Fungal appressoria as organs for sensing plant-derived signals and delivering protein and small-molecule effectors to host cells – FUNAPP

• Analysis of fungal gene function by over-expression, targeted mutagenesis and random insertional mutagenesis
• In planta expression assays for detecting suppression or induction of plant immunity by candidate effectors
• Analysis of fungal secondary metabolites by LC-MS, NMR
• Analysis of fungal gene expression by RT-qPCR and RNA-Seq
• Localisation of appressorial and secreted proteins in planta using fluorescent protein tagging and immunocytochemistry; Epi-fluorescence and confocal microscopy; Transmission electron microscopy, cryo-fixation and freeze-substitution
• Genome sequencing, assembly and gene annotation; prediction of effectors, secondary metabolism gene clusters; Comparative genomics for identification of host-specific effectors
• Use of fungal fluorescent reporter strains to screen plant components for signals reprogramming fungal gene expression

All equipment has been purchased and installed and four staff were appointed (2 Post-doc, 1 Engineer and 1 PhD student). New office and laboratory spaces were organized in BIOGER and equipped. Initial results from the first 4 months of the project are described in the confidential progress report.

Pre-invasive sensing of the host by pathogens is likely to be a widespread phenomenon in nature, potentially even in human fungal diseases, and its inhibition should lead to a full arrest of the infection. A better understanding of host perception by plant pathogenic fungi, infection morphogenesis and the early deployment of molecular weapons such as effector proteins and metabolites is likely to suggest novel strategies for the control of many economically damaging crop diseases through the rational design of fungicides, or the identification of novel criteria for breeding disease resistant plants.

Not applicable in 6 month report

Fungal plant pathogens are a major threat to global food security. For example, every year between 10 and 30% of the rice crop is destroyed by the rice blast fungus, Magnaporthe oryzae. Similarly, the anthracnose fungus Colletotrichum graminicola causes annual losses on maize amounting to 1 billion dollars in the USA alone. Colletotrichum and Magnaporthe use similar strategies to invade host plants: a special organ, the appressorium, is used to break into living host cells, where intracellular hyphae then develop.

The interaction between the brassica pathogen C. higginsianum and the model plant Arabidopsis provides a genetically tractable system for studying fungal pathogenicity and plant immune responses.Previously we found that the transcriptome of C. higginsianum appressoria undergoes massive reprogramming in response to plant contact compared to appressoria differentiated on artificial surfaces. This suggests that these cells function as sensing organs for plant signals, allowing the pathogen to prepare for subsequent host invasion. Appressoria also appear to be factories for the synthesis and secretion of a large array of secondary metabolites and protein effectors. A nanoscale pore (200nm diameter) in the appressorium base may serve as both a window for host sensing and for the focal delivery of effectors and small-molecules into host cells (Kleemann et al. 2012).

Building upon these novel findings, this research project will use a combination of genetic, genomic and cellular biology approaches to explore the role of appressoria in early infection focusing on secreted proteins and secondary metabolites produced by appressoria, the nature of the plant signals sensed by appressoria and how they are perceived.