Staff Publications

Staff Publications

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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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    General aspects on diagnostic methods: viruses, micro-organisms
    Huttinga, H. - \ 1997
    In: Diagnosis and Identification of Plant Pathogens / Dehne, H.W., Adam, G., Diekmann, M., Frahm, J., Mauler-Machnik, A., van Halteren, P., Dordrecht : Kluwer Academic Publishers - p. 15 - 16.
    Expression of potato leafroll virus ORF0 induces viral disease-like symptoms in transgenic potato plants
    Wilk, F. van der; Dullemans, A. ; Hans, F. ; Heuvel, J.F.J.M. van den; Huttinga, H. - \ 1997
    Expression of potato leafroll virus ORF 0 induces viral disease-like symptoms in transgenic potato plants.
    Wilk, F. van der; Houterman, P. ; Molthof, J. ; Hans, F. ; Dekker, B. ; Heuvel, J. van den; Huttinga, H. ; Goldbach, R. - \ 1997
    Molecular Plant-Microbe Interactions 10 (1997). - ISSN 0894-0282 - p. 153 - 159.
    Molecular basis of the interactions between luteoviruses and aphids
    Hogenhout, S.A. ; Verbeek, M. ; Hans, F. ; Houterman, P.M. ; Fortass, M. ; Wilk, F. van der; Huttinga, H. ; Heuvel, J.F.J.M. van den - \ 1996
    Agronomie 16 (1996). - ISSN 0249-5627 - p. 167 - 173.
    Sensitivity of indexing procedures for viruses and viroids
    Huttinga, K. - \ 1996
    Advances in Botanical Research 23 (1996). - ISSN 0065-2296 - p. 59 - 71.
    Potato leafroll virus : molecular analysis and genetically engineered resistance
    Wilk, F. van der - \ 1995
    Agricultural University. Promotor(en): R.W. Goldbach; H. Huttinga. - S.l. : Van der Wilk - ISBN 9789054854616 - 119
    luteovirus - plantenziekten - plantenvirussen - genetische modificatie - recombinant dna - plantenveredeling - ziekteresistentie - plaagresistentie - luteovirus - plant diseases - plant viruses - genetic engineering - recombinant dna - plant breeding - disease resistance - pest resistance

    The nucleotide sequence of the genomic RNA of potato leafroll virus (PLRV) was elucidated and its genetic organization deduced (Chapter 2). Six open reading frames (ORFs) were shown to be present on the genome. Both the PLRV coat protein gene and the RNA- dependent RNA polymerase gene were identified by interviral sequence comparison. The PLRV genomic organization was shown to be highly similar to that of beet western yellows virus (BWYV) and except for the ORF1 products all PLRV and BWYV coded proteins displayed an extensive amino acid sequence homology.

    In order to obtain resistance following the principle of pathogen-derived resistance, the PLRV coat protein gene was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and used to transform potato (Chapter 3). Upon analysis of the transgenic plants obtained it was shown that, although transgenic transcripts were abundantly present in the plant tissues, the presence of transgenic coat protein could not be detected. The transgenic potato plants were shown to be susceptible to PLRV infection but contained significant lower virus titers as compared to infected wild-type potato plants. To enhance the translational expression of the coat protein gene the sequences flanking the start codon were modified to a theoretically optimized context (Chapter 4). Potato plants were transformed with the altered coat protein gene and analyzed for the presence of transgenic coat protein. Despite of the induced mutations transgenic protein could not be detected. The results from inoculation experiments with PLRV were identical to those obtained with the transgenic plants containing the unaltered coat protein gene, the transgenic plants containing less viral antigen than infected wild-type plants.

    To investigate the role of the PLRV ORF1 product (P1) in the viral infection process and to define its intracellular location in infected plant cells, the protein was expressed in Escherichia coli and in the baculovirus expression system and used to raise an antiserurn (Chapter 5). Expression of P1 proved to be difficult, possibly due to a toxic effect imparted by the protein. Using an antiserurn raised against a recombinant P1 fusion protein, it was determined that P1 did not accumulate in infected plant tissues to detectable levels.

    To further investigate the function of the ORF1, its sequence was transformed into potato (Chapter 6). Surprisingly, the transgenic plants expressing detectable levels of ORF1, transcripts displayed an altered phenotype closely resembling that of virusdiseased plants. Plants expressing a modified and therefore untranslatable, version of the ORF1, sequence were phenotypically indistinguishable from wild-type control plants, indicating that the expression of the P1 protein induced virus disease-like symptoms. The transgenic potato plants containing the ORF1, sequence were analyzed for possibly acquired resistance (Chapter 7). Upon infection one plant line showed to be highly resistant while all other plant lines were susceptible to PLRV-infection similar to wild-type plants. The resistance obtained expressed itself as near immunity, only under high inoculation pressure a low percentage of the plants became infected.

    Characteristics of a resistance-breaking isolate of potato virus Y causing potato tuber necrotic ringspot disease.
    Heuvel, J.F.J.M. van den; Vlugt, R.A.A. van der; Verbeek, M. ; Haan, P.T. de; Huttinga, H. - \ 1994
    European Journal of Plant Pathology 100 (1994). - ISSN 0929-1873 - p. 347 - 356.
    Transgenic potato plants expressing PLRV ORF1.
    Wilk, F. van der; Molthoff, F. ; Heuvel, J. van den; Dekker, B. ; Goldbach, R. ; Huttinga, H. - \ 1993
    In: Abstract 9th Int. Congr. Virology, Glasgow, UK - p. 341 - 341.
    Engineering resistance against potato virus Y
    Vlugt, R.A.A. van der - \ 1993
    Agricultural University. Promotor(en): R.W. Goldbach; H. Huttinga. - S.l. : Van der Vlugt - ISBN 9789054850847 - 111
    plantenziekten - plantenvirussen - solanum tuberosum - aardappelen - potyvirus - planten - immunisatie - geïnduceerde resistentie - genetica - genetische variatie - evolutie - soortvorming - immunogenetica - genetische modificatie - recombinant dna - plant diseases - plant viruses - solanum tuberosum - potatoes - potyvirus - plants - immunization - induced resistance - genetics - genetic variation - evolution - speciation - immunogenetics - genetic engineering - recombinant dna

    Potato virus Y is the type species of the potyvirus genus, the largest genus of the plant virus family Potyviridae. The virus causes serious problems in the cultivation of several Solanaceous crops and although certain poly- and monogenic resistances are available, these can not always be employed, e.g. R y genes in potato cv. 'Bintje'. The aim of the research described in this thesis was to establish new forms of resistance against PVY by genetic modification of host plants. One such form of genetic engineered resistance is 'coat protein-mediated resistance', whereby expression of a viral coat protein (CP) in a transgenic plant may confer resistance against infection with the homologous virus, and some closely related viruses.

    At the start of this investigation no sequence data on the RNA genome of PVY were available, therefore cDNA synthesis and subsequent sequence determination was performed to obtain the necessary PVY CP gene sequence as well as additional sequences from the 3'-terminal region of the viral genome (Chapter 2 and Van der VIugt et al., 1989). This enabled the determination of the exact taxonomic position of the PVY N('tobacco veinal necrosis strain') isolate used in these experiments, among other PVY isolates from at least two different strains. Detailed comparisons of the PVY NCP and 3'-non translated (3'-NTR) sequences with those from a large number of geographically distinct PVY isolates that became available during the course of this investigation, showed that these sequences, in addition to distinguish between different potyvirus species (Ward and Shukla, 1991; Frenkel et al., 1989), can also be used for the distinction between strains of one potyvirus (Chapter 3, Van der VIugt et al., 1992a). Several strain specific amino acid sequences in the CPs and nucleotide sequences in the 3'-NTRs could be discerned, that are possibly involved in virulence and/or symptom expression. Further experiments are required to elucidate the precise biological significance of these sequence motifs. Interestingly the sequence comparisons as complied in Chapter 3 also confirmed the high levels of CP and 3'-NTR sequence identity between the PVY isolates at one hand and one putative isolate of pepper mottle virus (PepMoV, Dougherty et al., 1985) at the other, as described previously (Van der VIugt et al., 1989; Van der Vlugt, 1992). Initially described as an atypical strain of PVY (PVY-S, Zitter, 1972) PepMoV was later found to be serologically and biologically distinct from PVY (Purcifull et al., 1973, 1975; Zitter and Cook, 1973). Recent determination of the complete genomic RNA sequence of a Californian isolate of pepper mottle virus (PepMoV-C; Bowman-Vance et al, 1992a,b) and comparisons between a Florida isolate of PepMoV and PVY (Hiebert and Purcifull, 1992) however, suggest that PepMoV represents a distinct potyvirus though more closely related to PVY than to any other potyvirus. Additional sequence information of other, biologically well characterized, isolates of PepMoV, like a virus isolate apparently intermediate between PepMoV and PVY (Nelson and Wheeler, 1978), will hopefully aid in establishing the exact taxonomic position of this pepper infecting virus in the genus Potyvirus. Generally it is to be recommended that of all virus isolates whose (partial) sequences are under investigation, precise origin and other relevant biological characteristics are also accurately documented.
    In contrast to all other viruses for which 'CP-mediated resistance' has been described sofar, potyviruses do not express their CPs from a distinct, separate gene but through proteolytic cleavage of a polyprotein precursor. This necessitated the
    addition of translational start signals, directly upstream of the CP encoding sequence, in order to enable expression of the PVY NCP in transgenic potato and tobacco plants. Potato tuber disc and tobacco leaf disc transformations with these constructs resulted in large numbers of transgenic plants (Chapters 4 and 5). Despite the fact that a large number of transgenic plants was tested for CP expression, using a highly sensitive enzyme-amplification based ELISA format, in none of the plants significant amounts of viral CP could be detected. Whether this is caused by the extra N-terminal methionine residue, or improper folding of the CP, resulting in decreased stability of the protein, or by inefficient protein extractions, possibly resulting from protein insolubility, is not known. It remains to be tested whether transformation of plants with a construct in which a functional protease domain is coupled to a potyviral CP with an intact protein processing sequence, will result in high levels of expression of the CP. For more practical purposes however, PVY CP expression levels appear not to be of significant importance since the protection against PVY, observed in the transgenic tobacco plants (Chapter 5 and 6), is apparently RNA-mediated, i.e. prima rily based on the presence of the CP encoding RNA rather than on the coat protein itself. Transgenic tobacco lines expressing PVY CP transcripts devoid of a translational start signal (CP -ATG), possess equal levels of protection against both mechanically inoculated virus and virus transmitted by the natural aphid vector Myzuspersicae (Chapter 5 and 6). It seems highly unlikely that the protection in these CP -ATGplants is based on minute amounts (i.e. less then 0.0001 % of the total soluble protein) of a truncated viral polypeptide since the presence of six translational stopcodons preceding the first in-frame AUG startcodon, 162 nucleotides down stream the 5'-end of the CP encoding sequence, will prevent expression of such a polypeptide.

    Analysis of the transgenic potato lines (Chapter 4) showed that most lines, as the transgenic tobacco lines, expressed CP specific RNA transcripts. Under the given greenhouse conditions, however, in none of the transgenic plants protection to PVY could be determined. In view of the results obtained with the transgenic tobacco lines, it may be anticipated that virus challenging of additional transgenic potato lines, under more optimal greenhouse conditions, will reveal similar levels of RNA-mediated virus resistance as observed in tobacco. For all practical purposes genetically engineered resistance based on the presence of RNA molecules is to be preferred over forms of resistance that are based on the expression of a (foreign) protein. Apart from being energetically more favourable for the plant, it is likely to aid in the acceptance of genetically modified crop plants by both politicians and the public, something which might, in the next few years, turn out to be the major obstacle in the successful application of plant transformation techniques.

    At this stage one can only speculate on the mechanism(s) on which this RNAmediated resistance is based. Transformation of plants with partial CP or other PVY Ngenomic sequences will help in identifying the protection mechanism(s) involved and show whether regions other than the CP-encoding domain can be equally effective in conferring virus resistance. If the resistance is based on a 'sense-RNA' effect, i.e. hybridization of the positive sense transgenic RNA to negative-sense viral RNA replication intermediates, thereby blocking further virus replication, the ribozyme technology might prove an efficient expansion of this genetically engineered type of resistance. Ribozymes, RNA sequences capable of specific and catalytic cleavage of other RNA-sequences, are able to cleave target RNAs efficiently and catalytically in vitro . The antiviral application of ribozymes in transgenic plants however has sofar demonstrated not to be very successful and reported protection levels are not yet exceeding those obtained with antisense RNAs (Edington and Nelson, 1992). Chapter 7 describes the design and synthesis of hammerhead ribozymes capable to cleave a highly conserved region from the PVY RNA dependent RNA-polymerase cistron. It was shown that the correct formation of the hammerhead cleavage complex, determined at least in part by the lengths of the antisense arms of the ribozyme, forms an important factor in the efficiency of cleavage. Cellular and full-length viral RNA molecules generally posses extended, unknown secondary structures which are likely to hamper precise formation of hammerhead structures, which requires bimolecular basepairing. Correct hammerhead formation and efficient cleavage of these RNAs will therefore require ribozymes with rather long basepairing arms. These long antisense arms however will make catalytic cleavage rather unlikely since complex dissociation will probably become the rate limiting factor. For this reason one can assume that ribozymes will only be successful when introduced into specific antisense RNA molecules, directed against the less abundant viral complementary strands, rather than as highly efficient RNA cleaving "enzymes".

    Expression of the potato leafrol luteovirus coat protein gene in transgenic potato plants inhibits viral infection.
    Wilk, F. van der; Posthumus-Lutke Willink, D. ; Huisman, M.J. ; Huttinga, H. ; Goldbach, R.W. - \ 1991
    Plant Molecular Biology 17 (1991). - ISSN 0167-4412 - p. 431 - 439.
    Transgenic plants expressing the coat protein gene of potato leafroll luteovirus.
    Wilk, F. van de; Huisman, M.J. ; Goldbach, R.W. ; Huttinga, H. - \ 1990
    In: Abstract 8th Int. Congr. Virology, Berlin - p. 454 - 454.
    Nucleotide sequence and organization of potato leafroll virus genomic RNA.
    Wilk, F. van der; Huisman, M.J. ; Cornelissen, B.J.C. ; Huttinga, H. ; Goldbach, R.W. - \ 1989
    FEBS Letters 245 (1989). - ISSN 0014-5793 - p. 51 - 56.
    Properties of viruses of the potyvirus group. 2. Buoyant density, S value, particle morphology, and molecular weight of the coat protein subunit of bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus, and potato virus Yn
    Huttinga, H. ; Mosch, W.H.M. - \ 1974
    Wageningen : Instituut voor Plantenziektenkundig Onderzoek (Mededeling Instituut voor Plantenziektenkundig Onderzoek No. 644) - 5
    Separation of long and short particles of tobacco rattle virus with polyethylene glycol
    Huttinga, H. - \ 1973
    (Wageningen) : [s.n.] (Mededeling / Instituut voor plantenziektenkundig onderzoek no. 613) - 4
    plantenziekten - plantenvirussen - nicotiana - tabak - tabaksratelvirus - virologie - methodologie - technieken - plant diseases - plant viruses - tobacco - Tobacco rattle virus - virology - methodology - techniques
    Properties of viruses of the potyvirus group. 1. A simple method to purify bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus and potato virus Yn
    Huttinga, H. - \ 1973
    Wageningen : Instituut voor Plantenziektenkundig Onderzoek (Mededeling Instituut voor Plantenziektenkundig Onderzoek No. 637) - 3
    Interaction between long and short particles of tobacco rattle virus
    Huttinga, H. - \ 1972
    Landbouwhogeschool Wageningen. Promotor(en): J.P.H. van der Want; A. van Kammen. - Wageningen : Pudoc - ISBN 9789022004197 - 80
    plantenziekten - plantenplagen - gewasbescherming - plantenziektekunde - afwijkingen, planten - nicotiana - tabak - plantenvirussen - tabaksratelvirus - plant diseases - plant pests - plant protection - plant pathology - plant disorders - nicotiana - tobacco - plant viruses - Tobacco rattle virus

    Tobacco rattle virus is a rod-shaped multiparticle virus.

    Short particles alone are not infectious, long ones are but give rise to the formation of incomplete virus. Mixtures of long and short particles induce the formation of complete virus. The interaction between long and short particles is not strain-specific: if long and short particles of different strains are inoculated together complete virus is also formed. These new strains have properties of both parent strains.

    The interaction between heterologous long and short particles explains why there are so many different tobacco rattle virus isolates and why no correlation can be found between classifications based on different characteristics.

    Interaction between components of pea early - browning virus
    Huttinga, H. - \ 1969
    Wageningen : [s.n.] (Mededeling / Instituut voor plantenziektenkundig onderzoek no. 526) - 5
    plantenziekten - plantenplagen - gewasbescherming - plantenziektekunde - afwijkingen, planten - pisum sativum - erwten - plantenvirussen - plant diseases - plant pests - plant protection - plant pathology - plant disorders - peas - plant viruses
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