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.

Construction of a Constitutively Activated Gα Mutant in the Maize Pathogen <i>Cochliobolus heterostrophus</i>

“…Other studies demonstrated that some similar phenotypes result from constant activation or silencing of the Gα subunit. In M. grisea activated and null mutants of the Gα subunit magB exhibited similar phenotypes in terms of reduced conidiation, sexual reproduction and virulence [8]. These findings were supported by a recent work in C. parasitica [7].…”

“…Comparative analysis of the phenotypic traits exhibited by fungal strains containing null or activated Gα alleles has been used by a number of laboratories to identify putative signaling-related functions [7][8][9][10]. Free Gβ may cause a signal in both the Gα null and activated mutant strains and the phenotypic traits of both strains are affected mainly by the manipulated Gα gene.…”

“…Site specific mutagenesis (such as Q204-L, G42-R and R178-C) designed to constitutively activate Gα signaling was reported in C. parasitica [7], M. grisea [8], N. crassa [9] and in A. nidulans [10]. Conversion of one of these amino acids abolishes GTPase activity, which in turn would constitutively activate G protein signaling.…”

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

“…Among these genes found to be important for early plant infection processes are several components of cAMP signaling and two mitogen-activated protein (MAP) kinase pathways. In M. oryzae, the cAMP-PKA pathway regulates the recognition of hydrophobic surfaces and initiation of appressorium formation (Mitchell and Dean, 1995;Fang and Dean, 2000). The Pmk1 MAP kinase pathway is required for appressorium formation and maturation .…”

Comparative In-Silico Study of β-Tubulin Proteins from Arthrobotrys musiformis for Resistance to Benzimidazole

“…fungi imperfecti, which lack the sexual mode of reproduction recombination becomes difficult. Therefore, in such cases strain improvement is normally achieved by mutagenesis [5] or protoplast fusion [6].…”