Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Horticultural Science
Non Technical Summary
Regulated protein secretion is a critical cellular response to pathogen attack in both plants and animals. Secretion of newly synthesized defense proteins is largely via the well-studied ER/Golgi pathway. However, in animal systems, unconventional, non-Golgi secretion of stress and pathogen response proteins has also been well documented. More recently, several labs, including our own, have accumulated sufficient data to suggest that non-Golgi secretion also occurs in plants. Analyses of pathogen-induced protein secretion in plants have identified a number of secreted proteins that lack a signal peptide for export by the conventional ER/Golgi pathway. These include normally cytoplasmic proteins that also have essential roles in extracellular defenses (e.g. mannitol dehydrogenase, MTD, and Cu/Zn superoxide dismutase, SOD), as well as proteins whose secretion via the Golgi would disrupt normal cellular processes (e.g. jacalin lectins, JLs). Because of their key roles in plant defenses, understanding the mechanisms behind leaderless protein secretion of these proteins could provide valuable tools for improving associated defenses. Thus, we propose to exploit the complementary approaches of microscopy and mass spectrometry (MS) to focus analyses of secretion kinetics and compartmentation and to elucidate details of the secretion of these leaderless proteins in response to salicylic acid (SA), an endogenous inducer of pathogen defense responses.We hypothesize that secretion of these normally cytosolic plant proteins, like secretion of leaderless proteins in animals, requires modification of the secreted protein itself or of interacting secretory pathway components. This, in turn, triggers interaction with secretory pathway components. Electron and confocal microscopy will be used to characterize changes in subcellular localization and associated cellular structures following SA treatment. We will then use protein crosslinking, immunopurification and a variety of MS approaches to identify specific proteins interacting with our target protein(s) at intervals immediately after exposure of cells to SA. Proteins in crosslinked complexes will further be assessed for the presence of SA-induced post-translational modifications (PTMs). The unique capabilities of an NSF MRI funded Orbitrap Velos Elite (ETD, HCD and high-res MSn) provide enhanced detection of interacting partners and related PTMs. Interactions will be verified using bimolecular fluorescence complementation (BiFC) microscopy.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Because the cell wall is a major interface between plant cells and the environment, rapid, regulated secretion of proteins into this extracellular space (the apoplast) is an important defense response (Grant and Lamb, 2006). Secretion of defense proteins in both plants and animals was long thought to be solely via an endoplasmic reticulum (ER)/Golgi-mediated pathway, with an N-terminal signal peptide directing the protein to the ER for routing, modification and subsequent secretion via the Golgi. However, since Auron et al.'s (1987) report that interleukin 1 (IL1b), a cytokine with no signal peptide, is secreted from human monocytes in response to infection, "leaderless" secretion of a number of important stress response proteins has been confirmed (Nickel and Rabouille, 2009). Although non-Golgi secretion has been verified in many eukaryotes, our report that the normally cytoplasmic enzyme, mannitol dehydrogenase (MTD) was secreted by tobacco in response to the endogenous plant defense signal salicylic acid (SA) was the first report of leaderless secretion in plants (Cheng et al 2009a).Regulated conversion of mannitol to mannose by MTD in the cytosol of plants such as celery (Apium graveolens) allows mannitol to be used as both a metabolite and an osmoprotectant (Stoop et al., 1996). There is now compelling evidence that mannitol and MTD also play roles in plant-pathogen interactions. Mannitol's proposed antioxidant activity (Shen et al., 1997) together with its secretion by fungal pathogens (Jennings et al., 1998; Velez et al., 2008) suggested that pathogens use mannitol as a defense against plant-produced reactive oxygen (RO) (Patel and Williamson, 2016). In response, pathogen-induced plant MTD was hypothesized to catabolize this mannitol, thus protecting the host's RO-mediated defenses (e.g. Jennings et al 2002, Williamson et al 2013). However, while fungal mannitol is secreted into the extracellular space (e.g. Joosten et al., 1990), MTD is cytoplasmic in uninfected celery (Zamski et al., 1996). If the plant MTD catabolizes fungal mannitol, then MTD and mannitol must be co-localized. In Cheng et al (2009a) we showed that MTD is, in fact, secreted in response to SA. This is important because MTD has no recognized ER/Golgi signal sequence, and its SA-induced secretion is not inhibited by brefeldin A (BFA), an inhibitor of Golgi-mediated protein transport. Golgi-mediated secretion of the pathogen response protein PR1a is, in contrast, inhibited by BFA. Thus, contrary to the model that PR-protein secretion occurs only via the ER/Golgi, MTD secretion appears to be by a non-Golgi process.The existence of leaderless secretion in animals is well accepted, and several general mechanisms have been described: (1) Direct translocation across the plasma membrane: fibroblast growth factor 1 (FGF-1) and IL1α, for instance, are translocated across the plasma membrane (PM) via either the chaperonin-like action of hydrophobic "release complexes" or through transmembrane channels (Jackson et al., 1992; Watanabe and Kobayasgi, 1994, respectively). (2) Endolysosomal pathways: normally cytoplasmic proteins such as IL-1β (Andre et al., 1999) are transported into intracellular vesicles (endolysosomes) via channels such as ATP-binding cassette (ABC) transporters. These vesicles then fuse with the cell membrane to release included proteins into the apoplast. (3) Exosome-mediated secretion: intracellular vesicles called endosomes are first generated by inward budding of the PM. Cytosolic proteins, such as HSP90 are then packaged into the endosomes by further inward budding to form multivesicular bodies. Next, these fuse with the PM to release the included, formerly cytosolic components (Yu et al., 2006). (4) Membrane blebbing: cytosolic secretory transglutaminase and galectins (Aumüller et al., 1999) are proposed to be packaged directly into membrane-derived blebs, or protrusions. The blebs detach and the included proteins released when the blebs rupture.Non-Golgi protein secretion is necessary in a number of cases. For instance, the presence of some secreted proteins in the ER/Golgi would disrupt ER functioning. For example, in animals, non-Golgi secretion of galectin1 keeps this galactose-binding lectin from binding glycolipids and glycoproteins in the lumen of the Golgi/ER (Hughes, 1999). Non-Golgi secretion is also needed if a protein has multiple functions, with each occurring in a different cellular compartment. HMGB1 (High mobility group box 1), for example, is normally nuclear, where it mediates DNA-binding complex assembly. Upon bacterial induction of monocytes, HMGB1 is secreted into the extracellular space where it acts as an endotoxin mediator or a cell differentiation signal (Müller et al., 2001). As previously discussed, MTD has normal roles in metabolism and osmoregulation in the cytoplasm. During infection, however, MTD has a very different role in the extracellular space. As MTD's role in central metabolism requires cytoplasmic localization, a nonclassical mechanism is needed to mediate its secretion in response to pathogen attack. In addition to MTD, there is growing confirmation that non-Golgi mechanisms are involved in the secretion of many plant proteins. For example, recent mass spectrophotometric (MS) analyses of SA-induced protein secretion not only confirms the previously reported presence of SOD in the secretome (Slabas et al., 2004; Oh et al., 2005, Cheng et al., 2009b), but suggests that many proteins whose secretion occurs soon after SA treatment lack a classical signal peptide, including plant lectins such as the jacalin lectins (Cheng et al 2009b).Our Ultimate Goal is to decode the mechanism of secretion for previously identified, non-Golgi secreted proteins. To date we have validated procedures described below, and used them to identify proteins 10 and 20 min post-treatment that not only interact with MTD during secretion, but also display significant quantitative and qualitative changes in response to SA (Ho, 2015). Many of these have been previously associated with other interacting protein complexes reported to be involved in trafficking via vesicular mechanisms. Finally, since many non-classically secreted proteins appear to be first modified in response to a stress initiated signal, enabling them to interact with relevant secretion machinery (Denny et al., 2000), analysis of posttranslational modifications potentially triggering secretion needs to be assessed.Objectives. Although a number of stress and pathogen-induced non-Golgi secretion mechanisms have been detailed in animals, none have been well characterized in plants. Because a number of key plant responses appear to involve such Golgi-independent, leaderless protein secretion (Ding et al 2014), understanding the mechanisms involved is critical if our goal is to improve pathogen resistance in plants. Thus we propose to elucidate early events in the secretion of several leaderless proteins previously identified as follows: Objective 1. Use immunoelectron, confocal microscopy and endomembrane markers to assess changes in localization of MTD, JL and Cu/Zn SOD in response to the endogenous SAR induction signal SA, and to identify the cellular structures associated with these proteins. Obj. 2. Use crosslinking, immunopurification and LC/MS/MS analysis to isolate and identify SA-triggered -protein interactors (i.e. potential components of the secretory mechanism) for JL and SOD as described for MTD above. Obj. 3. Use previously proven, epitope-tagged MTD constructs expressed in Arabidopsis as well as assessment of the native MTD in celery and JL and SOD in Arabidopsis to characterize potential secretion-associated protein modifications (in both secreted proteins and interactors). Obj. 4. Confirm interactions identified in Objective 3 using Bimolecular Fluorescence Complementation (BiFC) and reverse IP interaction analyses.
Project Methods
Objective 1. Assess changes in MTD, JL and SOD localization using immunoelectron and confocal microscopy. These two complementary approaches will be used to assess changes in the subcellular localization of MTD, JL and SOD in response to treatment with SA. We previously used electron microscopy (EM) and anti-MTD antibodies to examine changes in MTD localization in response to SA (Cheng et al, 2009b). These analyses, however, were performed on samples harvested and fixed several hours after treatment. By this time, MTD was essentially all secreted. Later proteomic analysis (Cheng et al., 2009a) showed that JL secretion peaked by 1 hour after SA treatment while SOD secretion peaked 2 h post-treatment. Further characterization (Blackburn et al, 2010; Ho, 2015) showed that MTD secretion also occurs rapidly, with unique MTD-secretory complexes appearing 10-15 min post-treatment. Thus, we will assess both JL and MTD localization at 10 min intervals during the first hour after treatment, while SOD will be assessed at 10 min intervals starting at 45 mins. Since MTD is a native celery protein, +/-SA-treated celery cell suspension cultures initially will be used for MTD localization. Sample preparation and electron microscopy will be performed as described (Cheng et al., 2009b). Alternatively, we will use MTD fused to a FLAG affinity tag as described in Ho (2015). As FLAG peptides are designed so an anti-FLAG monoclonal antibody has high specificity, it is well suited for validating subcellular location. Extensive use of FLAG-tagged 35S-MTD constructs in the U.S. labs has shown FLAG does not disrupt either normal SA-mediated secretion of MTD or MTD enzyme activity (Wang et al. 2008, Cheng et al., 2009b, Ho 2015).Fluorescent protein fusion to target proteins followed by confocal microscopic analysis will be used as a complementary approach to EM by the Galili lab to assess intracellular localization of MTD, JL and SOD (Michaeli et al., 2014). Studies show that fusion of fluorescent proteins does not hinder protein secretion (Bozkurt et al., 2011). For instance, we will generate transgenic Arabidopsis plants expressing MTD, fused in-frame to the mRFP1 protein under control of a 35S promoter. This fusion will be used because unlike GFP, mRFP1 is stable and fluoresces in the relatively acidic pH of the plant apoplast (Samalova et al., 2006). mRFP1 will be fused to either the N- or C-terminal of MTD, and both fusions checked for MTD activity (Stoop et al., 1995) as well as secretion using confocal microscopy and immunoblot analysis of soluble apoplastic components (Vanacker et al., 1998). To avoid potential problems resulting from excessive expression, several lines will be screened, and - plants expressing appropriate levels will be used for characterization. Sub-cellular localization of the fusion proteins will be compared between +/-SA treated plants at the indicated time points, and any specific subcellular labeling will be validated by comparison with known organelle markers. Although combining electron and confocal microscopy maximizes the chances of success, we can also compare EM or confocal results with classical cell fractionation analyses.Objective 2. Identify protein interactions associated with leaderless secretion. Because protein-protein interactions are likely initiated by pathogen attack, we will compare protein complexes isolated from cells treated with + and - SA a described by Cheng et al. (2009b) and harvested at the timepoints indicated in Objective 1. Since protein complexes can destabilize during extraction, interacting complexes will be stabilized by reversible formaldehyde cross-linking and cytosolic and microsomal subcellular fractions prepared as described (Piersma et al, 2013). Crosslinked complexes will be affinity copurified (CoIPed) using anti-FLAG, or anti-JL or anti-SOD bead-linked polyclonals (Covance, RTP, NC). Unbound protein is removed by washing, and bead-linked antibody-bound complexes (formaldehyde cross-linked, interacting proteins) are eluted with room temperature SDS sample buffer, then heated (or not) prior to separation by SDS-PAGE. The presence of interacting proteins in unheated samples results in the appearance of a complex that is shifted to a higher molecular mass region of the gel; heating, in turn, reverses crosslinking and returns the "bait" protein (MTD, SOD or JL) to its noncomplexed molecular weight. Bands containing our bait protein are identified by parallel immunoblot analysis. The gel region containing mobility shifted proteins in unheated samples (i.e. proteins interacting with the bait in vivo) are excised, subjected to in-gel trypsinization, analyzed by LC/MS/MS, and the resulting data searched against a protein database derived from the complete A. thaliana genome to identify component proteins (Ho, 2015). Finally in order to minimize false positives, "nonspecific" interactions identified by parallel analysis of untreated, uninduced and wildtype controls are subtracted from the list of identified +/-SA interactors. Using these LC/MS/MS approaches we have successfully identified a number of potential cytosolic MTD interactors (Ho, 2015). These analyses will be expanded to include additional timepoints and cellular fractions to provide a more comprehensive assessment of the complex formation in the cytosol, and since secreted proteins likely interact with membrane and cytosolic proteins, both cellular fractions will be assessed.Objective 3. Characterize SA-mediated, post-translational modifications of secreted proteins and their interactors. Since stress initiated posttranslational modification of many non-classically secreted proteins apparently enables them to interact with relevant secretion machinery (Denny et al., 2000), analysis of SA-triggered posttranslational modifications (PTMs) will be assessed. Since secreted proteins could also interact with membrane-bound and cytosolic proteins, both cytosolic and membrane fractions will be assessed. Given the abundance of MTD in celery, celery cells will initially be used for assessing MTD PTMs while Arabidopsis will be used for analysis of JL and SOD PTMs. Finally, our ability to isolate MTD-interacting protein complexes (Objective 2) provides targeted enrichment of both potentially modified bait proteins, and their interactors. This potentially provides a viable means of assessing modifications in this diverse and initially unknown interactor population and provides us the ability to assess the hypothesis that modification of interactors might be a trigger for secretion.Cells will be treated as described above (Cheng et al., 2009a,b) and a comprehensive MS-based approach used to discover the extent and nature of SA-induced PTMs. Proteins will be trypsin digested and the resulting peptides analyzed by LC/MS/MS. Additional proteases (Lys-C, Asp-N) will be used to generate overlapping peptide maps, enabling sequence coverage of regions not accessible with trypsin. In addition to direct analysis, peptides containing specific PTMs of interest can be enriched using affinity approaches such as immobilized metal ion affinity chromatography (IMAC) to improve the detection of low stoichiometry modifications (e.g. Herring et al., 2015).Objective 4. Verify and characterize interacting proteins. Manual inspection as used above, combined with reverse immunoblotting using antibodies to putative interactors, as well as bioinformatic analysis (e.g. STRING v10 protein interaction analysis software, string-db.org)(Szklarczyk et al., 2015) will be used to screen putative interactors. We would next assess and verify selected interactors using; a) bimolecular fluorescence complementation (BiFC) (Michaeli et al 2014) to confirm protein interactions identified by co-immunoprecipitation, or if appropriate b) reverse IP using interactors identified by immunoaffinity MS in Objective 2 as the bait.