Rac1 is a small GTPase involved in actin cytoskeleton organization and polarized cell growth in many organisms. In this study, we investigate the biological function of MgRac1, a Rac1 homolog in Magnaporthe grisea. The Mgrac1 deletion mutants are defective in conidial production. Among the few conidia generated, they are malformed and defective in appressorial formation and consequently lose pathogenicity. Genetic complementation with native MgRac1 fully recovers all these defective phenotypes. Consistently, expression of a dominant negative allele of MgRac1 exhibits the same defect as the deletion mutants, while expression of a constitutively active allele of MgRac1 can induce abnormally large conidia with defects in infection-related growth. Furthermore, we show the interactions between MgRac1 and its effectors, including the PAK kinase Chm1 and NADPH oxidases (Nox1 and Nox2), by the yeast two-hybrid assay. While the Nox proteins are important for pathogenicity, the MgRac1-Chm1 interaction is responsible for conidiogenesis. A constitutively active chm1 mutant, in which the Rac1-binding PBD domain is removed, fully restores conidiation of the Mgrac1 deletion mutants, but these conidia do not develop appressoria normally and are not pathogenic to rice plants. Our data suggest that the MgRac1-Chm1 pathway is responsible for conidiogenesis, but additional pathways, including the Nox pathway, are necessary for appressorial formation and pathogenicity.

Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.

Unilateral cross-incompatibility (UCI) is a unidirectional inter/intra-population reproductive barrier when both parents are self-compatible. Maize Gametophyte factor1 (Ga1) is an intraspecific UCI system and has been utilized in breeding. However, the mechanism underlying maize UCI specificity has remained mysterious for decades. Here, we report the cloning of ZmGa1P, a pollen-expressed PECTIN METHYLESTERASE (PME) gene at the Ga1 locus that can confer the male function in the maize UCI system. Homozygous transgenic plants expressing ZmGa1P in a ga1 background can fertilize Ga1-S plants and can be fertilized by pollen of ga1 plants. ZmGa1P protein is predominantly localized to the apex of growing pollen tubes and may interact with another pollen-specific PME protein, ZmPME10-1, to maintain the state of pectin methylesterification required for pollen tube growth in Ga1-S silks. Our study discloses a PME-mediated UCI mechanism and provides a tool to manipulate hybrid breeding.