Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis

Abstract

The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance to P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by ndr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class.

Figure 1

Sporulation levels of different P. parasitica isolates on eds1 mutant lines and wild-type Arabidopsis accessions. Conidiospore suspensions (4 × 10⁴/ml) of each isolate were sprayed onto 2-week-old seedlings. Spores were harvested from leaves and counted after 7 days of incubation (see Methods). For each P. parasitica isolate tested, the genetically susceptible accession is marked with an asterisk and the resistant accession with the corresponding RPP gene. The experiment was repeated on 1-week-old seedlings with similar results.

Figure 2

Pathogen development and disease symptom expression in wild-type and eds1 plants inoculated with P. parasitica or P. syringae. Development of P. parasitica isolate Emco5 is shown in A–C. Leaves were stained with lactophenol–trypan blue 5 days after inoculation and viewed under a light microscope to reveal pathogen mycelium and necrotic plant cells. (Bar = 50 μm.) Disease phenotypes of wild-type Ws-0 and Ws eds1-1 leaves inoculated with P. syringae strain DC3000 or DC3000 expressing avrRps4 are shown in D–G. Leaves were photographed 5 days after infiltration with suspensions of 1 × 10⁵ bacteria/ml. (A) Ler wild-type, showing a discrete necrotic lesion (n) of plant cells surrounding an Emco5 penetration site. Resistance is conferred by RPP8. (B) Ler eds1-2, showing mycelium growing beyond the penetration site (p) but surrounded by a trail of necrotic plant cells (n). (C) Ws-0, lacking RPP8, is fully susceptible to Emco5. The mycelium forms haustoria (h) and grows systemically without associated plant cell death. (D) DC3000 expressing avrRps4 causes no disease symptoms in Ws-0 due to resistance conferred by RPS4. (E) DC3000 expressing avrRps4 causes severe disease symptoms in leaves of eds1-1. (F) DC3000 containing no functional avr gene elicits mild disease symptoms in Ws-0 leaves. (G) Disease symptom development in eds1-1 leaves inoculated with DC3000 is more rapid than in Ws-0 leaves.

Figure 3

Growth of different P. syringae strains in leaves of Ws-0 or Ws eds1-1. Leaves were infiltrated with suspensions of DC3000 containing an empty vector or expressing different avr genes, as indicated, and bacteria were recovered from leaves at various times after inoculation (see Methods). Results in A and B represent data from two separate experiments. These tests were repeated twice with similar results.

Figure 4

Growth of different P. syringae strains in leaves of Col-0 or Col ndr1-1. Leaves were inoculated with DC3000 containing an empty vector, DC3000 expressing avrRpt2, or DC3000 expressing avrRps4, as indicated, and growth of bacteria was measured as in Fig. 3. Tests were repeated twice with similar results.