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Cytokinins as key regulators in plant-microbe-insect interactions: connecting plant growth and defence
Giron, D. ; Frago, E. ; Glevarec, G. ; Pieterse, C.M.J. ; Dicke, M. - \ 2013
Functional Ecology 27 (2013)3. - ISSN 0269-8463 - p. 599 - 609.
extracellular invertase - messenger-rnas - nodule organogenesis - herbivore attack - tobacco plants - green islands - getting ready - responses - nicotiana - immunity
Plant hormones play important roles in regulating plant growth and defence by mediating developmental processes and signalling networks involved in plant responses to a wide range of parasitic and mutualistic biotic interactions. Plants are known to rapidly respond to pathogen and herbivore attack by reconfiguring their metabolism to reduce pathogen/herbivore food acquisition. This involves the production of defensive plant secondary compounds, but also an alteration of the plant primary metabolism to fuel the energetic requirements of the direct defence. Cytokinins are plant hormones that play a key role in plant morphology, plant defence, leaf senescence and sourcesink relationships. They are involved in numerous plantbiotic interactions. These phytohormones may have been the target of arthropods and pathogens over the course of the evolutionary arms race between plants and their biotic partners to hijack the plant metabolism, control its physiology and/or morphology and successfully invade the plant. In the case of arthropods, cytokinin-induced phenotypes can be mediated by their bacterial symbionts, giving rise to intricate plantmicrobeinsect interactions. Cytokinin-mediated effects strongly impact not only plant growth and defence but also the whole community of insect and pathogen species sharing the same plant by facilitating or preventing plant invasion. This suggests that cytokinins (CKs) are key regulators of the plant growth-defence trade-off and highlights the complexity of the finely balanced responses that plants use while facing both invaders and mutualists.
Genome composition of 'Elatior'-begonias hybrids analyzed by genomic in situ hybridisation
Marasek Ciolakowska, A.R. ; Ramanna, M.S. ; Laak, W.A. ; Tuyl, J.M. van - \ 2010
Euphytica 171 (2010)2. - ISSN 0014-2336 - p. 273 - 282.
hordeum-vulgare-l - lycopersicon-esculentum - chromosome elimination - cytogenetic analysis - bulbosum l - plant dna - differentiation - extraction - nicotiana - progenies
Interspecific hybridization of various tuberous Begonia species hybrids with Begonia socotrana results in so-called 'Elatior'-begonias hybrids (B. x hiemalis Fotsch). In our study, genomic in situ hybridization (GISH) has been employed to assess the genome composition in eleven 'Elatior'-begonias hybrids and their ancestor genotypes. Genomic DNA of tuberous Begonia was sonicated to 1-10-kb fragments, labelled by nick translation with digoxigenin-11-dUTP and used as a probe whereas B. socotrana DNA was autoclaved to 100 bp fragments and used as block. The genome of tuberous Begonia was clearly pronounced in 'Elatior'-begonias when the probe concentration was similar to 3.75 ng/mu l (150 ng/slide), with 30 times the excess of B. socotrana blocking DNA and stringency of post hybridization washings at 73% (0.1x SSC at 42A degrees C). In 'Elatior'-begonias hybrids GISH distinguished two groups comprising short (0.6-1.03 mu m in length) and relatively longer chromosomes (1.87-3.88 mu m) which represent B. socotrana and tuberous Begonia genomes, respectively. The number of chromosomes derived from tuberous Begonia ranged from 14 to 56 and for B. socotrana from 7 to 28 which suggest the presence of different ploidy levels in analyzed 'Elatior'-begonia hybrids. Intergenomic recombination has not been detected through GISH in hybrids analyzed. Genomic in situ hybridization turned out to be useful to identify the genome constitution of 'Elatior'-begonia hybrids and thus gain an insight into the origins of these cultivars. This knowledge on the ploidy level and genome composition is essential for further progress in breeding Begonias.
Characterization of genes coding for small hypervariable peptides in Globodera rostochiensis
Bers, N.E.M. van - \ 2008
University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - [S.l.] : S.n. - ISBN 9789085049579 - 229
globodera rostochiensis - plantenparasitaire nematoden - peptiden - genen - genexpressie - solanum tuberosum - arabidopsis - nicotiana - gensplitsing - gastheer-pathogeen interacties - moleculaire interacties - plant parasitic nematodes - peptides - genes - gene expression - gene splicing - host pathogen interactions - molecular interactions
Plant parasitic nematodes secrete a cocktail of effector molecules, which are involved
in several aspects of the interaction with the host, eg. in host defense suppression, in
migration and in feeding cell formation. In this thesis, we performed the first study on
10 novel peptide genes, believed to be important for parasitism of the potato cyst
nematode, Globodera rostochiensis. Nine of the peptide genes described here belong
to the SECPEP gene family. The SECPEP genes are all expressed in the dorsal
esophageal gland, which is one of the main sites for the production of effector
molecules. This, together with the predominant expression in preparasitic and early
parasitic juvenile nematodes, makes it very likely that the SECPEPs code for effector
peptides essential for succesful infection and feeding site formation.
In chapter 2, we show that diversifying selection is a likely driver of the molecular
evolution of the SECPEPs. The sequences of the mature peptides appear to be highly
diverse, while the non)coding 3’UTR and intronic regions as well as the region coding
for the signal peptide for secretion are relatively conserved. In fact, a pairwise
comparison of the SECPEPs reveals no significant sequence similarity between family
members at all. In chapter 5 we further speculate on a possible role for RNA)editing as
a mechanism to yield hypervariability in the SECPEPs, because the sequence diversity
at the transcript level significantly exceeds that of the genomic locus. Chapter 5 further
elaborates on the analysis of trans)splicing in SECPEP1 transcripts. We show that
SECPEP1 transcripts are trans)spliced to a surprising diversity of novel spliced)leader
sequences. The first approach to unravel the role of the members of the SECPEP family
in plant parasitism, is described in chapter 4. We generated transgenic potato and
Arabidopsis plants expressing SECPEP3 while using the CaMV 35S promotor. The
phenotype associated with SECPEP3 in both potato and Arabidopsis plants includes a
reduction of root growth and an alteration of the leaf morphology. The SECPEP3
peptide harbors several sequence motifs first found in the cyclin)dependent kinase
inhibitors ICK1/KRP1, SIM and Smr1. We, therefore, suggest a role for SECPEP3 in cell
cycle alteration in nematode feeding site formation. Although the SECPEP genes show
only a low level of primary sequence similarity, all code for positively charged,
hydrophilic peptides with a C)x)G γ)core motif (chapter 2). These are characteristics
typical for host defense peptides, and in chapter 6 we investigate whether these
characteristics are also found for other peptides involved in plant)parasite interactions.
We show that a considerable number of these effector peptides share a positive
charge, hydrophilicity and C)x)G γ)core motif with the SECPEPs, and we speculate on a
role for the positive charge in peptide)ligand interaction.
In chapter 3 we describe the NEMPEP peptide, secreted by G. rostochiensis. NEMPEP
is also a positively charged, hydrophilic peptide with a C)x)G γ)core motif, although it is
genetically unrelated to the SECPEP gene family. During the life cycle of G.
rostochiensis, the expression pattern of NEMPEP reveals a striking regulation. NEMPEP
is highly expressed in preparasitic juveniles and in the parasitic life stages after initial
feeding cell formation. However, NEMPEP expression was hardly detectable in the
juveniles just after entering the plant root. Several disease resistance genes condition
nematode resistance at the onset of parasitism. The downregulation of NEMPEP at
exactly this timepoint could be a strategy to avoid recognition by the host’s immune
system. In planta expression of NEMPEP, as a fusion to GFP, shows that NEMPEP
accumulates in the nucleolus of tobacco cells. Potato plants transformed with
35S::NEMPEP were slow at forming roots and the internodes between the leaflets were
shortened. This, together with a reduced transformation efficiency, led us to
hypothesize a role for NEMPEP in cytokinin signaling (Chapter 3).
Currently, there are two models regarding the functional role of the SECPEPs and
NEMPEP. The first one concerns a role as an antimicrobial peptide, which could protect
the host plant against secondary infections by opportunistic microbes. As a competing
hypothesis, the high hydrophilicity of the peptides may point to a role as peptide
hormone. As such, they may be involved in redirecting cell cycle or hormonal regulation
upon feeding cell formation.
Metabolic engineering of monoterpene biosynthesis in plants
Lücker, J. - \ 2002
University. Promotor(en): L.H.W. van der Plas; H.A. Verhoeven; Harro Bouwmeester. - S.l. : S.n. - ISBN 9789058087171 - 158
nicotiana - petunia - citrus limon - monoterpenen - biosynthese - genetische modificatie - metabolisme - transgene planten - plantenfysiologie - monoterpenes - biosynthesis - genetic engineering - metabolism - transgenic plants - plant physiology
<FONT FACE="Times" SIZE=2></font><FONT FACE="Times"><p>Monoterpenes are a large group of compounds that belong to the terpenoid family of natural compounds in plants. They are small, volatile, lipophilic substances of which around one thousand different structures have been identified. Monoterpenes are involved in plant-insect, plant-microorganism and plant-plant interactions. Many monoterpenes, such as menthol, carvone, limonene and linalool, are of commercial interest as they are commonly used in foods, beverages, perfumes and cosmetics and in many cleaning products. In flowers they also contribute to the characteristic scent. Monoterpene synthases and subsequent modifying enzymes such as cytochrome P450 hydroxylases, dehydrogenases, reductases and isomerases are responsible for the production of the variety of different carbon skeletons of monoterpenes that are found in nature. In this thesis the use of genetic engineering to introduce or alter the production of monoterpenes by plants was explored.</p><p>Initially, as described in Chapter 2, <em>S</em> -linalool synthase from <em>Clarkia breweri</em> was introduced in Petunia plants regulated by a constitutive promoter. Expression was obtained in all tissues analysed, but formation of linalool was restricted to leaves, sepals, corollas, stems and ovaries, and could not be detected in nectaries, roots, pollen and style. Although it was expected that the formation of linalool would result in an alteration of the scent of the plants, no linalool was detected in the headspace. Instead, all the <em>S</em> -linalool produced was efficiently converted by an endogenous glucosyltransferase present in the petunia tissues to the non-volatile <em>S</em> -linalyl-<FONT FACE="Symbol">b</font>-D-glucopyranoside. These results showed that genetic engineering of plants for monoterpene biosynthesis is possible, but that it can lead to unexpected conversions of the produced metabolites by endogenous enzyme activities.</p><p>In order to obtain new monoterpene synthases for the genetic engineering of plants, a cDNA library was made of the fruit peel of lemon, a plant species producing many different monoterpenes. From this library four different monoterpene synthases were obtained as described in Chapter 3, which together showed to be responsible for more than 90% of the total number of components present in lemon oil. The product specificity of the enzymes could be analysed after heterologous expression in <em>Escherichia coli</em> . Two of the four cDNA-encoded enzymes were producing (+)-limonene, the main component present in lemon. One cDNA-encoded enzyme was mainly producing (-)-<FONT FACE="Symbol">b</font>-pinene and the fourth cDNA-encoded enzyme was mainly producing<FONT FACE="Symbol">g</font>-terpinene. The latter two enzymes were both producing traces of multiple side products as well. Also other properties of the heterologously expressed enzymes were determined, which are described in Chapter 3.</p><p>Three monoterpene synthases responsible for the production of different main products were chosen for the genetic engineering of <em>Nicotiana tabacum</em> 'Petit Havana' SR1, described in Chapter 4. The wild type of this tobacco variety produces one monoterpene, linalool that is only emitted from the flowers. After the transformation with the three monoterpene synthases and subsequent crossings, a plant was obtained that emitted all the three main products of the three introduced monoterpene synthases in addition to the endogenous linalool in the flowers. The levels of limonene,<FONT FACE="Symbol">b</font>-pinene and<FONT FACE="Symbol">g</font>-terpinene emitted from the leaves and flowers of the plant were higher than the level of the endogenous monoterpene. Also the side products of the monoterpene synthases were detected. The extensive modification of the volatile profile of the tobacco plants that we obtained indicates that there is a sufficient amount of substrate available to the introduced enzymes.</p><p>In Chapter 5 the transgenic tobacco plant emitting the products of three monoterpene synthases, was used in a subsequent transformation experiment in order to modify the already introduced pathway. A second step in the pathway was introduced by transformation of the plant material with a limonene-3-hydroxylase isolated from spearmint, which is supposed to be localised in the endoplasmatic reticulum (ER) in the cytosol of the plant cells, while the primarily introduced monoterpene synthases were most likely localised in the plastids in the transgenic plants. The introduction of the cytochrome P450 monoterpene hydroxylase and the resulting formation of the hydroxylated product of (+)-limonene, (+)- <em>trans</em> -isopiperitenol demonstrates that there is intracellular trafficking of limonene from the plastids to the ER in the cytosol. That this trafficking mechanism would be present in plants normally producing these hydroxylated monoterpenes could be expected, but that it is apparently also present in plants not specialised for the production of these compounds is an exciting discovery. Apart from the production and subsequent emission of high further oxidised conversion product isopiperitenone was detected. In addition, an increase in the <em>p</em> -cymene level and the formation of the new products 1,3,8- <em>p</em> -menthatriene and 1,5,8- <em>p</em> -menthatriene were detected. The occurrence of these latter two products and the increase of the <em>p</em> -cymene level could be a consequence of the metabolic engineering of the biosynthetic route into a cell compartment not adapted to the production of these compounds. Endogenous enzymes and pH differences were suggested to be the main cause the formation of these products.</p><p>Chapter 6 discusses the various strategies followed for the metabolic engineering of monoterpene biosynthesis in this thesis and by other groups. Functional implications are discussed such as ecological and physiological consequences of the new metabolites for the transgenic plants. The commercial aspects and interesting opportunities for further research are also discussed.
Source-sink relations in transgenic tobacco with modified trehalose metabolism : a comparative labelling study with the stable isotopes 13C and 15N of wildtype and five transgenic types
Visser, A.J.C. de; Leeuwen, P.H. van; Pot, C.S. - \ 1999
Wageningen : AB-DLO (Note / Agricultural Research Department, Research Institute for Agrobiology and Soil Fertility 179) - 32
plantengroeiregulatoren - genetische modificatie - nicotiana - trehalose - vloeistoffen (liquids) - sapstroom - distributie - plant growth regulators - genetic engineering - liquids - sap flow - distribution
Management of broomrape (Orobanche cernua) in tobacco (Nicotiana tabacum)
Dhanapal, G.N. - \ 1996
Agricultural University. Promotor(en): Paul Struik; M. Udayakumar; S.J. ter Borg. - S.l. : Dhanap␁al - ISBN 9789054855712 - 183
parasitaire planten - epifyten - nicotiana - tabak - orobanchaceae - gewasbescherming - plagenbestrijding - ziektebestrijding - parasitic plants - epiphytes - tobacco - plant protection - pest control - disease control
<br/>Tobacco is an important commercial crop in India. India is the third largest tobacco producing country in the world. Tobacco is cultivated in an area of 0.428 million ha. Non- Virginia tobaccos such as bidi tobacco constitute about 65% of the total tobacco area in the country.<p>Broomrape <em>(Orobanche cernua)</em> is <em></em> a debilitating holoparasitic weed in all tobacco growing areas in India, with a devastating effect on the crop. In India, yield loss in tobacco ranges from 30 - 70%; at present hand weeding is the only practice in India applied to control the parasite.<p>With this background, several field and laboratory experiments were conducted in Karnataka State, Southern India, to study the germination biology and to develop a suitable method to induce the germination of the parasite, and to develop a technology by integrating agronomic and chemical approaches to control the parasite at different phases.<p>The germination phase of the parasite is a critical period. The seed bank of the parasite cae be reduced by stimulating the germination through chemicals, natural host stimulants or both. GR24 (a strigol analogue) at 1.0 and 0.1 ppm, was the standard to assess potential germination. Of the other chemicals, gibberellic acid at 10 and 20 ppm was most effective. The stimulating effects of host plants were significant even when GR24 was applied. Suicidal germination of the parasitic seeds triggered by growing trap crops reduced the weed population and the growth of the host plants was hastened due to green manuring effect of trap crops. Therefore, including a trap crop in the rotation may reduce the problem. Sunhemp <em>(Crotalaria juncea</em> L.) and greengram <em>(Vigna radiata</em> L.) are promising trap crops in a cropping system containing bidi tobacco in areas where tobacco is grown in a long growing season.<p>Chemical control by (systemic) herbicides is also an option. Maleic hydrazide (MH) reduced broomrape spikes at 0.25 - 0.75 kg a.i./ha applied at 30 or 40 days after transplanting (DAT) tobacco. Higher tobacco yields were obtained with 0.25 kg a.i./ha MH, which was on par with the hand weeding treatment both in "infested" and "non- infested" tobacco plants. Higher concentrations of MH were toxic to tobacco crop. Glyphosate at 0.50 kg a.i./ha applied at 60 DAT and imazaquin at 0.01 kg a.i./ha applied at 30 DAT reduced the broomrape population by almost 80% and increased tobacco leaf dry weight by more than 40% compared to the control treatment. Imazapyr and EPTC were less effective.<p>Swabbing natural plant oils killed the bud and stem parts of the parasite by suffocation. Neem, coconut and sunflower oils showed quick knock-down effects in killing the bud part, whereas neem oil did not kill the stem part of the parasite. Niger, castor and mustard oils appeared to be (somewhat) less effective.<p>In general, there is a negative linear relation between broomrape infestation and tobacco yield, with a very large (negative) regression coefficient.<p>No single method is effective in controlling the parasite. The seed bank of the parasite should be minimized in a phased manner by integrating cultural and chemical methods of control. Therefore, an integrated management strategy is the best perspective to control broomrapes in a crop wherever it is problematic.<p>Key words: bidi tobacco, broomrape, chemical control, <em>Crotalaria juncea,</em> gibberellic acid, germination stimulants, GR24, herbicides, integrated weed control, natural plant oils, <em>Orobanche cernua,</em> parasitic weed, suicidal germination, trap crop, <em>Vigna radiata.</em>
Sympton development, X-body formation and 126-kDa-protein in plants infected with tobacco mosaic virus
Wijdeveld, M.M.G. - \ 1990
Agricultural University. Promotor(en): J. Bruinsma; R.W. Goldbach. - S.l. : S.n. - 105
plantenziekten - plantenvirussen - nicotiana - tabak - tabaksmozaïekvirus - plantenziektekunde - misvormingen - fasciatie - plant diseases - plant viruses - tobacco - Tobacco mosaic virus - plant pathology - malformations - fasciation
<p><TT>Upon infection with tobacco mosaic virus (TMV) sensitive tobacco varieties develop systemic mosaic symptoms in the developing leaves. These symptoms are the visible result of the interaction of the virus with its host and the nature and the severity of the symptoms are determined by the genetic constitutions of both partners. Their interaction involves several stages, including virus entry, replication and spread, the latter of which are dependent on viruscoded proteins. How the virus induces symptoms remains unclear.</TT><p><TT>TMV codes for proteins of 183, 126, 30 and 17.5 kDa, that are necessary for replication (183 kDa and presumably 126 kDa), cell- to-cell transport (30 kDa) and long-distance transport, as well as for coating the viral RNA (17.5 kDa). Virus multiplication leads to cellular disturbances in the chloroplasts, cytoplasm and nucleus, as reviewed in chapter 1. Since viral multiplication most likely occurs in the cytoplasm, changes in the nucleus seem to be secondary and might be related to the developmentally- controlled appearance of mosaic symptoms. As shown by Van Telgen (Changes in chromatinassociated proteins of virus-infected tobacco leaves, thesis Agricultural University Wageningen, 1985), during the development of systemic mosaic symptoms the viral non-structural 126-kDa protein was present among the chromatin- associated proteins in fractionated leaf homogenates. The aim of the present investigation was to study the implications of this finding.</TT><p><TT>An antiserum was raised against a fusion protein of <em>E.coli</em> β-galactosidase and amino acids 339-852 of the 126-kDa-protein. By immunoelectron microscopy of sections from systemically infected leaves the viral protein was not detectable in nuclei but, instead, was found to be present in cytoplasmic inclusions, characterized by the presence of tubules, and designated X- bodies. In embedded purified nuclear preparations from systemically infected leaves similar amorphous structures, most likely X-bodies,</TT>I<TT>were present and specifically labelled. In contrast, using antibodies against tobacco histones, only nuclei were labelled. Antibodies against viral coat protein labelled crystalline virus inclusions in the cytoplasm and did not react with nuclei. Light microscopic analysis indicated that Xbodies were almost always associated with nuclei. Thus, the presence of X- bodies in nuclear preparations seemed to result from adherence of the X-bodies to the nuclei and the viral 126-kDa protein appeared to be confined to the X-bodies (Chapter 2).</TT><p><TT>To study the time course of X-body occurrence in relation to symptom development, a largely synchronized infection was employed. Plants were infected by differential temperature treatment and virus multiplication in systemically infected leaves was initiated by transfer of the plants from 4 to 25°</TT>C. An ELISA assay was developed to quantify the amount of viral 126-kDa protein present. In leaves up to 4 cm in length the accumulation of 126-kDa protein was followed in relation to viral multiplication, the development of X- bodies and the formation of symptoms. Both 126-kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h.<p>By electron microscopy small X-bodies, weakly labelled with antibodies against the 126- kDa protein, were detected as early as 24 h after transfer but were not found to be associated with nuclei. Thereafter, X-bodies increased in size and 126-kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves developing mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations support the idea that association of X-bodies with nuclei is connected with symptom induction when the leaf is invaded by the virus early in its development (Chapter 3).<p>TMV strains inducing symptoms ranging from severe mosaic to virtually none were used to further test this hypothesis. The RNAs of strains <em>flavum</em> , V4 and masked encoded in vitro proteins of similar sizes as those of the common strain W U1. Antisera against the coat and 126-kDa proteins of W U1. recognized corresponding proteins of the three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coat<TT>protein in virus crystals. The 126-kDa protein was localized in large X-bodies associated with nuclei in <em>flavum</em> -infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked- infected plants. Neither X-bodies nor 126-kDa proteins were observed in V4-infected tissue. Since 126-kDa protein from V4 was - at least <em>in vitro</em> -not more sensitive to proteolysis than that from <em>flavum</em> , accumulation of the protein in X-bodies in the latter might have resulted from excess synthesis. These results are consistant with the hypothesis that the size and location of the X-bodies determine symptom expression (Chapter 4).</TT><p><TT>Further TMV strains were used to probe the generality of this conclusion. Strains U2 and U5, inducing mild mosaic symptoms, were selected because of their presence in chloroplasts, and Holmes' ribgrass (HR) because it is a distantly related strain that produces a symptomless systemic infection at 30</TT>°C.<TT>Strains U2 and U5 coded for proteins of 126 kDa, while the strain HR coded for a protein of 130 kDa. Strikingly, these proteins were not recognized by antisera against the protein from W U1. Electron microscopic analysis of tissues infected with U2 and U5 established the presence of virus clusters in the cytoplasm as well as in chloroplasts. In scattered cells infected with HR virus clusters were found adjacent to nuclei and chloroplasts. X- bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U1-infected tomato displaying severe mosaic symptoms. The induction of X- bodies thus appears to be characteristic of some strains of TMV only. Since other strains induce mosaic symptoms without X-bodies being present, the occurrence of X-bodies can, at most, be associated with the severity of the symptoms induced by the former strains (Chapter 5).</TT><p><TT>In Chapter 6 the significance of X-bodies in TMV infection is discussed. A function as the site of virus replication seems unlikely because some strains multiply to substantial levels without X-bodies being present. As discussed in chapter 4 their occurrence might be explained as resulting from a build up of 126-kDa proteins. Their close association with nuclei in systemically infected leaves suggests that they might influence nuclear activity. The rate at which association occurs and the developmental stage of the leaf at that</TT><TT>time appear to be factors that play a role in symptom expression in tobacco infected with M W U1. or flavum, and masked. Other mechanisms must also play a role, as evidenced by symptoms expressed by strains that do not induce X-bodies.</TT><p><TT></TT>
Changes in chromatin-associated proteins of virus-infected tobacco leaves
Telgen, H.J. van - \ 1985
Landbouwhogeschool Wageningen. Promotor(en): J. Bruinsma, co-promotor(en): L.C. van Loon. - Wageningen : Van Telgen - 99
chromatine - nicotiana - plantenziekten - plantenvirussen - solanaceae - tabak - tabaksmozaïekvirus - chromatin - plant diseases - plant viruses - tobacco - Tobacco mosaic virus
<p/>Symptoms of viral infections in plants often resemble disturbances in growth and development. Therefore, symptoms appear to result from an interference of the virus with the regulation of growth and development of the host plant. Particularly the non-histone chromatin- associated proteins are considered to be the regulators of specific gene expression. The aim of the present study was to elucidate whether upon infection of a plant with a virus, alterations occur in the non-histone chromatin-associated protein composition of the leaves.<p/>A survey of the literature on viral pathogenesis in plants, the properties of chromatin- associated proteins, and their possible role in the regulation of specific gene expression is given in Chapter 1.<p/>In Chapter 2 we looked for changes in the chromatin-associated protein composition of leaves from virus-infected tobacco plants. As a model system the combination 'Samsun' tobacco-tobacco mosaic virus (TMV) was used. In this combination, mosaic symptoms develop in the newly emerging leaves, the mosaic consisting of alternating patches of light-green and dark-green tissue. To ensure recovery of representative amounts of nuclei from the whole leaf and not predominantly from either light green or dark green tissue, known procedures for the isolation of nuclei had to be modified. By homogenizing the leaves in a large volume of buffer and repeated grinding of the homogenate, up to 45% of the nuclei present in the leaves were freed from the cell debris. From these nuclei chromatin was purified, and chromatin-associated proteins were dissociated from the DNA in buffer containing urea and high salt. Analysis of these proteins in SDS-containing polyacrylamide gels revealed a single consistent alteration upon TMV infection, being the induction of a new protein of about 116 kDa. The use of two-dimensional polyacrylamide gel electrophoresis showed a second alteration to occur. This second change consisted of the appearance of a new protein of about 20 kDa. This protein was serologically identified as TMV coat protein.<p/>Further investigations into the effects of both mosaic-inducing and necrotic symptoms- Producing viruses on the chromatin- associated protein constitution of different tobacco cultivars, revealed that of the viruses tested only cucumber mosaic virus (CMV) induced any specific alterations. A 'green' isolate induced a single new protein of about 28 kDa, and the 'yellow' strain P6 one of about 29 kDa. These new proteins co-migrated in SDS- containing polyacrylamide gels with the coat proteins of the respective CMV strains. CMV was also the only virus - apart from TMV - that induced mosaic symptoms. In combinations resulting in localized or systemic necrosis, no changes were observed. Thus it turned out that only if virus infection results in the development of systemic mosaic symptoms, discrete changes in the chromatinassociated protein profile occur (Chapter 3).<p/>The presence of viral coat protein associated with host chromatin represents virus specificity of the induced change. The presence of the 116 kDa protein exclusively after TMV infection was also connected to the virus. It was not induced in uninfected plants during senescence of fully-grown leaves or upon ageing of detached leaves (Chapter 3). Since a primary role for coat protein in the induction of symptoms is unlikely, we further concentrated on the TMV-induced 116 kDa protein.<p/>Whereas TMV was detected in upper leaves that become infected systemically already 96 h after inoculation of lower, fully-expanded leaves, the 116 kDa protein became discernable in the systemically-infected leaves only between 120 to 144 h after inoculation. This moment coincided with the appearance of visible symptoms (vein clearing). At that time the 116 kDa protein was also detectable, both in the soluble protein and in the sedimentable membrane fractions. However, the 116 kDa protein was found to be preferentially associated with, on the one hand, the membranous fraction and, on the other hand, the chromatin. Based on the amount of the protein in the different fractions and on morphometric analysis of tissue sections, it was calculated that the concentration in the chromatin was about eight-fold higher than in the cytoplasm. Moreover, in contrast to TMV coat protein, its dissociation from chromatin required sodium chloride. This indicates that the 116 kDa protein is bound more tightly to the chromatin than TMV coat protein. These observations strongly suggest that the 116 kDa protein may play a regulatory role in gene expression, analogous to the non-histone chromatin proteins (Chapter 4).<p/>Since TMV is known to code for a protein of a similar molecular mass, it was investigated whether the new 116 kDa chromatin-associated protein from mosaic-diseased tobacco leaves is identical with the TMV-coded 126 kDa protein. In SDS-containing polyacrylamide gels the 116 kDa protein comigrated with the 126 kDa translational product synthesized in vitro from TMV RNA. Furthermore, limited digestion of the 116 kDa polypeptide and the 126 kDa translational product with protease V8 yielded the same peptide fragments, indicating that both proteins are identical (Chapter 5).<p/>In Chapter 6 two-dimensional gel electrophoretic patterns of chromatin-associated proteins from cultivars and species of <em>Nicotiana</em> with different genetic constitutions were compared, using an improved protein preparation procedure. Notably the application of phenol extraction, followed by precipitation of proteins with ethanol, yielded highly concentrated protein samples. This procedure resulted in sharper and more intense protein spots on the gels than before. In all <em>Nicotiana</em> species and cultivars examined, <em>90%</em> of the chromatin-associated proteins appeared identical. Among the 10%, differing polypeptides no specific polypeptide spot(s) could be associated with the presence of the gene <em>N,</em> that governs hypersensitivity towards TMV.<p/>Chapter 7 discusses the possible role of the 116 kDa protein in symptom development. It is proposed that the 116 kDa polypeptide may act as a repressor of plant genes that specifically function early in leaf development. This hypothesis is based on the observation that inhibition of DNA-dependent RNA synthesis with actinomycin D induces vein clearing and mosaic in developing leaves similar to the symptoms induced by TMV in leaves not over 15 mm in length at the moment of inoculation. Recent results by Haseloff <em>et al</em> . (Proc. Natl. Acad. Sci. USA 81, 4358-4363 (1984)) indicate that two similarly-sized translational products of the serologically unrelated plant viruses alfalfa mosaic virus and brome mosaic virus have approximately 20-30% amino acid homology with the TMV-coded 116 kDa polypeptide. Such observations suggest that other plant viruses may use similar strategies to interact with their hosts.
Early experiments in agroforestry; colonial tobacco cultivation with tree fallows in Samatra, Indonesia
Wiersum, K.F. - \ 1983
International Tree Crops Journal 2 (1983). - p. 313 - 321.
geschiedenis - sumatra - nicotiana - zwerflandbouw - tabak - agroforestry - history - shifting cultivation - tobacco
Het 'vuil' in de tabak
Dijkstra, J. - \ 1982
Gewasbescherming 13 (1982)4/5. - ISSN 0166-6495 - p. 11 - 22.
nicotiana - plantenziekten - plantenvirussen - tabak - tabaksmozaïekvirus - plant diseases - plant viruses - tobacco - Tobacco mosaic virus
Historisch overzichtsartikel over de bevindingen in het onderzoek naar tabaksmozaiekvirus
Regulation of ethylene biosynthesis in virus-infected tobacco leaves
Laat, A.M.M. de - \ 1982
Landbouwhogeschool Wageningen. Promotor(en): J. Bruinsma. - Wageningen : De Laat - 88
plantenziekten - plantenvirussen - nicotiana - tabak - acc - metabolisme - ethyleen - biosynthese - plant diseases - plant viruses - tobacco - metabolism - ethylene - biosynthesis
During the hypersensitive reaction of tobacco <em>(Nicotiana tabacum</em> L.) cv. Samsun NN to tobacco mosaic virus (TMV), the appearance of local lesions was accompanied by a large burst of ethylene. Biosynthesis of both basal and virus-stimulated ethylene production was investigated <em>in vivo</em> by labeling experiments, the use of specific inhibitors, and the determination of the concentration of the probable precursor and intermediates.<p/>Determination by labeling of the role of a specific compound as a precursor in a particular biosynthetic pathway may be complicated. By comparing the specific radioactivities of, on the one hand, the endogenous precursor pool, and, on the other hand, the product involved, a quantitative estimate of precursor-product conversion can be obtained. However, this ratio is altered by unequal distribution of the labeled precursor and/or the product formation within an individual plant or animal, within a specific organ or tissue, or even within cells, leading to erroneous conclusions.<p/>The main biosynthetic pathway of ethylene in plant tissues has been established as methionine ->S-adenosylmethionine (SAM) ->1-aminocyclopropane-l-carboxylic acid (ACC) ->ethylene. In this research we investigated the role of methionine as ethylene precursor in a pathological situation suchas a hypersensitive reaction. Non-infected and hypersensitively- reacting tobacco leaves were labeled with L-(U- <sup><font size="-1">14</font></SUP>C)methionine by petiolar uptake. Under these conditions the labeled methionine was retained mainly in the veins, whereas the virus- induced ethylene production was restricted to the immediate vicinity of the developing lesions in interveinal tissues, preventing the assessment of the importance of methionine as the ethylene precursor (Chapter I). This complication was avoided by labeling leaves by vacuum infiltration. A comparison of the specific radioactivities of the methionine pool and the ethylene produced by non-infected or hypersensitively-reacting leaves, was highly indicative of methionine being the only ethylene precursor in both cases.<p/>By using aminoethoxyvinylglycine (AVG), a specific inhibitor of methionine-derived ethylene synthesis, and by determination of endogenous concentrations of ACC, methionine was further demonstrated to be the only precursor of ethylene in both noninfected and TMV-infected Samsun NN tobacco. Even the small amount of ethylene emanated in the presence of AVG was derived from methionine.<p/>Endogenous concentrations of both methionine and SAM remained constant up to 4 days after inoculation. Furthermore, exogenously applied methionine or SAM did not increase ethylene production in non-infected leaf discs, although both precursors were directly available for ethylene production. In contrast, ethylene production was increased severalfold upon incubation of leaf discs in solutions of ACC. Thus, ethylene production in tobacco was not regulated at the level of the concentration or availability of either methionine or SAM, but was primarily limited at the level of ACC production (Chapter III).<p/>The sharp peak in ethylene production near the time of lesion appearance was preceded by a strong rise in ACC production, peaking 8 h earlier. As a result, ACC accumulated in the tissue. Only after lesions had become macroscopically visible, the capacity of the leaf to convert ACC to ethylene increased severalfold, associated with a sharp decrease in ACC content and a large rise in ethylene evolution. Thus, virus-stimulated ethylene production during a hypersensitive reaction turned out to be regulated at the level of both the production of ACC and its conversion to ethylene (Chapter III).<p/>Investigation of genetically different host/virus combinations revealed that an increased ethylene production after virus infection was determined neither by the genetic constitution of the host plant, nor by the properties of the infecting virus, but was related exclusively to the type of symptoms expressed. No rise in ACC production occurred in combinations leading to systemic mosaic symptoms.<p/>The rise in ACC production in hypersensitively-reacting combinations depended on both RNA and protein synthesis, suggesting the ACC-synthase to be synthesized de novo. So far efforts to find an ACC- synthase- inducing factor have failed: the increase in ACC production could not be mimicked by local membrane damage, and no ACC-synthasestimulating agent could be isolated from leaves with developing lesions.<p/>Light inhibited the conversion of ACC to ethylene via (part of) the photosynthetic system. Inhibiting protein synthesis during the shift from light to darkness abolished the increase in ACC conversion, indicating that the enzyme is synthesized de novo. However, the rapid decrease upon a shift from darkness to light cannot be easily explained and may involve both active degradation and/or inactivation (Chapter V).<p/>After primary infection of hypersensitively-responding plants the ACC-converting capacity was increased systemically within the plant. As. no ACC accumulated upon challenge inoculation of systemically-resistant leaves. acquired resistance may be related to the increased capacity to convert ACC to ethylene (Chapter IV).<p/>The involvement of virus-stimulated ethylene production in virus localization was further investigated by studying effects of temperature, light conditions, and leaf age on both ethylene production and lesion size (Chapter VI). In non-infected leaves, both endogenous and ACC-stimulated ethylene production increased with increasing temperature up to 35°C, and decreased with increasing leaf age. Light inhibited only the conversion of ACC to ethylene. Temperature, light and leaf age similarly affected the pattern of virus - stimulated ethylene production; enhanced localization of the virus in old leaves was associated with a sharp peak in ethylene production near the time of lesion appearance. In contrast, large lesions developed in continuous light or in young leaves, where virus-induced ethylene production increased only gradually from lesion appearance onwards. Hence, an early burst of ethylene and the virus localizing reaction are closely connected.<p/>The expression of the <em>N</em> gene in Samsun NN tobacco, which confers hypersensitivity towards all strains of TMV, does not occur above 28°C. From experiments in which temperature was shifted from 20° to 30°C and back, the <em>N</em> gene was demonstrated to be involved only in the initiation, and not in the realization of the hypersensitive reaction. <em>N</em> -gene activity is required for at least 6 h between 16 and 24 h after inoculation for both stimulation of ethylene production and local lesions to develop.
Tabaksteelt in Nederland
Anonymous, - \ 1981
Wageningen : Pudoc (Literatuurlijst / Centrum voor Landbouwpublikaties en Landbouwdocumentatie no. 4525)
bibliografieën - nederland - nicotiana - tabak - bibliographies - netherlands - tobacco
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 - tobacco - cowpeas - cell structure - proteins - cells - viruses - plant diseases - plant pests - plant protection - plant pathology - plant disorders - rna viruses
<p/>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.<br/>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 <em>in</em><em>vitro</em> 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.<br/>The TMV multiplication runs synchronously in all protoplasts that are infected. Therefore, proteins synthesized in small amounts upon infection, may be thus detected.<br/>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: <em>Nicotiana Tabacum</em> 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.).<br/>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 <em>g</em> 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 <em>g</em> supernatant (chapter 4.).<br/>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 ( <em>Vigna unguiculata</em> (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 <sup>5</sup> 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 <sup>5</sup> 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.<br/>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.).<br/>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.<br/>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.<br/>The polypeptides of molecular weight 170,000, 116,500, 72,000 and coat protein were present in the 31,000 x <em>g</em> 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 <em>g</em> supernatant fraction (see table 6. l., chapter 6.).<br/>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.<br/>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.).<br/>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.<br/>A polypeptide of 170,000 daltons is synthesized <em>in vitro</em> 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 <em>in vitro</em> 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.<br/>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 <em>in vitro</em> 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.<br/>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 <em>g</em> 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 <em>g</em> 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 <em>g</em> 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).<br/>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.<br/>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.).<p/>
Mutability of the self-incompatibility locus and identification of the S-bearing chromosome in Nicotiana alata
Gastel, A.J.G. van - \ 1976
Landbouwhogeschool Wageningen. Promotor(en): J.H. van der Veen, co-promotor(en): D. de Nettancourt. - Wageningen : Pudoc - ISBN 9789022006030 - 89
plantkunde - nicotiana - voortplanting - solanaceae - tabak - zelfcompatibiliteit - mutaties - loci - triploïdie - aneuploïdie - botany - reproduction - tobacco - self compatibility - mutations - triploidy - aneuploidy
<p/>γrays, X rays, fast neutrons and ethyl methanesulfonate (EMS) were used for inducing mutations at the self-incompatibility locus of <em>Nicotiana alata.</em><p/>Chronic gamma irradiation and EMS treatment neither induced selfcompatibility mutations nor led to changes from one S allele to another. X rays and fast neutrons induced many self-compatibility mutations, but did not generate new self-incompatibility alleles.<p/>Triploid individuals were male sterile.<p/>Tri(S)allelic aneuploid plants were self-incompatible because heterogenic di(S)allelic pollen grains are not functional.<p/>Self-compatibility in pollen-part mutants with and without a centric fragment was explained by complementation of the mutant S allele by a fragment or duplication. Deviations of expected segregation ratios were explained by lethality of S homozygotes.<p/>It was shown that the longest unsatellited acrocentric chromosome is the S-bearing chromosome.
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
Purification of cytoplasmic ribosomes by column chromatography
Venekamp, J.H. ; Kliffen, C. ; Mosch, W.H.M. - \ 1973
Wageningen : Instituut voor Plantenziektenkundig Onderzoek (Mededeling Instituut voor Plantenziektenkundig Onderzoek No. 651) - 3
chromatografie - ribosomen - phaseolus vulgaris - pisum sativum - nicotiana - lolium multiflorum - chromatography - ribosomes
Additional data on the ultrastructure of inclusion bodies evoked by sharka (plum pox) virus
Bakel, C.H.J. van; Oosten, H.J. van - \ 1972
Wageningen : [s.n.] (Mededeling / Laboratorium voor virologie. Landbouwhogeschool no. 87) - 8
cellen - nicotiana - organellen - plantenziekten - afwijkingen, planten - plantenziektekunde - plantenplagen - gewasbescherming - plantenvirussen - pruimen - pruimedanten - prunus domestica - taxonomie - tabak - virussen - cells - organelles - plant diseases - plant disorders - plant pathology - plant pests - plant protection - plant viruses - plums - prunes - taxonomy - tobacco - viruses
The relation of polyphenoloxidase and peroxidase to symptom expression in tobacco var. "Samsun NN" after infection with tobacco mosaic virus
Loon, L.C. van; Geelen, J.L.M.C. - \ 1972
Wageningen : [s.n.] (Mededeling / Laboratorium voor virologie. Landbouwhogeschool no. 83) - 12
nicotiana - plantenziekten - afwijkingen, planten - plantenziektekunde - plantenplagen - gewasbescherming - plantenvirussen - tabak - plant diseases - plant disorders - plant pathology - plant pests - plant protection - plant viruses - tobacco
Pathogenese en symptoomexpressie in viruszieke tabak : een onderzoek naar veranderingen in oplosbare eiwitten
Loon, L.C. van - \ 1972
Landbouwhogeschool Wageningen. Promotor(en): J.P.H. van der Want; A. van Kammen. - Wageningen : Laboratorium voor Virologie, Landbouwhogeschool - 152
plantenziekten - plantenplagen - gewasbescherming - plantenziektekunde - afwijkingen, planten - nicotiana - tabak - plantenvirussen - cum laude - plant diseases - plant pests - plant protection - plant pathology - plant disorders - tobacco - plant viruses
The nature and the severity of the reaction of a plant to infection by a virus are determined by the genetic properties of both virus and hostplant. The genetic information of both is expressed in the formation of proteins. Our aim was to gain insight into the specificity of the interaction between virus and hostplant by investigating the biochemical mechanism of pathogenesis and symptom expression. Hence, we attempted to show if differences in protein constitution between noninfected and infected plants could indeed be established and whether such differences can be regarded as characteristic of the infecting virus or of the hostplant. In this study we made use of tobacco varieties that differ in reaction type to tobacco mosaic virus (TMV) and of strains of this virus that are distinguished by the symptoms they induce in tobacco. As the multiplication of TMV most probably takes place in the cytoplasm, the soluble protein fraction was investigated. Previous investigations have emphasized alterations in enzyme activities as a result of infection. In contrast to this, our attention was focused on changes in the electrophoretic pattern of protein bands induced by infection, not manifesting themselves immediately under the form of enzymatic activity.

By using electrophoresis in 5, 7.5 and 10% polyacrylamide gel, over fifty different protein components in the soluble protein fraction from leaves of Nicotiana tabacum could be distinguished. No differences in protein patterns were observed between noninfected plants of the varieties Samsun and Samsun NN, although Samsun plants react to infection with TMV W U1 by formation of systemic mosaic symptoms, while Samsun NN plants - which contain the factor N from N. glutinosa - develop local lesions at temperatures below 25°. However, in the two varieties different and characteristic changes in protein patterns appeared upon infection. Apart from a number of quantitative changes, one new band was present in mosaic-diseased Samsun plants four weeks after infection. This band was identified as the free coat protein of the virus by co-electrophoresis and serology. A reduction in the electrophoretic mobility of the major band was also recorded.

In the inoculated leaves of Samsun NN plants four new protein components (I-IV) were present one week after infection. These new components are not related to TMV coat protein. In N. glutinosa one new band was induced and two bands increased markedly after infection with TMV W U1, while one other band disappeared. These bands differed in electrophoretic mobility from the new bands observed after infection of Samsun NN plants. Therefore, none of the new components I-IV can be regarded as product of the Hg chromosomes, that are derived from N. glutinosa and contain the factor N. This was further substantiated by patterns from Samsun plants showing semi-systemic yellow ringspot symptoms as a result of infection with TMV HR. In this combination, both free TMV HR coat protein and the new components I-IV were apparent.

In Samsun plants infected by TMV W U1 or TMV HR only a limited number of quantitative changes was observed. Contrary to this, Samsun NN plants infected by TMV W U1 showed a considerable number of quantitative changes, most of which did not appear in the two combinations mentioned earlier. The extent of these changes correlated with the lesion density on the leaves.

When systemic mosaic symptoms were induced in both Samsun and Samsun NN plants as a result of infection with TMV W U1 at 30°, identical changes in protein patterns were observed for both varieties. These changes were the same as those in the combination TMV W U1 - Samsun at 20° and those in the combination TMV W U1 - Samsun EN, in which identical symptoms are induced. on the other hand, the formation of local lesions on the variety Samsun EN upon infection with TMV HR led to the appearance of the new components I- IV. It follows that in the combination TMV - tobacco the changes in soluble proteins are connected with the type of symptoms - either mosaic or local lesions produced, and that in all cases they are hostplant dependent.

The induction of local or systemic necrosis on Samsun and Samsun NN tobacco with tobacco necrosis virus (TNV), tobacco rattle virus (TRV) and potato virus Y n(PVY n) always led to the appearance of the four new components in both varieties,
but the relative proportions of the bands varied with the variety used, and with the characteristic of the virus to remain local or become systemic. Relatively low concentrations of the four new components were observed after infection with cucumber
mosaic virus, although no necrosis developed in these combinations. Bands I and II were present after infection with PVY o, that only causes mild mottling. Although potato virus X (PVX) induces a similar mottling, the new components were not detected in plants infected by this virus. In none of the combinations the presence of virus-specific proteins could be established.

Many of the quantitative changes observed in the various combinations occurred under different conditions and evidently represented general reactions, as similar changes were detected after cutting or freezing of the leaves. Same changes, however, were characteristic of the type of symptoms produced after virus infection. Cutting or freezing of the leaves or production of artificial necrosis by spraying with HgCl 2 induced no new components. The greater part of the quantitative changes occurring as a result of necrosis induced by virus infection were not observed in HgCl 2 induced necrosis either. So necrosis due to virus infection and artificially induced necrosis can be clearly distinguished by the accompanying changes in the soluble protein fraction. Since cutting or freezing of the leaves induces changes that are only partly similar to those observed when necrosis is induced by virus infections, ageing and injury seem to be only minor facets of the metabolic alterations underlying this type of symptom.

In the combination TMV W U1 - Samsun NN the four new components first appeared at the onset of necrosis, and the bands increased in intensity with time. By five clays after inoculation band I ceased to increase, whereas bands II, III and IV increased up to day 14. From day 7 onward, the four bands were also present in the young leaves that had developed after inoculation and neither showed symptoms nor contained virus. In these leaves also, the bands increased in intensity with time. These bands were also present in the young leaves that had developed after inoculation with TNV or TRV.

In the combination TMV W U1 - Samsun NN the amount of the four new components correlated with lesion density. The increase in intensity of the bands was reduced by treatment of the leaves with actinomycin D (MD) two days after inoculation. AMD inhibited the incorporation of 35S- methionine and 14C-leucine in the soluble protein fraction by 56-60%. Infection with TMV in itself also strongly inhibited synthesis of soluble proteins. These two effects appeared to be at least additive. However, the inhibition of the amount of the new components by AMD always amounted to less than 50%. Although preferential synthesis of the new components could not be demonstrated by electrophoresis of labeled proteins, the ratios of the radioactivities incorporated into protein from infected and noninfected plants point to de novo synthesis which can be only partly inhibited by AMD.

The new components are not isoenzymes; of thirty enzymes studied, and do not contain carbohydrate, lipid or RNA. Their strong colouration with coomassieblue may indicate a high content of basic amino acids.

A possible relation between the occurrence of these new components in young, developing leaves not containing virus, and the ability of these leaves to react with the formation of small lesions after (a second) inoculation with a virus that induces local lesions, was further investigated. There appeared to be a distinct correlation between the presence of the new components and the state of systemic acquired resistance. However, when eluates from gel slices that contained the four new components were applied to the plants simultaneously or 24 hours before inoculation with TMV, no effect on number or size of the lesions could be demonstrated. On the other hand, the multiplication of TMV in leaves that developed after inoculation of Samsun plants with TNV did appear to be inhibited to a considerable extent as a result of the first infection.

Ammonium sulfate fractionation of purified protein fractions from noninfected and TMV IV U1-infected Samsun NN plants revealed two other new components. Upon gelfiltration on Sephadex G 100, in addition to the new components I-IV, another eight, more slowly migrating components were detected. These twelve new components all have molecular weights between 10,000 and 20,000. A number of these were observed as quantitative changes upon electrophoresis of unfractionated extracts. During electrophoresis of extracts from noninfected plants separation due to differences in molecular size prevailed. Therefore, the new components differ from the majority of the other soluble proteins from tobacco leaves by their relatively small charges.

In addition to these twelve new components, a new ribonuclease and a new peroxidase isoenzyme were detected. The peroxidase isoenzyme was distinguished by a low pH optimum and a far greater affinity towards guaiacol than towards o -diphenols. It was induced in all combinations in which necrosis due to virus infection occurs, and to a small extent after infection with PVX and PVY o. In the combination TMV W U1 - Samsun NN maximal activity was reached when symptom development could be considered complete. The new isoenzymes were not present in young, symptomless leaves not containing virus.

On the basis of data given in the literature, it can be envisaged that the new components might also be present in tobacco plants which, upon TMV infection, react with systemic mosaic symptoms, but in that case only after the multiplicaticn of the virus has stopped. Therefore, it seems possible that both in hypersensitively reacting tobacco plants and in plants that react with systemic mosaic symptoms, the appearance of the new components is connected with a slowing down of viral synthesis through a direct or indirect inhibition of virus multiplication. Their presence, together with the occurrence of increased peroxidase activity - that is correlated with a decreased rate of lesion enlargement - might be responsible for the expression of acquired resistance to subsequent inoculation. However, this phenomenon could also be explained by inhibition of the formation of a specific protein which may not, or in turn may result in the induction of new proteins and enzymes.

The induction of the hypersensitive reaction and of necrosis by viruses seems to be governed by a common mechanism. Presumably TMV W U1 in Samsun tobacco represses this mechanism. This would enable the virus to multiply and spread throughout the plant.

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 - tobacco - plant viruses - Tobacco rattle virus
<p/>Tobacco rattle virus is a rod-shaped multiparticle virus.<p/>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.<p/>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.
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