A linear motif in the N terminus of flagellin triggers a signaling cascade that induces defense activation. The ectodomain of TLR5 binds to flagellin, resulting in the activation of MAPKs, which initiate a series of events that culminate in transcriptional activation of genes providing resistance against pathogens. Plants also possess a receptor similar to TLR5, called FLS2 (FLAGELLIN SENSING RECEPTOR 2), which binds to the N terminus of flagellin and activates MAPK signaling and gene expression. FLS2 and its homologs are required for host resistance against fungi, bacteria and viruses.
To test whether FLS2 is necessary for long-distance transport of flg22, we applied a mixture of 10 mM TAMRA-flg22 and 10 mM FAM-scrambled peptide to a filter paper disc placed on the adaxial side of leaves of WT Col plants and examined movement by fluorescence microscopy after 1 h. Efficient transport of flg22 was observed in vascular tissues, but not in non-vascular cells or leaf blades. In contrast, FAM-scrambled flg22 was not mobile. The results confirm that FLS2 is necessary for accumulation and transport of flg22 in plant tissue.
To further characterize the role of FLS2 in flg22 transport, we infiltrated Col leaves with a solution containing 5 mM TAMRA-flg22 or 50 mM MG132 and 50 mM BDM and measured the movement of flg22 by fluorescence microscopy after 2 h. The majority of flg22 was present in the cytoplasm, but not the stroma, of the leaves infiltrated with MG132 and BDM. In contrast, a small fraction of the flg22 infiltrated with TAMRA-flg22 colocalized with the endocytic marker FM4-64 in MVBs and prevacuolar compartments in BDM-treated plants.