Article Figures & Data
Figures
- Figure 1.
Physical Delineation of RPP14.
(A) Alignment of YAC clones relative to molecular markers flanking RPP14. Individual YAC clones were positioned by hybridization with the RFLP flanking markers ve021 and pAT3-89.1. End probes from the YACs (solid ellipses for the right ends and open ellipses for the left ends) were obtained as described in Methods and used to orient the YAC clones.
(B) Two YAC end probes, 4D9LE and 8D7LE, were used to isolate overlapping P1 clones. Two of the P1 clones, P1-37C7 and P1-53P2, which are marked with asterisks, were found to overlap. Random sequence of these clones identified a fragment, 37C7-5 (striped boxes), with homology to RPP5. (C) DNA gel blot analysis of five Arabidopsis accessions reveals a small, polymorphic gene family. The blot was hybridized at high stringency and probed with the 37C7-5 fragment. DNA from the accessions Landsberg erecta (L), Col-0 (C), Ws-0 (W), Nd-1 (N), and Oystese (O) were digested with EcoRI and HindIII as indicated.
rec., recombinant.
- Figure 2.
Physical Organization of RPP1/10/14 Candidate Genes in Ws-0.
(A) Three different genes containing 37C7-5–homologous sequence were identified and assembled into two contigs. Only informative clones are shown. Minimal constructs for Hom-A (psub-Hom.A), Hom-B (psub-Hom.B), and Hom-C (psub-Hom.C) were derived from ws-cos65.1, ws-cos69.12, and ws-phage1.1 (see Methods). EcoRI restriction sites are indicated by open circles and HindIII sites by filled circles. The 1.1-kb HindIII probe from the 5′ end of Hom-A that was used in the DNA gel blot analysis shown in (B) is indicated with an asterisk. Thick black arrows represent positions of the genes (from ATG to stop codon).
(B) DNA from the Ws-0 (W) and Col-0 (C) Arabidopsis accessions was digested with HindIII or EcoRI. The filter was probed with a 1.1-kb HindIII fragment from the 5′ end of Hom-A. The identity of fragments derived from the Hom-A, Hom-B, and Hom-C genes is indicated at left. Molecular markers are indicated at right. Hybridizing bands derived from Hom-D are also shown.
- Figure 3.
Response Phenotypes of Arabidopsis Leaves 6 Days afte Inoculation with P. parasitica Isolate Noco2.
Nine-day-old seedlings were spray inoculated with a suspension of conidiosporangia. The phenotypes were monitored macroscopically with a hand lens and microscopically by observing lactophenol trypan blue–stained leaves. h, haustorium; m, mycelium; n, necrotic plant cell.
(A) and (E) Resistance in wild-type Ws-0 specified by RPP14. Noco2 mycelium is effectively contained within a discrete cluster of plant necrotic cells at the site of attempted penetration.
(B) and (F) Resistance in a CW84 line that is homozygous for T-DNA containing the minimal Hom-A construct (see Figure 2A). Resistance is as effective as in wild-type Ws-0.
(C) and (G) Resistance in a CW84 line that is homozygous for T-DNA carrying Hom-B (see Figure 2A) is less effective than is wild-type Ws-0. A low level of pathogen asexual sporulation is visible, and limited mycelium development was accompanied by a trail of necrotic plant cells.
(D) and (H) High levels of pathogen asexual sporulation are visible in the broadly compatible line CW84. Extensive mycelium growth and haustorial development occurred in the absence of plant cell necrosis.
- Figure 4.
Structure of the RPP1 Gene Family Members and Their Predicted Proteins.
(A) Sequence features of genes RPP1-WsA, RPP1-WsB, and RPP1-WsC. E1 to E8 represent exons 1 to 8. The predicted ATG and the stop codons are shown. Exons showing high homology are indicated with symbols as given in the key.
(B) Schematic comparison of the RPP1 protein family of related R gene products. The percentage of amino acid sequence identity between RPP1-WsA and RPP1-WsB, RPP1-WsC, RPP5, N, and L6 is shown. Intron positions are marked by arrowheads. Similar protein domains are indicated with identical shading according to the key. The TIR domain has similarity with the cytoplasmic domains of Toll and IL-1R (see also Figure 5). The NB-ARC domain contains motifs that constitute an NBS and domains with homology to APAF-1 and CED-4, regulators of cell death. LRR corresponds to the C-terminal part of the proteins that contain the LRRs. N termini correspond to the extension of the proteins observed in RPP1-WsB and RPP1-WsC. Signal domain (SD) is observed in RPP1-WsA and L6.
- Figure 5.
Alignment and Hydrophobicity Plot of the TIR Domains of RPP1-WsA, RPP1-WsB, RPP1-WsC, RPP5, N, and L6.
(A) Prettybox representation of a Pileup analysis (Genetics Computer Group, Madison, WI) of the TIR region of RPP1-WsA (exon 1), RPP1-WsB (exons 1 and 2), RPP1-WsC (exons 1 and 2), RPP5 (exon 1), N (exon 1), and L6 (exon 1). The N-terminal extensions of the RPP1 family and L6 proteins relative to RPP5 and N are shaded and indicated by an arrow. The asterisk indicates the position of the introns in RPP1-WsB and RPP1-WsC. Black boxes indicate identity and gray boxes similarity. Dashes are gaps introduced by the program to optimize the alignment.
(B) Hydrophobicity analysis of the sequences shown in (A). The N-terminal extensions are shaded and indicated by an arrow. Analysis was performed as detailed in Methods.
- Figure 6.
Structural Domains of the RPP1 Protein Family and Analysis of Their LRRs.
(A) The RPP1 family of proteins can be divided into several domains. The TIR domain (exon 1 in RPP1-WsA, RPP5, N, and L6; exon 2 and exon 3 in RPP1-WsB and RPP1-WsC) is similar to the cytoplasmic domains of Drosophila Toll and mammalian interleukin-1 receptors. The NB-ARC domain (exon 2 in RPP1-WsA, RPP5, N, and L6; exon 4 in RPP1-WsB and RPP1-WsC) has similarity to the nematode CED-4 and mammalian APAF-1 proteins, which are both activators of apoptotic proteases (Van der Biezen and Jones, 1998). The LRR domain (exons 3 to 6 in RPP5; exons 3 and 4 in N and L6; exon 4 in RPP1-WsA; and exon 6 in RPP1-WsB) is envisaged to mediate specific protein–protein interactions. Residues corresponding to the β-strand/β-turn structural motif in the porcine ribonuclease inhibitor are delimited by vertical lines in the RPP1-WsA sequence. Identical amino acids between RPP1-WsA, RPP1-WsB, and RPP1-WsC are shown in black. Amino acids conserved in only two of the proteins are shown in blue. Different amino acids in all three proteins are shown in red. The conserved kinase-1a (P loop) domain (GPPGIGKTT), the kinase-2 domain (FLVLDE), the kinase-3a domain (FGPGSR), and the hydrophobic motif conserved in this class of proteins (ELPLGL) are underlined. An 11–amino acid deletion in the Ws-rpp10 mutant is highlighted by the upper box within the NB-ARC domain. A deletion of one LRR in RPP1-WsC LRR is highlighted by the lower boxes. The arrow in the TIR domain indicates the start of homology among the RPP1-WsA, RPP1-WsB, and RPP1-WsC proteins.
(B) Amino acid differences within the LRR domains between the RPP1 family of proteins. The number of different amino acids, the number of amino acids used, and the percentage of difference are indicated. Amino acids changes are more prevalent in the predicted β-strand/β-turn structural motif that is predicted to interact with the cognate ligand (Jones and Jones, 1997).
(C) Synonymous and nonsynonymous nucleotide substitutions in different regions of the coding sequences of the RPP1 gene family. The values shown are calculated as described previously (Parniske et al., 1997).
Tables
- Table 1.
Interaction Phenotypes of Different Wild-Type, Transgenic, and Mutant Arabidopsis Accessions after Inoculations with Five Isolates of P. parasitica
Arabidopsis Line No. of Lines Analyzed No. of Resitant Lines to the Following P. parasitica Isolates Noco2 Emoy2 Cala2 Maks9 Emco5 Ws-0 1 1 1 1 1 0 Col-0 1 0 1 1 NTa 0 CW84 1 0 0 0 0 0 Ws-0 rpp10 1 1 1 0 NT NT CW84b ws-cos65.1 6 6 6 6 6 0 psub–Hom-A 12 11 11 11 11 0 psub–Hom-Bc 12 12 12 0 12 0 psub–Hom-C 12 12 0 0 0d 0 CW84–Hom-A × edsl F2e 2 0 0 0 NT 0 -
↵a NT, not tested.
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↵b The constructs used for transformation are shown in Figure 2A. The complete cosmid ws-cos65.1 was used in the transformation.
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↵c The interaction phenotypes in most of these lines were characterized by a low level of P. parasitica sporulation.
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↵d Only four lines were tested for the interaction phenotype.
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↵e A Ws-0 edsl mutant was used in the cross (Parker et al., 1996).
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