Mechanistic Insights into Immunosuppressive Subtilase A (IssA)-Mediated Flagellin Immune Suppression in the Arabidopsis Root Microbiome

Abstract

Plants have an innate immune system that is triggered in response to microbe-associated molecular patterns (MAMPs). Flg22 is a MAMP that varies by species and is found in the FliC subunit of bacterial flagella. Despite having flagella, some Arabidopsis thaliana Columbia-0 (Col-0) root commensals, including Dyella japonica MF79, reside on A. thaliana roots without triggering the immune system. Immunosuppressive subtilase A (IssA) was identified in Dja MF79 as an actor in this immune suppression. This paper focuses on the top 25 suppressors from the Dangl labs collection of 165 A. thaliana root associated bacteria and finds no correlation between strength of root growth inhibition (RGI) suppression and GUS suppression. Furthermore, by incubating flg22 with Dja MF79 supernatant, I show that IssA alters flg22 such that it is no longer perceived by the plant. Additionally, homology was utilized to identify the presence of IssA homologs in 22 of the 25 top RGI suppressors. Genetic deletion revealed that IssA homologs contribute to immune suppression in both Ralstonia sp. CL21 and Luteibacter sp. MF22. This data suggests IssA is an important mechanism by which commensals amongst multiple phyla suppress activation of the flg22 immune response in A. thaliana. Furthermore, preliminary research on suppressive Curtobacterium spp. that lack IssA homologs reveals that Curtobacterium spp. is not altering flg22. Therefore, other mechanisms of A. thaliana immune suppression are yet to be discovered. A. thaliana pCYP71A12::GUS and A. thaliana WER::FLS2-GFP are commonly used in studies that identify if the immune system is active in response to flg22. Both plant lines have drawbacks that make the generation of new immune identifying plant lines valuable. For instance, A. thaliana pCYP71A12::GUS lines involve staining and destaining steps, which extend the GUS assay by two days, and produce qualitative results of blue or white root tips. On the other hand, pWER::FLS2-GFP plant lines are used most commonly in Root Growth Inhibition (RGI) assays, which take two weeks to complete. This paper begins the process of generating plant lines that link promoters that are transcriptionally active in response to flg22 with mKOK, a fluorescent reporter.