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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.

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1-and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.Magnaporthe grisea is an ascomycete pathogen of important cereal crops, such as rice, barley, and wheat. It causes rice blast, one of the most severe fungal diseases of rice throughout the world (33, 35). The fungus infects rice plants in a sophisticated manner, like many other foliar pathogens. Germ tubes produced from conidia differentiate into specialized infection structures called appressoria and then use the enormous turgor pressure generated in appressoria for plant penetration (8). Mature appressoria develop thin penetration pegs to physically pierce the host surface and enter plant epidermal cells. At early stages, the penetration peg contains high concentrations of actin filaments (3) and recruits the rice cell membrane to form the extrainvasive hyphal membrane, while infection hyphal growth and its co-opt plasmodesmata for cell-to-cell movement (18). It has been hypothesized that actin and cytoskeleton elements may be involved in the reestablishment of polarized growth by determining the penetration site and in stabilizing the tip of the penetration peg (15). After penetration, the peg differentiates into infectious hyphae that grow inter-and intracellularly and result in development of blast lesions.Signal transduction pathways that regulate infection-related morphogenesis have been extensively studied in M. grisea during the past few years (34, 41). In general, cyclic AMP (cAMP) signaling is involved in surface recognition and initiation of appressorium formation (1,20,22,44). However, appressorium formation is regulated by the Pmk1 mitogen-activated protein kinase pathway (25,42,46). The pmk1 deletion mutant fails to form appressoria and is nonpathogenic. One putative transcription factor regulated by PMK1 is MST12, which is homologous to Saccharomyces cerevisiae Ste12 and essential for pathogenesis (27). The mst12 deletion mutant forms melanized appressoria that have normal appressorium turgor but fail to develop penetration pegs, probably due to cytoskeleton defects i...

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