Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.
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