Publications by authors named "Alon Savidor"

32 Publications

A tecpr2 knockout mouse exhibits age-dependent neuroaxonal dystrophy associated with autophagosome accumulation.

Autophagy 2020 Dec 10:1-14. Epub 2020 Dec 10.

Departments of Biomolecular Sciences, The Weizmann Institute of Science , Rehovot, Israel.

Mutations in the coding sequence of human were recently linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder involving intellectual disability, autonomic-sensory neuropathy, chronic respiratory disease and decreased pain sensitivity. Here, we report the generation of a novel CRISPR-Cas9 knockout () mouse that exhibits behavioral pathologies observed in SPG49 patients. mice develop neurodegenerative patterns in an age-dependent manner, manifested predominantly as neuroaxonal dystrophy in the gracile (GrN) and cuneate nuclei (CuN) of the medulla oblongata in the brainstem and dorsal white matter column of the spinal cord. Age-dependent correlation with accumulation of autophagosomes suggests compromised targeting to lysosome. Taken together, our findings establish the knockout mouse as a potential model for SPG49 and ascribe a new role to TECPR2 in macroautophagy/autophagy-related neurodegenerative disorders.
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http://dx.doi.org/10.1080/15548627.2020.1852724DOI Listing
December 2020

ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration.

Nat Cell Biol 2020 11 12;22(11):1346-1356. Epub 2020 Oct 12.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.
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http://dx.doi.org/10.1038/s41556-020-00588-4DOI Listing
November 2020

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

Front Microbiol 2020 10;11:189. Epub 2020 Mar 10.

Departmant of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.

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 serovar -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 and might represent a common mechanism in other Gram-negative bacteria.
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http://dx.doi.org/10.3389/fmicb.2020.00189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075815PMC
March 2020

Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress.

DNA Repair (Amst) 2019 11 24;83:102674. Epub 2019 Jul 24.

Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, 76100, Israel. Electronic address:

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.
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http://dx.doi.org/10.1016/j.dnarep.2019.102674DOI Listing
November 2019

Chlorophyll catabolism precedes changes in chloroplast structure and proteome during leaf senescence.

Plant Direct 2019 Mar 20;3(3):e00127. Epub 2019 Mar 20.

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture The Hebrew University Rehovot Israel.

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll-binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra-plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.
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http://dx.doi.org/10.1002/pld3.127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508775PMC
March 2019

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

Front Plant Sci 2019 5;10:428. Epub 2019 Apr 5.

Plant Stress and Germplasm Development Unit, USDA-ARS, Lubbock, TX, United States.

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.
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http://dx.doi.org/10.3389/fpls.2019.00428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459962PMC
April 2019

High Levels of CO Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

Appl Environ Microbiol 2019 01 13;85(1). Epub 2018 Dec 13.

Department of Postharvest and Food Sciences, Agricultural Research Organization (ARO), The Volcani Center, Rishon LeZion, Israel

During nonventilated storage of carrots, CO gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of and The lactic acid bacterium secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when was incubated under high CO levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the dextransucrase-encoding genes and during the first 4 to 8 h of exposure to high CO levels compared to aerated conditions. A phylogenetic analysis of YL48 revealed a high similarity to other genes harbored by different species. The ecological benefit of dextran production under elevated CO requires further investigation. However, this study implies an overlooked role of CO in the physiology and fitness of in stored carrots, and perhaps in other food items, during storage under nonventilated conditions. The bacterium is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of .
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http://dx.doi.org/10.1128/AEM.00473-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293096PMC
January 2019

The Disordered Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated Granules.

Proteomics 2018 11 8;18(21-22):e1800076. Epub 2018 Aug 8.

Department of Molecular Genetics, Weizmann Institute of Science Department of Molecular Genetics, 76100, Rehovot, Israel.

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.
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http://dx.doi.org/10.1002/pmic.201800076DOI Listing
November 2018

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

PLoS Pathog 2018 01 29;14(1):e1006880. Epub 2018 Jan 29.

School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel.

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-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 Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana 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 Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.
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http://dx.doi.org/10.1371/journal.ppat.1006880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805367PMC
January 2018

The genetic basis for the adaptation of E. coli to sugar synthesis from CO.

Nat Commun 2017 11 22;8(1):1705. Epub 2017 Nov 22.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel.

Understanding the evolution of a new metabolic capability in full mechanistic detail is challenging, as causative mutations may be masked by non-essential "hitchhiking" mutations accumulated during the evolutionary trajectory. We have previously used adaptive laboratory evolution of a rationally engineered ancestor to generate an Escherichia coli strain able to utilize CO fixation for sugar synthesis. Here, we reveal the genetic basis underlying this metabolic transition. Five mutations are sufficient to enable robust growth when a non-native Calvin-Benson-Bassham cycle provides all the sugar-derived metabolic building blocks. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO fixation cycle toward biomass (prs, serA, and pgi), or in key regulators of carbon metabolism (crp and ppsR). Using suppressor analysis, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the enzymatic constraints that are essential to the metabolic stability of autocatalytic cycles and are relevant to future efforts in constructing non-native carbon fixation pathways.
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http://dx.doi.org/10.1038/s41467-017-01835-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700066PMC
November 2017

BcXYG1, a Secreted Xyloglucanase from , Triggers Both Cell Death and Plant Immune Responses.

Plant Physiol 2017 Sep 14;175(1):438-456. Epub 2017 Jul 14.

Department of Molecular Biology and Ecology of Plants, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

In search of cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a strain overexpressing produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.
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http://dx.doi.org/10.1104/pp.17.00375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580746PMC
September 2017

Diadenosine tetraphosphate (Ap4A) - an E. coli alarmone or a damage metabolite?

FEBS J 2017 07 9;284(14):2194-2215. Epub 2017 Jun 9.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.

Under stress, metabolism is changing: specific up- or down-regulation of proteins and metabolites occurs as well as side effects. Distinguishing specific stress-signaling metabolites (alarmones) from side products (damage metabolites) is not trivial. One example is diadenosine tetraphosphate (Ap4A) - a side product of aminoacyl-tRNA synthetases found in all domains of life. The earliest observations suggested that Ap4A serves as an alarmone for heat stress in Escherichia coli. However, despite 50 years of research, the signaling mechanisms associated with Ap4A remain unknown. We defined a set of criteria for distinguishing alarmones from damage metabolites to systematically classify Ap4A. In a nutshell, no indications for a signaling cascade that is triggered by Ap4A were found; rather, we found that Ap4A is efficiently removed in a constitutive, nonregulated manner. Several fold perturbations in Ap4A concentrations have no effect, yet accumulation at very high levels is toxic due to disturbance of zinc homeostasis, and also because Ap4A's structural overlap with ATP can result in spurious binding and inactivation of ATP-binding proteins. Overall, Ap4A met all criteria for a damage metabolite. While we do not exclude any role in signaling, our results indicate that the damage metabolite option should be considered as the null hypothesis when examining Ap4A and other metabolites whose levels change upon stress.
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http://dx.doi.org/10.1111/febs.14113DOI Listing
July 2017

Database-independent Protein Sequencing (DiPS) Enables Full-length Protein and Antibody Sequence Determination.

Mol Cell Proteomics 2017 06 27;16(6):1151-1161. Epub 2017 Mar 27.

From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot;

Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.
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http://dx.doi.org/10.1074/mcp.O116.065417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461544PMC
June 2017

Label-free deep shotgun proteomics reveals protein dynamics during tomato fruit tissues development.

Plant J 2017 Apr 22;90(2):396-417. Epub 2017 Mar 22.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel.

Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.
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http://dx.doi.org/10.1111/tpj.13490DOI Listing
April 2017

Communication between viruses guides lysis-lysogeny decisions.

Nature 2017 01 18;541(7638):488-493. Epub 2017 Jan 18.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.
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http://dx.doi.org/10.1038/nature21049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378303PMC
January 2017

Adipose-Induced Retroperitoneal Soft Tissue Sarcoma Tumorigenesis: A Potential Crosstalk between Sarcoma and Fat Cells.

Mol Cancer Res 2016 12 12;14(12):1254-1265. Epub 2016 Sep 12.

Department of Surgery, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.

Previous data demonstrated that high retroperitoneal visceral fat content increases retroperitoneal soft-tissue sarcoma (RSTS) local recurrence and patients' mortality. Most RSTS tumors initiate and recur within visceral fat. The objective of the current study was to evaluate potential paracrine effects of visceral fat on RSTS. A xenograft model was used to evaluate in vivo effects of human visceral fat on STS growth. Tissue explants were prepared from visceral fat, and their conditioned medium (CM) was utilized for various in vitro experiments designed to evaluate growth, survival, migration, and invasion of STS and endothelial cells. Visceral fat-secreted protumorigenic factors were identified by mass spectrometry. The in vivo experiments demonstrated a significant increase in STS tumor growth rate when SK-LMS-1 leiomyosarcoma cells were colocalized with human visceral fat compared with subcutaneous injection of cancer cells only. The in vitro model demonstrated that visceral fat CM increased STS cellular growth and reduced doxorubicin-induced apoptosis. Visceral fat also enhanced STS cellular migration and invasion. In addition, visceral fat CM significantly increased endothelial cell tube formation, suggesting its role as a proangiogenic factor in the STS tumor microenvironment (TME). Using a robust proteomic approach, liquid chromatography and tandem mass spectrometry resolved various molecules within the visceral fat CM, of which a subset was associated with protumorigenic biologic processes. These results suggest that visceral fat directly interacts with STS cells by secreting specific adipokines into the TME, thus augmenting STS tumor cell proliferation and invasiveness. Fat-induced STS molecular deregulations should be studied to identify new potential prognostic and therapeutic targets.

Implications: Visceral fat induces protumorigenic effects, in STS, through various secreted factors that should be investigated to improve our understanding of adipose-cancer cell interactions. Mol Cancer Res; 14(12); 1254-65. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0131DOI Listing
December 2016

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories.

J Virol 2016 Nov 14;90(21):10039-10047. Epub 2016 Oct 14.

Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel

The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.
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http://dx.doi.org/10.1128/JVI.01319-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068508PMC
November 2016

Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling.

Cell 2015 Dec;163(6):1428-43

Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

Host-microbiome co-evolution drives homeostasis and disease susceptibility, yet regulatory principles governing the integrated intestinal host-commensal microenvironment remain obscure. While inflammasome signaling participates in these interactions, its activators and microbiome-modulating mechanisms are unknown. Here, we demonstrate that the microbiota-associated metabolites taurine, histamine, and spermine shape the host-microbiome interface by co-modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream anti-microbial peptide (AMP) profiles. Distortion of this balanced AMP landscape by inflammasome deficiency drives dysbiosis development. Upon fecal transfer, colitis-inducing microbiota hijacks this microenvironment-orchestrating machinery through metabolite-mediated inflammasome suppression, leading to distorted AMP balance favoring its preferential colonization. Restoration of the metabolite-inflammasome-AMP axis reinstates a normal microbiota and ameliorates colitis. Together, we identify microbial modulators of the NLRP6 inflammasome and highlight mechanisms by which microbiome-host interactions cooperatively drive microbial community stability through metabolite-mediated innate immune modulation. Therefore, targeted "postbiotic" metabolomic intervention may restore a normal microenvironment as treatment or prevention of dysbiosis-driven diseases.
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http://dx.doi.org/10.1016/j.cell.2015.10.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665753PMC
December 2015

Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

Environ Microbiol Rep 2015 Dec;7(6):990-1004

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.
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http://dx.doi.org/10.1111/1758-2229.12346DOI Listing
December 2015

Dysregulated miRNA biogenesis downstream of cellular stress and ALS-causing mutations: a new mechanism for ALS.

EMBO J 2015 Nov 1;34(21):2633-51. Epub 2015 Sep 1.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.
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http://dx.doi.org/10.15252/embj.201490493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641530PMC
November 2015

MS1-based label-free proteomics using a quadrupole orbitrap mass spectrometer.

J Proteome Res 2015 Apr 24;14(4):1979-86. Epub 2015 Mar 24.

†de Botton Institute for Protein Profiling and ‡Ilana and Pascal Mantoux Institute for Bioinformatics, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 76100, Israel.

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-N. 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.
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http://dx.doi.org/10.1021/pr501045tDOI Listing
April 2015

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

PLoS One 2015 16;10(3):e0120194. Epub 2015 Mar 16.

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361053PMC
January 2016

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

Mol Plant Microbe Interact 2015 Jan;2015(1):1-12

1 Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel.

The plant pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterium responsible for wilt and canker disease of tomato. While disease development is well characterized and diagnosed, molecular mechanisms of Cmm virulence are poorly understood. Here, we identified and characterized two Cmm transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of Cmm. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. While 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 Cmm and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type Cmm and the two mutants under infection-mimicking 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 Cmm virulence.
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http://dx.doi.org/10.1094/MPMI-02-14-0018-R.testissueDOI Listing
January 2015

Clavibacter michiganensis subsp. michiganensis Vatr1 and Vatr2 transcriptional regulators are required for virulence in tomato.

Mol Plant Microbe Interact 2014 Oct;27(10):1035-47

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 Δvatr2 mutant showing a more dramatic effect than Δvatr1. 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 infection-mimicking 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.
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http://dx.doi.org/10.1094/MPMI-02-14-0061-RDOI Listing
October 2014

Quantification of proteins by label-free LC-MS(E.).

Methods Mol Biol 2014 ;1156:223-36

Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, 76100, Israel.

Quantitative proteomics by LC-MS/MS is a widely used approach for quantifying a significant portion of any complex proteome. Among the different techniques used for this purpose, one is by use of Data Independent Acquisition (DIA). We present a descriptive protocol for label-free quantitation of proteins by one DIA method termed LC-MS(E), which facilitates large-scale quantification of proteins without the need for isotopic labelling and with no theoretical limit to the number of samples included in an experiment.
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http://dx.doi.org/10.1007/978-1-4939-0685-7_15DOI Listing
December 2014

Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici.

Mol Plant Microbe Interact 2012 Oct;25(10):1350-60

University of Tennessee, Knoxville, TN, USA.

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.
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http://dx.doi.org/10.1094/MPMI-02-12-0028-RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551261PMC
October 2012

The Clavibacter michiganensis subsp. michiganensis-tomato interactome reveals the perception of pathogen by the host and suggests mechanisms of infection.

J Proteome Res 2012 Feb 20;11(2):736-50. Epub 2011 Dec 20.

Department of Molecular Biology and Ecology of Plants, Tel Aviv University , Tel Aviv 69978, Israel.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes wilt and canker disease of tomato (Solanum lycopersicum). Mechanisms of Cmm pathogenicity and tomato response to Cmm infection are not well understood. To explore the interaction between Cmm and tomato, multidimensional protein identification technology (MudPIT) and tandem mass spectrometry were used to analyze in vitro and in planta generated samples. The results show that during infection Cmm senses the plant environment, transmits signals, induces, and then secretes multiple hydrolytic enzymes, including serine proteases of the Pat-1, Ppa, and Sbt familes, the CelA, XysA, and NagA glycosyl hydrolases, and other cell wall-degrading enzymes. Tomato induction of pathogenesis-related (PR) proteins, LOX1, and other defense-related proteins during infection indicates that the plant senses the invading bacterium and mounts a basal defense response, although partial with some suppressed components including class III peroxidases and a secreted serine peptidase. The tomato ethylene-synthesizing enzyme ACC-oxidase was induced during infection with the wild-type Cmm but not during infection with an endophytic Cmm strain, identifying Cmm-triggered host synthesis of ethylene as an important factor in disease symptom development. The proteomic data were also used to improve Cmm genome annotation, and thousands of Cmm gene models were confirmed or expanded.
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http://dx.doi.org/10.1021/pr200646aDOI Listing
February 2012

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Authors:
Brian J Haas Sophien Kamoun Michael C Zody Rays H Y Jiang Robert E Handsaker Liliana M Cano Manfred Grabherr Chinnappa D Kodira Sylvain Raffaele Trudy Torto-Alalibo Tolga O Bozkurt Audrey M V Ah-Fong Lucia Alvarado Vicky L Anderson Miles R Armstrong Anna Avrova Laura Baxter Jim Beynon Petra C Boevink Stephanie R Bollmann Jorunn I B Bos Vincent Bulone Guohong Cai Cahid Cakir James C Carrington Megan Chawner Lucio Conti Stefano Costanzo Richard Ewan Noah Fahlgren Michael A Fischbach Johanna Fugelstad Eleanor M Gilroy Sante Gnerre Pamela J Green Laura J Grenville-Briggs John Griffith Niklaus J Grünwald Karolyn Horn Neil R Horner Chia-Hui Hu Edgar Huitema Dong-Hoon Jeong Alexandra M E Jones Jonathan D G Jones Richard W Jones Elinor K Karlsson Sridhara G Kunjeti Kurt Lamour Zhenyu Liu Lijun Ma Daniel Maclean Marcus C Chibucos Hayes McDonald Jessica McWalters Harold J G Meijer William Morgan Paul F Morris Carol A Munro Keith O'Neill Manuel Ospina-Giraldo Andrés Pinzón Leighton Pritchard Bernard Ramsahoye Qinghu Ren Silvia Restrepo Sourav Roy Ari Sadanandom Alon Savidor Sebastian Schornack David C Schwartz Ulrike D Schumann Ben Schwessinger Lauren Seyer Ted Sharpe Cristina Silvar Jing Song David J Studholme Sean Sykes Marco Thines Peter J I van de Vondervoort Vipaporn Phuntumart Stephan Wawra Rob Weide Joe Win Carolyn Young Shiguo Zhou William Fry Blake C Meyers Pieter van West Jean Ristaino Francine Govers Paul R J Birch Stephen C Whisson Howard S Judelson Chad Nusbaum

Nature 2009 Sep 9;461(7262):393-8. Epub 2009 Sep 9.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02141, USA.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.
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http://dx.doi.org/10.1038/nature08358DOI Listing
September 2009

Cross-species global proteomics reveals conserved and unique processes in Phytophthora sojae and Phytophthora ramorum.

Mol Cell Proteomics 2008 Aug 3;7(8):1501-16. Epub 2008 Mar 3.

Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory Oak Ridge, Oak Ridge, Tennessee 37830, USA.

Phytophthora ramorum and Phytophthora sojae are destructive plant pathogens. P. sojae has a narrow host range, whereas P. ramorum has a wide host range. A global proteomics comparison of the vegetative (mycelium) and infective (germinating cyst) life stages of P. sojae and P. ramorum was conducted to identify candidate proteins involved in host range, early infection, and vegetative growth. Sixty-two candidates for early infection, 26 candidates for vegetative growth, and numerous proteins that may be involved in defining host specificity were identified. In addition, common life stage proteomic trends between the organisms were observed. In mycelia, proteins involved in transport and metabolism of amino acids, carbohydrates, and other small molecules were up-regulated. In the germinating cysts, up-regulated proteins associated with lipid transport and metabolism, cytoskeleton, and protein synthesis were observed. It appears that the germinating cyst catabolizes lipid reserves through the beta-oxidation pathway to drive the extensive protein synthesis necessary to produce the germ tube and initiate infection. Once inside the host, the pathogen switches to vegetative growth in which energy is derived from glycolysis and utilized for synthesis of amino acids and other molecules that assist survival in the plant tissue.
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http://dx.doi.org/10.1074/mcp.M700431-MCP200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2500229PMC
August 2008

Expressed peptide tags: an additional layer of data for genome annotation.

J Proteome Res 2006 Nov;5(11):3048-58

Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, USA.

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here, we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six-frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well-annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed a high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well-annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller subdatabases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While approximately 76% of Phytophthora EPTs supported the current annotation, a portion of them (7.7% and 12.9% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.
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http://dx.doi.org/10.1021/pr060134xDOI Listing
November 2006