1.10.7S
A G PROTEIN ALPHA SUBUNIT FROM COCHLIOBOLUS HETEROSTROPHUS INVOLVED IN MATING AND APPRESSORIUM FORMATION

B GILLIAN TURGEON1, BA HORWITZ2, TANYA M SANDROCK1, SHUN-WEN LU 1, AMIR SHARON3, VLADIMIR RITTER 2, and O. C. YODER 1

1Dept. of Plant Pathology, Cornell University, Ithaca, NY 14853; 2 Dept. of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel; 3 Botany Dept., Tel Aviv University, Tel Aviv 69978, Israel

Background and objectives
Heterotrimeric GTP-binding (G) proteins belong to the GTPase superfamily and are involved in transducing extracellular signals from activated membrane receptors to a variety of intracellular targets (effectors) via a cascade of interacting proteins. A particular G protein can be uniquely involved with one set of receptor/effector molecules or it can be promiscuous, transducing signals from two or more activated receptors. Some alpha subunits are highly conserved [1], a feature that has facilitated cloning. In several cases involving plant pathogenic fungi, at least one G alpha protein is involved in two complex developmental processes: pathogenesis and mating. For Cryphonectria parasitica, the G alpha protein encoded by the gene cpg-1 is required for both virulence and female fertility, whereas a second gene, Magnaporthe grisea, one of them (magB) is required for appressorium formation, pathogenicity to rice, and perithecium production when heterozygous in a cross; mutations in magA and magC caused mild effects on mating and/or conidiation but did not interfere with appressorium formation or virulence [3]. Similarly, of four G alpha protein encoding genes in Ustilago maydis, mutations in three had no apparent phenotype, but disruption of one (gpa3 appears to participate in multiple signaling pathways.
Our investigations of Cochliobolus heterostrophus have centered around its abilities both to parasitize corn and to undergo sexual reproduction. To investigate possible signal transduction pathways involved in these processes, we cloned a G alpha protein-encoding gene ( CGA1 ), deleted it from the genome, and examined ability of deletion mutants to mate and infect corn.

Results and conclusions
The deduced amino acid sequence of CGA1 showed similarity to G alpha proteins from other filamentous fungi and suggested that CGA1 is a member of the G alpha i class. Mutants had reduced ability to form appressoria on glass surfaces and on corn leaves, but nevertheless caused lesions on corn plants like those of wild type. Mutants were female sterile; sexual development was completely abolished when the mutant allele was homozygous in a cross. Ascospores produced in crosses heterozygous at Cga1 were all wild type. The signal transduction pathway represented by CGA1 appears to be involved in developmental pathways leading to either appressorium formation or mating; in sexual development CGA1 is required for both fertility and ascospore viability. The phenotypes caused by CGA1 deletion are similar to those caused by inactivation of magB in M. grisea [3]. A significant difference between the two systems is that magB mutants of M. grisea have reduced virulence corresponding to the reduction in appressorium formation, whereas cga1 mutants of C. heterostrophus have no detectable change in virulence. One interpretation of this result is that appressoria are not critical for infection by C. heterostrophus,. This could explain why a mutation affecting a signal transduction pathway causes reduced appressoria production in both fungi but reduced virulence only in M.grisea..

References
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2. Gao S, Nuss DL, 1996. Proc. Natl. Acad. Sci. 93, 14122-14127.
3. Liu S, Dean RA, 1997. Mol. Plant-Microbe Interact. 10, 1075-1086.
4. Regenfelder E, Spellig T, Hartmann A, Lauenstein S, Boelker M, Kahmann R, 1997. EMBO J. 16, 1934-1942.