The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotide-gated ion channel

Abstract

Gene-for-gene disease resistance typically includes a programmed cell death response known as the hypersensitive response (HR). The Arabidopsis thaliana dnd1 mutant was previously isolated as a line that failed to produce the HR in response to avirulent Pseudomonas syringae pathogens; plants homozygous for the recessive dnd1-1 mutation still carry out effective gene-for-gene resistance. The dnd1-1 mutation also causes constitutive systemic resistance and elevated levels of salicylic acid. In the present study, a positional cloning approach was used to isolate DND1. DND1 encodes the same protein as AtCNGC2, a cyclic nucleotide-gated ion channel of previously unknown organismal function that can allow passage of Ca(2+), K(+) and other cations [Leng, Q., Mercier, R. W., Yao, W. & Berkowitz, G. A. (1999) Plant Physiol. 121, 753-761]. By using a nahG transgene, we found that salicylic acid is required for the elevated resistance caused by the dnd1 mutation but that removal of salicylic acid did not completely eliminate the dwarf and loss-of-HR phenotypes of mutant dnd1 plants. A stop codon that would severely truncate the DND1 gene product was identified in the dnd1-1 allele. This demonstrates that broad-spectrum disease resistance and inhibition of the HR can be activated in plants by disruption of a cyclic nucleotide-gated ion channel.

Figure 1

Summary of positional cloning experiments leading to isolation of the DND1 gene. (A) CHS1, pCIT1243, and nga106 are previously mapped molecular genetic markers on Arabidopsis chromosome 5. pCIT1243 and nga106 were used to identify BAC clones carrying wild-type Col-0 genomic DNA inserts. Inserts from four BACs were subcloned to create a binary cosmid library; some of these cosmids were used as additional restriction fragment length polymorphism markers to refine the genetic interval containing DND1. Three cosmids that complement the dnd1 mutation were identified of 43 tested in a focused shotgun complementation project. (B) Higher-resolution complementation experiments using more precise subclones identified additional complementing and noncomplementing clones, including other clones not shown, that implicated an ≈5-kb region as the DND1 locus. This region encodes a previously identified cDNA named AtCNGC2. Cosmids 1H3 and 2A1 are shown in both A and B. E, EcoRI site; X, XbaI site.

Figure 2

Size-complementation of mutant dnd1 plants by addition of the cloned wild-type DND1 locus. Col-0 dnd1-1/dnd1-1 plants transformed with cosmids 1A8 or 1H2, or other complementing cosmids (not shown), corrected the dnd1 dwarf rosette phenotype.

Figure 3

Complementation of the reduced-HR and constitutive resistance phenotypes of dnd1 plants by transformation with cloned wild-type DND1. (A) HR scores for leaves inoculated with P. syringae pv. glycinea Race 4 carrying cloned avrRpt2 (filled bars) or an empty plasmid vector (open bars). HR tissue collapse scored on a scale of 0–5 (0 = no visible collapse; 5 = confluent collapse of inoculated tissue; values are mean ± SE). Data are shown for T₂ plants derived from hemizygous primary transformants; 1A8 (dwarf) and 1H2 (dwarf) represent control sets of T₂ segregants that returned to the dnd1/dnd1 dwarf rosette size because of loss (via segregation) of the DND1 transgene. (B) Growth of virulent P. syringae pv. tomato DC3000 in leaf tissue of inoculated plants. Col-0, wild type; 1H3, Col-0 dnd1/dnd1 line carrying wild-type DND1 because of transformation with cosmid 1H3; dnd1, Col-0 dnd1/dnd1; 1H3 (dwarf), dwarf-sized T₂ plants from a Col-0 dnd1/dnd1 T₁ transformant that was hemizygous for the introduced 1H3 construct.

Figure 4

Loss of resistance in salicylate hydroxylase plants. Leaves were inoculated uniformly with virulent P. syringae pv. tomato DC3000 (open symbols) or Pst DC3000 avrRpt2⁺ (filled symbols). Squares, Col-0 dnd1/dnd1 line; circles, Col-0 nahG⁺ B15 line; triangles, Col-0 dnd1/dnd1, nahG⁺/nahG⁺ line. Leaf population data are shown as mean ± SE.