Periodic Reporting for period 1 - MIREDI (Mechanisms of Immune Receptor Diversification in Cereals)
Reporting period: 2017-09-01 to 2019-02-28
Protein domains integrated into NLRs are considered putative, homologous decoys of plant proteins, targeted by pathogen effectors. We undertook a new approach to build on the previous NLR-ID pipelines developed in our lab.This new approach incorporated a reciprocal BLAST analysis comparing the entire NLR complement from the plant genomes on public databases, including Phytozome, Plant Ensemble, and Refseq against all of the predicted proteins from any plant species.This analysis allows to identify putative plant proteins targeted by pathogens, which represent potential susceptibility molecules that pathogens exploit to establish disease. Using the A. thaliana protein accessions, we mapped NLR-IDs to plant metabolic and signalling pathways. This analysis successfully enabled identification of a series of pathways represented by multiple NLR-ID homologues. We have cloned nine NLR-IDs that we are currently challenging with putative corresponding effectors from economically important bacterial, fungal, and insect pathogens of wheat to identify functional NLRs for future study.
Finally, we have identified and cloned a candidate NLR platform that was able to signal independently of other paired NLRs in heterologous Nicotiana benthamiana. We are currently performing deletions and domain swap analysis to test whether this platform can generate new functional fusions.
To improve our yeast two hybrid screens beyong the state of the art, we designed a bait capture to target effectors predicted from Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici (provided by Diane Saunders at JIC) and wheat blast Magnaporthe oryzae (provided by Nick Talbot, Exeter). DNA sequence of predicted effectors was extracted, conserved effectors with over 90% similarity were collapsed to prevent unbalanced enrichment of different effectors. Effector sequence with low complexity was masked to prevent design of baits with off target hits. Then filtering steps where carried out to remove any which were cross hitting to the wheat nuclear, chloroplast or mitochondrial genome. Baits which had low specificity to target sequence were also removed. Finally baits where manually designed to effectors which had no baits after filtering.