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Crop breeding aims to balance disease resistance with yield; however, single resistance (R) genes can lead to resistance breakdown, and R gene pyramiding may affect growth fitness. Here we report that the rice Pigm locus contains a cluster of genes encoding nucleotide-binding leucine-rich repeat (NLR) receptors that confer durable resistance to the fungus Magnaporthe oryzae without yield penalty. Among these NLR receptors, PigmR confers broad-spectrum resistance, whereas PigmS competitively attenuates PigmR homodimerization to suppress resistance. PigmS expression, and thus PigmR-mediated resistance, are subjected to tight epigenetic regulation. PigmS increases seed production to counteract the yield cost induced by PigmR. Therefore, our study reveals a mechanism balancing high disease resistance and yield through epigenetic regulation of paired antagonistic NLR receptors, providing a tool to develop elite crop varieties.

Understanding how pathogenic fungi adapt to host plant cells is of major concern to securing global food production. The hemibiotrophic rice blast fungus Magnaporthe oryzae, cause of the most serious disease of cultivated rice, colonizes leaf cells asymptomatically as a biotroph for 4-5 days in susceptible rice cultivars before entering its destructive necrotrophic phase. During the biotrophic growth stage, M. oryzae remains undetected in the plant while acquiring nutrients and growing cell-to-cell. Which fungal processes facilitate in planta growth and development are still being elucidated. Here, we used gene functional analysis to show how components of the NADPH-requiring glutathione and thioredoxin antioxidation systems of M. oryzae contribute to disease. Loss of glutathione reductase, thioredoxin reductase and thioredoxin peroxidase-encoding genes resulted in strains severely attenuated in their ability to grow in rice cells and that failed to produce spreading necrotic lesions on the leaf surface. Glutathione reductase, but not thioredoxin reductase or thioredoxin peroxidase, was shown to be required for neutralizing plant generated reactive oxygen species (ROS). The thioredoxin proteins, but not glutathione reductase, were shown to contribute to cell-wall integrity. Furthermore, glutathione and thioredoxin gene expression, under axenic growth conditions, was dependent on both the presence of glucose and the M. oryzae sugar/ NADPH sensor Tps1, thereby suggesting how glucose availability, NADPH production and antioxidation might be connected. Taken together, this work identifies components of the fungal glutathione and thioredoxin antioxidation systems as determinants of rice blast disease that act to facilitate biotrophic colonization of host cells by M. oryzae.

Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs in which ~20% of the RNA originated from IH in first-invaded cells. We analyzed these IH RNAs relative to control mycelial RNAs using M. oryzae oligoarrays. With a 10-fold differential expression threshold, we identified known effector PWL2 and 58 candidate effectors. Four of these candidates were confirmed to be fungal biotrophy-associated secreted (BAS) proteins. Fluorescently labeled BAS proteins were secreted into rice cells in distinct patterns in compatible, but not in incompatible, interactions. BAS1 and BAS2 proteins preferentially accumulated in biotrophic interfacial complexes along with known avirulence effectors, BAS3 showed additional localization near cell wall crossing points, and BAS4 uniformly outlined growing IH. Analysis of the same infected-tissue RNAs with rice oligoarrays identified putative effector-induced rice susceptibility genes, which are highly enriched for sensor-transduction components rather than typically identified defense response genes.

Magnaporthe oryzae, the causative organism of rice blast, infects cereal crops and grasses at various stages of plant development. A comprehensive understanding of its metabolism and the implications on pathogenesis is necessary for countering this devastating crop disease. We present the role of the CorA magnesium transporters, MoAlr2 and MoMnr2, in development and pathogenicity of M. oryzae. The MoALR2 and MoMNR2 genes individually complement the Mg2+ uptake defects of a S. cerevisiae CorA transporter double mutant. MoALR2 and MoMNR2 respond to extracellular Mg2+ and Ca2+ levels and their expression is elevated under Mg2+ scarce conditions. RNA silencing mediated knockdown of MoALR2 (WT+siALR2, Δmnr2+siALR2 and ALR2+MNR2 simultaneous silencing) drastically alters intracellular cation concentrations and sensitivity to metal ions. MoALR2 silencing is detrimental to vegetative growth and surface hydrophobicity of mycelia, and the transformants display loss of cell wall integrity. MoALR2 is required for conidiogenesis and appressorium development, and is essential for infection. Investigation of knockdown transformants reveal low cAMP levels and altered expression of genes encoding proteins involved in MoMps1 cell wall integrity and cAMP MoPmk1 driven MAP Kinase signaling pathways. In contrast to MoALR2 knockdowns, the MoMNR2 deletion (Δmnr2) shows increased sensitivity to CorA inhibitors as well as altered cation sensitivity, but has limited effect on surface hydrophobicity and severity of plant infection. Interestingly, MoALR2 expression is elevated in Δmnr2. Impairment of development and infectivity of knockdown transformants and altered intracellular cation composition suggest that CorA transporters are essential for Mg2+ homeostasis within the cell, and are crucial to maintaining normal gene expression associated with cell structure, signal transduction and surface hydrophobicity in M. oryzae. We suggest that CorA transporters, and especially MoALR2, constitute an attractive target for the development of antifungal agents against this pathogen.

The blast fungus Magnaporthe grisea causes a serious disease on a wide variety of grasses including rice, wheat, and barley. Rice blast is the most serious disease of cultivated rice and therefore poses a threat to the world's most important food security crop. Here, I review recent progress toward understanding the molecular biology of plant infection by M. grisea , which involves development of a specialized cell, the appressorium. This dome-shaped cell generates enormous turgor pressure and physical force, allowing the fungus to breach the host cuticle and invade plant tissue. The review also considers the role of avirulence genes in M. grisea and the mechanisms by which resistant rice cultivars are able to perceive the fungus and defend themselves. Finally, the likely mechanisms that promote genetic diversity in M. grisea and our current understanding of the population structure of the blast fungus are evaluated.

The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG_Ef1, which demonstrated low M values across heterogeneous tissues. By contrast, metabolic pathway genes MGG_Fad (FAD binding domain-containing protein) and MGG_Gapdh (Glyceraldehyde-3-phosphate dehydrogenase) performed poorly, due to their lack of expression stability across samples.

The order Magnaporthales comprises about 200 species and includes the economically and scientifically important rice blast fungus and the take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthales with their type species and available gene and genome resources. The polyphyletic Magnaporthe 1972 is proposed for suppression, and Pyricularia 1880 and Nakataea 1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

Conidiation of phytopathogenic fungi is a key developmental process that plays a central role in their life cycles and in epidemics. However, there is little information on conidiation-induced molecular changes in the rice blast fungus Magnaporthe oryzae. As a first step to understand conidiogenesis in this fungus, we measured genome-wide gene expression profiles during conidiation using a whole genome oligonucleotide microarray. At a two-fold expression difference, approximately 4.42% and 4.08% of genes were upregulated and downregulated, respectively, during conidiation. The differentially expressed genes were functionally categorized by gene ontology (GO) term analysis, which demonstrated that the gene set encoded proteins that function in metabolism, cell wall biosynthesis, transcription, and molecule transport. To define the events of the complicated process of conidiogenesis, another set of microarray experiments was performed using a deletion mutant for MoHOX2, a stage-specific transcriptional regulator essential for conidial formation, which was expressed de novo in a conidiation-specific manner in M. oryzae. Gene expression profiles were compared between the wild-type and the ΔMohox2 mutant during conidiation. This analysis defined a common gene set that was upregulated in the wild-type and downregulated in the ΔMohox2 mutant during conidiation; this gene set is expected to include conidiation-related downstream genes of MoHOX2. We identified several hundred genes that are differentially-expressed during conidiation; our results serve as an important resource for understanding the conidiation, a process in M. oryzae, which is critical for disease development.

WRKY transcription factors are crucial regulatory components of plant responses to pathogen infection. In the present study, we report isolation and functional characterization of the pathogen-responsive rice WRKY30 gene, whose transcripts accumulate rapidly in response to salicylic acid (SA) and jasmonic acid (JA) treatment. Overexpression of WRKY30 in rice enhanced resistance to rice sheath blight fungus Rhizoctonia solarii and blast fungus Magnaporthe grisea. The enhanced resistance in the transgenic lines overexpressing WRKY30 was associated with activated expression of JA synthesis-related genes LOX, AOS2 and pathogenesis-related (PR)3 and PR10, and increased endogenous JA accumulation under the challenge of fungal pathogens. WRKY30 was nuclearlocalized and had transcriptional activation ability in yeast cells, supporting that it functions as a transcription factor. Together, our findings indicate that JA plays a crucial role in the WRKY30-mediated defense responses to fungal pathogens, and that the rice WRKY30 seems promising as an important candidate gene to improve disease resistance in rice.

Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus.