Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes

Abstract

The identification of two Arabidopsis thaliana genes involved in determining recessive resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum, is reported. Dominant (RRS1-S) and recessive (RRS1-R) alleles from susceptible and resistant accessions encode highly similar predicted proteins differing in length and which present a novel structure combining domains found in plant Toll-IL-1 receptor-nucleotide binding site-leucin-rich repeat resistance proteins and a WRKY motif characteristic of some plant transcriptional factors. Although genetically defined as a recessive allele, RRS1-R behaves as a dominant resistance gene in transgenic plants. Sequence analysis of the RRS1 genes present in two homozygous intragenic recombinant lines indicates that several domains of RRS1-R are essential for its resistance function. Additionally, RRS1-R-mediated resistance is partially salicylic acid- and NDR1-dependent, suggesting the existence of similar signaling pathways to those controlled by resistance genes in specific resistance.

Figure 1

Map-based cloning of the RRS1-S locus from A. thaliana Col-5 accession. (A) Contig of the four A. thaliana yeast artificial chromosome (YAC) clones CIC2A12, CIC11H2, CIC4H11, and CIC11A11 spanning the RRS1 locus. (B) Contig of cosmid clones generated from BAC clones T29K4 and T25P9 that span the RRS1 locus from the YAC contig. (C) Structural organization of the B1 cosmid. The direction of transcription of the three genes present on B1 is indicated by arrows. (D) Resistant Nd-1 plants were transformed with cosmid B1 or with different constructs derived from it (M13, M14, and B1MT9, black lines) and tested for their response to strain GMI1000. Similar constructs were also generated from Nd-1 cosmid H and used to transform susceptible Col-5 plants (H8, H10, and HMTB2, gray lines). For each construct, the number of independent transgenic lines tested, their genetic background, and their responses to strain GMI1000 (R, resistant; S, susceptible) are indicated on the right.

Figure 2

Schematic representation of RRS1 allelic structures in accessions Col-5 and Nd-1 and in two intragenic recombinant lines. Filled and open boxes indicate exons corresponding to a Col-5 and a Nd-1 genetic background, respectively. Introns are indicated as gaps. Localization of TIR, NB-ARC [nucleotide binding adapter shared by APAF-1 R proteins and CED-4 (14, 15)], LRR, nuclear localization signal (NLS), and WRKY (consensus domain of WRKY protein family) domains on the genes is indicated. In the RRS⁵⁴⁰ gene, the recombination event occurred between nucleotides 13018 (Col-5) and 13408 (Nd-1) and between nucleotides 13403 and 13686 in RRS⁵⁶⁶ gene (as indicated by *). The response to strain GMI1000 of homozygous plants at the RRS1 locus is indicated on the right (R, resistant; S, susceptible).

Figure 3

Amino acid comparison of RRS1-R and RRS1-S. The open boxes indicate the different conserved domains. The six imperfect LRR domains are underlined and the amino acids differing between RRS1-R and RRS1-S are shown as black boxes.

Figure 4

Internal bacterial growth curves of R. solanacerum strain GMI1000 in Col-5 plants (●), CH1.2, a selected transgenic Col-5 line containing the cloned RRS1-R gene (○), and Nd-1 plants (■). Root inoculations with strain GMI1000 were performed as described (31).

Figure 5

Effect of salicylic acid and ndr1–1 on RRS1-R-mediated resistance to R. solanacearum. Plants were inoculated with strain GMI1000. Bacterial growth was estimated as described (16) at the time of inoculation, 7 days after inoculation at which time 75–100% of Col-5 leaves were wilted and no symptoms were visible on Nd-1, Nd-1/NahG, and RRS1-R/ndr1–1 plants (T7), and 12 days after inoculation at which time 75–100% of Nd-1/NahG and RRS1-R/ndr1–1 leaves were wilted whereas no symptoms were detectable on Nd-1 plants (T12).