Functional characterisation of candidate Fusarium effectors

The new fusarium candidate effector study will involve the following experiments:

1. A rigorous bioinformatics analysis of the reference PH-1 genome, the newly generated F. graminearum (Fg) pan genome (n=24 isolates), several published and unpublished in planta and in vitro transcriptomics data sets (Affymetrix or RNA seq derived) and phylogenetic analyses to identify and rank a list of candidate genes that possess the hallmarks of various types of fungal effectors. Protein structure prediction analysis will also be performed and regular reviews of the effector literature will be used to augment this candidate gene list.
2. Depending on the effector hypothesis being investigated, the functional testing of each candidate Fg effector genes will be done using one or more bioassays from the range available. The bioassays to be used will included (a) transient over-expression In Nicotiana benthamiana leaves using the PEAQ vector and agroinfiltrations, (b) barley stripe mosaic virus mediated virus induced over-expression (BSMV-VOX) in wheat floral tissue followed by Fg inoculation, (c) stable over-expression in transgenic Arabidopsis plants, (d) individual effector gene deletions in Fg and then floral and coleoptile inoculations as well as various in vitro tests.
3. To explore the potential mechanisms underlying the in planta expression of fungal effectors, gene expression clustering, MEME analysis, Nsite and Transfac analyses of the promoter, intron and 3'UTR sequences will be done to identify potential regulatory sequences responsible for the co-ordination of in planta expression. Included in these analyses will be the sequences of genes residing in the 72 predicted secondary metabolite clusters (SMCs) in Fg a sub-set of which are also known to be specifically upregulated in planta.
4. Existing Fg candidate effector leads, for example FgSSP32, FgSSP33 and potentially FgSSP5 will be explored by the addition N or C terminal fluorescent protein tags, with or without nuclear localisation sequences (NLS) and transforming into Fg. The resulting transformants will be used to explore in detail in planta temporal and spatial expression patterns in wheat and Arabidopsis and the potential trafficking of effectors ahead of the colonising hyphal front via the plasmodesmata (aka Magnaporthe orzyae infections). The wheat coleoptile assay and confocal microscope analyses will be particularly useful for these studies.
5. To explore the in planta expression of effectors, promoter/ regulatory element fluorescent protein tags constructs will be generated, transformed into wildtype Fg to explore the spatial and temporal patterns of gene expression in wheat and Arabidopsis.
6. To identity the potential host targets of effectors several routes will be considered. These include a yeast two hybrid screen of a suitable host library (potentially either N. benthaminana, early Fg infected wheat floral tissue or early infected Arabidopsis tissue (ecotype Landsberg erecta), or the screening of a N. benthaminana collection where each of the receptor-like kinase genes (RLK) has been disrupted in function through a T-DNA insertion event. The later would be done in collaboration with the University of Nanjing.