Flg22 Enters Vascular Tissue Through Endocytosis and FLS2-Mediated Entry

Flg22 is a 22-amino acid peptide that shares characteristics of flagellins from pathogen-associated molecular patterns (PAMs), and activates unique innate immune responses in N. benthamiana.

During plant development and defense, diverse signaling peptides enter cells through receptor-mediated entry, which may contribute to transport of these peptides to neighboring cells or distal tissues, as in the case of flg22. In the present study, we showed that flg22 is transported to vascular tissue via endocytosis and FLS2-mediated entry, and that this movement is specific.

FLS2-mediated entry into a plant cell was required for long-distance transport of flg22 to vascular tissue in Col plants. Adding inhibitors of FLS2 internalization, such as MG132 and 2,3-butanedione monoxime (BDM), simultaneously with TAMRA-flg22 prevented initial peptide binding/uptake in Col leaves, but did not affect flg22 accumulation.

In addition, flg22 did not accumulate in Col vacuoles after co-expression of fls2 and bak1 with TAMRA-flg22 (Fig. 6A, B). Similarly, FLS2-mediated internalization of flg22 was not inhibited in Col leaves treated with MG132 and BDM or fls2 and bak1 without the inhibitors (Fig. 6C, D).

To assess whether flg22 could enter vascular tissue via endocytosis, we infiltrated a leaf disc from Col with fluorescently labeled flg22 and a dye, prior to floating it on a fluorophore-labeled flg22/dye solution for a period of time. After infiltration, confocal microscopy images with differential interference contrast (DIC) and red and green fluorescence were obtained.

FAM-flg22, a fluorescence-sensitive molecule that is detectable in plasma membrane/endosome vesicles, was colocalized with FM4-64 in vascular vesicles of infiltrated Col leaf after 1 h of incubation (Fig. 5A). Two hours after infiltration, FAM-flg22 was detectable in vascular cells of the infiltrated area, but was rarely detected outside.


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