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PubMed1994

Molecular biology of resistance to potato virus X in potato.

It has been proposed that plants express resistance to pathogens when the product of a resistance gene interacts with an elicitor molecule produced by the pathogen. Although there is one instance with tobacco mosaic virus (TMV) in which virus resistance is known to act through the same type of mechanism, it is not known whether this model accounts generally for resistance interactions with plant viruses. To address this issue the interactions of resistance genes in potato with potato virus X (PVX) have been analysed at the molecular level. PVX is an RNA virus that is affected by three different types of resistance locus in various potato cultivars. By using recombinant isolates of PVX, incorporating components of strains or mutant viruses able to overcome or avoid the effects of the resistance loci, we have identified different regions of the viral genome that determine the outcome of the resistance interaction. This information has allowed us to investigate the resistance in detail. For example, with the resistance specified by the Rx locus, it has been shown that the coat protein is an avirulence determinant and elicitor of an induced resistance. This resistance acts by reducing virus accumulation in the inoculated cell. Although the recognition component of the resistance is highly specific, the induced response is apparently non-specific and is effective against viruses unrelated to PVX in cells doubly inoculated with PVX and a second virus. The recognition function of Rx is also expressed in Gomphrena globosa which is a non-host plant of PVX. Based on these data, we propose that virus resistance fits the paradigm of resistance to fungal and bacterial pathogens and that there are similarities between the mechanism of cultivar specific resistance and non-host resistance to pathogen attack. Further analysis of the mechanism of the non-specific response phase may ultimately allow genetic engineering of broad-spectrum virus resistance in crop plants.

Plant Diseases↗

PubMed1979

[Methodological hints on the gas chromatographic determination of kelevan and kepone in potatoes, potato foliage and soil].

The gas chromatographic determination of Kelevan, an ethyllaevulinic acid derivative of Kepone, requires its conversion to Kepone. Thermal decomposition in the injection block of the gas chromatograph at 45 degrees C. has proved to be well suited for routine work. The sample is extracted with acetonitrile; the extract is purified and concentrated under reduced pressure. The separation of Kelevan from Kepone already metabolically produced is possible by column chromatography (silica gel; elution with benzene and 5% acetone in benzene). The determination of pre-existent Kepone and of Kelevan (after thermal conversion to Kepone) was achieved by means of a Pye-Unicam gas chromatograph. The limits of detection are 0.002 p.p.m. for Kepone, and 0.003 p.p.m. for Kelevan. The recovery of Kelevan and Kepone added to control samples (for every 10 determinations with different amounts added to potatoes, potato foliage and soil samples) ranges from 63 to 88%; the standard derivation, from +/- 1.7 to 7.3%.

Chlordecone↗

PubMed1987

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells.

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.

Journal Article↗

PubMed1997

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

Carrier Proteins↗

PubMed1994

Small-scale field tests with transgenic potato, cv. Bintje, to test resistance to primary and secondary infections with potato virus y.

The coat protein (CP) gene of the potato virus Y (PVY) strain N605 has been cloned into a plant binary expression vector and introduced into the potato variety Bintje. The transformed lines, Bt6, that contained two copies of the CP gene showed complete resistance to the homologous strain PVY-N605 and a good resistance to the related strain PVY-O803 in the greenhouse. The good resistance of Bt6 to primary and secondary infections by PVY was confirmed in two successive field tests where the virus was transmitted by its natural aphid vector.

Animals↗

PubMed1991

RFLP mapping on potato chromosomes of two genes controlling extreme resistance to potato virus X (PVX).

Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance gene Rx1 mapped to the distal end of chromosome XII, whereas Rx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping of Rx1. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species. Rx2 was mapped by adopting this strategy.

Blotting, Southern↗

PubMed1990

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis.

A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.

Animals↗

PubMed1985

Optimisation of conditions for infection of isolated potato protoplasts with potato virus X.

Conditions for the reproducible infection of potato protoplasts with PVX were determined. Successful virus infection was achieved when potato protoplasts at a concentration of 3 X 10(5) cells/ml were incubated with 20 micrograms/ml PVX and 2 micrograms/ml Poly-L-ornithine (MW 100,000) for 15 minutes at 0 degrees C. Numbers of protoplasts infected varied between 30 and 50 per cent. Preincubation of PVX with PLO for a minimum of 10 minutes was essential for good infection. The type and molecular weight of polycation used had a major effect on virus infection. PLO at 100,000 MW was the most efficient.

Cell Survival↗

PubMed1997

Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato.

Open reading frame 4 (ORF4) of the potato virus X (PVX) genome encodes an 8K protein which is a part of the "triple gene block" and is known to play a role in the cell-to-cell movement of the virus in infected plants. To locate the 8K protein and further elucidate the mechanism of cell-to-cell transport of PVX, antibodies were raised against the 8K protein and used to localize this protein in PVX-infected tobacco and in transgenic potato plants expressing the 8K protein both by subcellular fractionation and by immunolabeling with colloidal gold. The results indicated that the 8K protein was localized to the cell wall.

Antibodies, Viral↗

PubMed2005

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato.

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K(i) values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.

Animals↗

PubMed1990

Comparative sequence of the helper component (HC) region of potato virus Y and a HC-defective strain, potato virus C.

Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code for a protein (PVC-HC) which is similar in molecular weight and immunological reactivity to the helper component protein of PVY (PVY-HC). PVC-HC, however, was inactive with respect to its ability to effect aphid transmission of either PVC or PVY. The 5'-terminal 2.7-kb regions of PVC and PVY were sequenced. Within the HC region there was 92% nucleotide homology between the two strains; comparison of the derived amino acid sequences revealed 24 amino acid differences. Comparison of the PVC-HC sequence with that of five potyviruses revealed 2 amino acid changes which were specific to PVC-HC. These amino acids are prime targets for mutational analysis of HC activity.

Amino Acid Sequence↗

PubMed1991

Replication of potato virus X RNA is altered in coinfections with potato virus Y.

Potato virus X (PVX) and potato virus Y (PVY) may coinfect tobacco to cause a classic synergistic disease. In the acute stage the disease is characterized by a dramatic increase in the accumulation of infectious PVX particles, with no corresponding increase or decrease in the accumulation of PVY. The accumulation of PVX genomic RNA and coat protein has been examined in doubly versus singly infected tobacco leaves. These experiments indicate that the levels of both viral components increase in doubly infected plants to about the same extent as the level of infectious PVX particles. The level of PVX subgenomic coat protein mRNA found associated with polyribosomes of synergistically infected plants is also increased to a similar extent. Pulse labelling experiments suggest that the increase in PVX coat protein is due to an increased rate of synthesis. The level of PVX (-) strand RNA template increases disproportionately in doubly infected tissue, to a level three times higher than that of the virion or its component parts. This result suggests that PVX/PVY synergism involves an alteration in the normal regulation of the relative levels of PVX (+) and (-) strand RNAs during viral replication.

Blotting, Northern↗

PubMed1997

The potato leafroll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C.

The 17 kDa protein (pr17), the phloem-limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274-282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid-binding, basic, C-proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+- and phospholipid-dependent, membrane-associated protein kinase. This protein kinase activity was inhibited by the addition of (19-36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell-companion cell complex of the phloem, by the activity of PKC-related, membrane-associated protein kinase activity.

Animals↗

PubMed1997

Molecular identification and relatedness of potato witches'-broom phytoplasma isolates from four potato cultivars.

Four isolates of potato witches'-broom phytoplasma, designated as PW1, PW2, PW3 and PW4, were established on four potato cultivars. The identity of each isolate was confirmed by PCR using two universal primer pairs and one specific primer set derived from phytoplasma of 16S rDNA sequences. The four isolate samples formed similar RFLP patterns after digestion of 1.2 kb PCR products with restriction endonucleases AluI, HhaI, RsaI and Sau3A. The direct DNA sequencing with the specific primer pair showed that there are no differences in the base sequences among PW1, PW2, and PW3 phytoplasma isolates and that PW4 is closely related to them. Thus, the four isolates were identified as members of the clover proliferation group.

Base Sequence↗

PubMed2000

Population biology of the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae), in two potato cropping systems in Israel.

The life cycle, within-field distribution, crop damage and impact of natural enemies of the potato tuber moth, Phthorimaea operculella (Zeller) were investigated in two potato cropping systems. The two systems differed in soil type (sandy vs. loess) which in turn affected the choice of cultivars, irrigation programme, insecticide application method (ground vs. aerial), and planting and harvest times. From mid-April to the end of May, almost twice as many moths were caught in pheromone traps in sandy than in loess fields. Highest infestation of tubers was found before harvest, and infestation was greater in loess than in sandy fields. Larval densities in foliage and tubers were significantly higher at the margins of the fields than in the centre. A significant positive correlation was found between adult catch and larval infestation on foliage in sandy fields but not in loess. Tuber infestation in sand was positively correlated with foliage infestation. No such correlation was detected in loess. Five parasitic wasps emerged from P. operculella larvae collected from commercial fields and volunteer plants: Diadegma pulchripes (Kokujev) and Temelucha decorata, (Gravenhorst) (Ichneumonidae) and Bracon gelechiae Ashmead and two other unidentified Braconidae. The most abundant predators at the field site were Coccinella septempunctata Linnaeus (Coccinellidae), Chrysoperla carnea Stephens (Chrysopidae), Orius albidipennis (Reuter) (Anthocoridae) and four ant species (Formicidae). Parasitism rate reached 40% and predation was estimated at 79%. Results are discussed with regard to the development of an integrated pest management programme for this important pest.

Animals↗

PubMed2003

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.

Amino Acid Sequence↗

PubMed1997

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes.

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50 degrees C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Base Composition↗

PubMed2005

The broadly insecticidal Photorhabdus luminescens toxin complex a (Tca): activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci.

Toxin complex a (Tca), a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens, has been found to be orally toxic to both the Colorado potato beetle, Leptinotarsa decemlineata, and the sweet potato whitefly, Bemisia tabaci biotype B. The 48 hour LC50 for Tca against neonate L. decemlineata was found to be 2.7 ppm, and the growth of 2nd instar L. decemlineata exposed to Tca for 72 hours was almost entirely inhibited at concentrations above 0.5 ppm. B. tabaci was highly susceptible to Tca as well; newly emerged nymphs that were artificially fed Tca developed poorly, or not at all. Tca concentrations between 0.1 and 0.2 ppm reduced the number of nymphs reaching the second instar by 50%. In addition, a preparation of Tca missing two prominent subunits, TcaAii and TcaAiii, was found to be at least as toxic to L. decemlineata and B. tabaci as Tca itself, indicating that the activity of Tca is not dependant on the presence of these subunits at the time of ingestion.

Animals↗