Role of Salicylic Acid and NIM1/NPR1 in Race-Specific Resistance in Arabidopsis

Corresponding author: Terrence P. Delaney, Cornell University, 360 Plant Science Bldg., Ithaca, NY 14853., tpd4{at}cornell.edu (E-mail)

Communicating editor: V. L. CHANDLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Salicylic acid (SA) and the NIM1/NPR1 protein have both been demonstrated to be required for systemic acquired resistance (SAR) and implicated in expression of race-specific resistance. In this work, we analyzed the role that each of these molecules play in the resistance response triggered by members of two subclasses of resistance (R) genes, members of which recognize unrelated pathogens. We tested the ability of TIR and coiled-coil-class (also known as leucine-zipper-class) R genes to confer resistance to Pseudomonas syringae pv. tomato or Peronospora parasitica in SA-depleted (NahG) and nim1/npr1 plants. We found that all of the P. syringae pv. tomato-specific R genes tested were dependent upon SA accumulation, while none showed strong dependence upon NIM1/NPR1 activity. A similar SA dependence was observed for the P. parasitica TIR and CC-class R genes RPP5 and RPP8, respectively. However, the P. parasitica-specific R genes differed in their requirement for NIM1/NPR1, with just RPP5 depending upon NIM1/NPR1 activity for effectiveness. These data are consistent with the hypothesis that at least in Arabidopsis, SA accumulation is necessary for the majority of R-gene-triggered resistance, while the role of NIM1/NPR in race-specific resistance is limited to resistance to P. parasitica mediated by TIR-class R genes.

IN angiosperms, resistance (R) genes confer race-specific or gene-for-gene resistance to a wide variety of pathogens. Plants containing a specific R gene are able to recognize pathogens that carry a corresponding avirulence (avr) gene, leading to the activation in the plant of a set of rapid defensive measures at the site of infection, which usually culminate in the generation of reactive oxygen species and localized cell death called the hypersensitive response (HR). Many R genes have been cloned from various plants and found to encode proteins that fall into a number of different classes (reviewed in DANGL and JONES 2001 ). The largest class includes proteins that have a predicted nucleotide binding site (NBS), which is thought to be important for downstream signaling (BENT 1996 ), and leucine-rich repeats (LRRs), which have been shown to be important for avr-protein recognition specificity (ELLIS et al. 1999 ; DODDS et al. 2001 ). NBS-LRR R-proteins can be divided into two subclasses that are based on the structure of their amino terminus: one subclass contains a coiled-coil (CC)-like domain (also called a leucine zipper domain), while the other contains a "TIR" domain that has homology to Drosophila Toll and human interleukin-1 transmembrane receptors (WHITHAM et al. 1994 ; PARKER et al. 1997 ).

Pathogen-triggered responses are often accompanied by induction of systemic defense responses that are active against a broad range of pathogens, including viruses, bacteria, and fungi. The best characterized of these is systemic acquired resistance (SAR), which is associated with accumulation of salicylic acid (SA) and a number of pathogenesis-related (PR) gene products (RYALS et al. 1996 ). Many physiological and genetic requirements for both race-specific and SAR have been determined in recent years, and in some cases both processes share these requirements. SAR has been shown to depend upon both SA accumulation and the NIM1/NPR1 protein, which facilitates a systemic response to pathogen-triggered SA accumulation (reviewed by DELANEY 1997 ). A variety of mutants that disrupt R gene function have been identified in Arabidopsis thaliana. These include mutants that compromise a single R gene (e.g., pbs1), as well as mutants, such as eds1, ndr1, pbs2, and pbs3 that show defects in responses to multiple R genes (CENTURY et al. 1995 ; PARKER et al. 1996 ; WARREN et al. 1999 ). Together, these different mutant classes implicate a hierarchical funneling of signals from specific inputs into a few common sets of defense responses. For example, LRR-NBS R genes in the TIR or CC class have been shown to require either EDS1 or NDR1, respectively, but not both (AARTS et al. 1998 ). In addition, some R genes, such as RPP7, RPP8, and RPP13-Nd, have been shown to act independently of both EDS1 and NDR1, implying the existence of as-yet-undefined R-gene signaling pathways (AARTS et al. 1998 ; MCDOWELL et al. 2000 ; BITTNER-EDDY and BEYNON 2001 ). Race-specific resistance has also been shown in some, but not all cases to depend upon the SAR effectors SA and NIM1/NPR1 (DELANEY et al. 1994 , DELANEY et al. 1995 ; SHAH et al. 1997 ; CLARKE et al. 2000 ; MCDOWELL et al. 2000 ; FEYS et al. 2001 ), but no correlation that would predict whether an R gene would require SA or NIM1/NPR1 on the basis of its protein structure or pathogen specificity has been established.

We wished to determine whether R-protein structure or pathogen specificity correlated with the requirement for SA accumulation or NIM1/NPR1 function. Therefore, we analyzed the effectiveness of both CC and TIR class R genes that recognize Peronospora parasitica and Pseudomonas syringae pv. tomato (Pst)-produced molecules in NahG and nim1/npr1 backgrounds. Individual R genes within NahG or nim1/npr1 plants were interrogated by inoculation with various avirulent Pst strains or P. parasitica isolates, and pathogen growth restriction was compared to that observed on wild-type controls. Our tests included the Pst-specific CC R genes RPM1 and RPS2, the TIR class gene RPS4 (BENT et al. 1994 ; MINDRINOS et al. 1994 ; GRANT et al. 1995 ; GASSMANN et al. 1999 ), and P. parasitica-specific RPP5 and RPP8, TIR and CC class genes, respectively. RPP5 and RPP8 were tested for SA dependence in two independently derived NahG backgrounds, and the effectiveness of these R genes in nim1/npr1 backgrounds was evaluated by testing whether P. parasitica resistance segregated with the appropriate R gene in nim1/npr1-selected F₂ plants derived from crosses between an R-gene-carrying accession and nim1/npr1 mutants in a susceptible accession. All R genes tested could be shown to require SA; however, only RPP5 was shown to require NIM1/NPR1.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plants and growth conditions:
A. thaliana accession Wassilewskija (Ws-0), Columbia (Col-0), and Landsberg erecta (Ler) were obtained from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH), Ws nim1-1 and Col NahG plants were described previously in DELANEY et al. 1994 , DELANEY et al. 1995 , and the Ws-NahG line (MOLINA et al. 1998 ) was provided by Syngenta (Research Triangle Park, NC). Ler NahG plants were obtained from Dr. Xinnian Dong (BOWLING et al. 1994 ), and npr1-2 plants were obtained from Dr. Jane Glazebrook (GLAZEBROOK et al. 1996 ). Crosses were performed by emasculating Ler flowers and applying pollen from the appropriate male parent to the stigma. The success of the crosses was evaluated by testing putative F₁ plants for heterozygosity at the dihydroflavonol 4-reductase (DFR) and g4539 cleaved amplified polymorphic sequence (CAPS) loci. Plants were grown at 22° in short-day conditions (14 hr light, 150 µE fluence provided by cool white fluorescent lamps) with 60% relative humidity in Cornell soil mix (BOODLEY and SHELDRAKE 1977 ), composed of 12 ft³ vermiculite, 7.6 ft³ peat moss, 4 ft³ perlite, 5 lb lime, and 4 lb Micromax micronutrient blend (Sierra Chemical, Milpitas, CA).

Molecular genotyping:
DNA for CAPS and SSLP analysis was extracted as described in KLIMYUK et al. 1993 . Amplification and cleavage of the PCR products was performed essentially as described (KONIECZNY and AUSUBEL 1993 ; BELL and ECKER 1994 ). Primers used include DFR, 5'-TGTTACATGGCTTCATACCA-3', 5'-AGATCCTGAGGTGAGTTTTTC-3'; and CER453919, 5'-ACGGCTTATAGTTGGGCAGTG-3', 5'-TTTTCGTGGTTTATATCGGGTCAA-3'.

Lactophenol trypan blue staining of P. parasitica:
To assess P. parasitica colonization of inoculated plants, leaves were stained with lactophenol trypan blue and cleared with saturated chloral hydrate, as described (UKNES et al. 1993 ). After the leaves had cleared, chloral hydrate was replaced with 70% glycerol for slide mounting. Whole leaves were analyzed and photographed with a MZ8 stereo microscope (Leica, Wetzler, Germany) and a PM-C 35-mm camera (Olympus, Melville, NY).

Pathogen inoculation and chemical elicitation:
P. parasitica isolate Noco2 (CRUTE et al. 1993 ) was provided by Jane Parker (The Sainsbury Laboratory, Norwich, UK) and Emco5 (HOLUB and BEYNON 1997 ) was provided by Jeff Dangl (University of North Carolina, Chapel Hill, NC). Noco2 and Emco5 were maintained on Col-0 or Ws-0 hosts, respectively, as described in UKNES et al. 1992 . Inoculum was prepared from plants 8 days postinfection by placing heavily sporulating leaves into water and gently vortexing; the spore suspensions (8 x 10⁴ conidiospores/ml) were misted onto Arabidopsis plants 15 days after sowing, using a compressed air paint sprayer (Preval; Precision Valve, Yonkers, NY), and plants were covered with a clear dome to maintain the high humidity that is optimal for P. parasitica germination and growth. Spores to be used in cotyledon assays were pelleted by centrifugation, resuspended in water (8 x 10⁴ conidiospores/ml), and then misted onto plants 5 days after sowing. Chemical induction of SAR was achieved by misting plants with a 0.33 mM suspension of 2,6-dichloroisonicotinic acid (INA; 0.25 mg/ml of a formulation containing 25% INA plus wettable powder), obtained from Syngenta.

P. syringae growth measurements:
P. syringae pv. tomato DC3000 strains were obtained from Dr. Brian Staskawicz (AARTS et al. 1998 ). Inoculation and quantification of P. syringae pv. tomato DC3000 was performed essentially as described in TORNERO and DANGL 2001 . Pots containing 2-week-old seedlings were inverted and the plants dipped in a suspension of DC3000 (OD₆₀₀ of 0.05) in 10 mM MgCl₂ and 0.02% (v/v) Silwet L-77; seedlings were then placed into a flat that was covered with a plastic dome for 1 hr to maintain humidity, after which the dome was removed. Two plants were then harvested per data point for bacterial quantification; four data points per time point were obtained for each interaction tested. Bacterial quantification was performed as described in TORNERO and DANGL 2001 .

RNA extraction and analysis:
Aerial plant tissue was cut off at the described time points and immediately frozen in liquid nitrogen, and RNA was extracted as in LAGRIMINI et al. 1987 . RNA gel-blot analysis was performed as described in UKNES et al. 1993 . Approximately 5.0 µg total RNA per sample was fractionated by electrophoresis on denaturing 1.2% agarose gels (1x MSE, 3% v/v formaldehyde; UKNES et al. 1993 ). RNA was transferred overnight in 6x SSC to NytranN nylon membranes (Schleicher and Schuell, Dassel, Germany) and then crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Probes were made using [-³²P]dCTP with a random primer labeling system (GIBCO/BRL, Carlsbad, CA) with Arabidopsis PR1 and PR2 cDNA probes (UKNES et al. 1992 ). The PDF1.2 template was amplified by PCR from genomic DNA with the following primers: 5'-CTCATGGCTAAGTTTGCTTCC-3' and 5'-AATACACACGATTTAGCACC-3'. Each probe was hybridized to a separate replicate blot containing equally loaded RNA samples. Overnight hybridizations and washes were performed at 65° as described by CHURCH and GILBERT 1984 . Radioactivity was detected using a phosphor screen and Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Role of SA and NIM1/NPR1 in race-specific resistance to P. syringae:
To test whether salicylic acid accumulation or NIM1/NPR1 is essential for transducing signals that originate from different Pst-specific R genes, we tested their effectiveness in wild-type, npr1-2, and salicylate-depleted, NahG-expressing Arabidopsis plants. The R genes tested included CC-NBS-LRR-class RPM1 and RPS2 (BENT et al. 1994 ; MINDRINOS et al. 1994 ; GRANT et al. 1995 ) and the TIR-class R gene RPS4 (GASSMANN et al. 1999 ). We inoculated accession Col-0, npr1-2, and NahG plants with Pst DC3000 expressing avrRpm1, avrRpt2, or avrRps4, bacterial avirulence genes that are recognized in the Col-0 accession by RPM1, RPS2, and RPS4, respectively. Growth of the three bacterial strains was significantly greater in the NahG background compared to wild-type plants, demonstrating that SA accumulation plays an important role in the efficacy of each of these R genes (Fig 1). However, resistance to all three avirulent DC3000 strains appeared to not be significantly compromised in npr1-2 plants, indicating that NIM1/NPR1 is not essential to confer robust race-specific resistance to Pst.

View larger version (32K): In this window In a new window Download PPT slide   Figure 1. Growth of P. syringae pv. tomato strains in leaves of npr1-2 and NahG plants. Wild-type (Col-0), Col npr1-2, and Col NahG plants were inoculated by vacuum infiltration with strain DC3000 expressing avrRpm1, avrRpt2, avrRps4, or empty vector alone. Growth of bacteria was assayed immediately following and 3 days after inoculation. Each data point represents the mean ±SE of four samples. The experiment was repeated three times with similar results.

Role of SA in race-specific resistance to P. parasitica:
We also analyzed whether SA accumulation was required for the ability of the TIR-class RPP5 and CC-class RPP8 R genes (PARKER et al. 1997 ; MCDOWELL et al. 1998 ) from Ler to confer resistance to P. parasitica. These tests were conducted by inoculating Ler NahG plants with P. parasitica Noco2 or Emco5, pathogen isolates that are recognized by RPP5 and RPP8, respectively (Table 1A). Hyphal growth was visualized using lactophenol trypan blue staining 10 days after inoculation of Ler NahG and control Ler plants. We found that Noco2 was able to colonize Ler NahG leaves, while Emco5 was not, indicating that RPP5, but not RPP8, requires SA accumulation for its action (Fig 2A). These results are consistent with those obtained by MCDOWELL et al. 2000 , who demonstrated that RPP8-mediated resistance was expressed in Col NahG plants carrying an RPP8 transgene, while RPP4-mediated resistance to Emoy2, which is likely mediated by an allele of RPP5 (VAN DER BIEZEN et al. 2002 ), was compromised. These tests with Ler NahG plants showed that RPP5-mediated resistance to Noco2 required SA accumulation, but RPP8-mediated resistance against Emco5 did not.

View larger version (102K): In this window In a new window Download PPT slide   Figure 2. Growth of P. parasitica in nahG-expressing plants. (A) Plants within a row were inoculated with the P. parasitica isolate shown. Compatible wild-type hosts Col and Ws are shown at the left, followed by parental Ler accessions (middle) and Ler-expressing NahG (right). (B, center) F1 plants from Ler x the susceptible host indicated. (right) F1 plants from crosses of Ler and Col NahG or Ws NahG plants. Hyphal growth was assessed by staining with trypan blue 10 days (A) or 8 days (B) after inoculation and comparing growth within a compatible host and control F1 hybrid not expressing nahG. The experiment was repeated three times with similar results.

In addition to testing RPP5 and RPP8 action in Ler NahG plants, we also assessed the SA dependence of the R genes in F₁ hybrid plants derived from Ler x Col NahG or Ler x Ws NahG crosses. These crosses enabled us to individually interrogate RPP5 or RPP8 in the presence of NahG by inoculating the plants with Noco2 or Emco5, respectively (Table 1B). In control crosses between Ler and wild-type Col-0 or Ws-0 plants, the F₁ plants were resistant to Noco2 or Emco5, respectively, due to action of the dominant heterozygous RPP5 or RPP8 loci from Ler (Fig 2B). By contrast, Ler x Col NahG F₁ plants allowed growth of Noco2, confirming the observation that RPP5 action requires SA accumulation. However, unlike Ler NahG plants, the F₁ hybrids from the Ler x Ws NahG cross showed extensive hyphal growth and significant sporulation after Emco5 inoculation, indicating that in the hybrids SA does play an important role in RPP8 signaling (Fig 2B). The difference observed between Ler NahG vs. Ler x Ws NahG hybrids in susceptibility to Emco5 is not simply due to differences in RPP8 copy number, because RPP8 heterozygous and homozygous F₂ plants derived from this same cross both fail to express resistance to Emco5 (data not shown). Therefore, in contrast to our observations of Ler NahG plants, in the hybrid plants, both RPP5 and RPP8 required SA accumulation to confer effective resistance to the test pathogens.

Role of NIM1/NPR1 in race-specific resistance to P. parasitica:
To assess the NIM1/NPR1 dependence of RPP5- and RPP8-initiated resistance, we analyzed a large number of nim1/npr1 F₂ plants derived from Ler x Col npr1-2 or Ler x Ws nim1-1 crosses, in which RPP5 and RPP8 would have segregated from their null alleles (Table 1C, Table 2, and Table 3). We chose to perform two separate crosses for these experiments because we found Emco5 growth to be significantly more robust on Ws-0 compared to Col-0, while Noco2 grows only on Col-0. To identify homozygous npr1-2 or nim1-1 plants from their respective F₂ population, we applied the NIM1/NPR1-dependent, SAR-inducing SA analog INA 3 days before inoculating the population with either Noco2 (for the Ler x Col npr1-2 F₂) or Emco5 (for the Ler x Ws nim1-1 F₂). Susceptible plants were known to be nim1/npr1 because of their inability to manifest INA-induced resistance to P. parasitica (CAO et al. 1994 ; DELANEY et al. 1995 ), while effective INA-induced resistance was observed in the normally Nim1⁺/Npr1⁺ compatible host controls (Col or Ws) in these experiments (data not shown). Further, susceptible plants must also lack effective R-gene action against the test pathogen either due to the absence of the cognate R gene or because an R gene present failed to function in the nim1/npr1 background, a determination that was the objective of this experiment. To determine whether RPP5 or RPP8 alleles were present in the susceptible nim1/npr1 plants, sporulating INA-treated plants were genotyped with molecular markers tightly linked to the respective RPP genes, and the frequency of the Landsberg allele of that marker was compared to the frequency seen in P. parasitica-susceptible Ler x Col-0 or Ler x Ws-0 F₂ plants that were not treated with INA. In F₂ plants from these control crosses, susceptible plants would not contain Ler alleles for molecular markers linked to effective Ler-derived R genes unless a recombination event between the R gene and the linked markers had occurred. We analyzed the simple sequence length polymorphism (SSLP; BELL and ECKER 1994 ) marker CER453919 to genotype the TIR-class RPP5 locus in Noco2-susceptible Ler x Col and INA-treated Ler x npr1-2 F₂ plants (Table 1C). This marker is within 100 kb of RPP5 and should thus be tightly linked to the RPP5-mediated resistance phenotype. We found an expected low frequency of Ler CER453919 alleles (2/60) in Noco2-susceptible Ler x Col-0 F₂ plants, while a significantly higher frequency of Ler CER453919 alleles (17/60) was found in Noco2-susceptible plants identified from an INA-treated Ler x Col npr1-2 F₂ population (Table 2). These findings indicate that RPP5-mediated resistance is generally compromised in a nim1/npr1 background and that NIM1/NPR1 therefore does play a significant role in enabling RPP5-mediated resistance. To examine functionality of the CC-class RPP8 gene in nim1/npr1 plants, we also assessed the occurrence of the RPP8-linked DFR CAPS (KONIECZNY and AUSUBEL 1993 ) marker, which is within 500 kb of the RPP8 locus. Of 56 alleles examined, we found no Ler DFR alleles (i.e., all were Ws-0 alleles) from Emco5-susceptible Ler x Ws F₂ plants, nor did we observe Ler DFR alleles among 46 chromosomes examined in the Emco5-susceptible Ler x Ws nim1-1 F₂ plants, as all 46 carried Ws-0 DFR alleles (Table 3). The similar low frequency of Ler alleles from the RPP8-linked DFR locus in the nim1-1 and NIM1 crosses demonstrates that RPP8-mediated resistance functions well in nim1-1 F₂ plants. Thus, RPP5 requires a functional NIM1/NPR1 protein to impart resistance, while RPP8 does not have this requirement.

To confirm the different reliance of these two RPP genes on NIM1/NPR1, F₃ plants were obtained from homozygous RPP5 npr1-2 and homozygous RPP8 nim1-1 F₂ plants (F₃ lines are henceforth referred to as R5n1 and R8n1, respectively) and tested for their ability to express resistance to Noco2 and Emco5 (Fig 3A). F₃ plants were known to be homozygous nim1/npr1 mutants, as they failed to express PR-1 3 days after treatment with INA (Fig 3B). For comparison, control RPP5 and RPP8 homozygous plants were obtained from corresponding wild-type F₂ populations (R5N1 and R8N1, respectively; both wild type for NIM1/NPR1). Extensive colonization of the R5n1 F₃ plants was seen compared to its wild-type R5N1 counterpart, while no colonization was seen in R8n1 or R8N1 F₃ plants, confirming the results seen in the genetic analysis of the F₂ population.

View larger version (71K): In this window In a new window Download PPT slide   Figure 3. RPP function in nim1/npr1 plants. (A) F3 homozygous RPP5/npr1-2 (R5n1) and RPP8/nim1-1 (R8n1) plants (right) were inoculated with the indicated P. parasitica strain. Parasite structures were stained with trypan blue 8 days after inoculation and compared to hyphal growth in the (left) compatible host and (center) wild-type F3 plants homozygous for RPP5/NPR1 (R5N1) or RPP8/NPR1 (R8N1). (B) RNA gel-blot analysis of PR1 gene expression 3 days following treatment with INA to confirm the nim1/npr1 status of these lines. The experiment was repeated three times with similar results.

Quantitative analysis of RPP requirements for SA and NPR1/NIM1:
To quantitatively assess RPP gene requirements for SA and NIM1/NPR1, we measured conidiophore production on cotyledons of young seedlings of various genotypes after inoculation with either Noco2 or Emco5 (Table 4). RPP5- and RPP8-expressing seedlings that contained or lacked NahG or NIM1/NPR1 function were inoculated 8 days after sowing, and the numbers of conidiophores per cotyledon were scored 8 days later. The results seen in these assays corroborate the findings described above: Ler NahG plants do not support Emco5 conidiophore production, while Ler x Ws NahG do, and RPP5-mediated resistance to Noco2 is compromised in both NahG and npr1-2 backgrounds. R5n1 seedlings are more susceptible to Noco2 than are R5N1 plants, while R8n1 does not allow Emco5 sporulation.

RPP5- and RPP8-mediated gene expression:
The observation that RPP5 and RPP8 differ in their requirement for NIM1/NPR1 led us to speculate about whether these two R genes induce different sets of defense genes. Therefore, we analyzed the expression of the well-characterized defense genes PR1, PR2, and PDF1.2 in Ler plants inoculated with P. parasitica isolate Noco2 or Emco5, which elicited RPP5- or RPP8-mediated resistance (Fig 4). We were able to see slight differences in the defense-gene expression profiles induced by each pathogen 1 and 2 days after inoculation. In our experience, the higher humidity and lower light intensity of our inoculation environment often leads to nonspecific elicitation of defense gene expression, and we see this response in this experiment most significantly 4 days after treatment. This background expression precluded reliable conclusions regarding gene expression 4 days postinoculation. We noted that 1 and 2 days after inoculation the well-characterized SA and NIM1/NIM1-dependent SAR genes PR1 and PR2 were induced much more strongly by RPP5 elicitation than by RPP8, while PDF1.2 showed greater induction in plants responding to a RPP8 signal. These differences imply that the RPP5- and RPP8-initiated signaling events leading to race-specific resistance initiate distinct downstream transcriptional responses.

View larger version (84K): In this window In a new window Download PPT slide   Figure 4. RPP-dependent induction of defense genes. Landsberg erecta plants were inoculated with 8 x 104 spores/ml of Noco2, Emco5, or mock inoculated with H2O. At 4 days after inoculation, Noco2- and Emco5-inoculated plants showed no evidence of infection. RNA was isolated from leaves 1, 2, and 4 days after inoculation. RNA gel blots were hybridized with radiolabeled PR1, PR2, or PDF1.2 probes as shown. Equal loading of each lane is demonstrated by ethidium bromide staining of rRNA.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We demonstrated that all five of the resistance genes tested in this study were compromised by diminished SA levels, regardless of whether they conferred resistance to bacterial or oomycete pathogens and regardless of whether those R genes contained a CC or TIR domain. By contrast, RPP5 is the only R gene we were able to show to be significantly compromised by mutations in NIM1/NPR1. While this study examined only a subset of known Arabidopsis R genes, our observations may form the basis of two broader generalizations: that the majority of Arabidopsis R genes require SA accumulation for full resistance activity and that NIM1/NPR1 may play a role only in resistance to P. parasitica mediated by TIR-class R genes. In support of this hypothesis, the RPP1 and RPP4 loci, which confer resistance to Noco2 and Emoy2, respectively, encode TIR-class R genes (PARKER et al. 1997 ; VAN DER BIEZEN et al. 2002 ) and resistance mediated by these loci has been shown to be compromised in nim1/npr1 seedlings (DELANEY et al. 1995 ; MCDOWELL et al. 2000 ), although VAN DER BIEZEN et al. 2002 noted that RPP4 is not significantly compromised in npr1-1 adult leaves. Also, RPP13-Nd is the second CC P. parasitica R gene to be cloned and was demonstrated to function independently of NIM1/NPR1 (BITTNER-EDDY and BEYNON 2001 ).

Our data support the hypothesis that functional homology exists for an important defense signal transduction pathway shared by plants and animals. The predicted NIM1/NPR1 protein product has similarity to Drosophila Cactus and human IB proteins (CAO et al. 1997 ; RYALS et al. 1997 ), which transduce signals initiated by the Toll and interleukin-1 receptors, respectively. Interestingly, both Cactus and IB are important for activation of the innate immune responses in these animals, much like NIM1/NPR1 is required for expression of SAR, a system with many similarities to animal innate immunity. Thus, our finding that an R gene containing TIR homology depends upon NIM1/NPR1 is consistent with the functional conservation of a TIR-like defense pathway in plants, providing evidence for a particularly ancient origin of this signaling pathway that would predate the divergence of plants and animals.

What role does NIM1/NPR1 play in RPP5- and RPP1-mediated resistance? While a number of Arabidopsis mutations that seem to directly impair the perception and response to R gene elicitation have been isolated, it is unlikely that NIM1/NPR1 plays such a central role in R gene signaling. The impairment of RPP5 by npr1-2 is less severe than that which results from SA depletion, implying that NIM1/NPR1 plays only a partial role in RPP5-initiated responses. It is plausible that, in addition to facilitating SAR, NIM1/NPR1-regulated gene induction is rapid enough to play a significant role in limiting the growth of incompatible P. parasitica. This is also consistent with the observation that npr1-2-compromised RPP5 resistance was also often associated with trailing necrosis behind the site of hyphal growth (data not shown), suggesting that the HR was elicited, but was too late or insufficient to halt pathogen growth. It is also possible that systemically induced genes regulated by NIM1/NPR1 act synergistically with TIR-class-initiated HR-related responses to prevent P. parasitica proliferation.

We should point out that RPP5-mediated resistance is weaker than that initiated by RPP8. While we rarely saw susceptible RPP5⁺ plants in our F₂ populations, the original characterization of this R gene noted that it was incompletely dominant (PARKER et al. 1993 ). We did occasionally see a similar phenomenon in F₁ plants: In rare instances, Ler x Col F₁ plants were found to be slightly more susceptible to Noco2 than were parental Ler plants (data not shown). In addition, we saw occasional sporulation of Noco2 on the RPP5 NPR1 F₃ seedlings. By contrast, we never saw sporulation on any RPP8-carrying plant of wild-type background in F₂ or F₃ plants. These observations are noteworthy because R genes of a single class may vary in their effectiveness, perhaps owing to the nature of interactions between particular R-gene and avr gene products. Therefore, it is possible that the differences we observed in NIM1/NPR1 dependence between RPP5 and RPP8 may be a consequence of the intensity of the resistance response initiated by those particular avr-R gene interactions rather than a qualitative difference in the resistance pathways initiated by R genes of differing structure. Further testing of TIR- and CC-class R genes will help establish whether R protein structure or its response potency is more predictive of its reliance upon NIM1/NPR1.

While a previous study found that RPP8 was functional in NahG-expressing plants (MCDOWELL et al. 2000 ), we demonstrated that, at least in certain genetic backgrounds, SA accumulation is necessary for RPP8 function. MCDOWELL et al. 2000 showed that an RPP8_Ler transgene could confer Emco5 resistance in a Col NahG background, and our own experiments demonstrated that Ler plants expressing the NahG transgene were not compromised in RPP8-specified resistance. However, in Ler x Ws NahG progeny, we found RPP8 to be impaired in conferring resistance to Emco5. There are a variety of possible explanations for this observation. In RNA gel-blot experiments, we found nahG mRNA levels to be significantly higher in the Ws NahG line compared to the Col NahG or Ler NahG lines used in these studies (our unpublished data), suggesting that the breakdown of RPP8 function in the Ws NahG line may be due to more efficient catabolism of SA in that line compared to the Ler NahG or Col NahG lines. Alternatively, the discrepancy between the two conflicting conclusions might result from quantitative genetic background effects that affect RPP8 efficacy, which may be more evident in an SA-depleted background. This possibility is supported by the observation that cotyledons of Ler x Ws F₁ seedlings are more susceptible to Emco5 than are Ler parent seedlings (Table 4), although they are still much less susceptible than Ler x Ws NahG F₁ seedlings. We do not believe that RPP8 heterozygosity is necessary to observe SA dependence, as we isolated RPP8-homozygous, NahG-expressing plants, which showed comparable levels of Emco5 susceptibility as did the heterozygous plants (data not shown).

If R genes of similar structure initiate similar or identical signal transduction pathways, we would expect them to have similar genetic requirements for their function. HRT is a closely related RPP8 paralog (92% amino acid identity), which confers resistance to turnip crinkle virus (COOLEY et al. 2000 ). Like RPP8, HRT-mediated resistance is compromised in a NahG background, but still functional in a nim1/npr1 background (KACHROO et al. 2000 ). While HRT and RPP8 confer resistance to very different pathogens, it seems likely that they initiate similar responses upon elicitation, given their extensive sequence similarity. The fact that both genes elicit SA-dependent, NPR1-independent resistance supports this hypothesis. KACHROO et al. 2000 found that a second locus, RRT, regulates HRT-mediated resistance. This locus may also prove to be important to RPP8-mediated resistance.

Interestingly, a number of mutants that constitutively exhibit SA-dependent, NIM1/NPR1-independent resistance have been identified (BOWLING et al. 1997 ; CLARKE et al. 1998 ; CLARKE et al. 2000 ). Such mutants may shed light on the biochemical events leading to SA-dependent, NIM1/NPR1-independent, race-specific resistance, if their resistance phenotype results from inappropriate expression of processes that are normally activated by R genes. In support of this idea, analysis of the constitutive PR gene expressers cpr1 and cpr6 showed that the defense phenotype associated with these two mutations requires EDS1 (CLARKE et al. 2001 ), a gene that is also required to transduce signals that originate from TIR-class R genes (AARTS et al. 1998 ).

There is significant evidence that SA plays roles in defense distinct from SAR. SHIRASU et al. 1997 demonstrated that while exogenous SA does not trigger programmed cell death (PCD) by itself, it is able to potentiate elicitor-triggered PCD at concentrations much lower than those shown to be sufficient to induce SAR. Also, the fungal toxin fumonisin B1 induces PCD in wild-type and npr1-1 plants but not in NahG-expressing plants (ASAI et al. 2000 ). Therefore, it seems plausible that in addition to its sufficiency in inducing SAR at high concentrations, at lower concentrations SA may be a necessary component of the programmed cell death response. SA-dependent, NIM1/NPR1-independent pathways are also important in regulating responses to pathogen elicitation. In a companion study, we described a number of Arabidopsis genes that require SA, but not NIM1/NPR1, for their pathogen-dependent induction (RAIRDAN et al. 2001 ). In addition, pathogen-elicited accumulation of camelexin, an Arabidopsis phytoalexin, requires SA, but not NIM1/NPR1 (ZHAO and LAST 1996 ). By combining the use of pathogens to interrogate individual R genes with the growing array of hosts containing defects in defense pathways, the genetic requirements for individual R-gene action will be revealed. Important questions persist as to the number of distinct signaling pathways that support race-specific resistance and how these pathways are shared or dedicated for specific pathogen defense responses.

	ACKNOWLEDGMENTS

We thank Dr. Brian Staskawicz for providing us with the P. syringae pv. tomato DC3000 strains, Dr. Jane Glazebrook for the Col npr1-2 seed, and Dr. Xinnian Dong for Ler NahG seeds. We gratefully acknowledge Dr. Alan Collmer and Wen Ling Deng for assistance with P. syringae experiments and Dr. Jonathan Jones for sharing unpublished results. G.J.R. was supported by a postgraduate fellowship from the National Science and Engineering Research Council of Canada. G.J.R. also received support from a DOE/NSF/USDA grant to the Research Training Group in Molecular Mechanisms of Plant Processes, the Field of Plant Biology at Cornell, and grants to T.P.D. T.P.D. acknowledges support from the National Science Foundation CAREER program (no. IBN-9722377) and U.S. Department of Agriculture NRICGP (No. 98-35303-6484).

Manuscript received November 13, 2001; Accepted for publication March 7, 2002.

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