Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.

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SummaryThe Kin1/Par-1/MARK kinases regulate various cellular processes in eukaryotic organisms. Kin1 orthologs are well conserved in fungal pathogens but none of them have been functionally characterized. Here, we show that KIN1 is important for pathogenesis and growth in two phytopathogenic fungi and that FgKin1 regulates ascospore germination and the localization of Tub1 b-tubulins in Fusarium graminearum.The Fgkin1 mutant and putative FgKIN1 S172A kinase dead (nonactivatable) transformants were characterized for defects in plant infection, sexual and asexual reproduction, and stress responses. The localization of FgKin1 and two b-tubulins were examined in the wild-type and mutant backgrounds. Deletion of FgKIN1 resulted in reduced virulence and defects in ascospore germination and release. FgKin1 localized to the center of septal pores. FgKIN1 deletion had no effect on Tub2 microtubules but disrupted Tub1 localization. In the mutant, Tub1 appeared to be enriched in the nucleolus. In Magnaporthe oryzae, MoKin1 has similar functions in growth and infection and it also localizes to septal pores. The S172A mutation had no effect on the localization and function of FgKIN1 during sexual reproduction.These results indicate that FgKIN1 has kinase-dependent and independent functions and it specifically regulates Tub1 b-tubulins. FgKin1 plays a critical role in ascospore discharge, germination, and plant infection.

In Magnaporthe oryzae, the Mst11-Mst7-Pmk1 MAP kinase pathway is essential for appressorium formation and invasive growth. To determine their roles in Pmk1 activation and plant infection, we characterized the two thioredoxin genes, TRX1 and TRX2, in M. oryzae. Whereas the Δtrx1 mutants had no detectable phenotypes, deletion of TRX2 caused pleiotropic defects in growth, conidiation, light sensing, responses to stresses and plant infection progresses. The Δtrx1 Δtrx2 double mutant had more severe defects than the Δtrx2 mutant and was non-pathogenic in infection assays. The Δtrx2 and Δtrx1 Δtrx2 mutant rarely formed appressoria on hyphal tips and were defective in invasive growth after penetration. Pmk1 phosphorylation was barely detectable in the Δtrx2 and Δtrx1 Δtrx2 mutants. Deletion of TRX2 affected proper folding or intra-/inter-molecular interaction of Mst7 and expression of the dominant active MST7 allele partially rescued the defects of the Δtrx1 Δtrx2 mutant. Furthermore, Cys305 is important for Mst7 function and Trx2 directly interacts with Mst7 in co-IP assays. Our data indicated that thioredoxins play important roles in intra-cellular ROS signalling and pathogenesis in M. oryzae. As the predominant thioredoxin gene, TRX2 may regulate the activation of Pmk1 MAPK via its effects on Mst7.

serovar Typhimurium is arguably one of the most studied bacterial pathogens and successful infection requires the delivery of its virulence factors (effectors) directly into host cells via the type III secretion systems (T3SSs). Central to pathogenesis, these effector proteins have been subjected to extensive studies over the years. Nevertheless, whether additional effectors exist remains unclear. Here we report the identification of a novel T3SS effector STM1239 (which we renamed SopF) via quantitative secretome profiling. Immunoblotting and β-lactamase reporter assays confirmed the secretion and translocation of SopF in a T3SS-dependent manner. Moreover, ectopic expression of SopF caused significant toxicity in yeast cells. Importantly, genetic ablation of led to strains defective in intracellular replication within macrophages and the mutant were also markedly attenuated in a mouse model of infection. Our study underscores the use of quantitative secretome profiling in identifying novel virulence factors for bacterial pathogens.

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Appressorium formation and invasive growth are two important steps in the infection cycle of Magnaporthe oryzae that are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase (MAPK) pathway. However, the molecular mechanism involved in the activation of Mst11 MAPK kinase kinase is not clear in the rice blast fungus. In this study, we functionally characterized the regulatory region of Mst11 and its self-inhibitory binding. Deletion of the middle region of Mst11, which contains the Ras-association (RA) domain and two conserved phosphorylation sites (S453 and S458), blocked Pmk1 activation and appressorium formation. However, the MST11(ΔRA) transformant MRD-2 still formed appressoria, although it was reduced in virulence. Interestingly, over 50% of its germ tubes branched and formed two appressoria by 48 h, which was suppressed by treatments with exogenous cAMP. The G18V dominant active mutation enhanced the interaction of Ras2 with Mst11, suggesting that Mst11 has stronger interactions with the activated Ras2. Furthermore, deletion and site-directed mutagenesis analyses indicated that phosphorylation at S453 and S458 of Mst11 is important for appressorium formation and required for the activation of Pmk1. We also showed that the N-terminal region of Mst11 directly interacted with its kinase domain, and the S789G mutation reduced their interactions. Expression of the MST11(S789G) allele rescued the defect of the mst11 mutant in plant infection and resulted in the formation of appressoria on hydrophilic surfaces, suggesting the gain-of-function effect of the S789G mutation. Overall, our results indicate that the interaction of Mst11 with activated Ras2 and phosphorylation of S453 and S458 play regulatory roles in Mst11 activation and infection-related morphogenesis, possibly by relieving its self-inhibitory interaction between its N-terminal region and the C-terminal kinase domain. In addition, binding of Mst11 to Ras2 may be involved in the feedback inhibition of cAMP signaling and further differentiation of germ tubes after appressorium formation.

The rice blast fungus Magnaporthe oryzae forms a specialized infection structure called an appressorium to breach the host-plant epidermis for successful infection. In this study, a mutant defective in appressorial penetration was isolated by a mutagenesis approach, in which an exogenous DNA fragment was found to be inserted into the first exon of MoCRC1. This gene encodes a putative carnitine-acylcarnitine carrier protein that is widely conserved among eukaryotic organisms. Deletion of MoCRC1 severely reduces appressorium turgor generation, appressorial penetration, and development of infection hyphae. The null mutant of MoCRC1 lost pathogenicity on intact and abraded host leaves. MoCRC1 was also found to be required for growth on minimal medium containing sodium acetate or olive oil. Moreover, the transformed MoCrc1-eGFP fusion protein was expressed throughout the infection process. Our results suggest that the carnitine-acylcarnitine carrier protein plays vital roles in appressorium-mediated infection and is essential for pathogenesis of M. oryzae and perhaps other phytopathogenic fungi.