Xiao-Lin Chen

N -Glycosylation of Effector Proteins by an α-1,3-Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity

This work identifies an α-1,3-mannosyltransferase that mediates the N-glycosylation of effector proteins in the rice blast fungus. The N-glycosylation is required to maintain protein stability and the chitin binding activity of the effector protein Slp1, factors that are essential for its effector function. Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.

A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae

Protein phosphatase 2A is a subgroup of widely conserved serine/threonine phosphatases and plays diverse roles in transcription, translation, differentiation, cell cycle, and signal transduction in many organisms. However, its roles in biotrophic and hemi-biotrophic phytopathogenic fungi remain to be investigated. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that was defective in vegetative hyphal growth. In the mutant, the T-DNA fragment was found to be inserted in the promoter region of a putative serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) gene MoPPG1. Deletion of MoPPG1 leads to severe defects in vegetative hyphal growth and conidiation. Conidia of the ∆Moppg1 null mutants were misshaped, and most of them were two-celled. The deletion mutants of MoPPG1 did not penetrate into host plant cells and failed to cause any disease lesions on rice leaves. Interestingly, significant reduction was found in the ∆Moppg1 null mutants in expression levels of several Rho GTPase family genes including MgCDC42, MgRHO3, and MgRAC1, which were important for pathogenesis of M. oryzae. Taken together, our results indicated that PP2Ac plays vital roles in asexual development and plant infection by regulating Rho GTPases in the rice blast fungus and perhaps other plant pathogenic fungi.

A carnitine–acylcarnitine carrier protein, MoCrc1, is essential for pathogenicity in Magnaporthe oryzae

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.

Glutamate synthase MoGlt1-mediated glutamate homeostasis is important for autophagy, virulence and conidiation in the rice blast fungus: Roles of MoGlt1 in pathogenicity and development

Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1-mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.

MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and β subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae.

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co-localized with peroxisomes during appressorial development. Compared with the massive vesicle-shaped peroxisomes formed in the wild-type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1-mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.

Plant Fungal Pathogenesis

1Department of Plant Pathology, China Agricultural University, Beijing 100193, China 2School of Environmental Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 3Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK, Canada S9H 3X2 4College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China 5Department of Plant Pathology and the Genome Center, University of California, Davis, CA 95616, USA

MoCps1 is important for conidiation, conidial morphology and virulence in Magnaporthe oryzae

Conidia play important roles in primary and secondary infections of airborne fungal pathogens. In this study, an insertional mutant with reduced capacity for conidiation was isolated from the rice blast fungus Magnaporthe oryzae. The mutant has a T-DNA insertion that disrupts a gene named MoCPS1. The deduced MoCps1 protein contains three AMP-binding domains. Gene complementation and gene knockout assays confirmed that MoCPS1 is important for conidiation. Conidia produced by the MoCPS1 deletion mutants are much more slender and longer than those produced by the wild-type strain. The Mocps1 mutants are less efficient in both appressorial penetration and invasive growth of infection hyphae, resulting in attenuated virulence toward host plants. MoCPS1 is highly expressed in a mature appressorium. Interestingly, the expression levels of several genes related to conidiation and pathogenicity have been significantly altered in the MoCPS1 deletion mutants. Taken together, our results indicated that MoCPS1 is important for conidiogenesis, conidial morphogenesis, and pathogenesis in the rice blast fungus.