Article Figures & Data
Figures
- Figure 1.
lra1 Mutants Do Not Respond to Inducible Expression of avrRpm1.
Three-week-old plants containing an inducible avrRpm1 expression system were sprayed with estradiol and stained with trypan blue 24 hr later. Trypan blue stains leaf veins and dead cells, revealing the region undergoing HR.
(A) Line a11 (RPM1, LRA1). Note the extent of cell death.
(B) Line a11r (rpm1-1, LRA1) contains the transgene from a11 crossed into the isogenic rpm1-1 allele. Note the lack of cell death.
(C) and (D) lra1-1 (RPM1, lra1-1) (C) and lra1-2 (RPM1, lra1-2) (D). The arrows point to areas of faint staining. Note that the lra1-1 mutant is Atrar1-21 and the lra1-2 mutant is Atrar1-22.
- Figure 2.
LRA1/PBS2 Is AtRAR1.
(A) Structure of the AtRAR1 gene. The predicted protein is portrayed in single-letter amino acid abbreviations. The mutations found in Atrar1-21 to Atrar1-28 are underlined. Nucleotides not present in the mRNA are shown in italic type.
(B) Relationship of Atrar1 mutations to the RAR1 protein domain structure. “C to Y” indicates a missense mutation resulting in a Cys-to-Tyr substitution. “STOP” indicates mutations that produce a stop codon. The allele number follows each mutation.
(C) AtRAR1 protein expression in the allelic series. Total proteins were extracted from each of the mutants and analyzed by protein gel blot analysis using anti-RAR1 serum. Equal loading was ensured by Ponceau staining. The top arrow indicates a molecular mass marker of 33 kD, and the bottom arrow indicates a molecular mass marker of 25 kD. Note that there is no detectable protein in the Atrar1-20 mutant or in the mutants produced by stop codons (Atrar1-21, -22, -24, -25, and -28), and there is less detectable protein where a mutation from Cys to Tyr has occurred (Atrar1-23, -26, and -27).
- Figure 3.
AtRAR1 and NDR1 Act Differently to Control R Function.
Wild-type and mutant Arabidopsis lines were inoculated with Pst DC3000 strains containing the indicated avr genes. On day 0 (white columns) and day 3 (black columns), bacteria were extracted from the plants and enumerated. Bacterial numbers are expressed as the logarithm of colony-forming units per milligram of fresh weight (cfu/mg FW). The average and se of four independent replicates are shown. These experiments were performed three times with similar results. ndr1-1 was described previously (Century et al., 1995).
(A) The susceptible control is a11r, an isogenic a11 derivative but in a rpm1-1 background (Grant et al., 1995).
(B) The susceptible control is the isogenic rps2-101C allele (Mindrinos et al., 1994).
(C) The susceptible control is the isogenic rps5-2 allele (Warren et al., 1998).
(D) The susceptible control is accession RLD (Hinsch and Staskawicz, 1996).
- Figure 4.
Staining of HR and Reactive Oxygen Intermediates in Atrar1 and Related Mutants upon Pst DC3000(avrRpm1) Inoculation.
Plants were inoculated as described in Table 1 on the right side of the leaf and stained with trypan blue 20 hr after inoculation ([A], [C], [E], [G], and [I]) or with DAB 1.5 hr after inoculation ([B], [D], [F], [H], and [J]). Trypan blue stains veins and dead cells dark blue, and DAB stains total peroxides as a brown precipitate, as indicated by the arrow in panel (B).
(A) and (B) a11.
(C) and (D) a11r.
(E) and (F) ndr1-1.
(G) and (H) Atrar1-21.
(I) and (J) Atrar1-21 ndr1-1.
- Figure 5.
Staining of HR Sites and Production of Reactive Oxygen Intermediates Is Altered during RPP7-Mediated Responses.
Plants were inoculated with Pp strain Hiks1 by spraying with an aqueous suspension containing 5 × 104 oospores/mL and then stained with trypan blue 5 days after inoculation ([A] to [E]) or with DAB 2 days after inoculation ([F] and [G]). The arrows in panels (F) and (G) point to sites of hyphae penetration.
(A) and (F) a11.
(B) rpp7.
(C) ndr1-1.
(D) and (G) Atrar1-21.
(E) Atrar1-21 ndr1-1.
- Figure 6.
Atrar1 and ndr1-1 Affect RPP7 Function Additively.
Plants were inoculated with Pp strain Hiks1 as described in Figure 5. Seven days after inoculation, the plants were stained with trypan blue, and the interactions sites were classified as HR, trailing necrosis (TN), and free hyphae (FH). A minimum of 591 interactions per genotype from three independent experiments are represented.
- Figure 7.
RPM1-myc in Atrar1 Mutants.
Plants were harvested at 5 weeks of age, and total extracts were prepared as described by Boyes et al. (1998). Arrows with numbers indicate the positions of molecular mass markers (kD). The arrows at both sides of the figure indicate the position of RPM1-myc. Three independent experiments were performed, and each gave similar results.
- Figure 8.
Genetic Requirements for the R Gene Functions Tested.
The font size reflects the relative contribution of each locus to the function of each R protein listed at top. A larger font implies that the null mutant compromises R function severely, and a smaller font implies a moderate effect. A locus placed in the same vertical orientation implies a single pathway. Note that the order of action can be inferred clearly only in the case of RPM1. Loci side by side imply that no relationship was determined, and split arrows represent parallel pathways.
Tables
No. of Leaves Exhibiting Visible HRa Bacterial Genotypeb Col-0 rar1-20 avrB::Ωc 1 of 53 2 of 45 avrRpt2 55 of 61 24 of 55 avrRpm1 59 of 59 12 of 60 avrPphB 65 of 68 17 of 60 ↵a Number of leaves showing visible tissue collapse out of the total number of leaves injected at 18 to 24 hr after injection.
↵b Pst strains expressing the indicated avirulence genes were injected at an OD600 of 0.075 (∼3.75 × 107 colony-forming units/mL).
↵c This strain expresses a nonfunctional avr gene and is unable to induce a visible HR in Col-0 plants.
