Genome-wide functional analysis reveals that infection-related fungal autophagy is necessary for rice blast disease

School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(37):15967-72. DOI: 10.1073/pnas.0901477106
Source: PubMed


To cause rice blast disease, the fungus Magnaporthe oryzae elaborates specialized infection structures called appressoria, which use enormous turgor to rupture the tough outer cuticle of a rice leaf. Here, we report the generation of a set of 22 isogenic M. oryzae mutants each differing by a single component of the predicted autophagic machinery of the fungus. Analysis of this set of targeted deletion mutants demonstrated that loss of any of the 16 genes necessary for nonselective macroautophagy renders the fungus unable to cause rice blast disease, due to impairment of both conidial programmed cell death and appressorium maturation. In contrast, genes necessary only for selective forms of autophagy, such as pexophagy and mitophagy, are dispensable for appressorium-mediated plant infection. A genome-wide analysis therefore demonstrates the importance of infection-associated, nonselective autophagy for the establishment of rice blast disease.

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    • "Appressorium development occurs in response to the hard, hydrophobic rice (Oryza sativa) leaf surface (Veneault-Fourrey et al., 2006; Saunders et al., 2010). The appressorium generates pressure by accumulating osmolytes, such as glycerol, to very high concentrations and uses autophagic cell death of the conidium to recycle cellular components to the developing appressorium (Veneault-Fourrey et al., 2006; Kershaw and Talbot, 2009). "
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    ABSTRACT: Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septin-mediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.
    The Plant Cell 11/2015; DOI:10.1105/tpc.15.00552 · 9.34 Impact Factor
    • "To test the function of PKC1 in M. oryzae, we initially attempted targeted gene replacement but this did not result in any null mutants, in spite of numerous attempts including utilisation of a Δku70 strain with enhanced frequency of homologous recombination (Kershaw and Talbot, 2009). We therefore decided to attempt gene silencing by means of RNA interference (RNAi), which has been reported in M. oryzae (Nakayashiki et al., 2005) and results in downregulation of gene expression, rather than complete loss of gene function. "
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    ABSTRACT: Protein kinase C constitutes a family of serine-threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation, and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of Magnaporthe oryzae. First, all attempts to generate a target deletion of PKC1, the single copy protein kinase C-encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2-encoding gene, MDL2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacementwith an analogue-sensitive PKC1(AS) allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expressionassociated with cell wall re-modelling, autophagy, signal transduction and secondary metabolism.When considered together, these results suggest protein kinase C is essential for growth and development of M. oryzaewith extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease. This article is protected by copyright. All rights reserved.
    Molecular Microbiology 07/2015; 98(3). DOI:10.1111/mmi.13132 · 4.42 Impact Factor
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    • "Interestingly, we found that Δfar1, Δfar2 and Δfar1Δfar2 double mutants were not impaired in lipid body mobilisation to the appressorium, or subsequent lipolysis during turgor generation in the appressorium. This study therefore provides evidence that lipid body mobilisation during appressorium development by M. oryzae, which occurs on the rice leaf surface in the absence of exogenous nutrients and is dependent on autophagy [4], [7], [9], [10], is regulated separately from lipid utilization and independently of Far1 and Far2. "
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    ABSTRACT: The rice blast fungus Magnaporthe oryzae causes plant disease via specialised infection structures called appressoria. These dome-shaped cells are able to generate enormous internal pressure, which enables penetration of rice tissue by invasive hyphae. Previous studies have shown that mobilisation of lipid bodies and subsequent lipid metabolism are essential pre-requisites for successful appressorium-mediated plant infection, which requires autophagic recycling of the contents of germinated spores and germ tubes to the developing appressorium. Here, we set out to identify putative regulators of lipid metabolism in the rice blast fungus. We report the identification of FAR1 and FAR2, which encode highly conserved members of the Zn2-Cys6 family of transcriptional regulators. We generated Δfar1, Δfar2 and Δfar1Δfar2 double mutants in M. oryzae and show that these deletion mutants are deficient in growth on long chain fatty acids. In addition, Δfar2 mutants are also unable to grow on acetate and short chain fatty acids. FAR1 and FAR2 are necessary for differential expression of genes involved in fatty acid β-oxidation, acetyl-CoA translocation, peroxisomal biogenesis, and the glyoxylate cycle in response to the presence of lipids. Furthermore, FAR2 is necessary for expression of genes associated with acetyl-CoA synthesis. Interestingly, Δfar1, Δfar2 and Δfar1Δfar2 mutants show no observable delay or reduction in lipid body mobilisation during plant infection, suggesting that these transcriptional regulators control lipid substrate utilization by the fungus but not the mobilisation of intracellular lipid reserves during infection-related morphogenesis.
    PLoS ONE 06/2014; 9(6):e99760. DOI:10.1371/journal.pone.0099760 · 3.23 Impact Factor
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