S PNINI-COHEN¹, A ZILBERSTEIN¹ and Z EYAL^1,2

¹Department of Plant Sciences, and ²Institute for Cereal Crops Improvement (ICCI), George S Wise Faculty of Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel

Background and objectives
Mycosphaerella graminicola (anamorph Septoria tritici) is a major pathogen of wheat in temperate regions, causing significant losses in yield. Cultivar-specific wheat-S. tritici isolate interactions have been reported [1]. Isolates revealing a consistent differential virulence on wheat cultivars may provide a tool to unravel genes involved in pathogenic specialization. S. tritici isolate ISR398 (ATCC 48507) produces few pycnidia (avirulent) on the wheat cultivar Seri 82 and high pycnidial coverage (virulent) on Shafir, and therefore has been chosen as a model for a random insertional inactivation of genes associated with isolate x cultivar specificity.

Results and conclusions
An efficient genetic transformation of S. tritici has been developed [2]. Co-transformation of two pUC-based plasmids separately carrying the E. coli hygromycin resistance gene (hph) and uidA (GUS) reporter gene, both driven by the Pgpd promoter from A. nidulans, yielded 398 mutants carrying multiple as well as single insertions. Two out of 164 HygR mutants differed in their pathogenic specificity from the original ISR398 isolate.

The 398D25 mutant (HygR, GUS+) showed a significant shift from low to high virulence on Seri 82. Molecular analysis showed that the D25 mutant contains a single insert with the hph and uidA genes, probably resulting from a recombination event between the homologous regions of the two plasmids that occurred prior to insertion into the fungal genome. The genomic margins of the integration locus were isolated by inverse PCR and used to construct plasmid vectors for a targeted insertion strategy designed to verify the linkage between the mutated locus and the virulent phenotype. The use of genomic fragments smaller than 1 kb as potential sites for the double-recombination events proved unsuccessful, leading only to random insertion events in unrelated genomic loci. When the isolate ISR398 was transformed with a plasmid that carried 1.3- and 1.7-kb genomic flanking regions, targeted insertions were obtained at a frequency of 25%. Pathogenic assays with the putative mutants are under way.

This approach has paved the way for the generation of additional mutants involved in events associated with the wheat S. tritici interaction.

References
1. Eyal Z, Levi E, 1987. Euphytica 36, 237-250.
2. Pnini-Cohen S, Zilberstein A, Schuster S et al., 1996. Phytopathology (suppl.), S40.