A proteomic investigation of \u3cem\u3ePhytophthora\u3c/em\u3e species using mass spectrometry and reverse genetics

Organisms in the genus Phytophthora are important plant pathogens, although understudied. Phytophthora was first brought into human awareness with the identification of P. infestans as the culprit for the Irish potato famine in the mid 1800s. Since then, over 80 Phytophthora species have been identified, many of which infect a wide variety of crops worldwide with devastating results. Traditionally, much of the work aimed at controlling Phytophthora diseases involved applied research. In recent years there has been a marked increase in molecular work on Phytophthora. This increase is evident not only from increased funding by agencies such as the National Science Foundation (NSF), but also from the type of research applied to Phytophthora for the first time. The first Phytophthora species to have their genomes sequenced were P. sojae and P. ramorum at 2004. Since then the genomes of two more Phytophthora species- P. capsici and P. infestans, were also sequenced. Availability of Phytophthora genome sequences provided us with the basis necessary for a proteomic investigation of these organisms. The study presented here represents the first large scale proteomic study of any Phytophthora species. Using mass spectrometry and available or newly developed bioinformatic tools we measured the proteomes of different asexual Phytophthora life stages. We also measured the protein complement of P. capsici infected tomato plants, the so called “interactome”, in order to gain an insight into the biological processes occurring in the pathogen during infection, and in the plant in response to the pathogen. We also used data from these proteomic experiments as a part of a novel approach aimed at improving the genome annotation of those Phytophthora species. Finally, we used different molecular techniques, including a reverse genetic technique called Targeted Induced Local Lesions in Genome (TILLING), to begin characterization of a few protein targets identified in those experiments. The accumulated data from all our experiments identified certain molecular processes, metabolic and others, that may explain the success of Phytophthora as a plant pathogen. The data from these experiments provides a platform on which future experiments can be based on to further characterize these interesting organisms

The Chloroplast Envelope Protease FTSH11 – Interaction With CPN60 and Identification of Potential Substrates

FTSH proteases are membrane-bound, ATP-dependent metalloproteases found in bacteria, mitochondria and chloroplasts. The product of one of the 12 genes encoding FTSH proteases in Arabidopsis, FTSH11, has been previously shown to be essential for acquired thermotolerance. However, the substrates of this protease, as well as the mechanism linking it to thermotolerance are largely unknown. To get insight into these, the FTSH11 knockout mutant was complemented with proteolytically active or inactive variants of this protease, tagged with HA-tag, under the control of the native promoter. Using these plants in thermotolerance assay demonstrated that the proteolytic activity, and not only the ATPase one, is essential for conferring thermotolerance. Immunoblot analyses of leaf extracts, isolated organelles and sub-fractionated chloroplast membranes localized FTSH11 mostly to chloroplast envelopes. Affinity purification followed by mass spectrometry analysis revealed interaction between FTSH11 and different components of the CPN60 chaperonin. In affinity enrichment assays, CPN60s as well as a number of envelope, stroma and thylakoid proteins were found associated with proteolytically inactive FTSH11. Comparative proteomic analysis of WT and knockout plants, grown at 20°C or exposed to 30°C for 6 h, revealed a plethora of upregulated chloroplast proteins in the knockout, some of them might be candidate substrates. Among these stood out TIC40, which was stabilized in the knockout line after recovery from heat stress, and three proteins that were found trapped in the affinity enrichment assay: the nucleotide antiporter PAPST2, the fatty acid binding protein FAP1 and the chaperone HSP70. The consistent behavior of these four proteins in different assays suggest that they are potential FTSH11 substrates

Loss of the Periplasmic Chaperone Skp and Mutations in the Efflux Pump AcrAB-TolC Play a Role in Acquired Resistance to Antimicrobial Peptides in Salmonella typhimurium

Bacterial resistance to antibiotics is a major concern worldwide, leading to an extensive search for alternative drugs. Promising candidates are antimicrobial peptides (AMPs), innate immunity molecules, shown to be highly efficient against multidrug resistant bacteria. Therefore, it is essential to study bacterial resistance mechanisms against them. For that purpose, we used experimental evolution, and isolated a Salmonella enterica serovar typhimurium-resistant line to the AMP 4DK5L7. This AMP displayed promising features including widespread activity against Gram-negative bacteria and protection from proteolytic degradation. However, the resistance that evolved in the isolated strain was particularly high. Whole genome sequencing revealed that five spontaneous mutations had evolved. Of these, three are novel in the context of acquired AMP resistance. Two mutations are related to the AcrAB-TolC multidrug efflux pump. One occurred in AcrB, the substrate-binding domain of the system, and the second in RamR, a transcriptional regulator of the system. Together, the mutations increased the minimal inhibitory concentration (MIC) by twofold toward this AMP. Moreover, the mutation in AcrB induced hypersusceptibility toward ampicillin and colistin. The last mutation occurred in Skp, a periplasmic chaperone that participates in the biogenesis of outer membrane proteins (OMPs). This mutation increased the MIC by twofold to 4DK5L7 and by fourfold to another AMP, seg5D. Proteomic analysis revealed that the mutation abolished Skp expression, reduced OMP abundance, and increased DegP levels. DegP, a protease that was reported to have an additional chaperone activity, escorts OMPs through the periplasm along with Skp, but is also associated with AMP resistance. In conclusion, our data demonstrate that both loss of Skp and manipulation of the AcrAB-TolC system are alternative strategies of AMP acquired resistance in Salmonella typhimurium and might represent a common mechanism in other Gram-negative bacteria.Peer Reviewe

Identification of a biomarker in cerebrospinal fluid for neuronopathic forms of Gaucher disease.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available

MS1-Based Label-Free Proteomics Using a Quadrupole Orbitrap Mass Spectrometer

Presented is a data set for benchmarking MS1-based label-free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1-intensity-based label-free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data were also used to compare different quantitative protein inference methods such as iBAQ and Hi-<i>N</i>. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385

Clavibacter michiganensis subsp michiganensis Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato

Savidor A, Chalupowicz L, Teper D, et al. Clavibacter michiganensis subsp michiganensis Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato. Molecular Plant-Microbe Interactions. 2014;27(10):1035-1047.The plant pathogen Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterium responsible for wilt and canker disease of tomato. Although disease development is well characterized and diagnosed, molecular mechanisms of C. michiganensis subsp. michiganensis virulence are poorly understood. Here, we identified and characterized two C. michiganensis subsp. michiganensis transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of C. michiganensis subsp. michiganensis. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Delta vatr2 mutant showing a more dramatic effect than Delta vatr1. Although both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type C. michiganensis subsp. michiganensis and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type C. michiganensis subsp. michiganensis and the two mutants under infectionmimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal-transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for C. michiganensis subsp. michiganensis virulence

The <i>Xanthomonas euvesicatoria</i> type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling

<div><p>The Gram-negative bacterium <i>Xanthomonas euvesicatoria</i> (<i>Xe</i>) is the causal agent of bacterial spot disease of pepper and tomato. <i>Xe</i> delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the <i>Xe</i> effector XopAU, which is conserved in numerous <i>Xanthomonas</i> species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. <i>Agrobacterium</i>-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by <i>Xanthomonas campestris</i> pv. <i>campestris</i>, but not by <i>Xe</i>. Protein-protein interaction studies in yeast and <i>in planta</i> revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 <i>in vitro</i> and enhanced its phosphorylation at multiple sites <i>in planta</i>. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2_K99R in <i>N</i>. <i>benthamiana</i> plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to <i>Xe</i> disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.</p></div

Expression of MKK2_K99R suppresses cell death mediated by XopAU.

<p><i>N</i>. <i>benthamiana</i> leaves were inoculated with <i>Agrobacterium</i> to co-express His-XopAU or an empty vector (EV) with MKK2-HA, MKK2_K99R-HA or GFP. Expression was driven by an estradiol-inducible system and 17β-estradiol was applied 24 h after agro-infiltration. (A) Photograph taken at 48 h after 17β-estradiol application. (B) Electrolyte leakage at 24 h and 48 h after 17β-estradiol application. A box plot displays 25^th, 50^th (middle line) and 75^th percentiles. (<i>n</i> = 5). Asterisks indicate a significant difference (Mann-Whitney U test, <i>p</i> value <0.05) relative to co-expression of His-XopAU with a GFP control. The experiment was repeated three times with similar results.</p

Elevation of GPNMB levels in CSF and brain of nGD patients.

<p>(A) Levels of GPNMB determined by LC-MS/MS in CSF of four type 3 GD patients. Results are means ± SEM. ** <i>p</i>< 0.01. (B) Levels of GPNMB in CSF of four type 3 GD patients determined by ELISA. Results are means ± SEM (n = 4). ** <i>p</i><0.01. (C) Western blot of GPNMB in CSF of control and a type 3 GD patient (sample designation 4). Results are from a typical experiment repeated 3 times. (D) Levels of GPNMB in nGD brain determined by ELISA(n = 3 for control, n = 6 for nGD (type 2 and type 3 patients). Results are means ± SEM, ** <i>p</i>< 0.01</p