Duplicated Flagellins in Pseudomonas Divergently Contribute to Motility and Plant Immune Elicitation

Flagellins are the main constituents of the flagellar filaments that provide bacterial motility, chemotactic ability, and host immune elicitation ability. Although the functions of flagellins have been extensively studied in bacteria with a single flagellin-encoding gene, the function of multiple flagellin-encoding genes in a single bacterial species is largely unknown. Here, the model plant-growth-promoting bacterium Pseudomonas kilonensis F113 was used to decipher the divergent functions of duplicated flagellins. We demonstrate that the two flagellins (FliC-1 and FliC-2) in 12 Pseudomonas strains, including F113, are evolutionarily distinct. Only the fliC-1 gene but not the fliC-2 gene in strain F113 is responsible for flagellar biogenesis, motility, and plant immune elicitation. The transcriptional expression of fliC-2 was significantly lower than that of fliC-1 in medium and in planta, most likely due to variations in promoter activity. In silico prediction revealed that all fliC-2 genes in the 12 Pseudomonas strains have a poorly conserved promoter motif. Compared to the Flg22-2 epitope (relative to FliC-2), Flg22-1 (relative to FliC-1) induced stronger FLAGELLIN SENSING 2 (FLS2)-mediated microbe-associated molecular pattern-triggered immunity and significantly inhibited plant root growth. A change in the 19th amino acid in Flg22-2 reduced its binding affinity to the FLS2/brassinosteroid insensitive 1-associated kinase 1 complex. Also, Flg22-2 epitopes in the other 11 Pseudomonas strains were presumed to have low binding affinity due to the same change in the 19th amino acid. These findings suggest that Pseudomonas has evolved duplicate flagellins, with only FliC-1 contributing to motility and plant immune elicitation. IMPORTANCE Flagellins have emerged as important microbial patterns. This work focuses on flagellin duplication in some plant-associated Pseudomonas. Our findings on the divergence of duplicated flagellins provide a conceptual framework for better understanding the functional determinant flagellin and its peptide in multiple-flagellin plant-growth-promoting rhizobacteria.