1.2.20
A NOVEL ARABIDOPSIS ACD6 MUTANT WITH GENE DOSE-DEPENDENT RESISTANCE TO PATHOGENS AND ACTIVATION OF MULTIPLE DEFENCE PATHWAYS

JT GREENBERG

Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

Background and objectives
The identification of genes that regulate multiple defence functions promises to shed light on signal transduction mechanisms used to confer pathogen resistance. Furthermore, defence regulatory genes may provide robust targets for the genetic engineering of disease resistance in diverse plants. Previously we identified one such gene, acd2 (accelerated cell death) that, when mutant, causes the activation of multiple defences including cell death, phytoalexin biosynthesis, cell wall alterations and salicylic acid (SA)-dependent defences [1]. In addition, Penninckx et al. [2] showed that acd2 mutations cause increased defensin and jasmonic acid (JA) levels. Thus the ACD2 gene appears to be involved in regulating multiple defence functions through several distinct signals. To identify additional defence regulatory genes, we have screened the M2 generation of EMS-mutagenized Arabidopsis for mutants with decreased disease symptoms after inoculation with the virulent pathogen P. syringae pv. maculicola 4326 (Psm4326). We reasoned that such mutants with decreased disease symptoms might also have reduced pathogen growth and possibly altered regulation of defences.

Results and conclusions
One mutant that exhibited reduced symptoms, acd6, was characterized in detail. When self-fertilized, the original acd6 mutant segregated in a dominant fashion for reduced symptoms after pathogen inoculation. After back-crossing four times, an F2 population was assayed for pathogen resistance and was found to segregate in a dominant fashion for such resistance as assayed directly by reduced growth of Psm4326. Two doses of the acd6 mutation conferred stunting, spontaneous punctate cell death patches on all green tissues, and even higher pathogen resistance than seen in the heterozyotes. Upon close examination, heterozygous acd6 mutants showed a mild cell death phenotype on some leaves in the absence of pathogens, but overall the plants appeared nearly normal in stature. Crosses between presumptive homozygotes (small plants with cell death patches) and wild-type plants yielded F1 plants that were resistant to pathogens and nearly normal in stature. Selfing of these F1 plants gave a pattern of 3:1 segregation of pathogen resistance to pathogen susceptibility. In contrast, plants that were small with cell death patches segregated as a single recessive trait. Analysis of both SA-dependent and SA-independent defence-related markers revealed that their levels were elevated in a gene-dose-dependent manner, suggesting that the ACD6 gene may be involved directly at an upstream point in the signalling pathway leading to the activation of multiple defences. Experiments to determine the relative contributions of known defence signal-transduction signals such as SA, ethylene and JA to the acd6 phenotype are under way.

References
1. Greenberg J, Guo A, Klessig D, Ausubel FM, 1994. Cell 77, 551-563.
2. Penninckx IAMA, Eggermont K, Terras FRG et al., 1996. Plant Cell 8, 2309-2323.