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PubMed1976

Purification and characterization of an a type phospholipase from potato and its effect on potato mitochondria.

A potato (Solanum tuberosum) phospholipid acyl-hydrolase, which - in the pH range 7.5 to 8.5-is at least 10,000 times more effective with phospholipids than with galactolipids, has been purified and characterized. It is a soluble enzyme readily distinguished from a neutral lipid lipase and a third lipid acyl-hydrolase which, while acting on phospholipid, shows a decided preference for glyceryl monoolein. The phospholipase in question has a pH optimum of 8.5, is stimulated by Ca(2+) at pH above 7.5 and inhibited by Ca(2+) at lower pH, is not dependent on detergents although stimulated by Triton X-100 to a moderate extent, and remains very active at temperatures close to zero. The phospholipids of intact potato mitochondria are highly susceptible to degradation by potato phospholipase, and it is suggested that this enzyme is involved in the extensive lipid breakdown which occurs in fresh potato slices following cutting, and in the deterioration of mitochondria during their preparation and aging.

Journal Article↗

PubMed2005

Variation in antibiosis ability, against potato pathogens, of bacterial communities recovered from the endo- and exoroots of potato crops produced under conventional versus minimum tillage systems.

The culturable component of bacterial communities found in the endoroot and associated exoroot (root zone soil) was examined in potatoes (Solanum tuberosum L.) grown under either conventional or minimum tillage systems. Bacterial species--abundance relationships were determined and in vitro antibiosis ability investigated to discover whether tillage practice or bacteria source (endo- or exoroot) influenced bacterial community structure and functional versatility. Antibiosis abilities against Phytophthora erythroseptica Pethyb. (causal agent of pink rot of potatoes), Streptomyces scabies (Thaxt.) Waksm. and Henrici) (causal agent of potato common scab), and Fusarium oxysporum Schlecht. Emend. Snyder and Hansen (causal agent of fusarium potato wilt) were selected as indicators of functional versatility. Bacterial community species richness and diversity indices were significantly greater (P = 0.001) in the exoroot than in the endoroot. While both endo- and exoroot communities possessed antibiosis ability against the phytopathogens tested, a significantly greater proportion (P = 0.0001) of the endoroot population demonstrated antibiosis ability than its exoroot counterpart against P. erythroseptica and F. oxysporum. Tillage regime had no significant influence on species-abundance relationships in the endo- or exoroot but did influence the relative antibiosis ability of bacteria in in vitro challenges against S. scabies, where bacteria sourced from minimum tillage systems were more likely to have antibiosis ability (P = 0.0151). We postulate that the difference in the frequency of isolates with antibiosis ability among endoroot versus exoroot populations points to the adaptation of endophytic bacterial communities that favour plant host defence against pathogens that attack the host systemically.

Agriculture↗

PubMed1987

Lesions of potato sprout and extracted potato sprout alkaloid toxicity in Syrian hamsters.

Hamsters were gavaged either dried potato sprout material, alkaloid extract of potato sprouts, or the marc from which the alkaloid fraction was extracted and then were examined for gross and microscopic lesions. Nine of 10 hamsters receiving dried potato sprout material and 3 of 5 hamsters receiving alkaloid extract had severe gastric and intestinal mucosal necrosis which was most severe in the glandular stomach, duodenum and proximal jejunum. All control hamsters gavaged with water and all hamsters gavaged with the potato sprout marc survived to the time of euthanasia and did not have gross or microscopic lesions.

Alkaloids↗

PubMed1999

Properties of extracellular polysaccharides of potato ring rot pathogen and corresponding recognition sites of potato cell walls.

The properties of extracellular polysaccharides of the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus (Cms) and the corresponding recognition sites isolated from cell walls of potato suspension cultures have been studied. Extracellular polysaccharides of Cms consist of 4-6 components, which differ greatly in molecular mass (from <1 kD to >700 kD), and are capable of formation of associates stabilized by electrostatic interactions in the presence of calcium. Using affinity column chromatography, sites possessing affinity for the total extracellular polysaccharide complex of Cms were isolated from cell walls of suspension cultures of three potato varieties with different resistance to the pathogen. The content of the receptor sites consisting of glycopeptides and sugars for the variety devoid of resistance was 10 times greater than that for the resistant variety. In the receptor fraction for the latter variety, only sugars were found. The molecular masses of the components of the receptor fraction of cell walls were from 39 to 86 kD. Polypeptides in the recognition sites for the resistant variety escaped detection in electrophoretic patterns. Study of the amino acid composition of the receptor sites of cell walls showed that the sites of the resistant variety contained trace amounts of only seven amino acids. In the sites of the receptive variety 14 amino acids were found, the content of polar amino acids being twice as large as that of nonpolar amino acids. Among polar amino acids, glutamine and glycine prevailed, whereas among nonpolar amino acids valine was dominant. We suggest that one of the reasons of variety-specific resistance of potato to Cms is the absence or a low content of the sites revealing the affinity for bacterial extracellular polysaccharides on the plant cell surface.

Actinomycetales↗

PubMed2004

Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor.

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.

Animals↗

PubMed2005

Nutritional properties of tubers of conventionally bred and transgenic lines of potato resistant to necrotic strain of Potato virus Y (PVYN).

The potential effect of genetic modification on nutritional properties of potatoes transformed to improve resistance to a necrotic strain of Potato virus Y was determined in a rat experiment. Autoclaved tubers from four transgenic lines were included to a diet in the amount of 40% and compared with the conventional cv. Irga. The experiment lasted 3 weeks and special attention was paid to nutritional properties of diets, caecal metabolism and serum indices. Genetic modification of potato had no negative effect on the chemical composition and nutritional properties of tubers, ecosystem of the caecum, activity of serum enzymes and non-specific defence mechanism of the rats. Obtained results indicate that transgenic potato with improved resistance to PVY(N): line R1F (truncated gene coding for PVY(N) polymerase in sense orientation), R2P (truncated gene coding for PVY(N) polymerase in antisense orientation), and NTR1.16 (non-translated regions of PVY(N) genome in sense orientation) are substantial and nutritional equivalence to the non-transgenic cultivar. Tubers of transgenic line NTR2.27 (non-translated regions of PVY(N) genome in antisense orientation) increased the bulk of caecal digesta and the production of SCFA as compared to tubers of the conventional cultivar and the other transgenic clones. Taking into account some deviations, it seems reasonable to undertake a long-term feeding study to confirm the nutritional properties of tubers of transgenic lines.

Animals↗

PubMedDate not supplied

[The effect of the 5'-leader of potato X-virus on expression of the gene for the potato Y virus membrane protein in transgenic Solanum tuberosum].

The 5'-leader of potato virus X (PVX) genomic RNA was successfully used to generate potato plants expressing the coat protein of the potato virus YN (Russian isolate) (PVY CP). Two expression cassettes were constructed that carried the PVY CP coding sequence along with the PVY 3'untranslated region under the control of the CaMV 35S promoter and poly(A) signal. The cassettes contained different sequences between the 35S promoter and the CP artificial AUG initiating codon of the coat protein: either a polylinker or a shortened variant of the PVX leader. Transgenic Belorusskii-3 potato plants generated by agrobacterial transformation were shown to express the PVY CP gene to different extents depending on the cassette integrated into the plant genome. The cassette containing the PVX leader provided detectable amounts of the coat protein in all tested plants, whereas plants carrying the cassette with the polylinker leader produced no coat protein above the Western detection threshold. Northern analysis revealed that the presence of the PVX leader in front of the PVY CP gene initiating codon resulted in a 10-fold increase in the PVY CP mRNA steady-state levels, thus suggesting an influence of the PVX leader on mRNA stability.

Base Sequence↗

PubMed1981

Continuous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato.

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum demissum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculated in vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continuous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis de novo of PSTV was substantiated by the incorporation of [3H]uridine and of [32P]orthophosphate into viroid RNA.

Cell Line↗

PubMed1986

Expression of resistance to potato virus X in isolated potato protoplasts.

Resistance to Potato Virus X (PVX) in potato cultivars is conferred by the genes Nx and/or Nb. The Nx gene is activated by group 3 strains of PVX. When protoplasts from the cultivars King Edward containing the Nx gene and Pentland Crown containing neither resistance gene were inoculated with a group 3 strain of PVX resistance was expressed in the King Edward protoplasts. Replication of PVX occurred in King Edward protoplasts but only at a very low level as compared with the level observed in the susceptible Pentland Crown protoplasts. Similar results were obtained when protoplasts obtained from leaf discs mechanically inoculated with PVX were used. Virus levels were assayed by immunofluorescence and ELISA. Maintenance of resistance in protoplasts of King Edward following inoculation with PVX RNA was confirmed suggesting that resistance probably operates at the transcription stage in replication. These results clearly demonstrate single gene resistance in potato protoplasts inoculated with PVX.

Immunity, Innate↗

PubMed2002

The Potato virus S resistance gene Ns maps to potato chromosome VIII.

The dominant allele Ns confers in potato resistance to Potato virus S (PVS). To identify the chromosomal location of Ns, we mapped the Ns-linked marker SCG17(448) and the ISSR marker UBC811(600) to linkage group VIII of the RFLP map of a population that did not segregate for Ns. The map position of the Ns locus on chromosome VIII was confirmed with the detection of linkage between Ns and three RFLP markers, GP126, GP189 and CP16, known to be located in a corresponding region on potato chromosome VIII. PCR-based assays were developed for these RFLP markers. The PCR primers specific for GP126 generated polymorphic products (STS marker). In the case of markers GP189 and CP16, informative polymorphism was revealed in the Ns population after digestion with the restriction enzymes HaeIII and HindIII, respectively. The genetic distance between Ns and the closest CP16 locus was 4.2 cM.

Journal Article↗

PubMed2004

Two allelic or tightly linked genetic factors at the PLRV.4 locus on potato chromosome XI control resistance to potato leafroll virus accumulation.

A novel locus for potato resistance to potato leafroll virus (PLRV) was characterized by inheritance studies and molecular mapping. The diploid parental clone DW 91-1187 was resistant to PLRV accumulation in both inoculated plants and their tuber progeny. The resistance to PLRV accumulation present in DW 91-1187 was not transmitted to any F1 offspring when crossed with a PLRV susceptible clone. Instead, one half of the F1 individuals exhibited undetectable amounts of PLRV as determined by ELISA during the primary infection assay, but accumulated PLRV in their tuber progeny plants. The other half was clearly infected both in the inoculated and tuber-born plants. The inheritance of resistance to PLRV accumulation may be explained by a model of two complementary alleles of a single gene ( PLRV.4) or by two complementary genes that are closely linked in repulsion phase. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers linked to the PLRV.4 locus were selected. The two complementary factors were closely linked in coupling phase to the alternative alleles UBC864(600) and UBC864(800) of DNA marker UBC864. These markers may be used for marker-assisted selection of genotypes having both factors for resistance to PLRV accumulation. The PLRV.4 locus was mapped to a central position on linkage group XI of the potato molecular map, where no resistance locus has been mapped previously.

Chromosome Mapping↗

PubMed2001

Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato.

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.

Cloning, Molecular↗

PubMed2001

Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.

Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

Cloning, Molecular↗

PubMed2004

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases.

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.

Acetates↗

PubMed2006

The potential of the fungus, Muscodor albus, as a microbial control agent of potato tuber moth (Lepidoptera: Gelechiidae) in stored potatoes.

Potato tuber moth (PTM), Phthorimaea operculella, is a serious pest of stored potato in most countries where potatoes are grown. Entomopathogens offer promise as alternatives to broad spectrum insecticides for management of this pest. The fungus Muscodor albus, which produces a mixture of antimicrobial volatile organic chemicals, was tested for its insecticidal activity against PTM. Adults and neonate larvae were exposed to volatiles generated by 15 or 30 g of M. albus rye grain culture plus water for 72 h in hermetically sealed 28.3 L chambers at 24 degrees C. Mean percent mortalities in adult moths exposed to 0, 15, and 30 g of fungal formulation were 0.9, 84.6, and 90.6%, respectively. Development to the pupal stage of PTM that were exposed as neonate larvae to 15 or 30 of M. albus culture was reduced by 61.8 and 72.8%, respectively, relative to controls.

Animals↗

PubMed2004

Pathogen-derived resistance in potato to Potato virus Y-aspects of stability and biosafety under field conditions.

Plants of three different potato cultivars/lines were transformed via Agrobacterium tumefaciens with a truncated NIb gene of a necrotic strain of Potato virus Y (PVY(N)) which had been C-terminally fused to enhanced blue-fluorescing protein. Resistance of the resulting transgenic clones was evaluated under glass house conditions using an NTN-strain of PVY. Four clones with the highest levels of resistance were chosen for further experiments. Their type of resistance was either recovery or extreme resistance. These clones and their resistance types were also characterised at the molecular level. Mechanisms other than post-transcriptional gene silencing seemed to be involved in the resistance which was not dependent on sequence homology between transgene and challenging virus. Stability of resistance was tested under field conditions. The plants usually became infected with PVY. Tubers of the clone with extreme resistance did not recover from infection whereas those from clones with the recovery type did. No influence of transgenic potatoes was apparent on aphid population numbers in test plots. Recombination events could not be detected at the RNA level between transgene and challenging virus.

Gene Silencing↗

PubMed2000

Extreme resistance to Potato leafroll virus in potato cv. Russet Burbank mediated by the viral replicase gene.

High levels of field resistance to Potato leafroll virus (PLRV; Genus: Polerovirus; Family: Luteoviridae) were achieved by expression of the unmodified, full-length PLRV replicase gene in potato plants cv. Russet Burbank. A high degree of resistance was also achieved, but less frequently, by expression of a truncated construct of the replicase gene. In limited testing, neither miss-frame nor antisense constructs of the replicase gene conferred resistance. The degree of resistance expressed among different transformant lines ranged from near immunity to full susceptibility. Resistance to the Colorado potato beetle (Leptinotarsa decemlineata Say) was combined with resistance to PLRV by expression of the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis in combination with the unmodified, full-length, viral replicase gene. Resistance was expressed as a reduced incidence of infection detectable by foliage symptoms or serological tests. Reduced incidence of infection was not associated with a decrease in virus antigen concentration in the few plants of resistant lines that became infected. Virus was not detected in the foliage of symptomless plants but was detected in progeny plants produced from the tubers of inoculated but symptomless test plants of some resistant lines. The resistance was effective under natural exposure and against plant-to-plant spread of PLRV by the aphid vector, Myzus persicae Sulzer. Three of the resistant lines selected in these studies were released and are now in commercial production.

Antiviral Agents↗

PubMed2004

Ultrastructure of the infection process of potato tuber by Helminthosporium solani, causal agent of potato silver scurf.

Silver scurf is an important postharvest disease affecting potato tubers worldwide, caused by Helminthosporium solani. In the present study, key steps of infection of potato tubers (cv. 'Dark Red Norland') by H. solani were described using transmission (TEM) and scanning electron microscopy (SEM). The fungus entered potato tubers mainly via hyphae, although germ tubes were also able to directly penetrate the tubers. An extracellular sheath was observed around hyphae growing over the surface of tubers and the host cell wall appeared lyzed at the point of penetration. Observations suggested that both mechanical and enzymatic processes are involved in periderm penetration. Hyphae of H. solani, 9 h after tuber inoculation, were present intracellularly mostly in the periderm and in some cortical cells. Two days after inoculation, host cells were invaded and both infected and neighbouring host cells showed signs of necrosis (disrupted cytoplasm, absence of typical organelles or endomembrane systems, collapsed peridermal cells) that were not observed in healthy control tubers. Four days after inoculation, completing the infection cycle, conidiophores emerged from peridermal cells directly by erupting through the host cell walls.

Helminthosporium↗