To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver protein... more To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the world's most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. DeltaMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment ...
Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic,... more Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic, posing a major challenge for sampling and data analysis. A protocol is described for the infection of the model grass species Brachypodium distachyon with Magnaporthe grisea (rice blast), together with modifications to extend the use to rice and barley. We outline a method for the preparation of long-term stocks of virulent fungal pathogens and for the generation of fungal inoculants for challenge of host plants. Host plant growth, pathogen inoculation and plant sampling protocols are presented together with methods for assessing the efficiency of both infection and sampling procedures. Included in the anticipated results is a description of the use of metabolite fingerprinting and multivariate data analysis to assess disease synchrony and validate system reproducibility between experiments. The design concepts will have value in any studies using biological systems that contain dynamic...
Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll)... more Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) of the rice-blast pathogen,Magnaporthe grisea (=Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ
Rice blast is caused by the fungus Magnaporthe grisea, which elaborates specialized infection cel... more Rice blast is caused by the fungus Magnaporthe grisea, which elaborates specialized infection cells called appressoria to penetrate the tough outer cuticle of the rice plant Oryza sativa. We found that the formation of an appressorium required, sequentially, the completion of mitosis, nuclear migration, and death of the conidium (fungal spore) from which the infection originated. Genetic intervention during mitosis
LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cyt... more LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cytoskeletal re-organisation and organ development in multi-cellular eukaryotes. Here, we report the characterization of four genes encoding LIM proteins in the rice blast fungus Magnaporthe oryzae. Targeted gene replacement of either the paxillin-encoding gene, PAX1, or LRG1 resulted in a significant reduction in hyphal growth and loss of pathogenicity, while deletion of RGA1 caused defects in conidiogenesis and appressorium development. A fourth LIM domain gene, LDP1, was not required for infection-associated development by M. oryzae. Live cell imaging revealed that Lrg1-GFP and Rga1-GFP both localize to septal pores, while Pax1-GFP is present in the cytoplasm. To explore the function of individual LIM domains, we carried out systematic deletion of each LIM domain, which revealed the importance of the Lrg1-LIM2 and Lrg1-RhoGAP domains for Lrg1 function and overlapping functions of the three LIM domains of Pax1. Interestingly, deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and SDS (sodium dodecyl sulfate). qRT-PCR analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis. When considered together, our results indicate that LIM domain proteins are key regulators of infection-associated morphogenesis by the rice blast fungus.
... In 344 Martin J. Gilbert, Darren M. Soanes and Nicholas J Talbot this method, linearized plas... more ... In 344 Martin J. Gilbert, Darren M. Soanes and Nicholas J Talbot this method, linearized plasmid DNA is transformed into the organism of choice in the presence of a restriction enzyme, which generates compatible or incompatible ends. ... 2000; Mikosch et al. 2001; Mullins et al. ...
Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of
Research in autophagy continues to accelerate, and as a result many new scientists are entering t... more Research in autophagy continues to accelerate, and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose. There are many useful and convenient methods that can be used
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since t... more In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have
To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver protein... more To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the world's most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. DeltaMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment ...
Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic,... more Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic, posing a major challenge for sampling and data analysis. A protocol is described for the infection of the model grass species Brachypodium distachyon with Magnaporthe grisea (rice blast), together with modifications to extend the use to rice and barley. We outline a method for the preparation of long-term stocks of virulent fungal pathogens and for the generation of fungal inoculants for challenge of host plants. Host plant growth, pathogen inoculation and plant sampling protocols are presented together with methods for assessing the efficiency of both infection and sampling procedures. Included in the anticipated results is a description of the use of metabolite fingerprinting and multivariate data analysis to assess disease synchrony and validate system reproducibility between experiments. The design concepts will have value in any studies using biological systems that contain dynamic...
Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll)... more Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) of the rice-blast pathogen,Magnaporthe grisea (=Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ
Rice blast is caused by the fungus Magnaporthe grisea, which elaborates specialized infection cel... more Rice blast is caused by the fungus Magnaporthe grisea, which elaborates specialized infection cells called appressoria to penetrate the tough outer cuticle of the rice plant Oryza sativa. We found that the formation of an appressorium required, sequentially, the completion of mitosis, nuclear migration, and death of the conidium (fungal spore) from which the infection originated. Genetic intervention during mitosis
LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cyt... more LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cytoskeletal re-organisation and organ development in multi-cellular eukaryotes. Here, we report the characterization of four genes encoding LIM proteins in the rice blast fungus Magnaporthe oryzae. Targeted gene replacement of either the paxillin-encoding gene, PAX1, or LRG1 resulted in a significant reduction in hyphal growth and loss of pathogenicity, while deletion of RGA1 caused defects in conidiogenesis and appressorium development. A fourth LIM domain gene, LDP1, was not required for infection-associated development by M. oryzae. Live cell imaging revealed that Lrg1-GFP and Rga1-GFP both localize to septal pores, while Pax1-GFP is present in the cytoplasm. To explore the function of individual LIM domains, we carried out systematic deletion of each LIM domain, which revealed the importance of the Lrg1-LIM2 and Lrg1-RhoGAP domains for Lrg1 function and overlapping functions of the three LIM domains of Pax1. Interestingly, deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and SDS (sodium dodecyl sulfate). qRT-PCR analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis. When considered together, our results indicate that LIM domain proteins are key regulators of infection-associated morphogenesis by the rice blast fungus.
... In 344 Martin J. Gilbert, Darren M. Soanes and Nicholas J Talbot this method, linearized plas... more ... In 344 Martin J. Gilbert, Darren M. Soanes and Nicholas J Talbot this method, linearized plasmid DNA is transformed into the organism of choice in the presence of a restriction enzyme, which generates compatible or incompatible ends. ... 2000; Mikosch et al. 2001; Mullins et al. ...
Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of
Research in autophagy continues to accelerate, and as a result many new scientists are entering t... more Research in autophagy continues to accelerate, and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose. There are many useful and convenient methods that can be used
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since t... more In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have
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