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Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.

Weak or transient protein-protein interactions (PPIs) are involved in a manifold of cellular processes in all living organisms, including plants. However, many of these interactions may remain undiscovered by co-immunoprecipitation (Co-IP) approaches due to their low binding affinities or transitory nature. Enzyme-mediated proximity-dependent in vivo biotin labeling can be a powerful strategy to efficiently capture weak and transient PPIs and has been successfully applied in different model angiosperm species. Here, we provide an optimized and robust protocol for biotin ligase-mediated proximity labeling for interactome mapping in the model liverwort Marchantia polymorpha., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays. Here, we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed vectors that enable Tn 7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location found ubiquitously across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas , Rhizobium ( Agrobacterium ), and Ralstonia . Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv. tomato DC3000 ( Pto -lux) reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana , Solanum lycopersicum , Nicotiana benthamiana , and Marchantia polymorpha . We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto -lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth, with a dynamic range of four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated the spatial patterns of Pto -lux growth in/on inoculated plant tissues. In conclusion, our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions., (© 2021 The Authors.)