NDR1, a Pathogen-Induced Component Required for Arabidopsis Disease Resistance

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A segregating population from a cross between Col-0 ndr1-1/ndr1-1 and La-er NDR1/NDR1 plants was used for genetic mapping. Molecular markers were obtained from the Arabidopsis Biological Resource Center (Ohio State University) and were mapped relative to NDR1. Flanking PCR markers nga162 (19) and gl1 (20) (22 cM apart) were used to rapidly prescreen large numbers of susceptible F₂ plants for recombinants. F₃ families from recombinant plants were then used to identify recombination events between RFLP markers tightly linked to NDR1, as well as to verify the genotype of the recombinants by disease assays (21).

Four YAC libraries (22) of Arabidopsis accession Col-0 genomic DNA were screened with 560B1 and pCIT1240 as hybridization probes. Plant DNA insert ends were isolated from YAC clones by inverse PCR and plasmid rescue as described (23), and were converted to RFLP markers for genetic mapping. The contig was extended until an overlapping set of YAC clones was constructed that linked the clones identified with 560B1 to those that hybridized with pCIT1240.

For construction of cosmid libraries, yeast DNA from clones CIC3D12 and CIC7E1 was partially digested with Sau 3A, size fractionated (to 15 to 20 kb), and cloned into the Bam HI site of the binary vector pCLD04541 (24). Clones carrying Arabidopsis DNA were isolated by hybridization with RFLP markers that cosegregated with NDR1. Cosmids were organized into overlapping contig islands by restriction analysis and hybridization patterns. The discovery of the 1.2-kb deletion in the area spanned by cosmid FH6 focused our efforts on cosmids in that region. Agrobacterium-mediated transformation of ndr1-1 plants was performed using the vacuum infiltration procedure of Bechtold et al. (25), with modifications as described by Bent et al. (24). T₁ seeds were surface-sterilized, and transformants were selected on agar-solidified Murashige-Skoog medium containing kanamycin (40 mg/ml). After 1 week, green plants were transferred to potting mix and inoculated after 3 to 5 weeks growth. The HR assay used to test for complementation is described by Kunkel et al. (26). Psm strain 4326 lacking an avr gene recognized by Arabidopsis plants does not cause a visible HR and was used as a negative control inoculation. Cosmids that lacked the region encompassed by the deletion (Fig. 1C) did not restore the HR resistance response in ndr1-1 plants. Thus, the region encoding NDR1 was delimited to a 3.5-kb section.

To sequence genomic DNA spanning the deletion in the ndr1-1 mutant, we sonicated cosmid FH6 DNA, then size-selected ∼1-kb fragments on agarose gels, purified them with GeneClean (Bio 101, Vista, CA), and subcloned them into the Eco RV site of pBluescript (Stratagene). Two Hind III fragments which spanned the deletion were isolated from FH6, radiolabeled, and used as probes against the subclones. DNA sequences from these subclones were obtained by the cyclic reaction termination method, using fluorescently labeled dideoxyribonucleotide triphosphates and following the instructions provided with the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer). DNA Sequencing Analysis, version 2.01 (Applied Biosystems) and SeqEd, version 1.03 (Gene Codes, Ann Arbor, MI) were used to process raw data. Sequences were aligned and analyzed with Sequencher 3.0 for the Macintosh.

ndr1-2 and ndr1-3 alleles were gifts from R. Innes.

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With hin1, there are three regions of similarity to NDR1: across amino acids 20 through 42 containing the first putative transmembrane domain there is 60% similarity, including conservative substitutions; across amino acids 73 through 85 there is 69% similarity; and from amino acids 113 through 118 there is 100% identity. When compared with clone NG2, from amino acids 146 through 167 there is 86% similarity to NDR1.

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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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We thank R. Innes for the generous gift of ndr1-2 and ndr1-3 mutants and P. Schulze-Lefert for critical reading of the manuscript. Supported by an NSF predoctoral fellowship to K.S.C., an NIH postdoctoral fellowship to A.D.S., and NSF grant MCB-9219959 to B.J.S.