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The feasibility of using the Saccharomyces cerevisiae genetic tools to get insights into the function of a plant-specific ubiquitin-ligase was examined. ATL2 is a potential ubiquitin-ligase of the RING-H2 type that was originally isolated as a conditionally toxic Arabidopsis cDNA when overexpressed in yeast. ATL2 is a member of an Arabidopsis family that comprises 80 proteins. After testing cDNAs from 25 ATL members for toxicity we found that in addition to ATL2 only ATL63 was toxic, suggesting specific interactions of each one of these two ATLs in yeast. We seek to identify suppressors of the ATL2 toxicity in yeast and we found that toxicity was suppressed by knock-out mutations on different components of the ubiquitination pathway. Suppression was achieved in four deubiquitinating enzyme mutants and in one ubiquitin-conjugating enzyme mutant. A model is proposed in which Ubc4 and ATL2 act together to target for degradation one or more essential yeast proteins, Doa4/Ubp4, Ubp6 and Ubp14 have a role in disassembling ubiquitin chains on the target proteins and Ubp15 protects ATL2 from auto-ubiquitination. We presuppose that our approach can be further utilized to analyze the function of this distinctive class of ubiquitin-ligases in yeast as well as in Arabidopsis.

To detect and ultimately isolate genes of Phytophthora capsici the expression of which is induced during its interaction with pepper, a comparative analysis of gene expression in the wild-type pathogenic fungus with expression in a non-pathogenic (Nop) mutant reduced in cutinase and esterase activities was performed by the differential display of mRNAs. Discrimination of fungal genes induced in planta, from plant genes induced in response to the pathogen, was accomplished by exposure of the mycelium to bare-rooted seedlings of pepper (Capsicum annuum) in sterile water, to allow the initiation of infection, and then physical removal of the induced mycelium. With six sets of primer combinations, eight cDNA fragments (representing fungal genes) were present in planta only for the pathogenic strain. RNA-blot analysis showed that the transcripts detected accumulated to detectable levels only at early stages of the interaction. Sequence analysis and database searches revealed homology of one of the cDNA clones to fungal cutinases. The 218 amino-acid sequence predicted from sequencing a genomic clone of P. capsici suggested a protein of molecular weight of 23 980 Da with similarity to fungal cutinases previously characterized. These results indicated that differential-display analysis is sufficiently sensitive to be applied for the detection and isolation of fungal genes induced during a plant-pathogen interaction.

Phytophthora capsici, P. citricola, P. cinnamomi and P. citrophthora were transformed without the removal of cell walls by particle acceleration with plasmids containing the beta-glucuronidase gene and hygromycin B resistance. Transformants were detected by histochemical and fluorometric beta-glucuronidase assays and confirmed by Southern-blot hybridization. It was found that the promoter of a plant virus is functional in Phytophthora. In addition, a method was designed to visually identify homogeneous transformed colonies, derived from zoospores of transformed multinucleated Phytophthora mycelia, based on blue color development on plates containing X-Gluc.