of the HR and there was no further growth of the pathogen (Fig. 2A) (9). By contrast there was no evidence of cellular H2O2 when Col-0 epidermal cells were penetrated by the pathogen (Fig. 2B), which grew to form masses of white mycelia and conidia on the leaf surface in 10 days (Fig. 2A). PR-1 transcripts accumulated in T-B6 plants 48 hours after inoculation, but not in inoculated Col-0 plants (Fig. 2B). Resistance was abolished in T-B6 plants containing salicylate hydroxylase, which converts salicylic acid to catechol (Fig. 2A). Resistance was also abolished in plants containing theRPW8 transgene from T-B6 crossed into theeds1-2/eds1-2 mutant background, but not in thendr1-1/ndr1-1 mutant background (Fig. 2A), indicating that EDS1, but not NDR1, was required for resistance.

RPW8.1 and RPW8.2 are similar to many of the characterized R genes in that they are naturally occurring, polymorphic, dominant alleles (1), occur in a gene cluster (1, 19, 21), and induce defense response associated with HR. Unlike other characterized R genes, RPW8.1 andRPW8.2 conferred resistance to a wide range of powdery mildew diseases of A. thaliana, reminiscent of that conferred by recessive alleles at the barley MLO locus (3). However, mlo-based resistance involves a spontaneous-lesion phenotype and cell wall apposition in epidermal tissues before pathogen attack (3), which is different from the HR phenotype triggered by R genes (1, 9). None of the tested powdery mildew pathogens could overcomeRPW8-mediated resistance, indicating thatRPW8-mediated resistance does not involve a gene-for-gene interaction (2, 21, 22), or possibly that each of these pathogens has an Avr gene that interacts withRPW8. The NH2-termini of NBS-LRR Rproteins play a role in the regulation of cell death (23), defense pathway-signaling (20), and in the determination of specificity (24). RPW8.1 and RPW8.2 had limited homology to the predicted NH2-terminus of an NBS-LRRR-like protein. RPW8.1 and RPW8.2 may therefore have some NBS-LRR R gene functions, to recognize powdery mildew pathogens, or to recruit NBS-LRR Rgene–mediated defenses for resistance. Alternatively RPW8.1 and RPW8.2 may interact with NBS-LRR R proteins to initiate resistance responses, possibly as targets for pathogen virulence factors that are guarded by NBS-LRR R proteins as has been proposed for Pto (25).

  • * To whom correspondence should be addressed. E-mail: j.g.turner{at}uea.ac.uk

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