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 invasion1,2,3. 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. ΔMgapt2 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 of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection.
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We thank C. Hawes and B. Martin for cryofixation and freeze substitution work, G.-L. Wang for supply of IR-68 seeds and T. Graham for supplying the BY4739, PFY3273A and DS94 yeast APT mutants. This study was supported by a grant to N.J.T. from the Biological Sciences and Biotechnology Research Council (BBSRC). Author Contributions Experimental work and data analysis were performed by M.J.G. and N.J.T. C.R.T. performed all immunological work and associated data analysis. G.E.W. performed electron microscopy.
Yeast complementation assay showing the Magnaporthe APT2 gene is unable to complement the cold sensitive phenotype of the Saccharomyces cerevisiae Δdrs2 mutant.
Influence of MgAPT2(p) expression on aminophospholipid internalisation in intact yeast cells. MgAPT2(p) is unable to restore the ability of drs2Δ to internalize phosphatidylserine.
Generation and virulence of ΔMgapt2 mutants. Targeted gene replacement strategy showing the generation of 6 gene replacement transformants. Subsequent plant infection assays and epidermal penetration assays show a significant reduction of ΔMgapt2 mutants to cause rice blast disease.
Electron micrographs showing the accumulation of Berkeley bodies in the yeast drs2Δ mutant.
The loss of MgAPT2 has no affect on endocytosis. Wild type and ΔMgapt2 conidia were stained with FM4-64 and show no significant difference in uptake kinetics.
The ΔMgapt2 mutant is impaired in its ability to secrete α-amylase. Using an anti-α-amylase antibody, immunogold experiments were conducted. The resulting electron micrographs show ΔMgapt2 is severely reduced in α-amylase secretion.
Plate assay showing ΔMgapt2 mutants are not compromised in their ability to secrete the enzyme trehalase.
Amino acid similarity and identity of MgAPT2 and the yeast aminophospholipid translocases.
Table showing the ability of the ΔMgapt2 mutant to grow on single carbon sources.
Summary of Magnaporthe strains used in the study.
Summary of yeast strains used in the study.
Comprehensive and detailed explanation of protocols used in the study together with references.
About this article
Nature Protocols (2006)