Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Non‐pathogenic association of L‐form bacteria (pseudomonas syringae pv. phaseolicola) with bean plants (Phaseolus vulgaris L.) and its potential for biocontrol of halo blight disease

L‐forms of the halo blight pathogen, Pseudomonas syringae phaseolicola, were maintained in a medium which suppressed cell wall synthesis. These L‐forms, unlike revertants (walled forms derived from unstable L‐forms) and cell walled (parent) organisms, did not elicit a hypersensitive response in tobacco leaves. Association of L‐forms with Phaseolus vulgaris was established by seed imbibition in L‐form suspensions compared with appropriate control treatments (5% mannitol or heat‐killed cells). Seedling emergence and plant growth was not affected by L‐form imbibition. The association was detected by agglutination assays using polyclonal antibody. The L‐form association was localized to the lower shoot tissue and was progressively lost with age of plants. Plants with associated L‐forms had vigour and shoot weights equivalent to controls and showed no disease symptoms. The cell walled form could not be isolated from plants showing positive agglutination. On challenge with the pathogen, plants associated with L‐forms showed significantly less disease symptoms than controls. Stem extracts, from associated plants, were inhibitory to in vitro cultures of both L‐forms and parent forms of Ps. syr. phaseolicola. These results indicate that L‐form associations confer induced systemic resistance to bean plants and might be developed as novel biocontrol systems.     

The emergence of transgenic potatoes as commercial products and tools for basic science

The potato has tremendous potential as a transgenic crop and is a good model system by which to analyse metabolic regulation and gene expression. The potato’s difficult genetics, but ease of genetic transformation and its clonal means of propagation make it ideal for applied agricultural molecular genetics. Thus, the next 4 years promise to put the potato (with a diversity of transgenic constructs expressed) in the limelight as many of the first transgenic agricultural products enter the marketplace.     

Structures of the α(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant toAleuria aurantia agglutinin: elucidation by1H-NMR spectroscopy

Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL₂) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz¹H-NMR spectroscopy. By this method, their antennae were found to be terminated either by (2-3 or 6)-linkedN-acetylneuraminic acid or (1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.Abbreviations NAc N-acetyl group - NGc N-glycolyl group - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Man mannose - Gal galactose - Fuc fucose - Con A concanavalin A - LCA Lens culinaris agglutinin - AAA Aleuria aurantia agglutinin - WGA Wheat germ agglutinin - RCA II Ricinus communis agglutinin II - PBS phosphate buffered saline, 0.01m Na₂HPO₄/0.14m NaCl, pH 7.2 - HPLC high performance liquid chromatography - EMEM Eagle's Minimal Essential Medium - Lec^R lectin resistant - MG -methylglycoside     

Phage resistance and altered growth habit in a strain of Streptococcus bovis

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.     

A chloramphenicol-streptomycin-resistance plasmid from a clinical strain of Staphylococcus sciuri and its structural relationships to other staphylococcal resistance plasmids

A 5.1-kb plasmid, designated pSCS12, isolated from a naturally occurring Staphylococcus sciuri conferred resistance to chloramphenicol (CmR) and streptomycin (SmR). Restriction endonuclease analyses of pSCS12 revealed partial structural homologies to the CmR-plasmids pC221 from S. aureus and pSCS1 from S. intermedius, to the SmR-plasmids pSAI-1 from S. hyicus and pS194 from S. aureus, as well as to the CmR/SmR plasmid pSK68 from S. aureus. Southern-blot hybridization with specific CmR- and SmR-gene probes confirmed these similarities and allowed the mapping of the CmR- and SmR-determinants in the S. sciuri plasmid pSCS12. These observations lead to the suggestion that CmR/SmR-plasmids, such as pSCS12, may have evolved from CmR- and SmR-plasmids by interplasmidic recombination.     

Resistance in the wild crop relatives Avena macrostachya and Hordeum bogdani to the aphid Rhopalosiphum padi

In previous screening tests the two wild crop relatives Avena macrostachya (Bal., ex Coss. et Dur.) and Hordeum bogdani (Wil.) demonstrated a high degree of resistance to the aphid Rhopalosiphum padi (L.). In a choice situation using wild and cultivated oats and barley, alate aphids settled in lower numbers on the wild species. The results were, however, variable in the Avena combination. Nymph production was significantly higher, development time shorter and adult weight higher on the cultivated varieties. From the third instar and onwards the excretion of honeydew was significantly lower on the resistant plants. In general the honeydew contained less than 1% free amino acids although excreta from H. vulgare contained 3.5%. The percentage of free amino acids found in the honeydew was similar for all plant species (5.2–7.6%) except for H. vulgare, on which the aphids excreted 22% of the amounts ingested. Amino acids excreted in high proportions on all plants included asparagine, -aminobutyric acid, glutamic acid, and glycine. Tissue sectioning did not reveal any obvious mechanical barriers to stylet penetration. The potential use of these wild species as sources for aphid resistance breeding in oats and barley is considered.

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Résumè Lors d'examens systématiques antérieurs, Avena macrostachya (Bal. ex Coss. & Dur.) et Hordeum bogdani (Wil.) ont présenté une résistance élevée au puceron Rhopalosiphum padi (L.). Lorsqu'ils avaient un choix comprenant de l'avoine et de l'orge cultivés, les pucerons ailés ont atterri en nombres moins importants sur les espèces sauvages. Les résultats étaient cependant variables dans le complexe avoine. La production de nymphes et le poids des adultes étaient plus élevés sur espèces cultivées, ainsi que la durée du développement était plus longue sur les espèces sauvages. A partir du troisième stade, l'excrétion de miellat a été significativement plus faible sur les espèces résistantes. En général, le miellat y contenait moins de 1% d'acides aminés bien que sur H. vulgare il en contînt 3,5%. Les pourcentages d'acides aminés libres du miellat étaient semblables sur toutes les plantes (5,2–7,6%), à l'exception de H. vulgare sur lequel les pucerons excrétaient 22% des taux ingérés. Les acides aminés excrétés en fortes quantités sur les différentes plantes, comprenaient l'asparagine, l'acide -aminobutyrique, l'acide glutamique et la glycine. Des coupes de tissus n'ont révélé aucun obstacle mécanique clair à la pénétration des stylets. Les possibilités d'utiliser ces espèces sauvages comme source de résistance aux pucerons dans la sélection de l'avoine et de l'orge ont été examinées.

     

Resistance of different lucerne cultivars to the pea aphid Acyrthosiphon pisum: influence of phloem composition on aphid fecundity

Sugars and amino acids were analysed in the phloem sap (sampling by the exudation method) of four clones of lucerne (Medicago sativa L.) characterized by their resistance to the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae). Differences in the qualitative amino acid balance are shown and a negative correlation between the reproductive rate of the aphid and the sugars/amino acids ratio is underlined. To demonstrate any causal relationship between these two variables, the reproduction of A. pisum on artificial diets copying the sap of the two extreme clones (resistant and susceptible) was measured. The results show that the resistance is not a simple nutritional effect; however, the amino acid balance contributes to the resistance exhibited by some cultivars.

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Résumé L'analyse (sucres et acides aminés) de la sève de 4 cultivars de luzerne (Medicago sativa) caractérisés pour leur résistance au puceron du pois (Acyrthosiphon pisum (Harris)) (Homoptera: Aphididae), est réalisée sur des exsudats de phloème. La teneur en sucres (exprimée en équivalent saccharose) montre d'importantes différences: rapport de 1 à 3 respectivement entre les clones sensible et résistant. La teneur globale en acides aminés exsudés varie en sens inverse, entraînant une corrélation négative pour les 4 clones étudiés entre le rapport sucre/acides aminés (0,6 à 3,6) et la résistance mesurée par le taux net de reproduction à 14 jours (78,5 à 11,2 larves/femelle). L'analyse des acidses aminés révèle de plus des différences qualitatives entre cultivars, notamment pour certains acides aminés essentiels méthionine, histidine, lysine, aromatiques.Le rôle de l'équilibre en acides aminés et en sacharose dans la résistance de certains cultivars au puceron du pois est apprécié par des mesures de la reproduction de A. pisum sur des milieux artificiels reproduisant le rapport sucre/acides aminés et/ou le spectre des acides aminés des 2 clones extrêmes (sensible et résistant). La résistance n'apparaît pas être le résultat d'une simple différence de la qualité nutritionnelle de la sève; la balance en acides aminés doit cependant participer pour une part à la résistance au puceron.

     

Negative interactions between Willamette mites and Pacific mites: possible management strategies for grapes

Willamette mites and Pacific mites are often negatively associated on grape vines. In particular, vineyards with early season infestations of the less damaging Willamette mite rarely develop high populations of the economically important Pacific mite. We report that Willamette mites had a negative effect on Pacific mite populations in both the greenhouse and field. In some experiments, the negative effect was more pronounced when vines had been damaged by previous feeding of Willamette mites and in other experiments concurrent feeding by both mite species was necessary to demonstrate a negative effect. Therefore, we cannot conclude if the mechanism of the response involves induced resistance against Pacific mites, more conventional interspecific competition, or both. Since predators were uncommon in our experiments, predator build up on Willamette mites did not cause the low Pacific mite population that we observed, although this mechanism may be important in many vineyards. Further, larger scale experiments are necesary to determine if growers can introduce Willamette mites to help control Pacific mites.

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Résumé E. willametti et T. pacificus sont souvent antagonistes dans les vignobles. En particulier, dans les vignobles contaminés tôt dans la saison par E. willametti,-dont les dégâts sont moins conséquents-, on observe rarement des populations élevées de T. pacificus, dont les dégâts sont économiquement importants. Nous avons constaté que E. Willametti a réduit les populations de T. pacificus, tant en serres qu'à l'extérieur. Dans quelques cas, l'effet antagoniste était plus marqué quand les vignes avaient été préalablement endommagées par l'alimentation de E. willametti. Dans d'autres expériences, la compétition alimentaire entre les deux espèces d'acariens était nécessaire pour produire un effet antagoniste. Cependant, nous ne pouvons pas en conclure que la réponse implique une résistance induite contre T. pacificus, ou une compétition interspécifique plus classique, ou les deux à la fois. Puisque les prédateurs étaient rares dans nos expériences, une explosion des prédateurs sur E. willametti n'a pu provoquer le faible niveau de population observé avec T. pacificus, bien qu'un tel méchanisme puisse être important dans certains vignobles. Quoi qu'il en soit, des expériences à plus grande échelle sont nécessaires pour déterminer si les vignerons peuvent introduire Eotetranychus willametti afin de limiter les populations de Tetranychus pacificus.