System-wide characterization of bZIP transcription factor proteins involved in infection-related morphogenesis of Magnaporthe oryzae.

The basic leucine zipper (bZIP) domain-containing transcription factors (TFs) function as key regulators of cellular growth and differentiation in eukaryotic organisms including fungi. We have previously identified MoAp1 and MoAtf1 as bZIP TFs in Magnaporthe oryzae and demonstrated that they regulate the oxidative stress response and are critical in conidiogenesis and pathogenicity. Studies of bZIP proteins could provide a novel strategy for controlling rice blast, but a systematic examination of the bZIP proteins has not been carried out. Here, we identified 19 additional bZIP TFs and characterized their functions. We found that the majority of these TFs exhibit active functions, most notably, in conidiogenesis. We showed that MoHac1 regulates the endoplasmic reticulum stress response through a conserved unfolded protein response pathway, MoMetR controls amino acid metabolism to govern growth and differentiation, and MoBzip10 governs appressorium function and invasive hyphal growth. Moreover, MoBzip5 participates in appressorium formation through a pathway distinct from that MoBzip10, and MoMeaB appears to exert a regulatory role through nutrient uptake and nitrogen utilization. Collectively, our results provide insights into shared and specific functions associated with each of these TFs and link the regulatory roles to the fungal growth, conidiation, appressorium formation, host penetration and pathogenicity.

Shared and distinct functions of two Gti1/Pac2 family proteins in growth, morphogenesis and pathogenicity of Magnaporthe oryzae.

Gti1/Pac2 are conserved family proteins that regulate morphogenic transition in yeasts such as Schizosaccharomyces pombe and Candida albicans, and they also control toxin production and pathogenicity in filamentous fungus Fusarium graminearum. To test the functions of Gti1/Pac2 paralogues MoGti1 and MoPac2 in the rice blast fungus Magnaporthe oryzae, we generated respective ΔMogti1 and ΔMopac2 mutant strains. We found that MoGti1 and MoPac2 exhibit shared and distinct roles in hyphal growth, conidiation, sexual reproduction, stress responses, surface hydrophobility, invasive hyphal growth and pathogenicity. Consistent with the putative conserved function of MoGti1, we showed that MoGti1-GFP is localized to the nucleus, whereas MoPac2-GFP is mainly found in the cytoplasm. In addition, we provided evidence that the nuclear localization of MoGti1 could be subject to regulation by MoPmk1 mitogen-activated protein kinase. Moreover, we found that the reduced pathogenicity in the ΔMopac2 mutant corresponds with an increased expression of plant defence genes, including PR1a, AOS2, LOX1, PAD4, and CHT1. Taken together, our studies provide a comprehensive analysis of two similar but distinct Gti1/Pac2 family proteins in M. oryzae, which underlines the important yet conserved functions of these family proteins in plant pathogenic fungi.

MoLys2 is necessary for growth, conidiogenesis, lysine biosynthesis, and pathogenicity in Magnaporthe oryzae.

Amino acid biosyntheses are complex but essential processes in growth and differentiation of eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, the lysine biosynthesis via the α-aminoadipate (AA) pathway involves several steps, including reduction of AA to AA 6-semialdehyde by AA reductase ScLys2. In filamentous fungus Penicillium chrysogenum, disruption of the LYS2 gene blocked the lysine biosynthesis but promoted the production of the secondary metabolite penicillin. In comparison, little is known about the function of AA reductase Lys2 in phytopathogenic fungi. We here characterized the functions of MoLys2, a homolog of ScLys2, from the rice blast fungus Magnaporthe oryzae. Our results showed that the ΔMolys2 mutants were auxotrophic for lysine. The ΔMolys2 mutants also exhibited drastic reduction in pathogenicity on rice, inducing small disease lesions. Microscopic examination of the lesions revealed that the invasive hyphae of ΔMolys2 mutants were mostly restricted to the primary infected leaf sheath cells. In addition, exogenous lysine restored the production of conidia and near wild-type appressoria differentiation, and rescued the defect of pathogenicity in conidia infection of detached barely and rice leaf sheath. Our results indicated that MoLys2 is necessary for lysine biosynthesis that affects growth, conidiogenesis, and pathogenicity of the fungus. This study does implicate the potential for targeting lysine biosynthesis for the development of novel fungicides against M. oryzae.

Histone acetyltransferase MoHat1 acetylates autophagy-related proteins MoAtg3 and MoAtg9 to orchestrate functional appressorium formation and pathogenicity in Magnaporthe oryzae.

Macroautophagy/autophagy is critical for normal appressorium formation and pathogenicity of the rice blast fungus Magnaporthe oryzae, but the molecular base of autophagy linked to pathogenicity remains elusive in this or other pathogenic fungi. We found that MoHat1, a histone acetyltransferase (HAT) homolog, had a role in the regulation of autophagy through the acetylation of autophagy related proteins MoAtg3 and MoAtg9. We also found that MoHat1 was subject to regulation by the protein kinase MoGsk1 that modulated the translocation of MoHat1 from the nucleus to the cytoplasm with the assistance of MoSsb1, a protein chaperone. The alternation of intracellular location affected MoHat1 in the modification of cytosolic autophagy proteins that maintained normal autophagy. Furthermore, we provided evidence linking acetylation of MoAtg3 and MoAtg9 by MoHat1 to functional appressorium development and pathogenicity. Together with the first report of MoAtg9 being subject to acetylation regulation by MoHat1, our studies depicted how MoHat1 regulated autophagy in conjunction with MoGsk1 and how normal autophagy was linked to appressorium formation and function and pathogenicity of M. oryzae. Abbreviations: A/Ala: alanine; AP: autophagosome; Atg genes/proteins: autophagy-related genes/proteins; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; DAPI: 4', 6-diamidino-2-phenylindole; D/Asp: aspartic acid; GFP: green fluorescent protein; GSK3: glycogen synthase kinase 3; HAT: histone acetyltransferase; Hsp70: heat-shock protein 70; IH: invasive hyphae; K/Lys: lysine; MMS: methyl methanesulfonate; Mo: Magnaporthe oryzae; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; R/Arg: arginine; S/Ser: serine; T/Thr: threonine; TOR: target of rapamycin; WT: wild type; YFP: yellow fluorescent protein.