Proteobacteria encode diverse flg22 peptides that elicit varying immune responses inArabidopsis thaliana

Abstract

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22 amino acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modelling. These data led us to hypothesize that most flg22 epitopes of the γ- and β-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. Flg22 peptides of the γ- and β-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and β-proteobacteria, a monophyletic group containing many common phytopathogens.

Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response

“…Mean photon counts were reduced by 71% and 76% with LRR1, by 79% and 83% with LRR3 and by 64% and 70% with LRR7, when compared to transgenic and wildtype controls, respectively. No response to the flg22-ɑ peptide was seen in any of the samples, as expected given this peptide is non-immunogenic to Arabidopsis 32 .…”

“…Representative LRR sequences corresponding to BdIRI1, 3, and 7 were expressed in Arabidopsis, and the impact on the native immune response was assessed using oxidative burst assays (Figure 4). Arabidopsis lines expressing any of the LRRs showed significantly impaired responses (p < 0.001, one way ANOVA) when leaf disks were exposed to the immunogenic flg22-y peptide 32 , compared to wildtype Arabidopsis controls or transgenic plants bearing empty plasmids. Mean photon counts were reduced by 71% and 76% with LRR1, by 79% and 83% with LRR3 and by 64% and 70% with LRR7, when compared to transgenic and wildtype controls, respectively.…”

“…Assays used a Synergy H1 Microplate Reader (BioTek Instruments, Inc.) using 1 s integration time, 60 min read time, with 2 min intervals measuring luminosity. Purified flg22 epitopes (EZBiolab, Parsippany, New Jersey, USA) included flg22-y (representative of the flg22 epitope of y-proteobacteria) and flg22-α (representative of the flg22 epitope of α-proteobacteria), which do and do not elicit an immune response in Arabidopsis, respectively 32 . The assays were conducted on extracts of wildtype Col-0, Col-0:BdLRR1, Col-0:BdLRR3, Col-0:BdLRR7, and a Col-0:pCambia1305.1 empty vector control with both epitope treatments and a non-epitope blank controls.…”

The right microbe-associated molecular patterns for effective recognition by plants

“…However, some microbes can avoid the detection by PRRs through mutating sequence of MAMPs. Mutations in flg22 that are not recognized by FLS2 was reported in some flagellated bacteria (Cheng et al, 2021). An α-proteobacterium Agrobacterium carrying a flg22 variant does not trigger FLS2mediated immune responses (Felix et al, 1999).…”

Natural variation of immune epitopes reveals intrabacterial antagonism

“…Using flg22 perception as a model, three general epitope outcomes have been described: immune activation, evasion, and antagonism. Many Gram-negative plant pathogens carry immunogenic epitopes, though some can evade perception (7,(30)(31)(32). MAMP evasion, also known as masking, occurs by accumulating sequence variation that prevents epitope binding (33,34).…”