Identification of Protein Biomarkers for Differentiating Listeria monocytogenes Genetic Lineage III.

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne illness characterized by septicemia, meningitis, encephalitis, abortions, and occasional death in infants and immunocompromised individuals. L. monocytogenes is composed of four genetic lineages (I, II, III, and IV) and fourteen serotypes. The aim of the current study was to identify proteins that can serve as biomarkers for detection of genetic lineage III strains based on simple antibody-based methods. Liquid chromatography (LC) with electrospray ionization tandem mass spectrometry (ESI MS/MS) followed by bioinformatics and computational analysis were performed on three L. monocytogenes strains (NRRL B-33007, NRRL B-33014, and NRRL B-33077), which were used as reference strains for lineages I, II, and III, respectively. Results from ESI MS/MS revealed 42 unique proteins present in NRRL B-33077 and absent in NRRL B-33007 and NRRL B-33014 strains. BLAST analysis of the 42 proteins against a broader panel of >80 sequenced strains from lineages I and II revealed four proteins [TM2 domain-containing protein (NRRL B-33077_2770), DUF3916 domain-containing protein (NRRL B-33077_1897), DNA adenine methylase (NRRL B-33077_1926), and protein RhsA (NRRL B-33077_1129)] that have no homology with any sequenced strains in lineages I and II. The four genes that encode these proteins were expressed in Escherichia coli strain DE3 and purified. Polyclonal antibodies were prepared against purified recombinant proteins. ELISA using the polyclonal antibodies against 12 L. monocytogenes lineage I, II, and III isolates indicated that TM2 protein and DNA adenine methylase (Dam) detected all lineage III strains with no reaction to lineage I and II strains. In conclusion, two proteins including TM2 protein and Dam are potentially useful biomarkers for detection and differentiation of L. monocytogenes lineage III strains in clinical, environmental, and food processing facilities. Furthermore, these results validate the approach of using a combination of proteomics and bioinformatics to identify useful protein biomarkers.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells.

BACKGROUND: Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically. OBJECTIVES: We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells. METHODS: A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0-200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA. RESULTS: Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 µM, 4 hours; P < 0.05). CONCLUSIONS: These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.

An apocrine mechanism delivers a fully immunocompetent exocrine secretion.

Apocrine secretion is a recently discovered widespread non-canonical and non-vesicular secretory mechanism whose regulation and purpose is only partly defined. Here, we demonstrate that apocrine secretion in the prepupal salivary glands (SGs) of Drosophila provides the sole source of immune-competent and defense-response proteins to the exuvial fluid that lies between the metamorphosing pupae and its pupal case. Genetic ablation of its delivery from the prepupal SGs to the exuvial fluid decreases the survival of pupae to microbial challenges, and the isolated apocrine secretion has strong antimicrobial effects in "agar-plate" tests. Thus, apocrine secretion provides an essential first line of defense against exogenously born infection and represents a highly specialized cellular mechanism for delivering components of innate immunity at the interface between an organism and its external environment.

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley.

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

Silver-coated magnetic nanocomposites induce growth inhibition and protein changes in foodborne bacteria.

Cytotoxicity concerns of nanoparticles on animal or human bodies have led to the design of iron oxide core nanocomposites, coated with elemental silver to allow their magnetic removal from bio-mixtures. Although the antimicrobial effect of silver is well-described, the effects of nanoparticles derived from silver on microorganisms remain unfolded. Here, we characterized a customized magnetic silver nanocomposite (Ag-MNP) and evaluated its effects on bacterial growth and protein changes. The Ag-MNP displayed both longitudinal and round shapes under High-Resolution Transmission Electron Microscopy imaging, while the Energy Dispersive X-ray Spectroscopy and X-ray diffraction analysis confirmed the presence of Ag, Fe3O4 (Magnetite) and FeO2 (Goethite). Optical density, bioluminescence imaging, and Colony Forming Unit assessments revealed that the presence of Ag-MNP induced strong dose-dependent bacteria (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and S. Anatum) growth inhibition. The TEM imaging showed penetration and infiltration of bacteria by Ag-MNP, leading to membrane degeneration and vacuole formation. The presence of Ag-MNP led to fifteen up-regulated and nine down-regulated proteins (P < 0.05) that are involved in cell membrane synthesis, inhibition of protein synthesis, interference with DNA synthesis, and energy metabolism inhibition. This study provides insights to develop alternative antimicrobials to treat foodborne pathogens with antibiotic resistance avoidance.

Proteomic Analysis of Arabidopsis pldα1 Mutants Revealed an Important Role of Phospholipase D Alpha 1 in Chloroplast Biogenesis.

Phospholipase D alpha 1 (PLDα1) is a phospholipid hydrolyzing enzyme playing multiple regulatory roles in stress responses of plants. Its signaling activity is mediated by phosphatidic acid (PA) production, capacity to bind, and modulate G-protein complexes or by interaction with other proteins. This work presents a quantitative proteomic analysis of two T-DNA insertion pldα1 mutants of Arabidopsis thaliana. Remarkably, PLDα1 knockouts caused differential regulation of many proteins forming protein complexes, while PLDα1 might be required for their stability. Almost one third of differentially abundant proteins (DAPs) in pldα1 mutants are implicated in metabolism and RNA binding. Latter functional class comprises proteins involved in translation, RNA editing, processing, stability, and decay. Many of these proteins, including those regulating chloroplast protein import and protein folding, share common functions in chloroplast biogenesis and leaf variegation. Consistently, pldα1 mutants showed altered level of TIC40 (a major regulator of protein import into chloroplast), differential accumulation of photosynthetic protein complexes and changed chloroplast sizes as revealed by immunoblotting, blue-native electrophoresis, and microscopic analyses, respectively. Our proteomic analysis also revealed that genetic depletion of PLDα1 also affected proteins involved in cell wall architecture, redox homeostasis, and abscisic acid signaling. Taking together, PLDα1 appears as a protein integrating cytosolic and plastidic protein translations, plastid protein degradation, and protein import into chloroplast in order to regulate chloroplast biogenesis in Arabidopsis.

Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting the Involvement of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

Antioxidant and angiotensin-I converting enzyme inhibitory activities of phenolic extracts and fractions derived from three phenolic-rich legume varieties.

Lentil, black soybean and black turtle bean are commonly consumed legumes of different genera, containing high phenolic contents, which are effective antioxidants and angiotensin-I converting enzyme (ACE) inhibitors. However, these legumes' phenolic compositions and ACE inhibition ability have not been compared. Crude water extract (CE) was semi-purified (SPE) and fractionated using column chromatography. Results showed that purification and fractionation could substantially increase phenolic contents and antioxidant capacities. Heating and variety had great effect on phenolic substances, antioxidant potential and mass yield of extracts and fractions. Only crude extracts showed potent ACE inhibitory activity. Black turtle bean's ACE inhibition potential was largely reduced by cooking. The order from low to high in terms of ACE inhibitory activity was black turtle bean < lentil < black soybean. Identification and quantification of individual phenolic compounds by UV spectroscopy and LC-MSn analysis confirmed 18, 22, and 14 compounds, respectively, for the three legumes.

Protein Composition of the Bovine Herpesvirus 1.1 Virion.

Bovine herpesvirus (BoHV) type 1 is an important agricultural pathogen that infects cattle and other ruminants worldwide. Acute infection of the oro-respiratory tract leads to immune suppression and allows commensal bacteria to infect an otherwise healthy lower respiratory tract. This condition is known as the Bovine Respiratory Disease (BRD). BoHV-1 latently infects the host for life and periodical stress events re-initiate BRD, translating into high morbidity and large economic losses. To gain a better understanding of the biology of BoHV-1 and the disease it causes, we elucidated the protein composition of extracellular virions using liquid chromatography-mass spectrometry analysis. We detected 33 viral proteins, including the expected proteins of the nucleocapsid and envelope as well as other regulatory proteins present in the viral tegument. In addition to viral proteins, we have also identified packaged proteins of host origin. This constitutes the first proteomic characterization of the BoHV virion.

Feedback Microtubule Control and Microtubule-Actin Cross-talk in Arabidopsis Revealed by Integrative Proteomic and Cell Biology Analysis of KATANIN 1 Mutants.

Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing.

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress.

UNLABELLED: Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. SIGNIFICANCE: Peanuts (Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.

Comparative proteomic study of Arabidopsis mutants mpk4 and mpk6.

Arabidopsis MPK4 and MPK6 are implicated in different signalling pathways responding to diverse external stimuli. This was recently correlated with transcriptomic profiles of Arabidopsis mpk4 and mpk6 mutants, and thus it should be reflected also on the level of constitutive proteomes. Therefore, we performed a shot gun comparative proteomic analysis of Arabidopsis mpk4 and mpk6 mutant roots. We have used bioinformatic tools and propose several new proteins as putative MPK4 and MPK6 phosphorylation targets. Among these proteins in the mpk6 mutant were important modulators of development such as CDC48A and phospholipase D alpha 1. In the case of the mpk4 mutant transcriptional reprogramming might be mediated by phosphorylation and change in the abundance of mRNA decapping complex VCS. Further comparison of mpk4 and mpk6 root differential proteomes showed differences in the composition and regulation of defense related proteins. The mpk4 mutant showed altered abundances of antioxidant proteins. The examination of catalase activity in response to oxidative stress revealed that this enzyme might be preferentially regulated by MPK4. Finally, we proposed developmentally important proteins as either directly or indirectly regulated by MPK4 and MPK6. These proteins contribute to known phenotypic defects in the mpk4 and mpk6 mutants.

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective.

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus.

Proteomic and biochemical analyses show a functional network of proteins involved in antioxidant defense of the Arabidopsis anp2anp3 double mutant.

Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

Integrative chemical proteomics and cell biology methods to study endocytosis and vesicular trafficking in Arabidopsis.

We present a comprehensive approach combining proteomics and cell biology to study vesicular trafficking in plants. Within this approach, we exploit chemical compounds inhibiting particular vesicular trafficking events in plant cells. Treatment of plants with these relatively specific inhibitors results in intracellular accumulation of proteins being transported by vesicles as well as in a change in abundance of regulatory proteins. Such pharmacological inhibition allows for identification of key proteins, and for further detailed functional investigation using cell biological, molecular biological, and biochemical methods used for validation of proteomic results.