Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsis genes RPS2 and RPM1

Abstract

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C-terminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cell using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

Figure 3

Subcellular localization of RPS2 by in vitro translation/translocation. (A) RPS2 appears to be cytoplasmic. RPS2 RNA (lanes 2–6) and β-lactamase RNA (a positive control for translocation; lanes 7–11) were translated either in the presence (lanes 3–6 and 8–11) or absence (lanes 2 and 7) of dog pancreatic microsomes. Lane 1 shows a no RNA control for translation. The reactions were treated with proteinase K (lanes 4 and 9) or fractionated by ultracentrifugation into precipitate (lanes 5 and 10) and supernatant (lanes 6 and 11) fractions after Na₂CO₃ treatment. The positions of molecular weight markers, RPS2, and β-lactamase are indicated on the left. (B) RPS2 detected in the precipitate fraction is an artifact. The microsomes were either included in the translation reaction as in the standard procedure (cotranslation; lanes 3 and 4) or added after the translation reaction was terminated with cycloheximide (posttranslation; lanes 1 and 2). The reactions were fractionated into precipitate (lanes 1 and 3) and supernatant fractions (lanes 2 and 4) after Na₂CO₃ treatment.

Figure 1

The I353K mutation in the putative transmembrane region of RPS2. The putative transmembrane region of RPS2 protein (Middle) (7) is compared with the corresponding region of N (Top) (23). Identical and similar residues between the two proteins are indicated by vertical lines and colons, respectively, between the two sequences. The putative transmembrane region of RPS2, predicted by alom (22), is underlined. The corresponding region of the I353K mutant is shown at bottom. The mutated amino acid residue in I353K and the corresponding residues in N and RPS2 are shown in boldface. The numbers indicate the amino acid residue numbers in each protein.

Figure 2

(A) The I353K RPS2 mutant gene can complement an rps2 mutant phenotype in transgenic plants. One-month old rps2-101C plants transformed with I353K (Left), with a rps2-101C mutant gene (Center), and with the RPS2 wild-type gene (Right) were inoculated at 0.5 × 10⁷ colony-forming unit/ml with Psp 3121 carrying a vector control (pLAFR3) or carrying avrRpt2. Only one-half of each leaf (arrowhead) was inoculated. The photographs were taken 20 hr after inoculation. (B) Transient expression of avrRpt2 causes a reduction of cointroduced GUS gene expression in RPS2 plants. Leaves of RPS2 wild-type (Right) and rps2-101C mutant (Left) plants were bombarded with biolistics carrying the avrRpt2 and GUS constructs. After a 27-hr incubation, the leaves were histochemically stained for GUS activity. The cells that express GUS enzyme at a high level are visualized as blue dots on the leaves.

Figure 4

avrRpt2 and avrB can elicit a specific resistance response when expressed in plants. RPS2 RPM1 (A), rps2 RPM1 (B), RPS2 rpm1 (C), and rps2 rpm1 (D) plants were bombarded with biolistics carrying the GUS and the indicated avr gene constructs. Each bar represents the mean value of four bombardment events. (Bars = SEM.)

Figure 5

Both avirulence and resistance genes can be analyzed by transient expression. rps2 rpm1 plants were bombarded with biolistics carrying the GUS gene, the indicated R gene, and the indicated avr genes. Each bar represents the mean value of four bombardment events. (Bars = SEM.)