Expression and silencing of cowpea mosaic virus transgenes
Sijen, T. - \ 1997
Agricultural University. Promotor(en): A. van Kammen; J. Wellink. - S.l. : Sijen - ISBN 9789054857235 - 133
koebonenmozaïekvirus - genexpressie - pleiotropie - genetische modificatie - recombinant dna - vigna - vignabonen - cowpea mosaic virus - gene expression - pleiotropy - genetic engineering - recombinant dna - vigna - cowpeas
Plant viruses are interesting pathogens because they can not exist without their hosts and exploit the plant machinery for their multiplication. Fundamental knowledge on viral processes is of great importance to understand, prevent and control virus infections which can cause drastic losses in crops. In this thesis, cowpea mosaic virus (CPMV) was studied. This virus consists of two, icosahedral particles that each carry a distinct single stranded RNA molecule of positive polarity. Several years of research have revealed much information on the genomic organisation, the strategy of gene expression and the multiplication processes of CPMV, which are described in Chapter 1, but also many aspects remain to be elucidated.
To study individual viral processes, like replication, encapsidation or cell to cell movement, transgenic plants can be generated that express individual viral genes like the replicase, coat protein or movement protein gene. A prerequisite in this approach is the presence of an efficient and reliable plant regeneration and transformation system. (CPMV) 5 natural host is the tropical grain legume cowpea, Vigna unguiculata, a plant species that is recalcitrant at regeneration. Although in experiments described in Chapter 2 fertile plants could be regenerated from nodal thin cell layer segments, the explants were not competent for Agrobacterium-mediated transformation. Possibly in further studies, these nodal explants could prove suited for another transformation method.
Therefore, tobacco, which is also a host for CPMV and highly competent for regeneration and transformation, was preferred as the species to generate transgenic plants carrying CPMV specific genes. Especially the CPMV movement proteins (MP) genes appealed to us for overexpression studies. CPMV cell to cell movement is enabled by the CPMV MPs that act to modify plasmodesmata. They are assumed to channel plasmodesmata with MP-containing tubular structures and through or with these tubules virus particles are transported to adjacent cells. To obtain more information on the plasmodesmatal modifications brought about by the MPs, transgenic tobacco plants were generated that carried the MP gene under the control of either a constitutive or an inducible 35S promoter. However, in none of these plants the MPs were expressed to detectable levels (Chapter 3). Using the potato virus X (PVX)-based expression vector, accumulation of CPMV MPs was observed in the form of tubular structures extending from the surface of infected protoplasts into the medium. These PVX-derivatives look promising for providing effective tools in future studies on the effects of the CPMV MPs in plants.
Studies on MP functioning could involve complementation experiments with a CPMV mutant that is defective in cell to cell movement. In experiments described in Chapter 4 is was analysed by a molecular approach whether the CPMV mutant N123, that was first described in 1976, could be used to this effect. As the basis of the N123 specific phenotype was found not only to rest in the movement protein gene but also in one of the two coat protein genes, this mutant seemed not very suitable for complementation studies. Presumably a recently developed CPMV mutant in which the MP gene has been replaced by the fluorescent marker protein GFP (green fluorescent protein), will be a more appropriate tool.
Transgenic Nicotiana benthamiana plants that were expressing either the CPMV MP or the replicase gene under the control of a constitutive promoter, were found to exhibit a resistant phenotype when inoculated with CPMV (Chapter 5). Protoplast studies revealed that the resistance occurred as full immunity and was maintained in the cell. Resistance was specific to viruses highly homologous to CPMV, and in addition it was found to be specifically directed against the replication of the CPMV segment of which the transgene was derived (Chapter 5). Pathogen derived resistance can be mediated either by the protein encoded by the transgene or by the transcribed mRNA. Protein -mediated resistance generally offers moderate protection against a broad range of viruses, while RNA-mediated resistance results in immunity at the cellular level. Resistance obtained in transgenic plants transformed with defective genes confirmed that an RNA-based mechanism was underlying the highly specific transgenic resistance against CPMV (Chapter 6).
Specifically in the resistant lines, the transgene mRNA steady state levels were low compared to the relative transgene nuclear transcription rates (Chapter 6). This indicated that resistance occurs from a specific, cytoplasmic RNA turnover mechanism. This process can be regarded as a post- transcriptional gene-silencing process, that is primarily induced on the transgene mRNAs but to which also incoming, homologous CPMV genomes fall victim. In addition, heterologous RNA molecules, like PVX genomes, that contain the sequences corresponding to the transgene, are eliminated (Chapter 6). By inserting sequences homologous to only parts of the transgene in the genome of PVX and studying the fate of these recombinant genomes, it was shown that the degradation process is primarily targeted to a defined region of the transgene mRNA, the 3' region. Further analyses revealed that degradation can occur at various sites within this 3' region and that not a specific sequence or structure is of predominant importance. We observed that small inserts, like of only 60 nucleotides, can tag recombinant PVX molecules for the elimination process, albeit with reduced efficiency, which suggested that the RNA turnover process carries quantitative features.
On the intruiging question why post-transcriptional gene-silencing is induced in only some of the transgenic lines, we revealed (Chapter 6) that the organisation of integrated transgene sequences has an important role. Transformation with a transgene containing a directly repeated MP gene, increased the frequency at which resistant lines arise to 60%, compared to 20% of resistant lines that occur upon transformation with a transgene with a single MP gene. Thus, the resistance process seems influenced by qualitative features of the integrated transgenes. Also, it was observed that resistance concurred with extensive methylation at the transcribed transgene sequences (Chapter 6), which could indicate an essential role of methylation at transcribed sequences in obtaining RNA-mediated pathogen derived resistance.
From these observations and from data described in literature, a model for RNA-mediated virus resistance was made and presented in Chapter 6. In Chapter 7, the post-transcriptional gene-silencing phenomenon is discussed in more details and in addition an approach is presented by which the process could be exploited to efficiently engineer virus resistance or study plant gene expression.
Photothermal regulation of phenological development and growth in bambara groundnut (Vigna subterranea (L.) Verdc.)
Linnemann, A.R. - \ 1994
Agricultural University. Promotor(en): M. Wessel, co-promotor(en): R. Rabbinge; E. Westphal. - S.l. : Linnemann - ISBN 9789054853350 - 123
vigna - vignabonen - licht - fotoperiodiciteit - vigna - cowpeas - light - photoperiodism
The photothermal regulation of phenological development and growth in bambara groundnut ( Vigna subterranea ) wa s studied to elucidate the crop's potential and limitations in current or future cropping systems. Eight accessions from the germplasm collection of the International Institute of Tropical Agriculture (IITA) were used for a preliminary assessment of sensitivity to photoperiod. In some accessions, photoperiods of 14 h or longer delayed or inhibited the onset of two developmental stages, flowering and podding, in comparison with photoperiods of 11 h or less. Within an accession, podding was always affected more than flowering. Microscopic studies in the laboratory enabled the cause of the delay or absence of the onset of podding under long photoperiods (14 h or more) to be identified as a check in the growth of fertilized ovaries. The minimal inductive period for the onset of podding was determined for two accessions differing in sensitivity to photoperiod. In the comparatively photoperiod-sensitive accession, the time of pod induction in relation to pod position on the plant was studied too. Three accessions were used to test whether the rate of progress towards flowering and the rate of progress towards podding could be described as functions of mean diurnal temperature and/or photoperiod. Finally, earlier indications that photoperiod influences growth as well as development were verified. The results of the experiments demonstrate the flexibility in the development of bambara groundnut, particularly in relation to changes in photoperiod. This flexibility largely explains why the crop can produce itself under marginal conditions in rain-fed areas.
|Cultivation of bambara groundnut (Vigna subterranea (L.) Verdc.) in Western Province, Zambia : report of a field study
Linnemann, A.R. - \ 1990
Wageningen : Agricultural University (Tropical crops communication / Wageningen Agricultural University, Department of Tropical Crop Science no. 16) - 34
vignabonen - teelt - cultuurmethoden - peulvruchten - vigna - zambia - cowpeas - cultivation - cultural methods - grain legumes
Early stages in cowpea chlorotic mottle virus infection
Roenhorst, J.W. - \ 1989
Agricultural University. Promotor(en): R.W. Goldbach; B.J.M. Verduin. - S.l. : Roenhorst - 99
plantenziekten - plantenvirussen - vigna - vignabonen - virussen - cellen - relaties - plant diseases - plant viruses - vigna - cowpeas - viruses - cells - relationships
Virussen zijn infectieuze eenheden, bestaande uit nucleïnezuren welke omgeven zijn door een eiwitmantel en eventueel een membraan. Voor wat betreft hun vermenigvuldiging zijn virussen afhankelijk van een levende gastheercel, waarbij ze over het vermogen moeten beschikken deze binnen te dringen, zich erin te vermenigvuldigen en zich vervolgens naar andere cellen te verspreiden.
Gedurende de laatste decennia is onze kennis over virussen en hun vermogen tot infectie enorm toegenomen. De opkomst van de moleculair- en celbiologische technieken heeft bijgedragen tot een gedetailleerde kennis van zowel de organisatie van het virale genoom, als de mechanismen die aan de expressie en replicatie ervan ten grondslag liggen. Daarnaast hebben biofysische studies informatie verschaft over de structuur en assemblage van het virale deeltje. De kennis over de initiële stadia van het infectieproces, het binnendringen in de gastheercel en de ontmanteling van het virale genoom bleef echter zeer schaars. Het feit dat in veel gevallen slechts enkele virusdeeltjes verantwoordelijk zijn voor infectie heeft vooral bestudering van de initiële interacties tussen virus en gastheercel aanzienlijk bemoeilijkt.
Dit proefschrift beschrijft het onderzoek verricht aan deze initiële stadia van virusinfecties bij planten. Twee vragen stonden hierbij centraal: (1) hoe en in welke vorm dringt een plantevirus een te infecteren cel binnen en (2) welk mechanisme is verantwoordelijk voor de ontmanteling van het virale genoom? Als model voor dit onderzoek is gekozen voor cowpea chlorotic mottle virus (CCMV) en geïsoleerde cowpea ( Vigna unguiculata ) mesophyl protoplasten. Een aantal eigenschappen van dit modelsysteem zijn weergegeven in de inleiding (Hoofdstuk l).
Met betrekking tot de initiële interacties tussen virus en gastheercel is voor zowel dierals plantevirussen een beknopt literatuuroverzicht gegeven van de tot nu toe bekende mechanismen (Hoofdstuk 2). Zowel op het niveau van de binding aan het celoppervlak, het binnendringen in de gastheercel, als ook op het niveau van de ontmanteling van het virale genoom, zijn verschillen en overeenkomsten tussen dier- en plantevirussen belicht. De meest opmerkelijke verschillen werden aangetroffen bij de binding en binnendringing van de gastheercel. Is voor de meeste diervirussen binding aan specifieke componenten van de plasmamembraan (receptoren) noodzakelijk voor penetratie, voor plantevirussen lijkt alleen een beschadiging van de plasmamembraan voldoende.
Om na te gaan in hoeverre ook voor plantevirussen (specifieke) interacties met membraancomponenten van belang zijn voor infectie van de cel, is de binding van CCMV aan cowpea protoplasten -bestudeerd in relatie tot binnendringing en infectie (Hoofdstuk 3). Uit de resultaten van dit onderzoek bleek dat de binding van CCMV aan geïsoleerde protoplasten gebaseerd is op (aspecifieke) elektrostatische interacties, terwijl penetratie afhankelijk is van mechanische beschadiging van de plasmamembraan. Tevens zijn aanwijzingen verkregen dat virusdeeltjes, welke middels endocytose worden opgenomen, niet betrokken zijn bij infectie van de cel. Uit deze gegevens werd geconcludeerd dat alleen die virusdeeltjes, die via membraanbeschadigingen direct in het cytoplasma van de gastheercel terecht komen, verantwoordelijk zijn voor infectie. Ten gevolge hiervan moet worden verondersteld dat ontmanteling van het virale genoom intracellulair plaatsvindt.
Aangezien voor het tabaksmozaïekvirus (TMV) inmiddels sterke aanwijzingen waren verkregen dat cytoplasmatische ribosomen een rol spelen bij ontmanteling van het genoom middels "cotranslational disassembly", werd nagegaan in hoeverre dit mechanisme ook van toepassing zou kunnen zijn op CCMV. "Cotranslational disassembly" veronderstelt dat ontmanteling plaatsvindt gelijk-tijdig met translatie van het virale genoom, waarbij in het geval van TMV het virusdeeltje vooraf zodanig behandeld moet worden dat het 5'-uiteinde van het RNA beschikbaar is voor initiatie van translatie.
Door in eerste instantie gebruik te maken van celvrije translatiesystemen werden aanwijzingen verkregen dat ook voor CCMV "cotranslational disassembly" een rol zou kunnen spelen bij de ontmanteling (Hoofdstuk 4). Na toevoeging van intacte virusdeeltjes aan dergelijke systemen werd synthese van virus specifieke eiwitten waargenomen. Bovendien kon de aanwezigheid van translationeel actieve virus-ribosoom complexen worden aangetoond.
De interactie tussen CCMV en ribosomen werd vervolgens nader geanalyseerd op eiwitniveau (Hoofdstuk 5). Hiertoe werden electroblots van ribosomale eiwitten, gescheiden onder denaturerende omstandigheden, geïncubeerd met virus, en werd het gebonden virus met behulp van immunologische methoden zichtbaar gemaakt. Ongeveer twintig eiwitten, behorend tot zowel de grote als kleine ribosomale subeenheden, bleken virus en/of viraal mantel-eiwit te binden. Dezelfde ribosomale eiwitten bleken eveneens betrokken te zijn bij binding van een aantal andere plantevirussen. Hoewel de beschreven experimenten hierover onvoldoende uitsluitsel geven, zou de waargenomen binding van virus aan deze ribosomale eiwitten op een functionele rol kunnen duiden.
Naast deze analyse op eiwitniveau is tevens gekeken naar de rol van "cotranslational disassembly" in de ontmanteling van CCMV in vivo (Hoofdstuk 6). Op verschillende tijdstippen na inoculatie werden met CCMV geïnoculeerde cowpea protoplasten gelyseerd en vervolgens geanalyseerd op de aanwezigheid van virus-ribosoom complexen. Inderdaad werden virus-ribosoom complexen aangetroffen, echter in tegenstelling tot in vitro, kon hun translationele activiteit in vivo niet worden aangetoond. Hiervoor wordt een aantal mogelijke verklaringen gegeven. Anderzijds benadrukten ook deze resultaten nogmaals de sterke affiniteit tussen CCMV en ribosomen, en werden aanwijzingen verkregen dat de vorming van virus-ribosoom complexen vooraf zou kunnen gaan aan een eventuele initiatie van "cotranslational disassembly".
Tenslotte zijn de in de diverse experimentele systemen verkregen gegevens samengevat in een model (Hoofdstuk 7, Figuur l). Dit model beoogt een beeld te geven van die gebeurtenissen die van toepassing zijn op de wellicht minder dan 0,01 % van de virusdeeltjes die verantwoordelijk zijn voor infectie van de cel. Met name daar waar het de interactie tussen virusdeeltje en ribosoom betreft, voorafgaand aan de initiatie van "cotranslational disassembly", bestaan nog veel vraagtekens. Een verdere ontrafeling van deze complexe interacties, alsmede het aantonen van "cotranslational disassembly" van CCMV in vivo. kunnen bijdragen tot een beter begrip van de initiële stadia van virusinfecties bij planten.
|Cultivation of Bambara groundnut (Vigna subterranea (L.) Verdc.) in Northern Nigeria : report of a field study
Linnemann, A.R. - \ 1988
Wageningen : Wageningen Agricultural University (Tropical crops communication no. 15) - 14
vignabonen - peulvruchten - nigeria - vigna - cowpeas - grain legumes
Localization of viral antigens in leaf protoplasts and plants by immunogold labelling
Lent, J.W.M. van - \ 1988
Agricultural University. Promotor(en): J.P.H. van der Want; B.J.M. Verduin. - S.l. : van Lent - 125
bromovirus - cellen - vignabonen - cytologie - histologie - methodologie - plantenziekten - plantenvirussen - relaties - technieken - vigna - virologie - virussen - bromovirus - cells - cowpeas - cytology - histology - methodology - plant diseases - plant viruses - relationships - techniques - vigna - virology - viruses
This thesis describes the application of an immunocytochemical technique, immunogold labelling, new in the light and electron microscopic study of the plant viral infection. In Chapter 1 the present state of knowledge of the plant viral infection process, as revealed by insitu studies of infected cells, is briefly reviewed. Until now, light and electron microscopic studies have merely described morphological changes in cells and tissue as a result of viral infection, but have failed to provide information on the functional role of these structures in the viral infection process and their association with viral components. A common cytopathological feature of many different plant viruses seems to be the induction of membranous vesicles or membranous bodies, which have been implicated in viral replication. However, only in a few cases some evidence was obtained with regard to the Intracellular location of viral replication and the association of replication and membranes. Available cytochemical techniques have apparently failed to provide a tool for the identification of virus particles and virus-encoded proteins within cellular structures. The Impact of a suitable detection techniques to elucidate the molecular processes of viral replication and transport insitu is obvious, as it would link findings obtained by invitro experiments to the events observed in the cell.
Immunogold labelling seems to provide such a tool for the tracing of antigens in light and electron microscopic preparations of biological specimens. Gold particles are excellent markers for electron microscopy, because of their high electron density which makes them appear as black dots In EM preparations. Furthermore, by a simple silver staining following gold labelling, viral antigens can be dete cted in semi-thin sections with the light microscope. The application of immunogold labelling for the light and electron microscopic localization of antigens is described in Chapters 2, 3, 4, 5, 6 and 7.
In Chapter 2 the preparation of homodisperse suspensions of colloidal gold particles is described. By adsorption of protein A to the surface of the gold particles, a marker (protein A-gold, pAg) is obtained which can be used for labelling antigen-antibody complexes. The specificity of the technique was demonstrated by gold labelling of antibodies bound to plant viruses in mixed suspensions of two viruses. Each virus was labelled using its homologous antiserum and pAg, and no significant cross-reaction with the other virus occurred. Simultaneous identification of two different viruses (CCMV and SBMV) with similar morphological appearance was achieved by double labelling with pAg-complexes containing gold particles of 7 and 16 nm, respectively. Immunogold labelling of viral antigens in suspension has been applied to distinguish between different serologically related viruses like strains of TMV (Pares and Whitecross, 1982), and the potyvirus sugarcane mosaic virus and maize dwarf mosaic virus (Alexander and Toler, 1986; 1985). A clear advantage of the immunogold labelling over conventional decoration of antigens is that the discrete gold particles allow quantification of the results.
The immunogold labelling of viral antigen in ultrathin sections of infected protoplasts is described in Chapter 3. Best results were obtained when the protoplasts were only mildly fixed with aldehydes, dehydrated and finally embedded in Lowicryl K4M at -30°C. The antigenicity of viral coat protein was well preserved. A disadvantage of the method is the limited preservation of cell structures, especially membranes due to extraction of lipids. Weibull etal. (1983) reported that approximately 50% of the lipid content of cells may be extracted, despite the low temperatures used in the Lowicryl K4M embedding procedure. Ashford etal. (1986) questioned the low temperature character of Lowicryl embedding, and found that during polymerization of the resin, temperature rises due to the exothermic nature of the reaction. With plant tissue (not protoplasts), low temperature dehydration and infiltration of the embedding resin must be prolonged, to allow sufficient penetration of the chemicals through the thick walls surrounding the plant cells, and this may result in even more extraction than reported by Weibull and colleagues. Rapid dehydration in ethanol and infiltration of plant tissue with a polar resin like LR White at ambient temperatures, therefore, seems to be a good alternative (Newman etal. , 1983; Causton, 1984; Newman and Jasani, 1984).
Light microscopic localization of viral antigen in semi-thin sections of LR White embedded plant tissue is described in Chapter 6. CCMV was successfully localized in petiolules of systemically inoculated cowpea plants by immunogold labelling and subsequent silver staining (immunogold/silver staining: IGSS). The silver stain could be observed in the light microscope by brightfield, darkfield and phase-contrast illumination. Most sensitive detection, however, was obtained with epi- illumination using polarized light (epipolarization microscopy). Combining epipolarization illumination with brightfield illumination allowed the simultaneous observation of silver stain and cell morphology.
Immunogold labelling and IGSS in combination with appropriate fixation and embedding of biological specimens, appear to be efficient and simple techniques for the insitu identification and localization of antigens, with many advantages over other immunochemical and cytochemical techniques, like ferritin- labelling, peroxidase-anti-peroxidase, immunofluorescence and autoradiography, which have only incidentally been used in plant virus research. Recently, Patterson and Verduin (1987) have reviewed the literature on the use of immunogold labelling in animal and plant virology, showing numerous fields of applications and discussing progress made in virus research. With respect to the technique the authors rightly concluded that immunogold labelling is a flexible technique with little limitation for the improvement of existing assays and the development of new ones.
Using immunogold labelling to identify and localize virus particles and coat protein, CCMV- infection in cowpea protoplasts was studied as function of the infection time. Observations with regard to virus entry into protoplasts are reported in Chapter 3. Upon inoculation aggregates of virus particles were observed attached to the plasmamembrane, or sometimes penetrating the plasmamembrane at places where the membrane appeared to be damaged. Virus was also found inside vesicles formed by invagination of the plasmamembrane. These vesicles with inoculum-virus particles were stable over long periods of time. Large vesicles (vacuoles) containing viral antigen were also detected at 24 h post-inoculation in protoplasts which were not infected by CCMV.
The mechanism by which plant viruses enter their host cells is still disputed (Shaw, 1986). Passage of the plasmalemma by endocytosis was suggested by Takebe (1975), and through pores or lesions by Burgess etal. (1973) and Watts etal. (1981). Our observations do not favour endocytosis to be the mechanism of virus entry leading to infection of the protoplasts as virus containing vesicles are stable. Recently, Roenhorst etal. (1988) presented data supporting a mechanism of virus entry by initial physical association of virus particles with the protoplast membrane and subsequent invasion of virus particles through membrane lesions. Such a mechanism may be also applicable to the cytoplasmic extrusions observed by Laidlaw (1987) after puncturing plant epidermal cells. The author suggested that virus particles may adsorb to the plasmalemma covering the extrusions, which are then withdrawn into the cell. Invasion of whole particles through membrane lesions may then be followed by a uncoating and initial translation (cotranslational disassembly) at the cytoplasmic ribosomes as suggested by Wilson (1985).
Ultrastructure of RNA-inoculated protoplasts was studied in sections of aldehyde- and osmium-fixed protoplasts (Chapter 4). Cytological alterations attributed to virus infection consisted of dilation of the endoplasmic reticulum (ER) and the formation of vesicles early in infection. Distended ER and vesicles seemed to form a kind of membranous area in the cytoplasm. In protoplasts fixed and embedded in Lowicryl K4M newly synthesized virus particles or coat protein were first localized in restricted areas of the cytoplasm at 6-9 h post-inoculation. The rough appearance of the cytoplasm in these areas suggested the presence of membranous structures like observed in osmium-fixed protoplasts. However, due to poor membrane preservation in Lowicryl embedded material this could not be proven. Within one protoplast several of these labelled areas were identified. At later stages of infection viral antigen was located throughout the cytoplasm, but also in the nucleus and in particular the nucleolus. No viral antigen was detected in or specifically associated with chloroplasts, mitochondria, microbodies and vacuoles. The specificity of gold labelling was demonstrated by quantification of the labelling density on sections of infected and non-infected protoplasts. These results indicate that CCMV coat protein synthesis and virus assembly take place in the cytoplasm of plant cells, but the involvement of cellular structures, in particular membranes, remains to be established. Protein synthesis and virus assembly may occur in certain restricted sites (compartments) in the cytoplasm possibly formed by the membranous bodies. Compartmentalization of the cytoplasm, creating different environments in the cell, may explain the occurrence of both disassembly and assembly in the same cell, and furthermore account for the phenomenon of specific assembly of viral RUA and homologous coat protein in cells infected with two related viruses like CCMV and BMV (Sakai etal. , 1983 ; Zaitlin and Hull, 1987). Whether RNA-replication also occurs in the same location as coat protein synthesis and virus assembly could be established by localization of non-structural virus encoded proteins involved in viral replication. However, antisera against these products of the CCMV-genome were not available. The function of CCMV coat protein or virus in the nucleus and especially the nucleolus is not known. Coat protein may have an affinity for ribosomal proteins and/or fulfill some functional role in the viral replication. Kim 1977 described the occurrence of filamentous inclusions (FI) in the nucleus often associated with the nucleolus. These FI were not found in the nuclei of cowpea protoplasts (this study) or tobacco protoplasts (Burgess etal. , 1974), but may be formed later in the infection by excess coat protein. Bancroft etal. (1969) showed the ability of CCMV-coat protein to form narrow tubules under specific conditions. The (FI) described by Kim (1977) may represent this type of coat protein aggregation, although the chemical composition of the (FI) is not yet known.
In Chapter 5 preliminary observations are reported on the localization of sites of CPMV replication in cowpea protoplasts, by in situ detection of coat proteins and non-structural proteins involved in viral replication and proteolytic processing. With regard to virus entry and subsequent locations of inoculum virus inside vesicles, similar phenomena were observed as in infection with CCMV. Infection of CPMV generates large inclusion bodies in the cytoplasm, consisting of membranous vesicles with fibrillary material and adjoining amorphous electron-dense material which have been observed as early as 12 h post- inoculation. Virus particles and/or coat protein were first detected 24 h after inoculation throughout the entire cytoplasm and in between the membranous vesicles and electron dense material. The 24K, 170K and their precursor proteins were exclusively localized in the electron dense material and not in association with the membranous vesicles or any other location in the cell. These results show that the electron-dense material consists at least in part of CPMV-encoded non-structural proteins and may represent a site for accumulation of non-functional proteins. The membranous vesicles have been implicated in viral RNA synthesis (Goldbach and Van Kammen, 1985). The failure to detect non- structural proteins in association with these membranes may be explained by either a low concentration of these proteins at the site of replication or by extraction of these proteins during the fixation and embedding procedure, despite the low temperature.
With IGSS the distribution of CCMV in cowpea plants was monitored at different times after systemic inoculation according to Dawson and Sehlegel (1976) (Chapters 6 and 7). No virus was detected at the time of temperature shift (t=0) in petiolule and leaves of plants subjected to 3 days of differential temperature treatment. Virus was first localized in phloem parenchyma cells of petiolule and veins at t=3 h and from there it spread to neighbouring tissues. Twenty four hours after systemic inoculation virus was located in the phloem, bundle sheath, cortex, but also in the cambium and some xylem cells. These results show that CCMV is transported from the inoculated primary leaves to the secondary leaves through the phloem, apparently following the route of metabolites. This finding is in agreement and further supports the generally accepted concept of plant virus long-distance transport through phloem. tissue (Matthews, 1982; Atabekov and Dorokhov, 1984). The failure to detect CCMV in differentiated sieve elements may indicate that the form in which the infectious entity is transported is another than virus particles (Atabekov and Dorokhov, 1984), or that the amount of virus transported through the sieve elements is below detectable levels. The true character of the synchrony of infection of leaf mesophyll cells obtained by differential temperature treatment is disputed. Infection of mesophyll tells may have been accomplished after shifting the plants to higher temperature by fast transport of infectious particles from the vascular tissue, as was also suggested by Dorokhov etal. (1981).
For the first time a suitable method for localization of antigens is available, which can be routinely applied for both light and electron microscopic study of the plant viral infection process. The application of the gold labelling technique in the localization of viral structural and non-structural proteins has been demonstrated, using CCMV- and CPMV-infections of plant cells as model system.
With regard to the technique, future work must be done on the improvement of the preservation of cellular structures, especially membranes, as these appear only poorly in Lowicryl embedded plant tissue even with dehydration, infiltration and polymerization at low temperatures. Alternatives, may be found in cryofixation and cryosectioning or freeze-substitution techniques.
With regard to the study of the plant viral infection process, the localization of virus-encoded proteins involved in replication and transport, but also the localization of plant viral nucleic acids by insitu hybridization, will contribute to the understanding of the mechanisms underlying these events. New biochemical techniques like the production of infectious transcripts from cloned viral cDNA (Ahlquist etal. 1984) enabling genetic manipulation of the viral genome, and integration of plant viral genes into the plant genome (Gardner etal. , 1984; Abel etal. , 1986) will supply future model systems for the study of virus-host interactions.
|Detection of viral antigen in semi-thin sections of plant tissue by immunogold-silver staining and light-microscopy.
Lent, J.W.M. van; Verduin, B.J.M. - \ 1987
Netherlands Journal of Plant Pathology 93 (1987). - ISSN 0028-2944 - p. 261 - 272.
koebonenmozaïekvirus - vignabonen - elisa - immunologische technieken - plantenziekten - plantenvirussen - kleuring - vigna - cowpea mosaic virus - cowpeas - elisa - immunological techniques - plant diseases - plant viruses - staining - vigna
Is a helper factor necessary for infection of cowpea protoplasts with blackeye cowpea mosaic virus?
Dijkstra, J. ; Beek, N.A.M. van; Lohuis, D. ; Helden, M. van; Meijer, R. - \ 1987
Netherlands Journal of Plant Pathology 93 (1987). - ISSN 0028-2944 - p. 43 - 47.
koebonenmozaïekvirus - vignabonen - plantenziekten - plantenvirussen - vigna - cowpea mosaic virus - cowpeas - plant diseases - plant viruses - vigna
De omstandigheden worden beschreven, waaronder een infectie van 'cowpea'-proplasten met het 'blackeye cowpea mosaic virus' mogelijk is
Infection of cowpea protoplasts with sonchus yellow net virus and festuca leaf streak virus
Beek, N.A.M. van - \ 1986
Landbouwhogeschool Wageningen. Promotor(en): J.P.H. van der Want; J.P.H. Dijkstra. - Wageningen : Van Beek - 67
cellen - vignabonen - plantenziekten - plantenvirussen - rhabdoviridae - vigna - virussen - cells - cowpeas - plant diseases - plant viruses - rhabdoviridae - vigna - viruses
The advantages of protoplast systems for plant virus research have been frequently reviewed (Zaitlin & Beachy, 1974; Takebe, 1975; Muhlbach, 1982; Sander & Mertens, 1984). Relatively little attention has been given to the limitations of such a system.Protoplasts do not exist under natural conditions. They lack a rigid cell wall and cell-to-cell connections are absent. Protoplasts are maintained in media that differ from the milieu in plant tissue with respect to nutrient composition, hormone balance, and, most importantly, tonicity. Several authors have documented the effects of osmotic stress in protoplasts of various sources (Lazar et al., 1973; Prevecz et al., 1978; Fleck et al. , 1982; Meyer & Aspart, 1983). These effects include altered nucleic acid and protein synthesis. Lazar et al. (1973) reported a more than ten-fold increase in RNase level upon protoplasts isolation. Although no reports have appeared that document effects of the altered physiological state of protoplasts on virus multiplication, extrapolation from the level of protoplast to plant must be done with caution.Our studies on Sonchus yellow net virus (SYNV) and Festuca leaf streak virus (FLSV) have been carried out with the aid of protoplasts, derived from the cowpea plant, a non-host for these viruses. Thus, our studies certainly are subject to the above stated limitations.In Chapter II we report on the infection of cowpea protoplasts with SYNV. The infection is mediated by polyethylene glycol, a compound that induces membrane fusion. Viral replication was demonstrated by the results of a biological assay, and it was shown that over 90% of the living protoplasts could be infected. Those SYNV particles, from which the envelope was removed, were much less efficiently introduced in protoplasts, indicating that fusion of viral envelope and protoplast membrane was an important mechanism for introduction. Conditions during the inoculation procedure were optimized. We showed that infection was blocked when a divalent cation chelating compound was included in the inoculation medium.A description at the ultrastructural level of the subsequent stages in the infection of cowpea protoplasts by SYNV is presented in Chapter III. The model for the budding process given by Francki (1973) is disputed. We observed virus particles in intermediate stages of budding that are highly suggestive for a simultaneous occurrence of coiling and budding as predicted by Peters & Schulz (1975). Budding of precoiled nucleoprotein strands as hypothesized by Francki (1973) was never observed.Budding of virus particles was prevented by tunicamycin. This led to the accumulation of massive amounts of coiled nucleoprotein strands and granular material in the nucleus. These structures were not seen in the cytoplasm, providing evidence that the inner nuclear membrane is the only site of assembly of SYNVAt later stages in infection, coiled nucleoprotein strands were observed free in the cytoplasm. They were shown to originate from mature particles as a consequence of fusion of their envelope with the endoplasmic reticulum membrane, followed by the release of the coiled nucleoprotein strand in the cytoplasm. The significance of these structures with respect to the decrease in infectivity of plant tissue and protoplasts in later stages of infection, and with respect to spread of the virus from cell-to-cell, is also discussed.In Chapter IV we show morphological stages of FLSV particles in cowpea protoplasts. Replication cannot be demonstrated formally since this virus has not been transmitted mechanically and a vector has not yet been identified. Thus, the infection of cowpea protoplasts with FLSV, until now only found in the monocotyledon Festuca gigantea has merits of its own,FLSV assembles at intracytoplasmic membranes. Striking similarities were noticed between the processes occurring in the cytoplasm of FLSV-infected protoplasts and those occurring in the nucleus of SYNV-infected protoplasts.A study on the protein synthesis of SYNV is presented in Chapter V. Four out of five structural proteins of SYNV (G, N, M 1 and M 2 ) were detected in infected protoplasts by gel electrophoresis of immunoprecipitates. The L protein could not be identified. The M 1 protein was shown to be phosphorylated and a polypeptide with a molecular weight of 38,000 was presumed to represent a less phosphorylated form of the M 1 protein. The G protein was proven to be glycosylated by N-glycosidical linked residues. The addition of 10 μg tunicamycin per ml incubation medium prevented glycosylation without markedly affecting protein synthesis. A nonglycosylated form of the G protein was not detected.
Molecular interactions during the assembly of cowpea chlorotic mottle virus studied by magnetic resonance
Vriend, G. - \ 1983
Landbouwhogeschool Wageningen. Promotor(en): T.J. Schaafsma, co-promotor(en): M.A. Heeminga. - Wageningen : Vriend - 121
cellen - vignabonen - plantenvirussen - vigna - virussen - cells - cowpeas - plant viruses - vigna - viruses
This thesis describes the application of 1 H- and 13 C- NMR, EPR, ST-EPR and calculational methods to study cowpea chlorotic mottle virus. This virus consists of RNA encapsidated by 180 identical protein subunits, arranged icosahedrally. The isolated coat protein of cowpea chlorotic mottle virus can be brougth into several well defined states of aggregation. This study could be carried out, because these stages can be produced in quantities sufficient to allow magnetic resonance measurements. All the results obtained are combined in the following model for the assembly proces of the virus:In this model a rigid protein core with a highly mobile, basic, N- terminal arm is invoked. This arm is the RNA binding part of the protein. The high mobility of this arm enhances the probability of interaction with the RNA and enables the protein to exhibit different modes of bonding to different local structures of the RNA. Upon binding the RNA, the N-terminal arm adopts a rigid a-helical conformation.In chapter two it is shown that virtually no mobility on a timescale faster than 10 -7 s can be observed in the virus.
In chapter three an EPR and ST-EPR study is presented on spin-labelled virus, From the results it is concluded that no anisotropic subunit mobility is present in the virus on the 10 -5 -10 -7 s timescale. Also in this chapter it is suggested that many ST-EPR results on maleimide- labelled proteins, in which anisotropic protein mobility was invoked for the interpretation of the spectra, are based on artefacts.
Chapter four shows that the N-terminal arm is the RNA binding part of the protein. This arm is found to be very mobile in the absence of RNA, whereas immobilization occurs upon binding RNA.
Chapter five is a small excursion to other plant viruses. It is shown that brome mosaic virus and belladonna mottle virus show the same behaviour as cowpea chlorotic mottle virus, whereas cowpea mosaic virus behaves completely different.
Chapter six deals with the molecular interactions during the assembly process. In this chapter it is shown that the arginine and lysine containing part of the N-terminal arm is responsible for the binding of oligo-nucleotides. It is suggested that a double stranded nucleotide conformation is required for proper interaction.
In chapter 7 a secondary structure prediction of the coat protein of CCMV is presented. The results indicate that the N-terminal arm shows no structural preference when the positive charges of arginine and lysine are retained, whereas in the absence of these positive charges there is a tendency towards α-helix formation. Also it is concluded that cowpea chlorotic mottle virus possesses the same β-role topology as southern bean mosaic virus, sattelite tobacco necrosis virus and tomato bushy stunt virus.
Chapter eight presents the results of an energy calculation study on the N-terminal arm. Although the method used contains too many approximations to allow detailed conclusions, the results of chapter seven are fully confirmed by these calculations.
In the model for the protein-RNA interaction in CCMV which is presented in chapter five, a random coil conformation for the N-terminal protein arm is invoked. In chapter nine an extension of this part of the model is presented. It is shown that the N-terminal possesses some time-averaged secondary structure (presumably an α-helix) in the absence of RNA. A conformational exchange process is observed in the N-terminal arm, in which about half 'of the residues participate. In empty capsids not all the N-terminal arms turn out to be mobile. 20-55% of the N-terminal arms is immobile on the NMR timescale in the empty capsids of the coat protein of CCMV.
Proteins synthesized in tobacco mosaic virus infected protoplasts
Huber, R. - \ 1979
Landbouwhogeschool Wageningen. Promotor(en): A. van Kammen. - Wageningen : Veenman - 103
tabaksmozaïekvirus - nicotiana - tabak - vigna - vignabonen - celstructuur - eiwitten - cellen - virussen - plantenziekten - plantenplagen - gewasbescherming - plantenziektekunde - afwijkingen, planten - rna-virussen - Tobacco mosaic virus - nicotiana - tobacco - vigna - cowpeas - cell structure - proteins - cells - viruses - plant diseases - plant pests - plant protection - plant pathology - plant disorders - rna viruses
The study described here concerns the proteins, synthesized as a result of tobacco mosaic virus (TMV) multiplication in tobacco protoplasts and in cowpea protoplasts. The identification of proteins involved in the TMV infection, for instance in the virus RNA replication, helps to elucidate the infection process in the plant cell. Not only virus coded proteins, but possibly also host coded proteins may play a part in the TMV multiplication.
Research on proteins encoded by the TMV RNA, carried out in cell-free protein synthesizing systems, has revealed that five polypeptides are synthesized under the direction of TMV (subgenomic) mRNAs (see table 1.2., chapter L). Whether the polypeptides, synthesized invitro with TMV RNA as messenger, are of functional significance for the TMV infection may only be determined by means of investigating TMV infected leaves and protoplasts.
The TMV multiplication runs synchronously in all protoplasts that are infected. Therefore, proteins synthesized in small amounts upon infection, may be thus detected.
The search for proteins sythesized in protoplasts as a result of TMV infection has long been hindered by the fact that various factors in the cultivation of the tobacco plants may adversely influence the quality of the protoplasts. The cultivation of the tobacco plants: Nicotiana Tabacum cv. L. Samsun, Samsun NN and Xanthi nc, could be standardized however, as described in chapter 2. When the tobacco plants were cultivated in this way, at least 50 % of the tobacco protoplasts could be infected with TMV and 70 % or more of the protoplasts survived the subsequent incubation period of 36 hours. This could be achieved every time the protoplasts were isolated. The intensity and quality of the light, the way of watering, the age of the tobacco plants and of the leaf, from which the protoplasts are isolated, among others, appeared to affect the quality of the protoplasts (chapter 3.).
The proteins, synthesized upon TMV infection, have to be distinguished among a great variety of host proteins. For this reason it is important to determine the incorporation of radioactive amino acids into protein synthesized as a result of TMV multiplication, in comparison with the incorporation into host proteins that are formed independently from the virus infection. Therefore the specific activity of TMV coat protein (cpm/mg protein) and of the proteins of the 27,000 x g supernatant fraction, synthesized in infected tobacco protoplasts were compared. It appeared that the specific activity of TMV coat protein was at least four times higher than of the proteins in the 27,000 x g supernatant (chapter 4.).
The proteins synthesized as a result of TMV multiplication were studied not only in tobacco protoplasts, but also in protoplasts from the primary leaves of cowpea ( Vigna unguiculata (L.) Walp. var. 'Blackeye Early Ramshorn'). The method used for the infection of tobacco protoplasts with TMV was not suitable for the infection of cowpea protoplasts with TMV. Best results were obtained when both protoplasts and virus were incubated in the presence of poly-D-lysine, for 7.5 min. before infection. The protoplasts were pre-incubated in 0.1 M potassium phosphate buffer (pH 5.4) at 0°C, at a concentration of 4 x 10 5 protoplasts/mI and 0.75 μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2 μg TMV/mI and 2 μg poly-D-lysine/ml. During infection the cowpea protoplasts were incubated together with TMV and poly-D-lysine in a concentration of 2 x 10 5 protoplasts/ml, 1 μg TMV/ml and 1 μg poly-D-lysine/ml, for 7.5 min, in the buffer mentioned above at 0°C. In this way 50 to 70 % of the cowpea protoplasts could be infected with TMV.
The course of TMV synthesis in cowpea protoplasts was comparable with that in tobacco protoplasts. The TMV multiplication in cowpea protoplasts was preceeded, however, by a period of 16 hours, during which the increase of TMV is slight, while the TMV multiplication in tobacco protoplasts was preceeded by a lag period of 8 hours. A possible explanation is that a much smaller amount of TMV particles penetrates into cowpea protoplasts during inoculation and/or starts to multiply than is the case in tobacco protoplasts (chapter 5.).
The proteins of TMV infected and mock-infected protoplasts were analysed therupon by means of SDS-polyacrylamide slabgel electrophoresis and the polypeptide patterns were visualized by autoradiography.
Ten polypeptides were distinguished, which are synthesized as a result of TMV multiplication in polypeptide patterns of proteins from infected tobacco protoplasts. The molecular weights were estimated to be 260,000, 240,000, 170,000, 116,500, 96,000, 90,000, 82,000, 72,000, 30,000 and 17,500 (coat protein). Polypeptides of similar molecular weight were absent or were present to much less extent in polypeptide patterns of proteins from mock-infected tobacco protoplasts. Many polypeptides were observed for reason that the detection capacity was improved by means of subcellular fractionation of the protoplast homogenates.
The polypeptides of molecular weight 170,000, 116,500, 72,000 and coat protein were present in the 31,000 x g supernatant fraction and the pellet fractions as well. The polypeptide of molecular weight of 30,000 was present exclusively in the pellet fractions. The other polypeptides were observed exclusively in polypeptide patterns of protein of the 31,000 x g supernatant fraction (see table 6. l., chapter 6.).
Eight polypeptides were observed, which were synthesized as a result of TMV multiplication in cowpea protoplasts. The molecular weights of the polypeptides were approximately 150,000, 116,500, 86,000, 72,000, 17,500 (coat protein), 16,000,14,000 and 10,000. Polypeptides of similar molecular weight were absent or present on a far less extent in polypeptide patterns of proteins from mockinfected cowpea protoplasts.
The polypeptides of molecular weight 116,500, 72,000 and coat protein were present in the 3 1,000 xg pellet and 3 1,000 xg supernatant. The other polyeptides were present exclusively in the 3 1,000 xg supernatant (table 7. l., chapter 7.).
It was assumed that the TMV coded polypeptides are similar in different hosts and, on the other hand, that the host polypeptides, synthesized upon TMV infection differ from host to host. When the TMV specific polypeptides, synthesized in infected tobacco protoplasts were compared with the specific polypeptides synthesized in TMV infected cowpea protoplasts, it appeared that only the polypeptides of molecular weight 116,500, 72,000 and coat protein are of similar size in both hosts (table 7.2., chapter 7). This is an indication that not only the polypeptide of 116,500 daltons and coat protein are TMV coded polypeptides, but that also the polypeptide of 72,000 daltons is encoded in the TMV RNA. It has not been reported that a polypeptide of this size is observed when TMV RNAs are translated in cell-free protein synthesizing systems.
A polypeptide of 170,000 daltons is synthesized in vitro under the direction of the TMV RNA. It appeared that the polypeptide synthesized in TMV infected tobacco leaves, has a slightly less electrophoretic mobility than the product of 170,000 daltons synthesized in vitro from TMV RNA as messenger. A polypeptide of similar electrophoretic mobility was present to a lesser extent in mockinfected tobacco protoplasts. Furthermore, a polypeptide of 170,000 daltons was not observed in TMV infected cowpea protoplasts. For these reasons it is likely, that the polypeptide of 170,000 daltons, synthesized in TMV infected tobacco protoplasts, is encoded in the genome of tobacco or is encoded in the TMV RNA, but then the polypeptide has no functional significance in the TMV multiplication process.
Further the polypeptide of 30,000 was observed only in TMV infected tobacco protoplasts, whereas a polypeptide of similar molecular weight was shown to be synthesized in vitro from a TMV subgenomic mRNA. The polypeptide of 30,000 daltons was detected exclusively in the polypeptide patterns of protein from the pellet fractions of TMV infected tobacco protoplasts. Polypeptide patterns of protein from corresponding fractions of cowpea protoplasts had a predominant, grey background. Due to this the polypeptide of 30,000 daltons may not be distinguished in TMV infected cowpea protoplasts, whereas the polypeptide of 30,000 daltons synthesized in TMV infected tobacco protoplasts can in fact be a polypeptide coded by TMV RNA. The other polypeptides synthesized in infected tobacco protoplasts or cowpea protoplasts as a result of TMV multiplication are presumably synthesized under the genome of tobacco or cowpea respectively.
Finally, it was attempted to examine in what way the polypeptides of 116,500 and 72,000 are involved in the TMV infection process. Both polypeptides were shown to be present in the 31,000 x g pellet of TMV infected tobacco and cowpea protoplasts. It was studied whether virus specific polypeptides of similar molecular weight can be observed in RNA-dependent RNA polymerase preparations isolated from the 31,000 x g pellet fraction of cowpea leaves infected with the cowpea strain of TMV (C-TMV). The RNA-dependent RNA polymerase preparations were isolated by extraction of the 31,000 x g pellet fraction and were further purified by means of subsequent DEAE-BioGel column chromatography and glycerol gradient centrifugation. The purification procedure used was the same procedure as described for the isolation of RNA-dependent RNA polymerase from cowpea leaves infected with cowpea mosiac virus (CPMV).
Four specific polypeptides of molecular weight of 98,000, 90,000, 72,000 and 46,000 were distinguished in RNA-dependent RNA polymerase preparations from C-TMV infected cowpea leaves, after glycerol gradient purifications. A polypeptide of molecular weight 116,500 was not observed. Polypeptides of molecular weights 72,000 and 46,000 were not found and those of molecular weights 98,000 and 90,000 were distinguished to a less extent in polypeptide patterns of preparations isolated in exactly the same way from mock-inoculated cowpea leaves.
RNA-dependent RNA polymerase activity was also observed in preparations isolated from mock-inoculated cowpea leaves. The specific activity (cpm/mg protein) of the preparation from mock-inoculated leaves was one sixth of the specific activity of the RNA-dependent RNA polymerase preparations from CTMV infected cowpea leaves. The RNA-dependent RNA polymerase activity in C-TMV infected cowpea leaves might therefore be attributed to the increase of one or several polypeptides, present already before inoculation. Since it was thought that the polypeptide of 72,000 daltons is a TMV coded polypeptide, it was examined which specific polypeptides are present in RNA-dependent RNA polymerase preparations isolated in a similar way from CPMV infected cowpea leaves. It appeared, that in addition to CPMV specific polypeptides, the polypeptides of molecular weight 98,000 and 90,000 were also observed in RNAdependent RNA polymerase preparations from CPMV infected leaves. The polypeptides of 72,000 and 46,000 daltons were distinguished only in preparations isolated from C-TMV infected cowpea leaves. These results suggest that the polypeptide of 72,000 daltons in involved is the synthesis of TMV RNA (chapter 8.).
Characterization of cowpea chlorotic mottle virus and its assembly
Verduin, B.J.M. - \ 1978
Landbouwhogeschool Wageningen. Promotor(en): A. van Kammen, co-promotor(en): J.P.H. van der Want. - Wageningen : [s.n.] - 134
bromovirus - vignabonen - in vitro - plantenziekten - plantenvirussen - replicatie - synthese - vigna - virussen - bromovirus - cowpeas - in vitro - plant diseases - plant viruses - replication - synthesis - vigna - viruses
This thesis decribes the conditions for isolation of cowpea chlorotic mottle virus (CCMV), its ribonucleic acid (RNA) and the coat protein, the characterization of the virus and its constituents (chapter 3, 4 and 5) and the dissociation and assembly behaviour of the virus (chapter 6 and 7).The aim of the investigation and a literature review pertaining to RNAprotein interactions, which are met with the tobamoviruses, the Leviviridae and the bromoviruses are given in chapter 1 and 2.CCMV, isolated and purified in the prescence of reducing agents such as ascorbic acid and mercaptoethanol, contained variable amounts of degraded RNA. At first RNA-2 was cleaved into two fragments but subsequently all RNA molecules were cleaved at random sites. The buoyant density in RbCl and the sedimentation coefficient of the virus remained unchanged i.e. the degraded RNA was still bound to the protein coat and did not change the stability of the nucleoprotein particles.The degradation of the RNA was stimulated when virus was incubated at 37°C in the presence of reducing agents such as mereaptoethanol. Besides reducing agents also oxygen and traces of metals appeared to play a role in the degradation process. Addition of chelating agents, such as 1 mM EDTA, to the homogenization buffer and the buffers in which the virus was kept, prevented in situ RNA degradation. Problably the degradation is caused by radicals, which are formed during the auto-oxidation of reducing agents by oxygen, catalysed by traces of metals (chapter 3).In chapter 4 a describtion is given of three coat protein isolation methods and the influence of the isolation method on the formation of pseudo top component (PT) i.e. an empty protein shell without RNA. By means of CaCl 2 RNA free coat protein could be isolated from virus, even when the virus particles contained exstensively degraded RNA. The formation of PT and its dissociation were pH dependent and both processes showed a remarkable hysteris effect. This effect can be explained by assuming two stable conformations of the coat protein.In chapter 5 the results of partial specific volume, ciruclar dichroism (CD) and sedimentation equilibrium measurements of CCMV are given. The apparent partial specific volume of the dissociated protein in 0.5 M CaCl 2 pH 7.5, mainly the dimer of the coat protein subunit, changed from 0.737 cm 3/g to 0.728 cm 3/g in 0.2 M NaCI, 0.01 M CaCl 2 pH 5.0, mainly PT. Calculation of the partial specific volume of CCMV from the experimentally determined volumes of RNA, 0.476 cm 3/g and coat protein, 0.745 cm 3/g in 0.2 M NaCl. 1 mM EDTA pH 5.0 resulted in a value of 0.660 cm 3/g, which is lower than the experimentally determined partial specific volume of CCMV, 0.719 cm 3/g. The difference is caused by RNA-protein interaction.The CD measurements of protein dimers and PT showed little difference between the secondary structure of both protein subunit aggregates. The α-helix content was in both cases smaller than 1%. The structure of the RNA, both free in solution and inside the virus particle showed a large amount of base pairing and base stacking. Small changes in the secondary structure occured. when the virus was swollen and dissociated.Chapter 6 describes the pH and ionic strength dependent dissociation of CCMV. Upon increasing the pH from 5.0 to 7.5 at 1 M NaCl, CCMV formed RNA-protein complexes, which sedimented slower than the intact virus particles but still retained an RNA-protein ratio identical to virus. When CCMV was incubated at pH 7.5 with increasing concentrations of NaCl, at first unfolding of the RNA occured, while all the protein subunits were still bound to the RNA, followed by a gradual release of protein subunits. In 1 M NaCI the RNA retained 4 to 8 protein subunits per RNA molecule. This RNA-protein complex is probably involved in the recognition of the protein by the viral RNA.In chapter 7 is described how this RNA-protein complex has been used for assembly of virus particles, 90% of which is stable in RbCl. These particles obtained after dissociation and reassociation of CCMV were characterised with respect to RNA content and compared with virus particles obtained after assembly of isolated RNA and coat protein.After centrifugation in a sucrose gradient both reassociated and assembled virus showed a band with a sedimentation coefficient of about 80 S at the main product. These particles contained RNA-1 and -2, comparable to the original virus preparation but less RNA-3 and -4. Two other classes of products were observed. On one hand a fraction sedimenting between 70 and 80 S, which contained mainly particles with RNA-3 and on the other hand a fraction with a sedimentation coefficient>110 S, in which some particles with RNA-1 and -2 occured, but mainly particles with RNA-3 and -4. Only 40% of the nucleoprotein particles assembled from isolated RNA and protein appeared to be stable in RbCl.Probably RNA-1 and RNA-2 can form stable virus particles by means of the RNA-protein complex, while RNA-3 also has to make a link with RNA-4, before a stable nucleoprotein particle sedimenting at 80 S is formed. The assembly products of CM are compared in this chapter with those of broad bean mottle virus and brome mosaic virus, two other bromoviruses.Possible assembly mechanisms, a model for the coat protein dimer and future assembly research are discussed in chapter 8.
Genetische analyse van cowpea-mozaiekvirusmutanten
Jager, C.P. de - \ 1978
Landbouwhogeschool Wageningen. Promotor(en): J.P.H. van der Want, co-promotor(en): A. van Kammen. - Wageningen : [s.n.] - 112
koebonenmozaïekvirus - vignabonen - genetica - vigna - virologie - virussen - cowpea mosaic virus - cowpeas - genetics - vigna - virology - viruses
In this thesis the genetic analysis of a plant virus with a divided genome is described.
In chapter 1 a review is given of RNA plant viruses with multipartite genomes (Table 1.1). If in plant viruses with heterogeneous RNA complements specific marker properties can be assigned to specific RNAs a functional specialization of these RNAs is suggested. Such a specialization provides evidence that the virus has a divided genome.
Location of genetic markers in multicomponent plant viruses may be accomplished by constructing in vitro new genotypes through the exchange of corresponding genome parts between strains of the virus which differ with respect to the marker property. Comparing the properties of the reciprocal new genotypes with those of the parent strains may indicate through which genome part the marker is transferred to the new genotypes.
Many markers have been located in a number of multicomponent plant viruses (Tables 1.2 and 1.3). The data indicate that the RNAs of these viruses are functionally specialized and thus are parts of split genomes.
Similar investigations with mutants of cowpea mosaic virus (CPMV) are reported here. CPMV has small icosahedral particles and a bipartite RNA genome. The two RNAs, with molecular weights of 1.37 and 2.02 x 10 6 are encapsidated in different particles. The two particle types can be separated by density gradient centrifugation, after which they are designated middle(M)- and bottom(B)-component. As they contain only part of the genome the purified components are not infectious alone but after mixing they are again highly infectious.
CPMV is well suited for research on the molecular aspects of virus multiplication. As with many vertebrate and bacterial viruses the use of defective mutants may be fruitful in this research. Therefore the isolation of such mutants of CPMV was one of the objectives of the work reported here.
In chapter 2 methods are described for infectivity tests, purification of virus and of the components and for isolation of the viral RNA. In addition, mutagenic treatment and isolation of mutants are described.
In chapter 3 the mutagenic treatment is discussed. Upon treatment with nitrous acid the virus rapidly lost its infectivity (Fig. 3.1). Inactivation of purified B-component was faster than inactivation of purified M-component (Fig. 3.1). Compared with virus, the inactivation of isolated RNA was very slow (Fig. 3.21, suggesting that with virus an effect on the coat protein was involved in inactivation. Thus the loss of infectivity of the virus would only partly reflect changes in the RNA. This is in accordance with the relatively low frequency with which mutants were found among local lesion isolates surviving the treatment of virus as compared with the mutation frequency of isolated RNA (Table 3.1). Therefore for the induction of mutants the treatment of RNA was preferred.
B-RNA was inactivated by nitrous acid at a faster rate than M-RNA (Fig. 3.3). Apparantly Inactivating deaminations occurred less in M-RNA than in B-RNA. Yet the number of mutants having mutations in their M-component was not lower than the number of mutants with a mutated B-component (Table 9.1).
In chapter 4 the isolation of the mutants is described and biological properties of the mutants are compared with those of the wild type.
Mutants were isolated from necrotic local lesions on Pinto beans. For maintaining mutants local lesion transfers on Pinto were preferred to bulk transfers in the systemic host Blackeye cowpea because with the latter method wild type virus often accumulated in the mutant population. This appearance of wild type virus was considered to result from reversion of the mutant genotype and subsequent preferential multiplication of the resulting wild genotype. The symptoms on four differential hosts are given; the kidney bean varieties Pinto and Noordhollandse Bruine and the cowpea varieties Blackeye Early Ramshorn and Early Red.
On the hypersensitive host Early Red, two spontaneous mutants were differentiated: S1 and S8. These mutants infected Early Red systemically giving mosaic and necrosis, respectively. Eight other mutants, designated by N-numbers, were isolated after nitrous acid treatment of virus or RNA. From one of these induced mutants (N142) a spontaneous submutant S15 was isolated.
Almost all induced mutants caused altered symptoms on all the differential hosts. On Pinto the local lesions were slightly smaller (N3, N142, S15) or much smaller (N123, N163, N168, N172), white instead of brown (N163, N168), or less necrotic (N140, N142).
The local spots on the primary leaves of Noordhollandse Bruine were smaller (N123, N163) and less necrotic (N163). With some mutants (N3, N57, N123) systemic infection of this bean variety did not occur.
In Blackeye the local and systemic symptoms were either milder (SI, N57, N163, N168, S15) or absent (N123, N140, N142, N172). One mutant (N3) produced a relatively large amount of top(T)- component, which consists of particles devoid of RNA (Fig. 4.1). Another mutant (N168) appeared to be temperature sensitive for the induction of symptoms (Fig. 4.21 and for virus production (Table 4.2). One mutant (N172), inducing no symptoms at either low or high temperature and having low virus production at low temperature, failed to produce virus at high temperature.
In Early Red the necrotic local lesions were either white instead of brown (N140, N168, N172) or they did not occur at all (N123). Four mutants caused smaller lesions on this cowpea variety (N163, N168, N172, S15). One nitrous acid mutant (N142) caused local chlorotic spots and systemic mosaic in Early Red, like the spontaneous mutant S1.
Chapter 5 describes the in vitro recombination (IVR) test. In this test heterologous combinations of components were composed by exchanging in vitro the purified components of mutants and wild type virus. Properties of the heterologous combinations were compared with those of the parent strains on the differential hosts. The test could be performed either by inoculating the mixtures of components directly on the differential hosts (Table 5.1; Fig. 5.2) or by inoculating the mixtures on the local lesion host Pinto and testing local lesion isolates taken from this variety on the differential hosts.
When each of the heterologous combinations had the phenotype of one of the parent strains it was clear which of the components was responsible for the differentiating propertie(s). In almost all IVR tests this proved to be the case.
Some mutants attained only low titres in Blackeye cowpeas and could not be purified in workable quantities (N123, N140, N142, N172). Thus purification of the components of these mutants was not possible and IVR tests could not be performed. Such mutants could be analyzed by the supplementation test which is described in chapter 6. In this test wild type components were separately added to homogenates of typical mutant local lesions from Pinto. When addition of wild type M-component restored the wild phenotype and addition of B-component did not (Fig. 6.2 en 6.3; Tables 6.1 - 6.41 it was concluded that the M-component carried one or more mutations. In the reciprocal case the B-component was considered to carry the mutation(s).
The supplementation test was less laborious than the IVR test but the interpretation of the results was also more equivocal. In supplementation tests the inoculum always contained three components: two of the mutant and one of the wild type. Whether the observed phenotype was the result of an interaction between all these components or only between two of them could not be established directly.
In chapter 7 experiments are described in which crude preparations of pairs of mutants were inoculated onto the differential hosts separately and after mixing. When the mutants carried their mutations in different components the unmutated components would together contain complete wild type genomes. In that case wild type infections would be initiated through reassortment of components (ROC) in the inoculum.
Subculturing local lesion isolates of virus from primary Blackeye leaves, inoculated with a mixture of N123 and N140 and showing local symptoms (Fig. 7.1), revealed that the virus resulting from this combination was indeed mainly wild type virus. This agreed with the results of supplementation tests with these mutants which showed that they carried their mutations for the absence of symptoms in Blackeye in the M- and B-component, respectively.
Normal wild type symptoms were also induced on Blackeye by a mixture of mutants (N123 and N142) having mutations in their M-components for absence of symptoms and for milder local and systemic symptoms, respectively. To explain this, the occurrence of two independent functional genetic units within the M-RNA was postulated. It was hypothesized that the mutants had their mutations in different functional units and could genetically complement each other in cells in which both M-components were present. Indeed local lesion isolates of virus from Blackeye plants doubly infected with these mutants mainly contained the M-component of either mutant.
Mutants may have mutations affecting the same phenotypic expression in both components. Occurrence of mutations in both components may be assumed if in IVR tests both heterologous combinations of components have phenotypes other than the parent strains or when in supplementation tests neither of the wild type components can completely restore the wild phenotype. In those cases the role of the mutations in the separate components may be evaluated by studying the properties of the two reciprocal hybrids of the mutant and the wild type. This is described in chapter 8 for the temperature sensitive mutant N168. Mutations in both components appeared to affect symptoms in three differential hosts (Table 8.1; Fig. 8.1). Temperature sensitivity was due to a mutation in the M-component. Recombining the components of the two hybrids mutually restored the genotypes of the wild type and the mutant N168.
In chapter 9 a general discussion and conclusions are given. Using four methods, i.e. in vitro recombination, supplementation, reassortment of components and isolation of hybrids, mutations in 11 mutants were located. Mutations affecting specific properties were found in both components, indicating that the components of CPMV are functionally specialized. This confirms that CPMV has a divided genome.
Phenotypic alterations assigned to the M-component were:
- smaller size and changed colour of local lesions on Pinto;
- smaller size of local spots and absence of systemic infection in Noordhollandse Bruine;
- smaller size and reduced chlorosis and absence of local spots and absence of systemic infection in Blackeye cowpeas;
- smaller size and even absence of local lesions in Early Red;
- production of a relatively large amount of T-component;
- temperature sensitivity.
Phenotypic alterations assigned to the B-component were:
- reduced necrosis of local lesions on Pinto;
- absence of systemic infection in Noordhollandse Bruine;
- reduced chlorosis of local spots and absence of local and systemic symptoms in Blackeye;
- changed colour of local lesions on Early Red and occurrence of systemic infection of this host;
- temperature sensitivity.
Apparently both components direct the formation of symptoms in all four hosts. Some changes were brought about by mutations in either component.
Research on the molecular basis of changes in phenotype may help to elucidate the process of virus multiplication and the distribution of functions between the virus components.
Structuur en eigenschappen van cowpea-mozaiekvirus
Geelen, J.L.M.C. - \ 1974
Landbouwhogeschool Wageningen. Promotor(en): A. van Kammen, co-promotor(en): J.P.H. van der Want. - s.l. : [s.n.] - 86
plantenziekten - plantenvirussen - vigna - vignabonen - koebonenmozaïekvirus - plant diseases - plant viruses - vigna - cowpeas - cowpea mosaic virus
Purified infectious preparations of cowpea mosaic virus (CPMV) consist of three centrifugal components with sedimentation coefficients of 58, 95 and 118 S. These are referred to as top (T), middle (M) and bottom (B) component. M and B are nucleoproteins containing 25 and 36% RNA respectively. All three components are isometric particles with a diameter of 20 nm and have identical capsids. Both nucleoproteins (M and B) are necessary for infectivity (Van Kamen, 1968) and some direct evidence for the distribution of the genetic properties has been given by Bruening (1969) and De Jager and Van Kammen (1970).
The molecular weights of the RNAs have been determined by three emperical methods which differ in the remaining secondary structure of the RNAs. From the sedimentation velocities after formylation of the RNAs (5% secondary structure remaining; Boedtker, 1968) values of 1.4 x 10 6 D (M-RNA) and 2.1 x 10 6 D (B-RNA) could be calculated. From the mobilities of the CPMV-RNAs compared to the mobilities of the Escherichia coli ribosomal RNAs upon electrophoresis in polyacrylamide gels under non denaturing conditions molecular weights of 1.55 x 10 6D (M-RNA) and 2.55 x 10 6D (B-RNA) could be calculated. If the molecular weights were determined by electrophoresis on polyacrylamide gels under denaturing conditions (8 M urea, 60°C) values of 1.37 x 10 6D (M-RNA) and 2.02 x 10 6D (B-RNA) were found. In 8 M urea at 60°C the secondary structure of the Escherichia coli ribosomal RNAs is completely eliminated (Reynders et al., 1973). The CPMV-RNAs are also completely denatured under these conditions as has been shown by hyperchromicity measurements (Fig. 3.3.). As the values determined by gel electrophoresis under denaturing conditions are not affected by differences in secondary structure between the marker-RNAs and the CPMV-RNAs, these molecular weights are the most reliable.
The capsid of CPMV is constructed from two different proteins. CPMV was degraded with 1% sodium dodecyl sulfate in the presence of 1% 2-mercaptoethanol. To prevent aggregation of the proteins it was necessary to protect the SH- groups of the protein with mercaptoethanol or to block these groups by carboxymethylation or performic acid oxidation. The molecular weights of the proteins were estimated by SDS-polyacrylamide gelelectrophoresis according to Weber and Osborn (1969). Molecular weights of 44,000 D (I) and 25,000 D (II) were found for the carboxymethylated proteins and 49,000 D (I) and 27,500 D (II) for the performic acid oxidized proteins. The SDS bound to the protein apparently does not eliminate differences in the conformation of the proteins. The two proteins are found in all three centrifugal components in equimolar ratio, indicating an identical capsid of T, M and B.
The particle weights of the centrifugal components have been determined by light-scattering and sedimentation-equilibrium centrifugation. By light-scattering particle weights of 4.5 x 10 6D, 5.6 x 10 6D and 6.1 x 10 6D, and by sedimentation-equilibrium particle weights of 3.80 x 10 6D, 5.15 x 10 6D and 5.87 x 10 6D were found for T, M and B respectively. The differences between the particle weights determined by light-scattering and by sedimentation-equilibrium are within the experimental error (± 5%) for M and B. The difference is larger for T. This is probably caused by aggregation or contamination with large particles. This will affect the particle weight obtained by light-scattering, but not the particle weight obtained by sedimentation-equilibrium if the contaminant or the aggregates precipitated. Although the differences between the values obtained by the two methods are not very large for M and B, the values of sedimentationequilibrium are probably the best, since the agreement of these particle weights with the molecular weights of the RNAs and the RNA- content of M and B is very good (M : 25 % RNA; Mw-RNA : 1 .37 x 10 6D and B : 36 % RNA ; Mw-RNA : 2.02 x 10 6D ). It can not be excluded, that some aggregation or contamination with large particles affects also the particle weights obtained by light-scattering of M and B, as all light-scattering values are consistently higher than the sedimentation-equilibrium values.
When purified preparations of CPMV are analysed by electrophoresis, two electrophoretic forms are seen : a slow (S) moving and a fast (F) moving electrophoretic form. Each of the electrophoretic forms consists of all three centrifugal components. S and F can be separated by electrophoresis in a sucrose gradient. If the proteins of a CPMV preparation consisting of S and F, are analysed by electrophoresis on polyacrylamide gels, three proteins are seen: one large protein (I) and two small proteins (II and III) (Fig. 4.2.). Protein II is specific for S, and protein III for F. Molecular weights of 25,000 D for protein II and 22,000 for protein III can be estimated by SDS-polyacrylamide gelelectrophoresis of the carboxymethylated proteins. The distance migrated by protein I of S (I S ) and the distance migrated by protein I of F (I F ) is the same. To check if there are differences between these two large proteins, the proteins of the separated electrophoretic forms were separated by gel filtration on Sephadex G 200 with 5 M urea as eluent, and the amino acid compositions determined (Table 4.3.). From this table it can be seen that there are not only differences between protein I and protein II and III, but that also protein I of S (I S ) and protein I of F (I F ) are clearly distinct. Both proteins of an eletrophoretic form are therefore characteristic for that form.
By incubation with proteolytic enzymes it is possible to increase the electrophoretic mobility of S. This increase in mobility of S is correlated with an increase in mobility of protein II (Fig. 5.1. and Fig. 5.2.). The differences between the molecular weights of protein II and III indicate a larger difference between these two proteins than 7 amino acids. When, as can be seen from Table 4.3., moreover protein I changes upon the conversion of S into F, it is clear, that the conversion of S into F is much more complicated than was supposed by Niblett and Semancik (1969). The properties of CM and the relationship between the centrifugal components, the electrophoretic forms and the proteins are summarized in Figure 10.1.
No differences can be seen in the electron microscope between the particles of S and F. This can be expected, as the conversion of S into F apparently does not lead to a reorganisation of the protein coat. A difference in molecular weight of approximately 2,500 D (estimated by SDS-polyacrylamide gelelectrophoresis) between protein II and III is not detectable in the electron microscope.
By three-dimensional image reconstruction of electron micrographs it was shown that the capsid of CPMV possesses 5, 3, 2 symmetry. Based on the properties of the proteins and the reconstructed image a model is proposed consisting of twelve pentamers of the large protein at the 5- fold positions and twenty trimers of the small protein at the 3-fold positions (Fig. 7.3., Crowther et al., in press). The geometrical figure traced out by the ridges of proteins joining 5-fold and 3-fold positions is approximately a rhombic triacontahedron.
Niblett and Semancik (1969, 1970) showed that S was less infectious than F, even though the RNA of S was more infectious and less degraded than the RNA of F. This is only true, if old preparations are tested. The infectivities of S and F are equal, provided fresh preparations are used. The infectivities of the RNAs of S and F are equal, and there is no appreciable sign of a difference in degradation, as judged by gel electrophoresis (Fig. 6.1.). After in vitro aging of S a loss in infectivity is observed; the initial infectivity can be restored by incubation with trypsin or chymotrypsin (Table 6.3.). Upon aging the mobility of S is not changed, but the mobility increases upon incubation with trypsin or chymotrypsin. Aging in vitro and incubation with trypsin or chymotrypsin affect neither the infectivity nor the mobility of F. Since no increase in mobility of S was observed upon aging, the charge is not decisive for the difference in infectivity between S and F, as has been suggested by Niblett and Semancik (1970). But it is likely that the amino acids which determine the difference between S and F, do play an important role in the loss of infectivity of S upon aging. Removal of these amino acids by incubation with proteolytic enzymes results in an restoration of the initial infectivity.
During storage partial conversion of S into F is sometimes observed. A fast conversion is seen with virus purified by the PEG/NaCl method from fresh leaves and essentially no conversion with virus purified by the butanol-chloroform method from frozen tissue (compare Fig. 5.1. and Fig. 5.3.). Essentially no increase in the mobility of S is observed also with preparations which have undergone an extra purification step, e.g. separation of the electrophoretic forms or separation of the centrifugal components. This partial conversion probably results from proteolytic enzymes contaminating the preparations, as incubation of CM in the soluble protein fraction or in the homogenate of infected Vigna leaves leads to the same conversion (Fig. 5.4.).
If the centrifugal components are centrifuged to equilibrium in CsCl gradients at neutral pH, eight components are found. These components are S and F of top component, S and F of middle component, S and F of a bottom component with a low buoyant density (B u ) and S and F of a bottom component with a high buoyant density (B l ). The difference in buoyant density between S and F of a centrifugal component (ca. 0.004 g/cm 3) is probably caused by a difference in the amount of CS +-ions bound to S and F, as the same difference in buoyant density is also found between S and F of top component (which consists of only protein). The buoyant density of all the components is dependent upon the pH. The density increases with increasing pH. The difference in density between pH 6.5 and pH 8 5 is approximately 0,005 g/cm 3. This density-shift is reversible and probably the result of an increased Cs +-ions binding with increasing pH.
Besides these small differences in buoyant density observed at different pH's and between the electrophoretic forms, there is a large difference in buoyant density between the two bottom components (about 0,040 g/cm 3). It is not clear what causes this difference. B u and B l have the same RNA-content, the same protein composition, the same sedimentation coefficient and the same contribution to the infectivity. When bottom component is centrifuged in CsCl solutions at pH 8.5 almost only B u is found and at pH 6.5 almost only B u (Fig. 8.3.). By increasing the pH from 6.5 to 8.5 or by increasing the length of the run at pH 6.5 (Fig. 8.4.) B u can be converted in B l .
The conversion from B u into B l is irreversible. A possible explanation for this phenomenon is as follows: the particles swell due to the high ionic strenght. The swelling causes an irreversible change in the conformation of the particles, resulting in an increased binding of Cs +- ions (B u changes to B l by increasing the length of the run at pH 6.5)Increasing the pH from 6.5 to 8.5 promotes the swelling. The process of the swelling to B u and the conversion of B u to B l is then so greatly accelerated that after about 20 hours centrifugation (the standard time in these experiments) almost no B u is left.
Parts of this thesis have been published earlier (Geelen et al., 1972; Geelen et al., 1973; Crowther et al., in press).
Lokalisatie van de RNA-replicatie van cowpea-mozaiekvirus
Assink, A.M. - \ 1974
Landbouwhogeschool Wageningen. Promotor(en): A. van Kammen; J.P.H. van der Want. - Wageningen : Pudoc, Centrum voor Landbouwpublicaties en Landbouwdocumentatie - ISBN 9789022005040 - 70
replicatie - dna-replicatie - plantenziekten - plantenvirussen - vigna - vignabonen - koebonenmozaïekvirus - replication - dna replication - plant diseases - plant viruses - vigna - cowpeas - cowpea mosaic virus
Cowpea mosaic virus (CPMV) is a spherical, multiparticle virus. The two viral RNAs are singlestranded and replicate by a double-stranded RNA. The virus induces cytopathic structures in cells of Vigna leaves upon infection.
Structures from cells were distributed over fractions after centrifugation on discontinuous sucrose gradients. The fractions were studied by electron microscopy. The distribution of replicating RNA of CPMV among the gradient-fractions was investigated by polyacrylamide-gel electrophoresis, by molecular hybridization, by electron microscopy and by pulselabel-experiments. A correlation was shown between the amount of replicating RNA of CPMV and the amount of cytopathic structures in the gradient-fractions.
Autoradiography revealed that the RNA replication of CPMV is associated with the vesicles in this cytopathic structure.
Voortgezette onderzoekingen naar de overerving van zaadhuidkleur en zaadhuidpatroon bij Vigna unguiculata (L.) walp
Boorsma, P.A. - \ 1972
Wageningen : [s.n.] (Celos rapporten no. 65) - 34
plantkunde - vignabonen - peulvruchten - suriname - vigna - botany - cowpeas - grain legumes - suriname - vigna
vergelijkend groei - analytisch onderzoek van de rijstcultivars IR 8 en Acorni; Lichtkonkurrentie bij Vigna unguiculata (L.) Walp. cv. African Red
Erdman, J.W. ; Schouten, H. - \ 1972
Wageningen : [s.n.] (Celos rapporten no. 78) - 49
vignabonen - oryza sativa - plantenveredeling - rijst - suriname - vigna - cowpeas - oryza sativa - plant breeding - rice - suriname - vigna
|Structure of the capsid of cowpea mosaic virus : the chemical subunit : molecular weight and number of subunits per particle
Geelen, J.L.M.C. ; Kammen, A. van; Verduin, B.J.M. - \ 1972
Wageningen : [s.n.] (Mededeling / Laboratorium voor virologie. Landbouwhogeschool no. 89) - 9
koebonenmozaïekvirus - vignabonen - plantenziekten - plantenvirussen - eiwitten - vigna - cowpea mosaic virus - cowpeas - plant diseases - plant viruses - proteins
Verkennende onderzoekingen naar de overerving van zaadhuidkleur en zaadhuidpatroon bij Vigna unguiculata (L.) Walp; Kiemproeven met stuifmeel van de bataat op vloeibare en vaste media
Sakiman, J.K.L. - \ 1971
Paramaribo : [s.n.] (CELOS rapporten no. 44) - 70
vignabonen - ipomoea batatas - suriname - zoete aardappelen - vigna - cowpeas - ipomoea batatas - suriname - sweet potatoes - vigna
Productie en verdeling van droge stof bij Sorghum Bicolor (L.) Moench CV. Martin onder invloed van de plantdichtheid; Produktie en verdeling van droge stof bij de cowpeacultivars blackeye en African red onder invloed van de plantdichtheid
Erdman, J.W. - \ 1971
Parimaribo : [s.n.] (CELOS rapporten no. 58) - 41
plantkunde - vignabonen - teelt - cultuurmethoden - sorghum bicolor - suriname - vigna - botany - cowpeas - cultivation - cultural methods - sorghum bicolor - suriname - vigna