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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

To better understand the genetic requirements for R gene-dependent defense activation in Arabidopsis, we tested the effect of several defense response mutants on resistance specified by eight RPP genes (for resistance to Peronospora parasitica) expressed in the Col-0 background. In most cases, resistance was not suppressed by a mutation in the SAR regulatory gene NPR1 or by expression of the NahG transgene. Thus, salicylic acid accumulation and NPR1 function are not necessary for resistance mediated by these RPP genes. In addition, resistance conferred by two of these genes, RPP7 and RPP8, was not significantly suppressed by mutations in either EDS1 or NDR1. RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1 which affect ethylene or jasmonic acid signaling. Double mutants were therefore tested. RPP7 and RPP8 were weakly suppressed in an eds1-2/ndr1-1 background, suggesting that these RPP genes operate additively through EDS1, NDR1 and as-yet-undefined signaling components. RPP7 was not compromised in coi1/npr1 or coi1/NahG backgrounds. These observations suggest that RPP7 initiates resistance through a novel signaling pathway that functions independently of salicylic acid accumulation or jasmonic acid response components.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

The ability of plants to resist pathogen colonization is often dependent upon the expression of naturally variable genes (referred to as R genes) conferring race-specific pathogen resistance ( Crute & Pink, 1996). Current models suggest that R gene-dependent defense responses are triggered by specific interactions between pathogen-encoded ligands and R gene-encoded receptors ( Keen, 1990). R gene action triggers a signal transduction cascade that in turn activates a suite of defense responses such as the rapid death of host cells (hypersensitive response; HR), localized tissue fortification, and antimicrobial gene expression ( Hammond-Kosack & Jones, 1996). Many R genes have been genetically defined and cloned, and the majority encode proteins with a consensus nucleotide binding site (NBS) and arrays of leucine-rich repeats (LRRs) ( Ellis & Jones, 1998). The conservation of these motifs in R proteins that respond to diverse pathogens suggests that R proteins might employ a limited number of signaling pathways.

Mutational screens in Arabidopsis have identified additional components of R gene-dependent resistance responses, including several loci that suppress the function of multiple R genes and are thus thought to encode signal transduction components (reviewed in Glazebrook et al. 1997 ). The ndr1 and eds1 loci were defined in screens for loss of race-specific resistance to strains of the bacterium Pseudomonas syringae or the oomycete Peronospora parasitica ( Century et al. 1995 ; Parker et al. 1996 ). EDS1 and NDR1 are each required for the function of different subclasses of NBS-LRR R genes ( Aarts et al. 1998 ; Century et al. 1995 ; Parker et al. 1996 ). In other words, the R genes suppressed by the ndr1 mutation are not affected by eds1 mutants, and vice versa. This proposed mutual exclusivity in function correlates with R protein structure rather than the types of pathogen recognized by the respective R gene products: eds1 suppresses NBS-LRR resistance proteins with N-terminal motifs similar to the cytoplasmic signaling domain of the Toll and Interleukin1 transmembrane receptors (TIR-NBS-LRR). Conversely, the ndr1 mutation suppresses NBS-LRR resistance proteins that contain an N-terminal leucine zipper rather than the TIR domain (LZ-NBS-LRR). These observations suggest a model in which EDS1 and NDR1 mediate distinct R gene-dependent signaling pathways ( Aarts et al. 1998 ).

One probable exception to this apparent rule was noted by Aarts and co-workers. The RPP8 gene, which encodes an LZ-NBS-LRR protein ( McDowell et al. 1998 ), is not suppressed by either ndr1 or eds1 single mutants, as predicted by the model. The eds1/ndr1 double mutant was not tested by Aarts et al. so the possibility that RPP8 signals defense additively through EDS1 and NDR1-dependent pathways was not ruled out. Alternatively, RPP8-mediated recognition could be transduced by signaling mechanisms operating independently of EDS1 or NDR1.

Salicylic acid (SA) is a key defense response component in Arabidopsis (reviewed in Durner et al. 1997 ). Transgenic plants expressing a bacterial salicylate hydroxylase protein that converts SA to catechol (encoded by the NahG gene) are compromised in systemic acquired resistance (SAR) (reviewed in Ryals et al. 1996 ). SA application rescues eds1 and ndr1 mutants, suggesting that these components act upstream or independently of SA ( Century et al. 1995 ; Parker et al. 1996 ). A second key SAR component was identified by mutations in the NPR/NIM1 gene ( Cao et al. 1994 ; Delaney et al. 1995 ). NPR1/NIM1 operates downstream of SA and encodes a probable transcription regulator ( Cao et al. 1997 ; Ryals et al. 1997 ). In addition, npr1/nim1 mutants and NahG suppress R genes that recognize specific isolates of the downy mildew pathogen P. parasitica or the bacterium P. syringae ( Cao et al. 1994 ; Delaney et al. 1994 ; Delaney et al. 1995 ). It has thus been proposed that SA-dependent regulatory components play a central role in R gene mediated ‘local’ resistance as well as SAR ( Delaney et al. 1994 ). However, the effect of NahG and npr1/nim1 on numerous R genes has not been tested, thus it is not known if SA accumulation and NPR1 function are universally required for R gene dependent defense induction.

Recent studies have revealed SA-independent resistance mechanisms in Arabidopsis, mediated by components of the jasmonic acid (JA) and ethylene (ET) response pathways (reviewed in Dong, 1998). For example, resistance to isolates of the necrotrophic fungi Alternaria brassisicola and Botrytis cinerea is compromised by a JA response mutant called coi1 but is unaffected by NahG or npr1( Thomma et al. 1998 ). In contrast, the same study revealed that resistance to a P. parasitica isolate is unaffected by coi1. The jasmonic acid insensitive mutant jar1 has also been shown to suppress resistance to Pythium irregulare, a soilborne oomycete which causes damping-off ( Staswick et al. 1998 ). Thus, SA-dependent resistance mechanisms could be triggered by biotrophs such as P. parasitica, while JA-dependent resistance pathways are triggered by necrotrophs. However, the effect of ET and JA response mutations on a large collection of genetically defined, race-specific R genes has not been tested.

In this study, we compared the effects of various single and double combinations of disease resistance mutations described above on resistance to eight isolates of P. parasitica, in each case conferred by a different RPP specificity. We found that RPP genes can differ markedly in their dependence upon mutationally defined signal transduction components, and that the RPP7 gene is not substantially affected by any of the Arabidopsis defense response mutants that have been defined to date.

Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

SA accumulation and NPR/NIM function are not necessary for downy mildew resistance conferred by several RPP genes

We inoculated Colombia (Col-0) lines containing either the NahG transgene or the npr1-1 allele with seven Col-incompatible isolates of P. parasitica that are recognized by distinct RPP specificities (as described in Experimental procedures). The effects of each mutant on resistance were measured as enhanced hyphal growth and asexual sporulation in mutant backgrounds relative to the wild-type resistance for each isolate (see Experimental procedures; Fig. 1). Resistance to Emoy2 and Cand5 appeared to be fully suppressed (heavy sporulation) in Col::NahG and partially suppressed (low to moderate sporulation) in Col-npr1-1 ( Figs 1 and 2). In contrast, resistance to Hiks1 was essentially unaffected in Col-npr1-1 and Col::NahG, suggesting that NPR1 function and SA accumulation are not necessary for RPP7 resistance. Mutations in the RPP7 gene conferred full susceptibility to Hiks1, demonstrating that resistance to this isolate in Col-0 is completely dependent upon RPP7 ( Figs 1 and 2, data not shown).

image

Figure 1. Asexual reproduction in wild-type and various mutant lines by P. parasitica isolates that are recognized by different RPP specificities.

Quantitative disease ratings are expressed as the mean number of sporangiophores per cotyledon. SE refers to standard error and N refers to the total number of cotyledons scored in two to three replicates. RPP8 is segregating 3 : 1 in the lines derived from the cross to Col::NahG lines, and the medium or heavily sporulating lines were omitted from the calculation of mean sporangiophores/cotyledon.

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image

Figure 2. Interaction phenotypes of P. parasitica isolates with wild-type Col and Col::NahG or Col-npr1-1.

(a) Each isolate is recognized by a different RPP gene. Cotyledons were photographed 7 days after inoculation.

(b) Interaction of the Hiks1 isolate with various defense response mutants. Cotyledons were stained 7 days after inoculation with Trypan Blue. Col-0 and eds1-2 exhibit hypersensitive cell death at infection sites with no hyphal growth, while Col-rpp7 supports profuse hyphal growth and oospore production. ndr1-1, NahG, npr1-1, eds/ndr exhibit patches of trailing necrosis indicative of a delayed resistance response.

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We also assessed the effect of NahG and npr1-1 on the RPP8 gene, which specifies resistance to the Emco5 isolate of Peronospora. The RPP8 allele in Col-0 is non-functional, therefore we tested the transgenic Col-0 plants expressing the RPP8 allele from Landsberg erecta (RPP8-Ler). Resistance to Emco5 in Col-0::RPP8 was unaffected by npr1-1 and only slightly suppressed by NahG. We observed only rare sporangiophore production and very limited hyphal growth in cotyledons of the latter ( Figs 1 and 2). Similarly, npr1-1 and NahG had very weak effects on RPP functions specifying resistance to the remaining isolates, Cala2, Wela3, Hind4 and Wand1. Pronounced wilting was observed for Wand1 and Hind4 in Col::NahG. This was associated with extensive colonization of tissue with hyphae in the absence of enhanced parasite reproduction.

Two previous papers reported that resistance to Wela was suppressed in Col::NahG and Col-npr1-1 ( Delaney et al. 1994 ; Delaney et al. 1995 ). The pathogen isolate used in our study, designated Wela3, was derived from a single sexual spore of the original mass Wela spore culture. Thus, the discrepancy in mutant phenotypes we report here may reflect segregation of avirulence determinants between these isolates.

Previous reports demonstrated partial or complete suppression of Ws-RPP1, Ws-RPP12 and RPS2 (resistance to P. syringae expressing avrRpt2) in NahG, and/or npr1/nim1 backgrounds ( Cao et al. 1994 ; Delaney et al. 1994 ; Delaney et al. 1995 ). Furthermore, application of SA or SA analogs, as well as over-expression of NPR1, is sufficient to prime resistance to P. parasitica ( Cao et al. 1998 ; Uknes et al. 1992 ). In contrast, our experiments demonstrate that SA accumulation and NPR1 function are not required for resistance specified by several RPP genes. This raises the question of whether SA- and NPR1-independent RPP genes, such as RPP7, activate a completely different resistance mechanism with distinct signaling components and downstream effectors, or whether they activate the same downstream effectors utilized by other RPP genes through an alternate signal transduction pathway that bypasses SA and NPR1. We intend to utilize DNA microarray analysis, further mutational analysis, and other approaches to test this question.

RPP7 and RPP8 are unaffected by eds1 or ndr1 mutation, and are only weakly suppressed by an eds1/ndr1 double mutant RPP8 function (in accession Landsberg erecta (Ler) was not strongly suppressed by either eds1-2 or ndr1-1 mutation, although a very slight shift towards susceptibility was observed ( Aarts et al. 1998 ). We observed previously that rpp8 loss of function alleles in Ler conferred only partial susceptibility to Emco5, even though at least one of the mutations (Ler-rpp8-4) causes a severe translational truncation that is probably a null allele ( McDowell et al. 1998 ). Thus, a second, weak R gene linked to RPP8 in Ler could contribute to resistance against Emco5, and may have clouded the interpretations of Aarts and co-workers. We tested the effect of ndr1 and eds1 mutations on RPP8 function (expressed as a transgene) in backgrounds (Col-0 and Wassilewskija (Ws) (Ws-0)) that are fully susceptible to Emco5 infection. RPP8 in either of these accessions provides strong resistance to Emco5 ( Fig. 1 and McDowell et al. 1998 ). We constructed an ndr1-1/RPP8 line in the Col background. An eds1 allele in Col has not been isolated, therefore we combined the Ws-eds1-1 allele ( Parker et al. 1996 ) with a Ws::RPP8 transgene (Experimental procedures). We observed that neither ndr1-1 nor eds1-1 had a significant effect on RPP8 transgene-dependent resistance ( Fig. 1) other than a very slight increase in hyphal growth. This observation extends the validity of previous observations that the cloned RPP8 functions independently of NDR1 and EDS1.

Resistance to Hiks1 conferred by RPP7 is not compromised by ndr1 ( Century et al. 1995 ), but the effect of eds1 mutation on RPP7 resistance has not been reported. We therefore tested the eds1-2 allele from Ler. Wild-type Ler is resistant to Hiks1 due to an RPP specificity that is either allelic with or closely linked to RPP7, based on the observation that no susceptible individuals were found in 2000 F2s from a Col-0 X Ler cross inoculated with Hiks1 (E.B. Holub, unpublished results). We inoculated Ler-eds1-2 seedlings and found no suppression of Hiks1 resistance in this background. Thus, RPP7 and RPP8 are distinct from other known Arabidopsis R genes in that they confer resistance independently of both NDR1 and EDS1.

To test whether eds1-2 and ndr1-1 have additive effects on RPP7 or RPP8 function, we bred a line homozygous for ndr1-1, eds1-2 and RPP8 (Experimental procedures). Inoculations of this line with Hiks1 and Emco5 revealed only a weak loss of resistance: moderate hyphal growth occurred in infected cotyledons, and light asexual sporulation was observed ( Figs 1 and 2). Thus, the combination of ndr1-1/eds1-2 has only a partial effect on RPP7 or RPP8 function. Our data, however, demonstrate for the first time that NDR1 and EDS1 can act in concert to at least partially transduce R function. Thus, the model of a simple dichotomy of signaling pathways subsequent to pathogen recognition based on R protein structure requires modification.

RPP7 is not affected by mutations in the ethylene or jasmonic acid response pathways

SA and JA signaling can be antagonistic (reviewed in Dong, 1998). The SA-independence of RPP7 therefore raised the possibility that it operates through a JA- and/or ET-dependent response pathway. We inoculated the Col-coi1-2 (A. Kloek and B. Kunkel, unpublished results), Col-ein2.1 and Col-jar1 ( Staswick et al. 1998 ) mutant lines with Hiks1. Each mutant exhibited wild-type RPP7 function. Thus, we conclude that RPP7 resistance is not dependent solely on the ET or JA response pathways. Double mutant combinations of coi1-2 with either Col::NahG or npr1-1 were also tested and they exhibited wild-type RPP7 function ( Fig. 1). This indicates that the SA, JA and ET-independence of RPP7 resistance cannot be explained by redundant utilization of SA-dependent and JA-dependent response pathways. We thus postulate the existence of a ‘third’ pathway whose components have not been genetically identified ( Fig. 3).

image

Figure 3. Model for RPP-dependent resistance regulation in Arabidopsis, inferred from the dependence or independence of RPP2, RPP4 and RPP7 on NDR1, EDS1, NPR1 and SA accumulation.

Branched pathways represented by dashed lines make relatively weak contributions to the expression of resistance.

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It should be noted that coi1 is a male-sterile line that can only be propagated as a heterozygote, thus all coi1-containing lines were segregating for the coi1 mutation. In each experiment we tested a large enough number of individuals (over 100) to have easily detected susceptible individuals in a segregating population.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

The data presented here, in combination with previous studies, suggest the existence of at least three mechanisms by which RPP gene-dependent pathogen recognition can be transduced into resistance responses. The first, exemplified by RPP2, requires EDS1 but not SA accumulation or NPR1. The second mechanism, exemplified by RPP4, requires EDS1 and SA accumulation and at least in cotyledons is partially dependent upon NDR1 and NPR1. The third mechanism, exemplified by RPP7 and probably RPP8, is partially dependent on the additive functions of NDR1 and EDS1 but is independent of SA accumulation and NPR1. Furthermore, previous studies demonstrated that RPP7 is not suppressed by other defense response mutations such as pbs1, pbs2, pbs3, pad1, pad2, pad3, pad4 or pad5 ( Glazebrook et al. 1997a; 1997b; Warren et al. 1999 ). We propose that RPP7 gene function is mediated by a novel regulatory network, either solely or in addition to previously described defense responses. We are conducting large-scale mutational screens to identify additional components of RPP7- and RPP8-dependent resistance, and have identified at least three new loci that suppress RPP7 function (J.M. McDowell et al. unpublished results).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

P. parasitica isolates

Seven isolates, each diagnostic for a different RPP specificity in Col-0, were used. Wild-type resistance in Col to each isolate is associated with rapid death of penetrated host cells (hypersensitive response), but varies in the restriction of parasite reproduction: three isolates (Cand5, Emoy2 and Hind4) produce low level asexual sporulation (mean 2–4 sporangiophores per cotyledon); one isolate (Cala2) produces a rare sporangiophore in less than 1% of seedlings; and the others (Hiks1, Wand1 and Wela3) never sporulate ( Fig. 1). Single RPP loci have been defined for resistance to Cala2 and Emoy2 (RPP2 and RPP4, respectively, on chromosome 4), and to Hiks1 and Wela3 (RPP7 and RPP6, respectively, on chromosome 1). Resistance to Cand5 and Hind4 appears to be digenic at distinct, independent loci for each isolate (C. Can and E. Holub, unpublished results); locus names have not been designated. Resistance to Wand1 is probably a single locus but unmapped.

Pathogenicity tests

P. parasitica isolates were maintained by weekly subculturing on susceptible recipient plants as described previously ( Dangl et al. 1992 ). Pathogen challenge inoculations were conducted by spraying 7-day-old seedlings with a spore suspension (5 × 104 spores ml−1 in dH2O). Seedlings were grown for 7 days at 16–18°, 8 h day length, 80–100% relative humidity. Asexual sporulation was visually assessed at 7 or 8 days after inoculation by counting sporangiophores on both sides of the cotyledon and classifying individual cotyledons as either N (no sporangia), L (1–10 sporangia), M (11–19) or H (20 or more). We used real numbers (0–10) for N and L cotyledons and assigned values of 15 (M) and 20 (H) to calculate the averages shown in Fig. 1. Plants were stained with lactophenol-trypan blue ( Koch & Slusarenko, 1990) by boiling for 3 min and continuing the incubation at room temperature overnight. Plants were then de-stained overnight in chloral hydrate and mounted in 70% glycerol for light microscopy.

Construction of RPP8 transgenic lines, crosses and mutant selection

One transgenic Col-0 line containing the pRPP8 plasmid construct ( McDowell et al. 1998 ) was used for crosses with Col-npr1-1 ( Cao et al. 1997 ), Col-ndr1-1 ( Century et al. 1997 ), and Col::NahG ( Weyman et al. 1995 ). A transgenic Ws-0 line containing RPP8 in the 9L9 cosmid construct ( McDowell et al. 1998 ) was used for crosses with Ws-eds1-1 ( Falk et al. 1999 ). In each case the transgenic line was shown by segregation analysis to contain a single transgene insertion locus. The eds1-2/ndr1-1/RPP8 triple homozygous line was selected from a Col-ndr1-1 X Ler-eds1-2 ( Falk et al. 1999 ) cross. Lines homozygous for Col-npr1-1, Col-ndr1-1, Ler-eds1-2 and Ws-eds1-1 were selected in the F2, F3 or F4 generation with PCR-based markers that distinguished between wild type and mutant alleles. Details of these markers are available upon request. We selected lines that were homozygous for the RPP8::Ler or Col::NahG transgene by progeny testing in the F3 or F4 generation for resistance to Basta or Kanamycin. We were unable to isolate F3 lines that were homozygous for both the RPP8 transgene and the Col::NahG transgene, and concluded that these transgenes must reside at linked loci. We were able to isolate a F4 family in which all tested individuals contained the Col::NahG transgene. This family segregated 3 : 1 for RPP8, and was used in the experiments described above.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

We gratefully acknowledge Andrew Kloek and Barbara Kunkel for the coi1-2, coi1/npr1 and coi1/Col::NahG lines. This research was funded by grants from the USDA-NRICGP to J.M.M (98-02482) and J.L.D. (99-35301-7848), and from the BBSRC to E.B.H.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof
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Note added in proof

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and Discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Note added in proof

Klessig and colleagues report that an RPP8-like resistance gene against Turnip Crinkle Virus called HRT requires SA, but not NPR1, ethylene or JA for its function. Mutations in eds1 and ndr1 have not been tested for their impact on HRT function. Nonetheless, members of the RPP8 family can differ in their genetic requirements for function (Kachroo et al. 2000 ). Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is Salicylic Acid dependent but NPR1, ethylene and jasmonate independent. Plant Cell, in press).