1.8.4S
IDENTIFICATION AND CHARACTERIZATION OF PATHOGENICITY GENES FROM THE RICE BLAST FUNGUS MAGNAPORTHE GRISEA

NJTALBOT, PV BALHADERE, MJ KERSHAW, KP DIXON, AJ FOSTER and DM SOANES

Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, UK

Background and objectives
Magnaporthe grisea causes a serious disease of cultivated rice and is responsible for serious crop losses throughout the developing world. It is also an experimentally tractable fungus for genetic manipulation, and this has allowed progress to be made in identifying genes required for the fungus to be able to cause disease. Our current objectives are to identify and characterize genes required for pathogenicity and to determine their regulatory interactions. In this way we hope to achieve a detailed understanding of fungal pathogenesis in a cereal pathogen of economic significance.

Results and conclusions
We have used three strategies to identify pathogenicity genes from M. grisea . Genes have been identified either based on their differential expression during pathogenesis, by identification of pathogenicity mutants, or by selecting candidate genes for targeted mutation based on physiological observations of M. grisea infection. The strategies have resulted in identification of a number of genes which encode determinants of successful disease establishment.

The first of these, MPG1, encodes a fungal hydrophobin which is expressed specifically during appressorium development and disease symptom production. Targeted deletion of MPG1 resulted in a reduction in the ability of the fungus to elaborate appressoria. This appears to be due to a defect in surface perception [1]. To understand the action of MPG1, we have determined its spatial expression patterns using the green fluorescent protein vital reporter expressed under control of the MPG1 promoter. This has shown specific expression of MPG1, occurs within developing appressoria, and is localized to these cells throughout the infection process. Significantly, expression of MPG1 in developing germ tubes was found only when germ tubes grew on hydrophobic surfaces conducive for appressorium formation. This is consistent with evidence showing secretion of the MPG1 hydrophobin at hydrophobic surface interfaces [1]. Cross-species complementation of an mpg1- mutant with seven other hydrophobin-encoding genes showed that the role of the MPG1 hydrophobin could be fulfilled by a variety of other hydrophobins when expressed as translational fusions to the MPG1 promoter. This indicates that hydrophobins are a closely related group of proteins and that MPG1 has evolved to fulfil its role in pathogenesis, due largely to its specific expression pattern during infection-related development. Further genes involved in pathogenesis have also been analysed by identifying pathogenicity mutants using REMI mutagenesis. These include at least two genes involved in the appressorium-mediated infection, and others involved in plant tissue colonization. We have determined that appressorium turgor in M. grisea is generated by accumulation of high concentrations of glycerol [2], and are currently isolating genes encoding components of the glycerol biosynthetic pathway. REMI mutants affected in appressorium function are currently being tested to determine whether turgor generation is compromised. Furthermore, the effect of mutations in regulatory genes such as NPR1, NPR2, PMK1 and CPKA on glycerol accumulation in developing appressoria is also being assessed, and results suggest involvement of a cAMP-dependent signalling pathway for appressorium function. In summary, a number of genetic components conditioning pathogenicity have been identified and are being characterized. This has led us to focus attention on appressorium development and function, and progress towards understanding these processes will be presented.

References
1. Talbot NJ, Kershaw MJ, Wakley, GE et al., 1996. Plant Cell 8, 985-999.
2. de Jong JC, McCormack BJ, Smirnoff N, Talbot NJ, 1997. Nature 389, 244-245.