Plant MDL proteins synergize with the cytokine MIF at CXCR2 and CXCR4 receptors in human cells.

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-CALMODULIN2 interaction by various in vitro and in vivo protein-protein interaction assays.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-calmodulin interaction by various in vitro and in vivo protein-protein interaction assays.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the fungal powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the powdery mildew disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calcium-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2 CT ), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2 CT LW/RR mutant variants in comparison to the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

Gene Gun-Mediated Transient Gene Expression for Functional Studies in Plant Immunity.

One major threat to plant cultivation are fungal pathogens, which can cause substantial yield losses in agriculture. As an example, cereal powdery mildew fungi such as the barley (Hordeum vulgare) pathogen, Blumeria graminis f. sp. hordei (Bgh), are among the ten most relevant fungal plant pathogens in molecular plant pathology and can lead to yield losses of up to 30%. Plant Mildew resistance Locus O (MLO) genes are required for successful colonization of plants by powdery mildew fungi. Accordingly, loss-of-function mlo mutants confer durable resistance against powdery mildew fungi in many plant species. In the case of barley, mlo-based resistance has been used for more than 40 years in agriculture without powdery mildew fungi effectively overcoming this kind of immunity. However, the molecular basis of mlo resistance and function(s) of the transmembrane Mlo protein(s) are still incompletely understood. The generation of transgenic barley plants to study the plant immune response and the involvement of Mlo therein is time-consuming and challenging. Therefore, transient gene expression via gene gun-mediated particle bombardment became a popular, easy, and efficient tool to investigate different aspects of plant defense responses in barley. Since Bgh fails to penetrate leaf epidermal cells of mlo mutants, single-cell complementation upon biolistic transformation resulting in (over-)expression of Mlo can be used to characterize the Mlo protein functionally in vivo. In this chapter, we describe in detail the gene gun-mediated transient expression of Mlo in barley leaf epidermal cells followed by powdery mildew inoculation and the subsequent microscopic evaluation. However, gene gun-mediated transient gene expression may be also used to address other research questions or to transform the epidermal tissues of other plant organs and/or species.

An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome.

BACKGROUND: Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses. RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species. CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.

Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Chemokine-like MDL proteins modulate flowering time and innate immunity in plants.

Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein-protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsismdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.

BACKGROUND: Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites. RESULTS: We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. CONCLUSION: Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

ABC transporter PEN3/PDR8/ABCG36 interacts with calmodulin that, like PEN3, is required for Arabidopsis nonhost resistance.

Nonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane-localized ATP-binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of pathogen-associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post-translational modification. Here, we investigated the PEN3 protein interaction network. We probed the Arabidopsis protein microarray AtPMA-5000 with the N-terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca(2+) sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca(2+) sensing to PEN3-mediated NHR. We demonstrated co-localization of PEN3 and CaM7, and we confirmed PEN3-CaM interaction in vitro and in vivo by PEN3 pull-down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants. Our study discloses CaM7 as a PEN3-interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca(2+) sensing to plant immunity.