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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1
    Ebert, Malaika K. ; Rangel, Lorena I. ; Spanner, Rebecca E. ; Taliadoros, Demetris ; Wang, Xiaoyun ; Friesen, Timothy L. ; Jonge, Ronnie de; Neubauer, Jonathan D. ; Secor, Gary A. ; Thomma, Bart P.H.J. ; Stukenbrock, Eva H. ; Bolton, Melvin D. - \ 2020
    Molecular Plant Pathology (2020). - ISSN 1464-6722
    Cercospora beticola - necrosis-inducing effector - virulence factor

    Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola–sugar beet disease process.

    SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual-function protein that facilitates infection while protecting from wheat-produced chitinases
    Liu, Zhaohui ; Gao, Yuanyuan ; Kim, Yong Min ; Faris, Justin D. ; Shelver, Weilin L. ; Wit, Pierre J.G.M. de; Xu, Steven S. ; Friesen, Timothy L. - \ 2016
    New Phytologist 211 (2016)3. - ISSN 0028-646X - p. 1052 - 1064.
    Parastagonosopora nodorum - Chitin - Host-selective toxin - Necrotroph - Necrotrophic effector - Programmed cell death (PCD) - Wheat (Triticum aestivum) chitinases

    SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases.

    FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici
    M'Barek, S. Ben; Cordewener, J.H.G. ; Tabib Ghaffary, M.S. ; Lee, T.A.J. van der; Liu, Z. ; Mirzadi Gohari, A. ; Mehrabi, R. ; America, A.H.P. ; Friesen, T.L. ; Hamza, S. ; Stergiopoulos, I. ; Wit, P.J.G.M. de; Kema, G.H.J. - \ 2015
    Fungal Genetics and Biology 79 (2015). - ISSN 1087-1845 - p. 54 - 62.
    host-selective toxins - mycosphaerella-graminicola - stagonospora-nodorum - septoria-tritici - rhynchosporium-secalis - cladosporium-fulvum - ptr toxa - hydrogen-peroxide - barley pathogen - tan spot
    Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.
    SnTox1, a Parastagonospora nodorum necrotrophic effector, elicits PCD while binding chitin to protect the pathogen from wheat chitinases
    Friesen, T.L. ; Liu, Z.H. ; Kim, Y.M. ; Gao, Y. ; Wit, P.J.G.M. de; Faris, J.D. - \ 2015
    In: Book of Abstracts 28th Fungal Genetics Conference. - - p. 209 - 210.
    Parastagonospora nodorum (Synonym: Stagonospora nodorum) is a destructive pathogen of wheat that induces yield and quality losses by causing necrosis on the leaves and glumes of wheat. P. nodorum is a necrotrophic specialist pathogen that secretes an arsenal of necrotrophic effectors (NEs) involved in inducing necrosis. SnTox1 was the first of seven NEs to be reported from P. nodorum and interacts directly or indirectly with the single dominant susceptibility gene Snn1. SnTox1 is recognized by Snn1 followed by the induction of a classical defense response involving programmed cell death (PCD), an oxidative burst, DNA laddering, and up regulation of several PR proteins including chitinases. However, this recognition results in wheat being susceptible to P. nodorum rather than resistant. Interestingly, the C-terminus of SnTox1 has homology to several plant chitin binding proteins and we have shown that SnTox1 does bind chitin. Therefore, we have expressed two wheat chitinases that are able to degrade the fungal cell wall in vitro to show that P. nodorum transformed other plant pathogens and non-pathogens with SnTox1 including Neurospora crassa (saprotroph), Pyrenophora teres f. teres (necrotrophic barley pathogen), and Cercospora beticola (hemi-biotrophic sugarbeet pathogen) and show that not only did some of these fungi become pathogens of wheat lines harboring Snn1, but all had increased protection from the wheat chitinases in vitro, exposing a secondary or possibly the primary function of SnTox1. The dual function of this protein explains the high prevalence of SnTox1, relative to other NEs in the P. nodorum global population and shows that necrotrophs harbor chitin binding proteins for their protection during pathogenesis.
    The parastagonospora nodorum necrotrophic effector SnTox1 elicits recognition in wheat and then protects from the resulting host defense response
    Friesen, T.L. ; Liu, Z. ; Kim, Y. ; Gao, Y. ; Wit, P.J.G.M. de; Faris, J.D. - \ 2014
    In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 24 - 24.
    CS-9.5 - Parastagonospora nodorum (Synonym: Stagonospora nodorum) is a destructive pathogen of wheat that induces yield and quality losses by causing disease on the leaves and glumes of wheat. P. nodorum is a necrotrophic specialist pathogen that secretes an arsenal of necrotrophic effectors (NEs) involved in disease induction. SnTox1 was the first of seven NEs to be reported from P. nodorum and interacts directly or indirectly with the single dominant susceptibility gene Snn1. SnTox1 is recognized by Snn1 followed by the induction of a classical defense response involving programmed cell death (PCD), an oxidative burst, DNA laddering and up regulation of several PR proteins, however, this recognition results in susceptibility to P. nodorum rather than resistance. Interestingly, in addition to inducing PCD that results in susceptibility, we have shown that SnTox1 is important in defending the pathogen against wheat chitinases that attack the cell wall of fungi, reducing growth. To demonstrate that SnTox1 was involved in protection from host-produced chitinases, we cloned and expressed several wheat chitinase genes to assess the effects of chitinases in the presence and absence of the SnTox1, showing that SnTox1 is highly important in protecting the pathogen from wheat chitinases. Additionally, we transformed non-pathogens of wheat with SnTox1 and showed that not only did some of these fungi become pathogens of wheat but all had increased protection from wheat chitinases. The dual function of this protein explains the high prevalence of SnTox1 , relative to other NEs in the P. nodorum global population.
    Investigations of how the necrotrophic specialist Parastagonospora nodorum is using the dual function necrotrophic effector SnTox1 to infect wheat
    Friesen, T.L. ; Liu, Z. ; Kim, Y. ; Gao, Y. ; Wit, P.J.G.M. de; Faris, J.D. - \ 2014
    In: Book of Abstracts Joint Meeting American Phytopathological Society and Canadian Phytopathological Society. - - p. 40 - S.
    Parastagonospora nodorum (Synonym Stagonospora nodorum) is a destructive pathogen of wheat that induces yield and quality losses by causing disease on both the leaves and glumes of wheat. P. nodorum is a necrotrophic specialist pathogen that secretes an arsenal of necrotrophic effectors (NEs) involved in disease induction. SnTox1 was the first of seven NEs reported from P. nodorum and was shown to interact directly or indirectly with the single dominant susceptibility gene Snn1. The SnTox1-Snn1 interaction induces several hallmarks of a defense response including an oxidative burst and DNA laddering, a classic apoptosis response, but results in susceptibility rather than resistance. Although several necrotrophic specialists are known to induce the defense response while causing disease, little is known about how necrotrophic specialists survive the harsh environment of the host defense response. SnTox1 contains homology to several plant chitin binding proteins and we have shown that SnTox1 localizes to the cell wall of mycelium in culture and that SnTox1 binds purified chitin. We have cloned and expressed several wheat chitinases and used them to show that SnTox1 not only induces PCD but that SnTox1 also has a second role of binding chitin in the fungal cell wall, resulting in protection from these wheat chitinases. The dual function of this protein explains the high prevalence of SnTox1, relative to other NEs in the P. nodorum global population.
    New broad-spectrum resistance to septoria tritici blotch derived from synthetic hexaploid wheat
    Tabib Ghaffary, M.S. ; Faris, J.D. ; Friesen, T.L. ; Visser, R.G.F. ; Lee, T.A.J. van der; Robert, O. ; Kema, G.H.J. - \ 2012
    Theoretical and Applied Genetics 124 (2012)1. - ISSN 0040-5752 - p. 125 - 142.
    mycosphaerella graminicola pathosystem - quantitative trait loci - aegilops-tauschii coss. - stagonospora nodorum blotch - multiple fungal pathogens - genetic-linkage map - bread wheat - durable resistance - rust resistance - common wheat
    Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most devastating foliar diseases of wheat. We screened five synthetic hexaploid wheats (SHs), 13 wheat varieties that represent the differential set of cultivars and two susceptible checks with a global set of 20 isolates and discovered exceptionally broad STB resistance in SHs. Subsequent development and analyses of recombinant inbred lines (RILs) from a cross between the SH M3 and the highly susceptible bread wheat cv. Kulm revealed two novel resistance loci on chromosomes 3D and 5A. The 3D resistance was expressed in the seedling and adult plant stages, and it controlled necrosis (N) and pycnidia (P) development as well as the latency periods of these parameters. This locus, which is closely linked to the microsatellite marker Xgwm494, was tentatively designated Stb16q and explained from 41 to 71% of the phenotypic variation at seedling stage and 28-31% in mature plants. The resistance locus on chromosome 5A was specifically expressed in the adult plant stage, associated with SSR marker Xhbg247, explained 12-32% of the variation in disease, was designated Stb17, and is the first unambiguously identified and named QTL for adult plant resistance to M. graminicola. Our results confirm that common wheat progenitors might be a rich source of new Stb resistance genes/QTLs that can be deployed in commercial breeding programs
    A highly conserved neutralizing epitope on group 2 influenza A viruses
    Ekiert, D.C. ; Friesen, R.H.E. ; Bhanha, G. ; Kwaks, T. ; Jongeneelen, M. ; Yu, W. ; Ophorst, C. ; Cox, F. ; Korse, H.J.W.M. ; Brandenburg, B. ; Vogels, R. ; Brakenhoff, J.P.J. ; Kompier, R. ; Koldijk, M.H. ; Cornelissen, A.H.M. ; Poon, L.L.M. ; Peiris, M. ; Koudstaal, W. ; Wilson, I.A. ; Goudsmit, J. - \ 2011
    Science 333 (2011)6044. - ISSN 0036-8075 - p. 843 - 850.
    monoclonal-antibodies - avian influenza - hemagglutinin - fusion - ph - recognition - vaccination - subtypes - peptide - season
    Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of VH1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the VH1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies
    Efficacy and mapping of resistance to Mycosphaerella graminicola in wheat
    Tabib Ghaffary, M.S. ; Faris, J.D. ; Friesen, T. ; Robert, O. ; Laurent, V. ; Lonnet, P. ; Margale, E. ; Lee, T.A.J. van der; Visser, R.G.F. ; Kema, G.H.J. - \ 2011
    In: Abstract book of 8th international symposium on Mycosphaerella and Stagonospora diseases of cereals, Mexico City, Mexico, 11-14 September 2011. - Mexico City, Mexico : Cimmyt - p. 51 - 51.
    Identification of a new resistance gene to septoria tritici blotch in wheat
    Tabib Ghaffary, M.S. ; Faris, J.D. ; Friesen, T.L. ; Kema, G.H.J. - \ 2010
    Septoria tritici blotch (STB) caused by the ascomycete Mycosphaerella graminicola is one of the most devastating foliar diseases of bread wheat in North-Western Europe, Centraland West Asia and also of durum wheat in North Africa
    Identification of a new resistance gene to septoria tritici blotch in wheat
    Tabib Ghaffary, M.S. ; Faris, J.D. ; Friesen, T.L. ; Kema, G.H.J. - \ 2010
    In: Abstracts of oral and poster presentations of the 8th international wheat conference, St.Petersburg, Russia, 1-4 June 2010. - St. Petersburg, Russia : N.I. Vavilov Research Institute of Plant Industry (VIR) - p. 320 - 320.
    Septoria tritici blotch (STB) caused by the ascomycete Mycosphaerella graminicola is one of the most devastating foliar diseases of bread wheat in North-Western Europe, Centraland West Asia and also of durum wheat in North Africa
    Identification of a new resistance gene to septoria tritici blotch in wheat
    Tabib Ghaffary, M.S. ; Fan's, J.D. ; Friesen, T.L. ; Kema, G.H.J. - \ 2010
    Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 151 - 151.
    tarwe - mycosphaerella graminicola - genetisch bepaalde resistentie - genen - germplasm - resistentieveredeling - wheat - mycosphaerella graminicola - genetic resistance - genes - germplasm - resistance breeding
    Door het screenen van lijnen en wilde verwanten van tarwe, is een nieuw resistentiegen tegen STB (Septoria tritici blotch) gevonden.
    Pre- and Postexposure Use of Human Monoclonal Antibody against H5N1 and H1N1 Influenza Virus in Mice: Viable Alternative to Oseltamivir
    Koudstaal, W. ; Koldijk, M.H. ; Brakenhoff, J.P.J. ; Cornelissen, A.H.M. ; Weverling, G.J. ; Friesen, R.H.E. ; Goudsmit, J. - \ 2009
    The Journal of Infectious Diseases 200 (2009)12. - ISSN 0022-1899 - p. 1870 - 1873.
    resistance - infection - h9n2
    New strategies to prevent and treat influenza virus infections are urgently needed. A recently discovered class of monoclonal antibodies (mAbs) neutralizing an unprecedented spectrum of influenza virus subtypes may have the potential for future use in humans. Here, we assess the efficacies of CR6261, which is representative of this novel class of mAbs, and oseltamivir in mice. We show that a single injection with 15 mg/kg CR6261 outperforms a 5-day course of treatment with oseltamivir (10 mg/kg/day) with respect to both prophylaxis and treatment of lethal H5N1 and H1N1 infections. These results justify further preclinical evaluation of broadly neutralizing mAbs against influenza virus for the prevention and treatment of influenza virus infections.
    Scatterometer-Derived Soil Moisture Calibrated for Soil Texture With a One-Dimensional Water-Flow Model
    Lange, R. de; Beck, R. ; Giesen, N. van de; Friesen, J. ; Wit, A.J.W. de; Wagner, W. - \ 2008
    IEEE Transactions on Geoscience and Remote Sensing 46 (2008)12. - ISSN 0196-2892 - p. 4041 - 4049.
    ers scatterometer - near-surface - assimilation - retrieval - validation - space
    Current global satellite scatterometer-based soil moisture retrieval algorithms do not take soil characteristics into account. In this paper, the characteristic time length of the soil water index has been calibrated for ten sampling frequencies and for different soil conductivity associated with 12 soil texture classes. The calibration experiment was independently performed from satellite observations. The reference soil moisture data set was created with a I-D water-flow model and by making use of precipitation measurements. The soil water index was simulated by applying the algorithm to the modeled soil moisture of the upper few centimeters. The resulting optimized characteristic time lengths T increase with longer sampling periods. For instance, a T of 7 days was found for sandy soil when a sampling period of I day was applied, whereas an optimized T-value of 18 days was found for a sampling period of 10 days. A maximum rmse improvement of 0.5% vol. can be expected when using the calibrated T-values instead of T = 20. The soil water index and the differentiated T-values were applied to European Remote Sensing (ERS) satellite scatterometer data and were validated against in situ soil moisture measurements. The results obtained using calibrated T-values and T = 20 did not differ (r = 0.39, rmse = 5.4% vol.) and can be explained by the averaged sampling period of 4-5 days. The soil water index obtained with current operational microwave sensors [Advanced Wind Scatterometer (ASCAT) and Advanced Microwave Scanning Radiometer-Earth Observation System] and future sensors (Soil Moisture and Ocean Salinity and Soil Moisture Active Passive) should benefit from soil texture differentiation, as they can record on a daily basis either individually or synergistically using several sensors. The proposed differentiated characteristic time length enables the continuation of the soil water index of sensors with varying sampling periods (e.g., ERS-ASCAT).
    Strategic combinations of satellite and ground-based data for large scale hydrological monitoring: the Volta basin as a 400,000 km2 observatory
    Giesen, N. van de; Friesen, J. ; Hafeez, M. ; Liebe, J. ; Andah, W. ; Andreini, M. ; Kunstmann, H. ; Marx, A. ; Burose, D. ; Moene, A.F. - \ 2004
    Baculovirus DNA replication
    Kool, M. - \ 1994
    Agricultural University. Promotor(en): R.W. Goldbach; J. Tramper; J.M. Vlak. - S.l. : Kool - ISBN 9789054852629 - 200
    baculovirus - kernpolyedervirussen - moleculaire genetica - replicatie - translatie - eiwitsynthese - baculovirus - nuclear polyhedrosis viruses - molecular genetics - replication - translation - protein synthesis

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous proteins for various applications (Luckow and Summers, 1988; Luckow, 1991). In both cases large-scale systems for the production of baculoviruses are important. Production in insect larvae is difficult to scale up and to control. Insect-cell cultures offer an attractive alternative. Moreover, in the case of pharmaceuticals and diagnostics in human and veterinary medicine insect-cell systems have to be applied since such systems are well defined.

    Due to the great interest in baculoviruses as biological insecticides and expression vectors for foreign genes, the molecular genetic aspects of especially the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV), the type member of the Baculoviridae, have been studied in much detail (Blissard and Rohrmann, 1990). Chapter 2 of this thesis presents, as of March 1994, an overview of the structural and functional organization of the AcMNPV genome. The genomes of AcMNPV (R.D. Possee, pers. comm.) and Bombyx mori MNPV (BmMNPV) (S. Maeda, pers. comm.) have been completely sequenced but are awaiting publication. In contrast to other large DNA viruses such as adenovirus, herpesviruses, and vacciniavirus (Fields and Knipe, 1990), the process of baculovirus DNA replication of AcMNPV is poorly understood. At the start of this study a few genes were found which were thought to be involved in AcMNPV DNA replication such as a helicase and a DNA polymerase. Sequences representing the origin of AcMNPV DNA replication were not known.

    Baculoviruses can be produced on a large scale in insect-cell cultures using batch (Maiorella et al., 1988), semicontinuous (Hink and Strauss, 1980) and continuous reactors (Kompier et al., 1988). Continuous production of wild-type (wt) AcMNPV and recombinants thereof was achieved in a system consisting of one bioreactor producing insect cells in series with a second bioreactor for virus infection and protein production (Kompier et al., 1988; Van Lier et al., 1992). After a few weeks of continuous operation, however, the productivity decreased to a low level. In the case of wt AcMNPV, the number of polyhedra per cell, the fraction of cells containing polyhedra, and the concentration of extracellular virus were found to be decreased (Kompier et al., 1988). Continuous production of an AcMNPV recombinant where the polyhedrin gene was replaced by the lacZ gene of Escherichia coli essentially gave the same results (Van Lier et al., 1992). The decrease of virus production was ascribed to a phenomenon known as passage effect (Tramper and Vlak, 1986), but the underlying mechanism remained unknown.

    Analysis of samples obtained from continuous bioreactor systems (Chapter 3) showed that with ongoing production a mutant AcMNPV became dominant. This mutant lacked about 43% of the original genome. The deleted DNA included the polyhedrin gene and several genes essential for DNA replication. The replication of the mutant appeared to be dependent on the presence of an intact helper AcMNPV. The passage effect in the continuous system is thus thought to be the result of interference between the deletion mutant and helper virus. These so-called defective interfering particles (DIPs) can only accumulate when the concentration of the intact virus is high enough to support the replication of these DIPs. Thus, for a successful continuous production of baculoviruses low multiplicities of infection should be used to avoid the accumulation of DIPs.

    One of the regions of the AcMNPV genome putatively involved in the generation of the DIPs is located in the EcoRI-C fragment of AcMNPV. Deletion mutants often lacked a considerable portion of Eco RI-C, but also maintained a consistent segment of this fragment that may be essential for replication and/or encapsidation. To investigate the genetic functions of the EcoRI-C fragment in the defective genomes and their possible role in the generation of these genomes, the nucleotide sequence of a 7.3 kilobase pair region of the right part of the Eco RI-C fragment was determined (Chapter 4). Eight putative open reading frames (ORFs) were identified and their respective amino acid sequences compared with a number of data libraries, The product of ORF 1227 corresponded with GP41, a virion protein, and its predicted protein sequence was found to be 55 amino acids longer at its C-terminus than reported previously (Whitford and Faulkner, 1992). The majority of ORF 1227, including the additional 55 amino acids, moreover, showed a high degree of homology with protein P40 of Helicoverpa zea SNPV, also a structural virion protein (Ma et al., 1993). Three other ORFs in the analyzed AcMNPV region showed homology with ORF's in the HzSNPV sequence, indicating that the general organization of this region is similar in both viruses, and possibly between MNPVs and SNPVs. However, no sequences have yet been identified within this region that may play a role in the generation and/or encapsidation of the DIPs.

    The generation and characterization of DIPs was further investigated in Chapter 5. Three small separate regions, representing only 5 % of the original AcMNPV genome, were found to be retained in DNA of defective genomes after 40 serial passages in insect cells with undiluted inocula. Independently, Lee and Krell (1992) showed that after 80 serial passages of AcMNPV, DIPs were found which contained tandem repeats of DNA, mainly derived from a small region of the AcMNPV genome, located in the Hin dIII-K fragment. Since all these defective genomes were still able to replicate in insect cells, although only with the help of intact virus, they must have retained essential cis-acting elements necessary for DNA replication. Therefore, a replication assay was developed to study whether these regions, retained in the defective genomes, contained cis -acting elements such as an origin ( ori ) of DNA replication. Transfection of Spodoptera frugiperda cells with plasmids containing these sequences followed by superinfection with intact helper AcMNPV resulted in amplification of these plasmids, as demonstrated by the Dpn I sensitivity assay. In order to demonstrate replicating activity of these plasmids, it appeared essential to transfect the cells well (24 h) before superinfection with helper virus, and for an optimal replication result the multiplicity used for superinfection had to be I or lower (Chapters 5 and 6). Using this assay seven putative origins of DNA replication were identified in the AcMNPV genome (Chapters 5, 6, and 7).

    Six of the seven putative ori's were found in the homologous regions hr 1, hr 2, hr 3, hr 4a, hr 4b, and hr 5 of AcMNPV (Chapter 6), which are interspersed along the genome (Cochran and Faulkner, 1983; Guarino et al., 1986). Recently, another hr region, hr 1a, has been identified in the AcMNPV genome, that could also serve as ori in a replication assay (Leisy and Rohrmann, 1993). Initial studies demonstrated that the hr regions function as enhancers for transcription, when placed in cis to the promoter of early baculovirus genes (Guarino etal., 1986; Guarino and Summers, 1986). Rodems and Friesen (1993) demonstrated that hr regions also function as enhancers in vivo . These results together with the data of this thesis imply that all hr's in AcMNPV may be bifunctional in vivo , i.e. have both enhancer and ori activity. Sequence analysis has shown that hr's contain two to eight 30 bp imperfect palindromes, interspaced by other repeated sequences, and that each palindrome contains a naturally occurring Eco RI site at its core (Guarino et al., 1986; Guarino and Summers, 1986). One copy of such a palindrome appeared to be sufficient for either enhancer function or ori activity (Guarino et al., 1986; Pearson et al. , 1992).

    In addition to the seven hr's, the Hin dIII-K fragment of AcMNPV was also found to carry a putative ori , although this fragment does not contain an hr region (Chapter 6). The Hin dIII-K ori had a complex structure (Chapter 7), resembling those of other large DNA viruses. This ori contained several regions, some of which were found to be essential for its activity, whereas others contain auxiliary sequences, that enhance ori activity. Sequence analysis of these regions identified several structures often found in other viral replication ori's , such as palindromes and other repeated motifs (DePamphilis, 1993). Recently an ori , also with a complex structure, but different from AcMNPV hr's, has been identified in another baculovirus, Orgyia pseudotsugata MNPV (OpMNPV) (Pearson et al., 1993).

    The individual role of all these ori's during viral DNA replication, and whether they are all active simultaneously in vivo , is unclear. Deletion of hr 5 from the AcMNPV genome or the closely related Bombyx mori MNPV (BmMNPV) genome had no effect on the replication of these viruses (Rodems and Friesen, 1993; Majima et al., 1993) . Also from the experiments with DIPs generated by serial passaging it can be deduced that not all the ori's are necessary for replication of the genome. After 40 serial, undiluted passages three small segments of the genome were predominantly found to be retained, harbouring only the hr 1, hr 3, and hr 5 regions (Chapter 5). Deletion of all hr's would indicate the importance of these regions for virus replication in vivo.

    The importance of the ori in the Hin dIII-K fragment is supported by sequence data of the corresponding region in the closely related BmMNPV (Kamita et al., 1993). Although most of the auxiliary sequences of this ori were found to be deleted in the BmMNPV genome, the essential part of this ori , containing the palindromes and the A/T rich region, was retained suggesting that these elements could not be deleted. These sequence data and the observation that after prolonged serial passage of AcMNPV (80 passages) large replicating DNA molecules are found in which repeated sequences from the Hin dIII-K fragment accumulate (Lee and Krell, 1992), may be a reflection of the importance of this region as genuine oriin vivo (Chapter 7).

    The occurrence of multiple ori's is not unique for baculoviruses, but has also been reported for herpesviruses and Chilo iridescent virus (CIV). The genome of herpes simplex virus I (HSV-1) contains three ori's , oriL , and two copies of ori , (for review, see Fields and Knipe, 1990) and it has been shown that the presence of a single ori , independent which one, is sufficient for replication (Longnecker and Roizman, 1986; Polvino-Bodnar et al. , 1987; Igarashi et al. , 1993). In CIV at least six putative ori's have been identified (Handermann et al. , 1992). It remains to be seen whether in the case of baculoviruses each of the eight putative ori's is necessary for viral replication. When the ori's are indeed functionally redundant, the presence of multiple ori's in the viral genome may increase the frequency of initiation and thus increase the speed of DNA replication. Analysis of intermediates of DNA replication may shed more light on the nature of in vivo ori's .

    The experiments in Chapter 6 also supported the view that a circular topology is a prerequisite for replication of ori -containing plasmids. Linear DNA, even if it contained an ori , did not replicate. These results are in line with the circular nature of baculovirus DNA and suggest a model for baculovirus replication involving a theta structure or a rolling circle. The latter model is supported by data of Leisy and Rohrmann (1993), who demonstrated that replicating plasmids form large concatemeric molecules. In addition, the finding of defective genomes with many reiterations (concatemers) of a 2.8 kbp segment, mainly mapping in the AcMNPV Hin dIII-K fragment (Lee and Krell, 1992), supported also a rolling circle as model for DNA replication.

    Not only cis -acting elements, but also trans -acting factors are important for DNA replication. Chapters 8 and 9 describe the functional mapping of AcMNPV genes required for DNA replication. A transient complementation assay was employed, in which, instead of AcMNPV infection, four co-transfected cosmid clones, encompassing almost the entire genome, provided all the essential trans -acting factors for plasmid DNA replication. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay was a valid and powerful approach to identify the AcMNPV replication genes. The assay was first used to define essential regions in the four cosmids (Chapter 8). Six essential regions were retrieved and these were further subcloned and tested (Chapter 9). Initially in this assay, plasmid replication appeared to be independent of the presence, in cis , of a viral ori , when cloned genes or viral DNA were used instead of complete virus to supply essential trans -acting factors (Chapter 8). However, this was caused by employing high gene copy numbers in the transfections (Chapter 9). As a consequence, a relative abundance of proteins is produced, which may lead to a saturation of specific origins with these proteins. The excess of proteins thus can bind to other originlike structures, even when the affinities are low, and hence cause replication of any plasmid.

    Nine genes involved in DNA replication were identified in the AcMNPV genome (Chapter 9). Six genes, specifying helicase, dna pol, ie-1, lef-1, lef-2, and lef-3 , were found to be essential, while three genes, p35, ie-2 , and pe38 , stimulated DNA replication. No stimulation was observed by the pcna -like protein gene. Two of the three identified stimulatory genes, ie-2 and pe38 , are known as transactivators for transcription (Carson et al. , 1988; Lu and Carstens, 1993), whereas the third stimulating gene, p35 , has previously been identified as inhibitor of virus-induced apoptosis in S. frugiperda cells (Clem et al. , 1991). However, the observation that infection with a p35 deletion mutant in Trichoplusia ni cells did not result in a reduction of virus production (Clem et al. , 1991) suggests that the stimulating effect of p35 in the transient replication assays is not based on activation of the replication process, but is due to inhibiting apoptosis, which may be induced by the expression of one or more of the replication genes.

    Of the six essential AcMNPV DNA replication proteins, putative functions could only be attributed for the helicase and DNA polymerase, based on their homology with other known helicases and DNA polymerases (Lu and Carstens, 1991; Tomalski et al. , 1988). Studying other viral systems, a number of striking similarities was noticed between Baculoviridae and Herpesviridae . Although these two viral families have traditionally been separated based on their different morphology and host specificity, they both have a large double stranded DNA genome, which replicates in the host cell nucleus, and has a circular form in at least one stage of their replication cycle. Their genomes may also replicate in a similar manner as transfection of origin-containing plasmids into infected cells resulted in large concatemers of input plasmid DNA (Leisy and Rohrmann, 1993; Hammerschmidt and Mankertz, 1991). Most strikingly, the number of essential replication genes is similar for both baculoviruses and herpesviruses. An attempt was made to relate the other four, hitherto unassigned, baculovirus replication proteins, IE-1, LEF-1, LEF-2, and LEF-3 with proteins involved in herpesvirus DNA replication (Chapter 10).

    Firstly, the sequences of replication proteins of five different herpesviruses were aligned, which resulted in the identification of a number of conserved motifs in these proteins. Many of these conserved motifs showed (distant) homology with the four baculovirus replication proteins and, most importantly, in the same linear spatial organization as in their putative herpesvirus homologues. Using these conserved motifs as markers the four replication proteins IE-1, LEF-1, LEF- 2, and LEF-3 of AcMNPV were aligned with herpesvirus homologues (Chapter 10). These alignments suggest that ie-1 codes for a single stranded DNA binding protein (SSB), lef-1 for a primase-associated protein, lef-2 for a DNA polymerase processivity factor, and lef-3 for a primase. The assignment to ie-1 to code for a SSB was further supported by the finding of a conserved known single stranded DNA binding sequence motif in six baculovirus IE-1, proteins, which is also found in many other prokaryotic and eukaryotic SSBs (Chapter 10). Further computer-assisted examination and biochemical analysis has to be done to confirm the suggested functions for the four baculovirus replication proteins.

    The similarity between Baculoviridae and Herpesviridae in DNA structure and mechanism of DNA replication, added to the employment of an identical kind and amount of essential replication genes, poses the question whether these two groups of viruses share a common lineage. On the basis of the mutation rate of the conserved baculovirus polyhedrin genes as compared to the insect species in which they occur (Vlak and Rohrmann, 1985) it has been postulated that baculoviruses are ancient viruses that have evolved along with the insects. The relationship among replication genes could imply that herpesviruses have evolved from baculoviruses along with their invertebrate hosts towards vertebrates. Alternatively, the emergence of herpesviruses may be the result of an independent, parallel evolutionary event in ancient vertebrates. Since viral DNA replication in nuclear environments is a conserved process, conserved host replication genes may have been. independently transduced into different ancient viral genomes.

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