Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9

Abstract

The Arabidopsis Ler-RPP27 gene confers AtSgt1b-independent resistance to downy mildew (Peronospora parasitica) isolate Hiks1. The RPP27 locus was mapped to a four-bacterial artificial chromosome interval on chromosome 1 from genetic analysis of a cross between the enhanced susceptibility mutant Col-edm1 (Col-sgt1) and Landsberg erecta (Ler-0). A Cf-like candidate gene in this interval was PCR amplified from Ler-0 and transformed into mutant Col-rpp7.1 plants. Homozygous transgenic lines conferred resistance to Hiks1 and at least four Ler-0 avirulent/Columbia-0 (Col-0) virulent isolates of downy mildew pathogen. A full-length RPP27 cDNA was isolated, and analysis of the deduced amino acid sequences showed that the gene encodes a receptor-like protein (RLP) with a distinct domain structure, composed of a signal peptide followed by extracellular Leu-rich repeats, a membrane spanning region, and a short cytoplasmic carboxyl domain. RPP27 is the first RLP-encoding gene to be implicated in disease resistance in Arabidopsis, enabling the deployment of Arabidopsis techniques to investigate the mechanisms of RLP function. Homology searches of the Arabidopsis genome, using the RPP27, Cf-9, and Cf-2 protein sequences as a starting point, identify 59 RLPs, including the already known CLAVATA2 and TOO MANY MOUTHS genes. A combination of sequence and phylogenetic analysis of these predicted RLPs reveals conserved structural features of the family.

Figure 1.

Map-based cloning of RPP27. Genetic map of RPP27 locus showing the molecular markers F12M16 and Nga280 that were initially used to define the mapping interval. cM, Centimorgan (A). B, The BAC contig spanning the RPP27 locus that was fine mapped with the markers shown above the bar. The numbers of recombinant individuals identified with the markers are shown below the bar. The black bar on the BAC clone F20D21 represents the Cf-like gene. C, This region was amplified from Ler-0, inserted into a binary vector to produce MT27, which was then introduced into Col-rpp7.1 plants. The cDNA was obtained and compared with genomic DNA to reveal the structure of the RPP27 gene. Untranslated regions are shown as gray, exons are shown as black, and introns are shown as white bars. D, Numbers below indicate the size of untranslated regions, exons, and introns.

Figure 2.

Pathogen development and interaction phenotypes of transformed and nontransformed plants inoculated with downy mildew isolate Hiks1. Cotyledons stained with DAB 1 d after inoculation and examined under a light microscope for H₂O₂ accumulation are shown in A and B (bar = 50 μm). A, Normal pathogen development and no H₂O₂ detection was observed in Col-rpp1.1. B, Accumulation of H₂O₂ was detected with DAB staining around the Hiks1 penetration sites in Col-rpp7 transformed with RPP27. Cotyledons stained with lactophenol-trypan blue 3 d and 7 d after inoculation and viewed under a light microscope to reveal pathogen mycelium and necrotic plant cells are shown in C and E (bar = 10 μm). C, Col-rpp7.1, shown with normal pathogen development, fully susceptible to Hiks1 3 d after inoculation. D, Col-rpp7.1::Ler-RPP27, showing mycelium growth beyond the penetration site but surrounded by a trail of necrotic plant cells 3 d after inoculation. E, Col-rpp7.1::Ler-RPP27, showing extensive mycelial growth and trailing necrosis with a conidiophore development, which was observed occasionally 7 d after inoculation.

Figure 3.

Predicted domain structure of RPP27. Domains A to G correspond with previous diagrams of Cf-9 (Jones et al., 1994; see text for descriptions). The B region is divided into B1, B2, and B3 to show the presence of its predicted TM region (B2, underlined). The site of the large deletion relative to Ler-0 is marked with an asterisk. The LRR consensus sequence appears boxed and aligned below the full sequence. The C region is shown divided into C1 (main LRR block), C2 (non-LRR island), and C3. The presence of the island of non-LRR sequence before the final four LRRs is a common element in RLPs and may be a structural hinge that allows the C1 block to adopt its correct conformation. The C3 region is highly conserved within the family and may be required for multiprotein complex formation.

Figure 4.

Multiple sequence alignment of C-terminal regions of RPP27, CLV2, Cf-9, and Cf-2. Sequences were cropped to show the C-terminal conserved region found across all Arabidopsis RLPs, which includes a section of the LRR region of the protein (domain C3) and continues past the variable (domain D), acidic (domain E), and transmembrane (domain F) domains to the C-terminal cytoplasmic peptide (domain G). Sequences were aligned using MAFFT. Identical and similar residues are displayed in black and gray boxes, respectively.

Figure 5.

Phylogenetic tree of the RLP family in Arabidopsis. Amino acid sequences of 59 RLPs from Arabidopsis, as well as Cf-2 and Cf-9, were aligned with MAFFT. The tree was generated from a truncated alignment consisting of the conserved C3-F domains. A total of 100 bootstrap replicates of this alignment were made. PHYLIP's Neighbor program was used to build the trees, and the Consense program generated the consensus tree and bootstrap values. Other bootstrapped trees (not shown) were built with parsimony and maximum likelihood methods from the full and truncated alignments. All trees were similar to this one, in that certain subfamilies appeared in every tree with high bootstrap values. The joining of these subfamilies in the higher nodes was inconsistent and invariably gave low bootstrap values. Note that At1g54480 is the Col-0 allele of RPP27.

Figure 6.

Distribution of RLPs in Arabidopsis relative to two subclasses of NB-LRR genes.