One of the first layers of active defense in plant-microbe interactions is based upon the recognition of pathogen associated molecular patterns (PAMPs). Although biochemically well studied, components of PAMP signaling await to be identified. Furthermore, emerging data point to a function of endocytosis in signaling (Chinchilla et al., 2007a; Geldner et al., 2007). Here, we conducted reverse and forward genetic approaches to identify components and to elucidate the role of endocytosis in PAMP signaling. Previous successful forward genetic approaches were refined to identify additional components in PAMP signaling (Gomez-Gomez and Boller, 2000). The sensitivity of the response to flg22 by seedling growth inhibition was enhanced by UV-B treatment (Logemann and Hahlbrock, 2002), and by employing a modified seedling growth inhibition assay on plates with reduced flagellin dosis. Arabidopsis thaliana ecotypes were inspected and most insensitive accessions were mutated in FLS2 alleles. Furthermore, screening a ã-irradiation population revealed several fli mutants (for flagellin-insensitive). Notably, only late PAMP responses such as callose deposition, seedling growth arrest and resistance to PtoDC3000 infection were impaired. The tested fli1-8 mutants were not allelic to FLS2 or BAK1, which suggests that yet unknown components of flg22 signaling are affected. While fli mutants were more susceptible to bacterial infection they appear more resistant to the oomycete Hyaloperonospora arabidopsis cv. Cala2. Taken together, potentially novel components involved in late PAMP responses were identified. FLS2 endocytosis is one of the flg22 responses and appears to contribute to flg22 signaling. We therefore tested several knock-out mutants in known endocytosis components for their response to flg22 and bacterial infection. While most mutants displayed wild-type-like flg22 responses, vps28-2, vps37-1, vps28-1 elch, and gnl1-1 exhibited enhanced susceptibility to PtoDC3000 infection. VPS28-2, VPS37-1, and VPS28-1 ELCH are components of the ESCRT I system responsible for sorting ubiquitinated proteins. GNL1 is an ARF GEF regulating vesicle trafficking at the Golgi and PM. To further delineate the role of endocytosis in plant immunity, a genetic screen for novel endocytosis mutants was established. Applying quantitative confocal microscopy 12 fel mutants (for FYVE-GFP endosome levels) with altered endosomal numbers in cotyledons were identified. Two selected mutants, fel4 with an increased endosome number and a few enlarged endosomes and fel5 with a reduced endosome number, were characterized in more detail. Both fel mutants displayed minor developmental defects, which did not co-segregate with the endosomal phenotype, and revealed unaltered endosomal levels in roots. In total, these approaches allowed us to isolate novel components involved in PTI and components regulating endocytosis in Arabidopsis. Map-based cloning will unravel the genetic identity of these mutants and elucidate how endocytosis contributes to immunity
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