New insights into the FLS2 trafficking and signaling pathway revealing a role for late defense responses in Arabidopsis immunity

Pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) enables plants to efficiently defend themselves against most pathogens. PTI is initiated by plasma membrane pattern recognition receptors like Flagellin Sensitive 2 (FLS2), which detects flg22 peptides derived from bacterial flagellin. Flg22 triggers cellular trafficking of FLS2 to endosomal compartments and several defense responses, including early responses such as closure of stomatal apertures or gene activation and late responses such as seedling growth arrest or callose deposition. Cellular trafficking is often regulated by ubiquitination. In order to find factors modulating FLS2 endosomal trafficking, we used the Pseudomonas syringe pv. tomato (Pto) DC3000 effector AvrPtoB to identify residues in FLS2, which are ubiquitinated by AvrPtoB. Using tandem mass-spectrometry we identified one ubiquitination site in the juxtamembrane domain of FLS2. FLS2 mutated in all juxtamembrane located lysines showed reduced ubiquitination in vitro, enhanced resistance to Pto DC3000 and increased FLS2 endosome numbers upon flg22 treatment. Ubiquitinated plasma membrane proteins are targeted to late endosomal compartments by the Endosomal Sorting Complex Required for Transport (ESCRT) in yeast and animals. Using confocal microscopy we observed flg22-dependent co-localization of FLS2 with ESCRT-1 positive vesicles. Furthermore, ESCRT-1 mutants vps28-2 and vps37-1 showed reduced flg22-triggered defense gene activation, loss of flg22-dependent stomatal closure and decreased numbers of FLS2 endosomes. Both mutants showed higher susceptibility to biotrophic pathogens, indicating a role of ESCRT-1 components in plant immunity. A second approach aimed to genetically dissect flg22 responses by analyzing a previously isolated flagellin insensitive 1 (fli1) mutant. Fli1 mutants were similar susceptible to Pto DC3000 than fls2-17 receptor mutants. Increased susceptibility of fli1 to Pto DC3000 correlated with higher expression of sugar starvation responsive genes during infections and reduced late flg22 responses. Early flg22 responses and transcriptional profiles three hours post infection resembled wild-type plants, suggesting a positive role of late PAMP responses in plant immunity. Genetic analysis showed that fli1 is recessive inherited and co-segregated with markers on the upper arm of chromosome 5. Sequence differences in fli1 predicted by whole genome sequence analysis were, however, shared with Ler wild-type plants, leaving the designation of fli1 to one gene open. In conclusion, these data provide good evidences for a role of late FLS2 endosomal trafficking and late flg22-repsonses as critical components of plant immunity against Pto DC3000