Investigating the role of truncated receptors in plant immunity

Overpopulation of the planet, climate change and the limitations in the available arable land are undoubtedly among the major problems of modern era that threaten global food security. Crop losses due to diseases in the field caused by plant pathogens account as one of the most daunting obstacles for food security in the 21st century. Engineering plants with durable resistance against pathogenic microbes is arising as a key strategy for sustainable food production. We aimed to unravel novel pathways for robust plant production and minimise crop losses to pathogenic infections by elucidating the role of uncharacterised “truncated” receptors in plant immunity. “Truncated” TIR-NB (TN) proteins are structurally similar to NLR immune receptors, without the LRR domain. Increasing evidence show that TN proteins may be important players in plant immune responses, possibly due to the role of the TIR domain. A transcriptomics study (Lewis et al., 2015) on Arabidopsis thaliana when challenged with the pathogenic bacteria Pseudomonas syringae pv. tomato (Pst DC3000) showed rapid upregulation of 4 TN genes from the same genetic locus: AT1G72920, AT1G72930, AT1G72940, and AT1G72950. RT-q-PCR confirmed that A. thaliana TN genes AT1G72920 and AT1G72940 are upregulated specifically in response to Pst DC3000, but not to the mutant strain Pst DC3000 hrpA that cannot deliver effectors, or in response to any of the other biotic and abiotic stresses tested. Analysis of TN promoter regulation showed that Pst DC3000 effectors HopQ1-1, HopAl1, HopB1, AvrPto, HopF2 and HopAB2 correlate with TN promoter induction when co-expressed in protoplasts. A study by (Mine et al., 2018) using chromatin immunoprecipitation (ChIP) showed that ERF6 transcription factor (TF) binds on AT1G72920 and AT1G72940 promoters, and suggests that more ERFs may regulate TN expression. Yeast expression vectors were generated with promoter AT1G72940 and versions of it with mutations on three motifs for TF recognition, to facilitate large scale yeast-one-hybrid screenings of a transcription factor library, to identify candidates that mediate TN promoter regulation. Pst DC3000 assays on knockout plants missing the TN locus did not reveal significant differences to wild type plants, possibly due to functional redundancy. A range of A. thaliana lines were generated, selected and characterised for constitutive and inducible overexpression of AT1G72940 to facilitate immunity assays and high-throughput experiments that can elucidate the role of AT1G72940 in plant immunity. Characterisation of overexpressing AT1G72940 lines showed that protein AT1G72940 is not detected beyond the early plant developmental stages, suggesting epigenetic silencing of transgene expression and/or protein degradation in adult plants. Preliminary results of Pst DC3000 bacterial growth indicate susceptibility to infection of the same plants when compared to wild type, which is inconsistent with the lack of protein detection. Nevertheless, the generation and selection of A. thaliana transgenic plants for AT1G72940 with many combinations of different promoters and tags offers many possibilities for future work in the field