The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor flagellin sensing2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)-sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b- and ARA6/Rab F1-positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.

(1) and found widespread application in plant virology either as tags for specific viral proteins during infection or as general reporters of cell infection (2, 3). While the study of plant viruses has benefited greatly from the use of GFP, it is known that FP-expressing viruses exhibit reduced infection efficiency that can negatively affect host range relative to the wild-type virus (4-6). The same is true for animal viruses, especially when FPs are attached to structural components of tightly packed virions (7). Moreover, the increased genetic load of plant viruses carrying a FP severely limits both local and systemic spread compared with that of the wild-type virus (6). Attempts have been made to overcome this problem in the extensively studied positive-strand RNA virus, Tobacco mosaic virus (TMV), through molecular evolution of the viral movement protein (MP), a protein critical for cell-tocell movement of the viral genome (6). An alternative strategy would be to utilize a smaller FP to reduce the genetic load. However, generation of smaller derivatives from GFP-based FPs is unlikely because the ␤-barrel structure of the protein is intrinsic to its function (8).Fluorescent peptide ligands represent promising smaller alternatives to GFP-based FPs (9). Yet, the necessity for an exogenous chemical substrate introduces limitations for this approach, particularly in plants where the cell wall poses an additional barrier to permeability (10), prompting us to search for other genetically encoded candidates. The utility of linear tetrapyrrole (bilin)-binding proteins as fluorescent probes in the near infrared region of the spectrum has been recognized (11), as has the potential for flavin-based fluorescent proteins as in vivo reporters (12). The latter are derived from photosensory modules known as light, oxygen or voltage sensing (LOV) domains present in a diverse range of photoreceptors from bacteria, fungi, and plants (13,14). UV/blue light is detected via the chromophore flavin mononucleotide (FMN) located within the LOV domain, giving the protein a weak intrinsic fluorescence with a maximal emission wavelength at 495 nm (15). Although used successfully to monitor bacterial cell populations (12, 16), the suitability of LOV-based FPs for studying protein localization and trafficking has not been investigated.In the present study, we examined whether LOV-based FPs could be used as fluorescent reporters of virus infection in plant cells because their relatively small size offers an advantage over GFP. Through the molecular evolution of plant-derived LOV domains, we have isolated a photoreversible FP that can be used effectively to track protein distribution within living cells. This FP, termed iLOV, outperformed GFP as a reporter of plant virus infection and movement, and conferred improved functionality over GFP when fused to proteins required for virus spread. iLOV, therefore, represents a new genetically encoded alternative to GFP-based FPs that exhibits greater utility for monitoring virus infection. Results a...

The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants.

Chitin acts as a pathogen-associated molecular pattern from fungal pathogens whose perception triggers a range of defense responses. We show that LYSIN MOTIF DOMAIN-CONTAINING GLY-COSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2), the Arabidopsis homolog of a rice chitin receptor-like protein, mediates a reduction in molecular flux via plasmodesmata in the presence of chitin. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not to the flagellin derivative flg22. Surprisingly, the chitin-recognition receptor CHITIN ELCITOR RE-CEPTOR KINASE 1 (CERK1) is not required for chitin-induced changes to plasmodesmata flux, suggesting that there are at least two chitin-activated response pathways in Arabidopsis and that LYM2 is not required for CERK1-mediated chitin-triggered defense responses, indicating that these pathways are independent. In accordance with a role in the regulation of intercellular flux, LYM2 is resident at the plasma membrane and is enriched at plasmodesmata. Chitin-triggered regulation of molecular flux between cells is required for defense responses against the fungal pathogen Botrytis cinerea, and thus we conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis.PAMP-triggered immunity | cell-to-cell communication