The Genomics of Cannabis and Its Close Relatives
Kovalchuk, I. ; Pellino, M. ; Rigault, P. ; Velzen, R. Van; Ebersbach, J. ; Ashnest, J.R. ; Mau, M. ; Schranz, M.E. ; Alcorn, J. ; Laprairie, R.B. ; McKay, J.K. ; Burbridge, C. ; Schneider, D. ; Vergara, D. ; Kane, N.C. ; Sharbel, T.F. - \ 2020
Annual Review of Plant Biology 71 (2020). - ISSN 1543-5008 - p. 713 - 739.
biosynthesis pathway evolution - Cannabaceae - Cannabis sativa L. - genomics - hemp - hops - Humulus lupulus - proteomics - Y chromosome
Cannabis sativa L. is an important yet controversial plant with a long history of recreational, medicinal, industrial, and agricultural use, and together with its sister genus Humulus, it represents a group of plants with a myriad of academic, agricultural, pharmaceutical, industrial, and social interests. We have performed a meta-analysis of pooled published genomics data, andwe present a comprehensive literature review on the evolutionary history of Cannabis and Humulus, including medicinal and industrial applications. We demonstrate that current Cannabis genome assemblies are incomplete, with sim10% missing, 10-25% unmapped, and 45S and 5S ribosomal DNA clusters as well as centromeres/satellite sequences not represented. These assemblies are also ordered at a low resolution, and their consensus quality clouds the accurate annotation of complete, partial, and pseudogenized gene copies. Considering the importance of genomics in the development of any crop, this analysis underlines the need for a coordinated effort to quantify the genetic and biochemical diversity of this species.
The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection
Vossenberg, Bart van de; Prodhomme, Charlotte ; Arkel, G. van; Gent-Pelzer, M.P.E. van; Bergervoet-van Deelen, J.E.M. ; Brankovics, Balázs ; Przetakiewicz, J. ; Visser, R.G.F. ; Lee, T.A.J. van der; Vossen, J.H. - \ 2019
Molecular Plant-Microbe Interactions 32 (2019)11. - ISSN 0894-0282 - p. 1536 - 1546.
avirulence factors - cell death - chytridiomycota - effector - fungus-plant interactions - Genomics - hypersensitive response - metabolomics - plant-pathogen interactions - population genetics - proteomics - resistance genes
Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host-resistance in a set of differential potato varieties. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum specific secretome of fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single copy gene and encodes a 376 amino acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.
Xylem Sap Proteomics Reveals Distinct Differences Between R Gene- and Endophyte-Mediated Resistance Against Fusarium Wilt Disease in Tomato
Lamo, Francisco J. de; Constantin, Maria E. ; Fresno, David H. ; Boeren, Sjef ; Rep, Martijn ; Takken, Frank L.W. - \ 2018
Frontiers in Microbiology 9 (2018). - ISSN 1664-302X
biocontrol - endophyte - exosomes - Fusarium wilt disease - NP24 - PR-5x - proteomics
Resistance (R) genes and endophytic organisms can both protect plants against pathogens. Although the outcome of both processes is the same, little is known about the commonalities and differences between both immune responses. Here we set out to phenotypically characterize both responses in the tomato-Fusarium pathosystem, and to identify markers to distinguish these responses at the molecular level. As endophyte Fusarium oxysporum (Fo) strain Fo47 was employed, which confers protection against various pathogens, including the vascular wilt fungus F. oxysporum f.sp. lycopersici (Fol). As R-gene conferring Fol resistance, the I-2 gene of tomato (Solanum lycopersicum) was used. Fol colonizes the xylem vessels of susceptible and I-2 resistant tomato plants, but only causes disease in the former. Fol was found to colonize the vasculature of endophyte-colonized plants, and could be isolated from stems of non-diseased plants co-inoculated with Fo47 and Fol. Because the xylem vessels form the main interface between plant and pathogen, the xylem sap proteomes during R gene- and Endophyte-Mediated Resistance (RMR and EMR) were compared using label-free quantitative nLC-MS/MS. Surprisingly, both proteomes were remarkably similar to the mock, revealing only one or two differentially accumulated proteins in the respective resistant interactions. Whereas in I-2 plants the accumulation of the pathogenesis-related protein PR-5x was strongly induced by Fol, the endophyte triggered induction of both NP24, another PR-5 isoform, and of a β-glucanase in the presence of Fol. Notably, over 54% of the identified xylem sap proteins have a predicted intracellular localization, which implies that these might be present in exosomes. In conclusion, whereas both resistance mechanisms permit the pathogen to colonize the vasculature, this does not result in disease and this resistance coincides with specific induction of two distinct PR-5 isoforms and a β-glucanase.
Use of proteomics to detect sex-related differences in effects of toxicants : implications for using proteomics in toxicology
Rietjens, Ivonne M.C.M. ; Vervoort, Jacques ; Maslowska-Górnicz, Anna ; Brink, Nico Van den; Beekmann, Karsten - \ 2018
Critical Reviews in Toxicology 48 (2018)8. - ISSN 1040-8444 - p. 666 - 681.
mode of action - proteomics - Sex related differences - state-of-the-art - toxicants
This review provides an overview of results obtained when using proteome analysis for detecting sex-based differences in response to toxicants. It reveals implications to be taken into account when considering the use of proteomics in toxicological studies. It appears that results may differ when studying the same chemical in the same species in different target tissues. Another result of interest is the limited dose-response behavior of differential abundance patterns observed in studies where more than one dose level is tested. It is concluded that use of proteomics to study differences in modes of action of toxic compounds is an active area of research. The examples from use of proteomics to study sex-dependent differences also reveal that further studies are needed to provide reliable insight in modes of action, novel biomarkers or even novel therapies. To eventually reach this aim for this and other toxicological endpoints, it is essential to consider background variability, consequences of timing of toxicant administration, dose-response behavior, relevant species and target organ, species and organ variability and the presence of proteoforms.
Purified Dietary Red and White Meat Proteins Show Beneficial Effects on Growth and Metabolism of Young Rats Compared to Casein and Soy Protein
Song, Shangxin ; Hua, Chun ; Zhao, Fan ; Li, Mengjie ; Fu, Qingquan ; Hooiveld, Guido J.E.J. ; Muller, Michael ; Li, Chunbao ; Zhou, Guanghong - \ 2018
Journal of Agricultural and Food Chemistry 66 (2018)38. - ISSN 0021-8561 - p. 9942 - 9951.
molecular nutrition - protein quality - proteomics - red meat - white meat
This study compared the effects of casein, soy protein (SP), red (RMP), and white meat (WMP) proteins on growth and metabolism of young rats. Compared to casein, the ratio of daily feed intake to daily body weight gain of rats was not changed by meat protein but reduced by SP by 93.3% (P < 0.05). Feeding RMP and WMP reduced the liver total cholesterol (TC) contents by 24.3% and 17.8%, respectively (P < 0.05). Only RMP increased plasma HDL-cholesterol concentrations (by 12.7%, P < 0.05), whereas SP increased plasma triacylglycerol, TC, and LDL-cholesterol concentrations by 23.7%, 19.5%, and 61.5%, respectively (P < 0.05). Plasma essential and total amino acid concentrations were increased by WMP (by 18.8% and 12.4%, P < 0.05) but reduced by SP (by 28.3% and 37.7%, P < 0.05). Twenty-five liver proteins were differentially expressed in response to different protein sources. Therefore, meat proteins were beneficial for growth and metabolism of young rats compared to casein and SP.
Genetical genomics of quality related traits in potato tubers using proteomics
Acharjee, A. ; Chibon, P.Y.F.R.P. ; Kloosterman, B.A. ; America, A.H.P. ; Renaut, J. ; Maliepaard, C.A. ; Visser, R.G.F. - \ 2018
Wageningen University & Research
genetical genomics - proteomics - protein QTL - potato quality traits
Background Recent advances in ~omics technologies such as transcriptomics, metabolomics and proteomics along with genotypic profiling have permitted the genetic dissection of complex traits such as quality traits in non-model species. To get more insight into the genetic factors underlying variation in quality traits related to carbohydrate and starch metabolism and cold sweetening, we determined the protein content and composition in potato tubers using 2Dâ€“gel electrophoresis in a diploid potato mapping population. Upon analyzing we made sure that the proteins from the patatin family were excluded to ensure a better representation of the other proteins. Results We subsequently performed pQTL analyses for all other proteins with a sufficient representation in the population and established a relationship between proteins and 26 potato tuber quality traits (e.g. flesh colour, enzymatic discoloration) by co-localization on the genetic map and a direct correlation study of protein abundances and phenotypic traits. Over 1643 unique protein spots were detected in total over the two harvests. We were able to map pQTLs for over 300 different protein spots some of which co-localized with traits such as starch content and cold sweetening. pQTLs were observed on every chromosome although not evenly distributed over the chromosomes. The largest number of pQTLs was found for chromosome 8 and the lowest for chromosome number 10. For some 20 protein spots multiple QTLs were observed. Conclusions From this analysis, hotspot areas for protein QTLs were identified on chromosomes three, five, eight and nine. The hotspot on chromosome 3 coincided with a QTL previously identified for total protein content and had more than 23 pQTLs in the region from 70 to 80Â cM. Some of the co-localizing protein spots associated with some of the most interesting tuber quality traits were identified, albeit far less than we had anticipated at the onset of the experiments.
Single-molecule protein sequencing through fingerprinting: computational assessment
Yao, Y. ; Docter, M.W. ; Ginkel, H.G.T.M. van; Ridder, D. de; Joo, C. - \ 2018
Delft University of Technology
computational biophysics - proteomics - single-molecule biophysics
Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.
FeedOmics, an approach to evaluate the functional properties of protein containing feed ingredients
Kar, Soumya K. - \ 2017
Wageningen University. Promotor(en): M.A. Smits; J.M. Wells, co-promotor(en): A.J.M. Jansman; D. Schokker. - Wageningen : Wageningen University - ISBN 9789463434461 - 254
compound feeds - ingredients - protein sources - proteins - functional properties - metabolism - feed formulation - protein digestion - proteomics - digestive tract - nutrition physiology - animal nutrition - livestock feeding - mengvoer - ingrediënten - eiwitbronnen - eiwitten - functionele eigenschappen - metabolisme - voersamenstelling - eiwitvertering - eiwitexpressieanalyse - spijsverteringskanaal - voedingsfysiologie - diervoeding - veevoeding
This thesis presents FeedOmics approach as a toolkit, to evaluate (novel) protein containing feed ingredients of different origin considering both their nutritional and functional value in terms of their capacity to support or modify nutrient supply, the animal’s physiology, tissue development and functioning. Such knowledge may contribute to introduce novel and/or alternative protein containing feed ingredients in the diet of livestock, thus creating a sustainable food supply for growing human population.
Enrichment proteomics challenges and perspectives : analysis of the N-glycoproteome and plasma membrane proteome in glycosylation mutants and plant-pathogen interactions
Song, Wei - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol; Twan America. - Wageningen : Wageningen University - ISBN 9789462578722 - 172
proteomics - glycoproteins - arabidopsis - plant-animal interactions - plant pathogens - plasma membranes - eiwitexpressieanalyse - glycoproteïnen - arabidopsis - plant-dier interacties - plantenziekteverwekkers - plasmamembranen
This thesis is based on two technology projects from the Centre for BioSystems Genomics (CBSG), entitled ‘Comparative proteomics on Plant Pathogen interactions through enrichment of the N-glycoproteome and tagged-glycoproteome’ (TD7) and ‘Plasma Membrane proteomics for Plant Pathogen interactions’ (TD5). In the former project we developed the protocol for isolation, identification and quantification of N-linked glycoproteins from plants and used it to obtain a comprehensive inventory of glycan-occupancy of Arabidopsis glycoproteins. In the second project, a protocol for the enrichment of plasma membrane (PM) fraction from plant material was developed and applied to study the role of the PM proteome in the interaction of plants with the plant pathogen Phytophthora infestans. Combined these activities have resulted in a thesis devoted to technical developments in label-free comparative enrichment proteomics, with validation in a number of different biological systems.
Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - biomarkers - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie
The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.
In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.
Proteomics as a tool to gain more insight into sub-lethal toxicological effects
Miller, Ingrid - \ 2016
Wageningen University. Promotor(en): Tinka Murk, co-promotor(en): A.C. Gutleb; T. Serchi. - Wageningen : Wageningen University - ISBN 9789462578210 - 182
proteomics - laboratory methods - sublethal effects - toxic substances - endocrine disruptors - food consumption - toxicology - animal experiments - eiwitexpressieanalyse - laboratoriummethoden - subletale effecten - toxische stoffen - hormoonverstoorders - voedselconsumptie - toxicologie - dierproeven
This thesis focuses on a modern analytical method, proteomics, to investigate its use in the field of toxicological research. Proteomics is a high resolution method which separates all proteins present in a sample at a clearly defined state and compares this pattern to another one, under slightly different conditions (e.g. after exposure to a chemical). Protein changes may give rise to or reflect disease/harm of the individual and can be attributed to alterations in body functions/regulation systems. Analysis conditions and different varieties of proteomic methods are explained, and a brief introduction given where proteomics is already applied in toxicology. A specific investigation has been performed with the flame retardant HBCD (i.e. hexabromocyclododecane). It is a compound that accumulates in lipid tissue from where it is only slowly removed. Its mechanism of action is not yet completely understood and sometimes seems to be contradictory. Rats were exposed to HBCD in very low doses for just one week and liver proteins were compared to those of unexposed animals. As HBCD is suggested to disturb the thyroid system, both healthy and hypothyroid rats were investigated, of both genders. In female rats, not in males, some specific liver protein changes were seen in glucose/carbohydrate and lipid metabolism, and also in some stress related proteins. Changes were not dependent on the thyroid function of the females. These results are in line with previous findings that female rats were more susceptible to HBCD than males. In a further step, protein patterns of unexposed animals of both genders were compared, revealing gender-dependent differences that exceeded the effects seen in any of the other comparisons, mainly in the pathways that were also affected by HBCD in females. A previous proteomic study on serum proteins has also shown clear gender-dependent concentration differences in rats. This underlines the importance of performing studies both in female and male individuals. The detection of considerable gender-dependent protein alterations confirms that proteomics is a biochemical tool with high sensitivity and large potential also in toxicological research.
Authentication of Closely Related Fish and Derived Fish Products Using Tandem Mass Spectrometry and Spectral Library Matching
Nessen, Merel A. ; Zwaan, Dennis J. van der; Grevers, Sander ; Dalebout, Hans ; Staats, Martijn ; Kok, Esther ; Palmblad, Magnus - \ 2016
Journal of Agricultural and Food Chemistry 64 (2016)18. - ISSN 0021-8561 - p. 3669 - 3677.
food authentication - mass spectrometry - proteomics - species identification - spectral libraries
Proteomics methodology has seen increased application in food authentication, including tandem mass spectrometry of targeted species-specific peptides in raw, processed, or mixed food products. We have previously described an alternative principle that uses untargeted data acquisition and spectral library matching, essentially spectral counting, to compare and identify samples without the need for genomic sequence information in food species populations. Here, we present an interlaboratory comparison demonstrating how a method based on this principle performs in a realistic context. We also increasingly challenge the method by using data from different types of mass spectrometers, by trying to distinguish closely related and commercially important flatfish, and by analyzing heavily contaminated samples. The method was found to be robust in different laboratories, and 94-97% of the analyzed samples were correctly identified, including all processed and contaminated samples.
Carboxypeptidase Z : an extracellular protein in zebrafish development
Kessels, M.Y. - \ 2015
Wageningen University. Promotor(en): Sacco de Vries; Johan van Leeuwen, co-promotor(en): Stefan Schulte-Merker; Sander Kranenbarg. - Wageningen : Wageningen University - ISBN 9789462575677 - 134
danio rerio - biologische ontwikkeling - carboxypeptidasen - skelet - eiwitten - eiwitexpressieanalyse - genexpressie - danio rerio - biological development - carboxypeptidases - skeleton - proteins - proteomics - gene expression
Historically the skeleton, in particular bone, was depicted as a rigid, inflexible, lifeless structure that readily breaks upon bending. We now know that bones in living organisms are complex, dynamic organs that combine toughness with flexibility. Astonishing is the fact that after their initial formation, bone structures are continuously remodeled (recycled and renewed), adapting to environmental demands at such a speed that in active healthy humans the distal part of the femur is completely replaced every six to twelve months. The zebrafish is a relatively new model organism in the field of skeletal development and primarily used as a powerful model for the identification of novel gene functions during skeletogenesis. At the molecular level, the biological similarity between zebrafish and humans is striking (Spoorendonk et al., 2010), but the actual skeletal composition of the zebrafish remained largely unknown.
The mechanical properties of the skeleton are largely dependent on the composition of proteins that are secreted into the extracellular matrix (ECM). In order to increase our understanding of normal skeletal development, it is necessary to identify and characterize changes in skeletal composition. The aim of this thesis was to analyze the composition of the extracellular matrix in zebrafish, in order to enable the identification of potential key regulatory proteins. By determination of the protein content in the zebrafish skeletal ECM with major changes in protein abundance during development we were able to identify various components of signaling pathways implicated in skeletogenesis. This first proteomic analysis of the zebrafish skeleton revealed the homology between the zebrafish and the skeleton of other vertebrate species including mammals. Our study provides a solid foundation for future studies on the composition and the regulation of the morphogenesis of the vertebrate skeleton.
After the identification of potential regulatory proteins in the developing zebrafish skeleton during the MS-based approach, the protein carboxypeptidase Z (Cpz) was selected for further analysis. This peptidase has previously been implicated in the Wnt signaling pathway, an elaborate pathway that regulates crucial aspects of development (Nusse and Varmus, 2012). Previous studies implicated a regulatory role for this peptidase through the processing of Wnt (Moeller et al., 2003; Wang et al., 2009). An analysis of the role of this peptidase in zebrafish was still lacking and therefore we mapped the spatio-temporal expression of cpz. We showed that expression of cpz is localized in and around juvenile zebrafish ossified structures. A more thorough analysis showed a complex expression pattern during early developmental stages that partially overlaps with that observed in other species. Furthermore, we provide a comparative view of cpz expression and the expression of its proposed ligand, Wnt4 (zebrafish Wnt4a and Wnt4b). Partial overlap with wnt4 expression provided the first evidence for a potential complementary function in the Wnt signaling pathway as observed in mammalian species.
In order to explore the role of cpz in zebrafish development, a loss-of-function mutant for zebrafish cpz was generated via TALEN-mediated mutagenesis. We show that mutant embryos display a variety of phenotypes during early development, most of which were similar to described defects in the Wnt/Calcium signaling pathway. These morphological phenotypes provide the first evidence for a connection between Cpz and regulation of the β-catenin independent Wnt signaling pathways which places the function of Cpz in a completely new perspective.
As a complement to these genetic studies an alternative approach was employed to examine the role of another pathway, the thyroid hormone system, in skeletogenesis. Thyroid hormones are required for skeletal development (Kim and Mohan, 2013), but to what extent thyroid hormone affects early bone development in zebrafish remained unclear. By exposing zebrafish embryos to the thyroid hormone triiodothyronine (T3) we showed that exposure accelerates ossification of craniofacial elements including the opercle and ceratohyal in a dose-dependent manner. This provides the first histological evidence of increased ossification in zebrafish due to thyroid hormone exposure which can be used as a starting point to explore the mechanism of thyroid hormone on skeletal development at a greater depth.
The zebrafish skeleton displays remarkable resemblance to that of other vertebrate species with regards to composition, regulatory components and hormonal response. The use of zebrafish as a model in future research will undoubtedly increase our understanding of vertebrate skeletal development and disease. In this thesis we provide a first insight in the extracellular protein content of the zebrafish skeleton, identify a role for the Cpz protein in β-catenin independent (non-canonical) Wnt signaling during development and shows the effect of thyroid hormone on ossification during early zebrafish development.
Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - passalora fulva - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - solanum lycopersicum - tomatoes - proteomics
Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.
Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.
In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.
In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.
In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.
In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.
Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.
Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.
One-carbon metabolism in acetogenic and sulfate-reducing bacteria
Visser, M. - \ 2015
Wageningen University. Promotor(en): Fons Stams. - Wageningen : Wageningen University - ISBN 9789462571730 - 210
anaërobe microbiologie - metabolisme - koolmonoxide - methanol - alcohol dehydrogenase - sulfaat reducerende bacteriën - genetische analyse - eiwitexpressieanalyse - anaerobic microbiology - metabolism - carbon monoxide - methanol - alcohol dehydrogenase - sulfate reducing bacteria - genetic analysis - proteomics
One-carbon metabolism in acetogenic and sulfate-reducing bacteria
Life on earth is sustained by the constant cycling of six essential elements: oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon. The continuous cycling of these elements is due to geo-chemical processes and the combined metabolism of all life on earth. Microorganisms like bacteria and archaea play a major role in this. This is also true for the carbon cycle. In this cycle carbon dioxide and methane are two important C-1 compounds present in the atmosphere. Carbon dioxide is the highest oxidative state of carbon while methane is the highest reduced form of carbon. The art to use light to produce organic compounds and conserve energy from the highest oxidative state of carbon is called photosynthesis and is performed by plants, algae and cyanobacteria. Photosynthesis is not the only system to fix carbon from carbon dioxide. Chemolithotrophs can fix carbon from carbon dioxide using inorganic electron donors, like hydrogen. Subsequently, fixed carbon can be used by other organisms, which also makes life possible for them. Microorganisms play a major role in the degradation of complex organic matter, producing smaller compounds including C-1 compounds. C-1 compounds other than carbon dioxide are e.g. carbon monoxide (CO), methanol and formate. Bacteria and archaea can utilize these relative simple compounds in the presence and absence of oxygen, alone and in cooperation with others (syntrophy). The complex and simple carbon compounds are finally oxidized to carbon dioxide, which closes the carbon cycle.
In addition to their importance to the carbon cycle, one carbon compounds like CO, methanol and formate are important for several applications. They are used as a building block for the production of chemicals. They are also used for bioremediation purposes and for wastewater treatment. Therefore, it is important to gain insight in the one carbon metabolism of microorganisms. The research described in this thesis focuses on the proteins and encoding genes involved in anaerobic degradation of C1 compounds by using genome and proteome analysis.
In Chapter 2 the genomes of two closely related sulfate-reducing bacteria, Desulfotomaculum nigrificans and D. nigrificans strain CO-1-SRB, are compared including their CO metabolism. Both the D. nigrificans type strain and strain CO-1-SRB can grow with CO. However, there are differences. The type strain can grow with 20% CO coupled to sulfate reduction in the presence of yeast extract, while strain CO-1-SRB can grow with 100% CO in the presence of yeast extract. Moreover, strain CO-1-SRB can grow with CO in the presence and absence of sulfate. It couples the oxidation of CO to carbon dioxide to hydrogen production. This conversion, the protein complex involved, and the genes coding for these proteins have been described before in other microorganisms. The genome of strain CO-1-SRB contains the genes coding for this protein complex while the genome of the D. nigrificans type strain does not. However, the genome of the type strain contains genes encoding two other CO dehydrogenases. This indicates that one or both are necessary for the type strain to grow with 20% CO. Additional research on the different CO dehydrogenases and their regulation is essential to assess if all different CO dehydrogenases can facilitate growth and how they are linked to for example creating a proton motive force for ATP production.
The methanol metabolism of anaerobic bacteria seems to differ more from that of methanogens than initially described. Methanogens use a methanol methyltransferase system that consists of two methyltranferases, methyltransferase 1 (subunits MtaB and MtaC) and methyltransferase 2 (MtaA). The methyl group from methanol is transferred to the MtaC subunit by MtaB. Subsequently, MtaA transports the methyl group from MtaC to coenzyme M. A genome and proteome analysis of the acetogenic bacterium Sporomusa strain An4 suggests that instead of MtaA a methyl-tetrahydrofolate methyltransferase is involved in the transport of the methyl bound to MtaC to tetrahydrofolate (Chapter 3).
Research done on the methanol metabolism of the sulfate-reducing bacterium Desulfotomaculum kuznetsovii also shows differences with that of methanogens (Chapter 5). The methanol methyltransferase system is vitamin B12 and cobalt dependent. D. kuznetsovii grows with methanol and sulfate, but can do this in presence and absence of vitamin B12 and cobalt. In the absence of vitamin B12 and cobalt D. kuznetsovii grows slower and reaches a lower optical density compared to growth in the presence of vitamin B12 and cobalt. This suggests that D. kuznetsovii can use both a methyltransferase system and a vitamin B12 and cobalt independent system for the degradation of methanol. Proteome results confirm this and suggest that the vitamin B12 and cobalt independent system consists of an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase. Moreover, the alcohol dehydrogenase seems to be involved in the oxidation of both methanol and ethanol (Chapter 5). The presence of two methanol degradation pathways give an ecological advantage to D. kuznetsovii in environments containing methanol and sulfate but limiting cobalt and vitamin B12 concentrations. Future research should elucidate if more sulfate-reducing bacteria, or perhaps even acetogenic bacteria, have two methanol degrading pathways.
Additional to the genome analysis of D. kuznetsovii to assess the genes coding for the proteins involved in the two methanol degradation pathways, the genome was also analyzed to assess genes encoding other degradation pathways (Chapter 4). This analysis shows many genes present in D. kuznetsovii are also present in Pelotomaculum thermopropionicum. P. thermopropionicum is known to degrade propionate in syntrophic interaction with a methanogen. D. kuznetsovii can also degrade propionate, but only coupled to sulfate reduction and not in syntrophy with methanogens. Moreover, P. thermopropionicum is not able to reduce sulfate. D. kuznetsovii is the only close related, non-syntrophic, propionate degrader of which the genome is available. Therefore, a genome comparison was performed between D. kuznetsovii and P. thermopropionicum to define the differences between a non-syntrophic and a syntrophic lifestyle. D. kuznetsovii misses membrane bound protein complexes like hydrogenases and an extra-cytoplasmic formate dehydrogenase. In order to expand the analysis between non-syntrophs and syntrophs, more genomes of propionate- and butyrate-degrading bacteria were included (Chapter 6). This extended analysis shows that the genomes of non-syntrophs do not contain genes coding for an extra-cytoplasmic formate dehydrogenase, in contrast to all syntrophs included in the analysis. This indicates the importance of this protein complex and the importance of formate as an interspecies electron carrier in syntrophic degradation of propionate and butyrate. Thanks to the extra cytoplasmic formate dehydrogenase the syntrophic bacteria can couple the degradation of propionate and butyrate to formate production. Subsequently, the formate is utilized by methanogens to produce methane. This keeps the formate concentration low, which is necessary for the entire process to be energetically favorable.
On the involvement of host proteins in Cowpea mosaic virus intercellular spread
Hollander, P.W. den - \ 2014
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Jan van Lent. - Wageningen : Wageningen University - ISBN 9789462570986 - 146
plantenvirussen - koebonenmozaïekvirus - plasmodesmata - planteiwitten - pathogenesis-gerelateerde eiwitten - viruseiwitten - virale transporteiwitten - chaperoninen - uitschakelen van genexpressie - genexpressie - eiwittransport - eiwitexpressieanalyse - plant viruses - cowpea mosaic virus - plasmodesmata - plant proteins - pathogenesis-related proteins - viral proteins - movement proteins - chaperonins - gene silencing - gene expression - protein transport - proteomics
Abstract of thesis Paulus den Hollander entitled “On the involvement of host proteins in Cowpea mosaic virus intercellular spread”.
Defence: 18th of November 13.30 h
Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement tubules inserted into the cell wall spanning plasmodesmata (PD) channels. These tubules are composed solely of viral movement proteins (MPs) and carry virus particles to neighbouring cells. The insertion of movement tubules into PD requires severe structural modification of these channels. These modifications are thought to rely on the concerted action of host and viral proteins. The aim of this thesis research has been to identify those host factors involved in the intercellular transport of CPMV.
Movement tubules collected from protoplasts, isolated plants cells without cell walls, were analysed by tandem mass spectrometry. This analysis revealed that seven host proteins associated specifically with these movement structures. Among these tubule-associated host proteins were CDC48-type AAA-proteins, heat shock protein (HSP) 60 and HSP70 isoforms. These three proteins were selected for further analysis in order to test their potential role in CPMV infection and spread. Targeted gene silencing of either CDC48 or HSP70 proteins caused a reduction in viral titres and also reduced the spread of CPMV in infected leaves. These results show that altered expression of these two tubule-associated host proteins influences the viral infection cycle.
Furthermore, interactions between the MP of CPMV and both plasma membrane intrinsic proteins (PIPs) and plasmodesmata-located proteins (PDLPs) were investigated using fluorescently labelled fusion proteins. An interaction between the MP and PIP-isoforms could not be established, however, MPs were found to co-localise with PDLP1 in PD. Using fluorescence lifetime imaging (FRET-FLIM) the molecular interaction between PDLPs and viral MPs was established in PD. This interaction was, however, only found in plant tissues and was not observed in protoplasts. This suggests that the interaction is restricted to structural context of the PD. Further experiments in protoplasts showed that co-localisation of MPs with PDLP1 was not required for the formation of movement tubules in these cells, which supports the hypothesis that the role of PDLP in plant virus intercellular transport is restricted to the PD.
Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family
Resjö, S. ; Ali, A. ; Meijer, H.J.G. ; Seidl, M.F. ; Snel, B. ; Sandin, M. ; Levander, F. ; Govers, F. ; Andreasson, E. - \ 2014
Journal of Proteome Research 13 (2014)4. - ISSN 1535-3893 - p. 1848 - 1859.
tandem mass-spectrometry - protein-kinases - arabidopsis-thaliana - in-vitro - proteomics - identification - expression - potato - organization - specificity
The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host–oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).
Vroege detectie van dracht bij koeien door Proteomics Biomerkers in melk = Early pregnancy detection using proteomics biomarkers in milk
Pas, M.F.W. te; Kruijt, L. ; Wit, A.A.C. de; Hulsegge, B. ; Riel, J.W. van; Heeres-van der Tol, J.J. ; Sulkers, H. ; Woelders, H. - \ 2014
Lelystad : Wageningen UR Livestock Research (Rapport / Wageningen UR Livestock Research nr. 747) - 10
melkkoeien - zwangerschap - eiwitexpressieanalyse - merkers - melkproductie - melkveehouderij - rundveehouderij - productiebeperkingen - optimalisatie - voortplantingsefficiëntie - dairy cows - pregnancy - proteomics - markers - milk production - dairy farming - cattle husbandry - production restrictions - optimization - reproductive efficiency
The aim of this study is to develop an accurate, fast, cheap, and reliable test to detect pregnancy before day 35 on the basis of markers in milk. The ultimate goal is to have a method that can be implemented in a practical setting.
A toolkit for the mzIdentML standard: the ProteoIDViewer, the mzidLibrary and the mzidValidator.
Ghali, F. ; Krishna, R. ; Lukasse, P.N.J. ; Martínez-Bartolomé, S. ; Reisinger, F. ; Hermjakob, H. ; Vizcaíno, J.A. ; Jones, A.R. - \ 2013
Molecular and Cellular Proteomics 12 (2013). - ISSN 1535-9476 - p. 3026 - 3035.
tandem mass-spectrometry - proteomics - software - parser - miape - rates
The Proteomics Standards Initiative has recently released the mzIdentML data standard for representing peptide and protein identification results, for example, created by a search engine. When a new standard format is produced, it is important that software tools are available that make it straightforward for laboratory scientists to use it routinely and for bioinformaticians to embed support in their own tools. Here we report the release of several open-source Java-based software packages based on mzIdentML: ProteoIDViewer, mzidLibrary, and mzidValidator. The ProteoIDViewer is a desktop application allowing users to visualize mzIdentML-formatted results originating from any appropriate identification software; it supports visualization of all the features of the mzIdentML format. The mzidLibrary is a software library containing routines for importing data from external search engines, post-processing identification data (such as false discovery rate calculations), combining results from multiple search engines, performing protein inference, setting identification thresholds, and exporting results from mzIdentML to plain text files. The mzidValidator is able to process files and report warnings or errors if files are not correctly formatted or contain some semantic error. We anticipate that these developments will simplify adoption of the new standard in proteomics laboratories and the integration of mzIdentML into other software tools. All three tools are freely available in the public domain.
Glossina hytrosavirus control strategies in tsetse fly factories: application of infectomics in virus management
Kariithi, H.M. - \ 2013
Wageningen University. Promotor(en): Just Vlak; Monique van Oers, co-promotor(en): A.M.M. Abd-Alla; G.A. Murilla. - Wageningen : Wageningen UR - ISBN 9789461737533 - 207
glossina pallidipes - dierenvirussen - speekselklierziekten - massakweek - virusziekten - ziektebestrijding - eiwitexpressieanalyse - genomica - glossina pallidipes - animal viruses - salivary gland diseases - mass rearing - viral diseases - disease control - proteomics - genomics
African trypanosomosis is a fatal zoonotic disease transmitted by tsetse flies (Diptera; Glossinidae); blood-sucking insects found only in sub-Saharan Africa. Two forms of trypanosomoses occur: the animal African trypanosomosis (AAT; nagana), and the human African trypanosomosis (HAT; sleeping sickness). Since there are no effective vaccines against trypanosomosis, tsetse fly eradication is the most effective disease control method. Tsetse flies can be effectively eradicated by the sterile insect technique (SIT), which is applied in an area-wide integrated pest management approach. SIT is an environmentally benign method with a long and solid record of accomplishments. SIT requires large-scale production of sexually sterilized male flies (by exposure to a precise and specific dose of ionizing radiation, usually from a 60Co or 137Ce source), which are sequentially released into a target wild insect population to out-compete wild type males in inseminating wild virgin females. Once inseminated by sterile males, the virgin females do not produce viable progeny flies. Importantly, these females do not typically re-mate. Ultimately, the target wild insect population can decrease to extinction. However, tsetse SIT programs are faced with a unique problem: laboratory colonies of many tsetse species are infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae). GpSGHV-infected flies have male aspermia or oligospermia, underdeveloped female ovarioles, sterility, salivary gland hypertrophy syndrome (SGH), distorted sex ratios, and reduced insemination rates. Without proper management, symptomatic GpSGHV infections (characterized by SGH symptoms) can cause collapse of Glossina colonies. To ensure colony productivity and survival, GpSGHV management strategies are required. This will ensure a sustained supply of sterile males for SIT programs. The aim of this PhD research was to investigate the functional and structural genomics and proteomics (infectomics) of GpSGHV as a prerequisite to development of rationally designed viral control strategies. A series of experiments were designed to: (i) investigate epidemiology and diversity of GpSGHV; (ii) identify GpSGHV proteome and how viral and host proteins contribute to the pathobiology of the virus; and (iii) investigate the interplay between GpSGHV, the microbiome and the host, and how these interactions influence the outcomes of viral infections. By relating GpSGHV and host infectomics data, cost-effective viral management strategies were developed. This resulted in significant reduction of GpSGHV loads and elimination of SGH from laboratory colonies of G. pallidipes.