In the past year, we have successfully completed and published our analysis of NLRs in 9 publicly available grass genomes that identified NLR clade prone to new exogenous domain fusions (Bailey et al Genome Biology 2018). In order to further test the effects of speciation, domestication and inbreeding on the number of NLRs in grasses, we acquired diverse germplasm of 5 species of Panicoideae. We designed an enrichment capture for targeted resequencing of NLRs, domestication genes and genome markers for wild, landraces and elite inbred members of the economically important crops of Maize, Sorghum bicolor and Setaria Italica. We obtained the seeds, extracted DNA from 80 samples across and Illumina libraries were prepared for capture and sequencing by Arbor bioscience using long read PacBio platform. In additiona, we prepared a set of 60 tetraploid wheat lines that include both domesticated and wild species for capture-based sequencing of NLRs. For wheat, we have analysed newly available genomes and prepared sequences to supplement existing wheat capture design. We already secured wheat DNA samples from our collaborators and are preparing them for sequencing. In addition, we are generating RNA sequencing data using newest long read technologies for a subset of species that represent independent polyploidization and hybridization events.
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.
