title: Site-directed mutagenesis of the magB gene affects growth and development in Magnaporthe grisea.
abstract: G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Galpha subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appressorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magB(G42R). This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magB(G42R) mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magB(G203R) (glycine 203 converted to arginine), was expected to block dissociation of the Gbetagamma from the Galpha subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magB(G203R) is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gbetagamma subunit in the signaling pathways regulating cellular development in M. grisea.
authors.affiliation: Department of Plant Pathology and Physiology and Clemson University Genomics Institute, Clemson University, SC 29634, USA.
authors.forename: E G
authors.initials: EG
authors.lastname: Fang
authors.forename: R A
authors.initials: RA
authors.lastname: Dean
mesh_terms: D000595:Amino Acid Sequence; D002454:Cell Differentiation / Q000235:genetics; D003433:Crosses, Genetic; D000242:Cyclic AMP / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics; D044385:GTP-Binding Protein alpha Subunits; D005800:Genes, Fungal; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D020082:Magnaporthe / Q000166:cytology / Q000235:genetics / Q000254:growth & development*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012275:Oryza / Q000382:microbiology*; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics*; D010957:Plasmids; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; C421031:magB protein, Magnaporthe grisea; D000242:Cyclic AMP; D020962:Heterotrimeric GTP-Binding Proteins