FM AUSUBEL, J DEWDNEY, J HEARD, PLOTNIKOVA, LT REUBER, J STONE, S VOLKO and P YORGEY

Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA

Background and objectives
A major focus of biochemical and molecular research in plant pathology has been the elucidation of the molecular mechanisms underlying pathogen recognition and the signalling cascades leading to the activation of defence responses. Until recently, the plant response to pathogen attack has been primarily studies using biochemical and physiological techniques. However, to identify regulatory factors and other defence response components that are not already correlated with a known biochemical or gene-induction response, it is necessary to screen directly for mutants that display enhanced susceptibility or resistance to pathogen attack.

Results and conclusions
We have carried out several screens for Arabidopsis thaliana mutants which exhibit enhanced disease susceptibility to infection by low inoculation doses of the virulent bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326 Glazebrook [1, 2]. To date, approximately 20 such eds mutants have been studied in detail, and 14 of these eds mutants have been subjected to complementation analysis. These mutations define 12 previously unknown EDS genes that do not correspond to previously known mutants such as npr1 or various pad mutants which also exhibit an eds phenotype. These data indicate that the screen for EDS genes is not saturated and that many factors involved in plant defence responses remain to be discovered.

Several observations suggest that the 12 EDS genes identified in our laboratory define a diverse set of previously unknown defence responses. Firstly, testing the eds mutants with several virulent pathogens revealed that most of the mutants have a distinguishable phenotype with respect to pathogen sensitivity. Secondly, all 12 eds mutants respond as wild-type and produce a hypersensitive response after infection by avirulent pathogens such as Psm ES4326 carrying the cloned avirulence gene avrRpt2, suggesting that none of the mutants affects genes in the gene-for-gene defence response pathway. Thirdly, although all 12 eds mutants are capable of mounting a systemic acquired resistance response, enhanced growth of Psm ES4326 is still apparent in the secondarily infected leaves of most of these eds mutants, indicating that the previously defined SAR pathway is also intact. Finally, the expression of several different pathogen-induced genes has been examined in eight of the 12 eds mutants. Among these eight eds mutants, only one mutant, eds5-1, displayed any significant difference from wild-type, expressing the pathogenesis-related gene PR1 to only about 10% of wild-type levels after infection with Psm ES4326. On the other hand, eds5-1 expressed wild-type levels of PR1 after treatment with salicylic acid or after the induction of SAR. A double eds5-1 npr1-1 mutant displayed an additive phenotype with respect to PR1 gene expression and susceptibility to P. syringae, as compared to either single mutant, implying that NPR1 and EDS5 act in distinct signalling pathways.

In addition to our work with Psm ES4326, we have shown that a powdery mildew, Erysiphe orontii isolate MGH, induces several known defence genes in wild-type Arabidopsis (ecotype Columbia) plants including PR1, BGL2 (PR2), PR5 and GST1. Upon screening the collection of 12 eds mutants for enhanced susceptibility to E. orontii MGH, we found that some, but not all, of these eds mutants show enhanced susceptibility. Investigation of defence gene induction in eds mutants revealed that in some cases enhanced susceptibility to E. orontii MGH correlated with failure to induce one or more defence genes in response to fungal infection. The fact that not all mutants showing enhanced susceptibility to bacterial pathogens are more susceptible to E. orontii MGH implies that it may be possible to isolate eds mutants that have specific defects in factors needed for defence against fungal pathogens.

References
1. Glazebrook J, Rogers EE, Ausubel FM, 1996. Genetics 143, 973-982.
2. Rogers EE, Ausubel FM, 1997. Plant Cell 9, 305-316.