‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Molecular aspects of the potato — Phytophthora infestans interaction

The fungal pathogenPhytophthora infestans is the causal organism of late blight, one of the most devastating diseases of potato. In the past, various aspects of the potato-P. infestans interaction have been studied extensively. In this paper we briefly review the current knowledge of the molecular events associated with the interaction and in addition we discuss a new approach for analyzing the molecular basis of pathogenicity ofP. infestans.     

Reproductive performance of nili-ravi buffaloes after a single injection of GnRH earlypost partum

Summary Sixty dairy buffaloes (second to fourth lactation) from a large buffalo farm were used to compare the effects of single intramuscular injections of 100 µg gondadotrophin releasing hormone (GnRH), 250 µg GnRH or saline given on day 14post partum. The buffaloes had calved at the end of the breeding season (December). Milk samples for progesterone determination were taken at the time of injection and then three times a week either until first insemination or until around day 90post partum. GnRH given at 14 dayspost partum resulted in quicker completion of uterine involution, earlier resumption of ovarian activity, shorter intervals between calving and conception and a better first service conception rate in non-suckled dairy buffaloes. Differences between the results obtained by a GnRH dose level of 100 µg and 250 µg were non-significant. In the post-treatment period cases of prolonged luteal activity were common in all groups of buffaloes. Therefore the sequential administration of GnRH and prostaglandin is suggested for the management of post-partum reproductive activity in problem herds.

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Desempeno Reproductivo De Bufalos Nili-Ravi Despues De Una Sola Inyeccion De GnRH Temprano Despues Del Post-Parto

Resumen Sesenta búfalos lecheros (segunda a cuarta lactancia) pertenecientes a un hato grande fueron utilizados para comparar los efectos de inyecciones intramusculares de 100 µg de GnRH, 250 µg y de solución salina, aplicadas 14 días después del parto. Los búfalos habian parido al final de la estación de pariciones (diciembre). Se tomaron muestras de leche para medir progesterona, en el momento de la inyección y después tres veces por semana hasta la primera inseminación, o hasta cerca de los 90 días post-parto. La GnRH administrada 14 días post-parto, dio como resultado una rápida involución uterina, más rápido retorno a la actividad ovárica, más cortos intervalos entre partos y concepción, y un mejor índice servicio-concepción en búfalos lecheros que no estaban amamantando. Los resultados de dosis de 100 µg y 250 µg no fueron significativos. En el período post-tratamiento, los casos de actividad luteal prolongada fueron comunes en todos los grupos de búfalos. Se sugiere entonces, la administración secuencial de GnRH y prostaglandina, parar el manejo de la actividad reproductiva post-parto en hatos problema.

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Performances De Reproduction Du Buffle Nili-Ravi Apres Une Injection Unique PrecocePost Partum De Gonadotrophine (GnRH)

Résumé Soixante bufflesses laitières (de la seconde à la quatrième lactation), appartenant à une grande ferme d'élevage de buffles, ont été utilisées pour comparer les effets d'une injection unique intramusculaire de 100 µg de gonadotrophine, ou de 250 µg du même produit ou d'une solution de sérum physiologique. Les bufflesses avaient mis bas à la fin de la saison de reproduction soit en décembre. Des prélèvements de lait ont été effectués pour déterminer le taux de progestérone au moment de l'injection d'une part, puis trois fois par semaine d'autre part jusqu'à la première insémination ou aux alentours d'une période de 90 jours après le part. La gonadotrophine, libérée sous forme d'hormone (GnRH), injectée 14 jours après la mise bas a eu pour effet un retour complet plus rapide de l'involution utérine et des intervalles plus courts entre la mise bas et la conception ainsi qu'un meilleur taux de conception à la première monte chez les femelles laitières non allaitantes. Les différences entre les résultats obtenus par une dose de 100/µg ou de 250/µg de gonadotrophine (GnRH) n'étaient pas significatives. Pendant la période post-thérapeutique, des cas d'activité lutéale prolongée ont été courants dans tous les groupes de bufflesses. En conséquence, l'administration séquentielle de GnRH et de prostaglandine est conseillée pour contrôler l'activité post-partum de reproduction dans les troupeaux qui présentent des problèmes de cet ordre.

     

The effect of Prussian blue and sodium-ethylenediaminetetraacetic acid on the faecal and urinary elimination of thallium by the dog.

After oral administration of sublethal doses of thallium (lower than 10 mg/kg) to dogs, 75.5 +/- 3.9 per cent of the dose was eliminated from the body--47 per cent faecal and 28 per cent urinary. Cumulative excretion reached 50 and 70 per cent after 10 and 25 days respectively, and was almost complete after 75 days. This was slightly lower at higher doses. The total body-burden of thallium, calculated from the cumulative excretion, decreased exponentially for at least the first 40 days with a half-time (T 1/2) of 6.5 days. Oral doses of Prussian blue after 10 days resulted in an acceleration of the thallium elimination from the body (T 1/2 2.5 days), an increase of the faecal (49 per cent) and a decrease of the urinary excretion (18 per cent). Nevertheless, the cumulative faecal and urinary thallium excretion was not influenced. Minor transitory influences of Prussian blue on the faecal thallium excretion could be observed up to day 26. After 40 days, Prussian blue could no longer influence the faecal thallium excretion. At no time did sodium-ethylenediaminetetraacetic (Na2EDTA) acid significantly change faecal or urinary thallium excretion.     

Inhibition of chitin synthesis by two 1-(2,6-disubstituted benzoyl)-3-phenylurea insecticides. II

The knowledge of the biochemical mode of action of 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea (diflubenzuron) is presented, explaining the insecticidal effect. Like its structural analog, 1-(2,6-dichlorobenzyl)-3-(3,4-dichlorophenyl)urea (Du 19111), it inhibits chitin synthesis in the cuticle of larvae. Virtually complete inhibition was demonstrable 15 min after the application of diflubenzuron. Neither diflubenzuron nor Du 19111 has any effect upon chitinase activity either in vivo or in vitro. The insecticidal effect upon the cuticle, therefore, must be explained as an inhibition of chitin synthesis and not as an activation of chitin degradation. In contrast to the action of Du 19111, no accumulation of N-acetylglucosamine occurs upon treatment of larvae with diflubenzuron. Similarities and differences in the mode of action of both compounds are discussed, together with other effects reported in the literature.     

山区农业面源污染的防治途径及对策

结合重庆市地形地貌特点和山区农业面源污染发生的因子，提出山区农业面源污染的防治对策。一方面必需结合当地的自然环境条件和经济发展水平提出农业面源污染的防治规划；另一方面必需结合生态农业建设这一主线，使物质、能量在生态系统内良性循环。紧紧抓住产生农业面源污染源的因子——化肥、农药、农田秸杆、畜禽粪便、农村生活废物等，提出科学的防治和减少途径。     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis>

L₁₁A-Fukushima (L₁₁A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L₁₁A-F and L₁₁A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L₁₁A-F appears to possess the properties necessary for practical use. To manage L₁₁A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L₁₁A-F preparations was established. Received 10 June 2002/ Accepted in revised form 30 October 2002     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Gene Flow Analysis of Phytophthora porri Reveals a New Species: Phytophthora brassicae Sp. Nov.

Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2 ⁸³, Ldh-2 ¹⁰⁴, Idh-1 ¹⁰⁸, Idh-1 ¹¹², and Idh-2 ⁹⁸), whereas six different alleles were observed at four isozyme loci (Ldh-2 ⁸⁵, Ldh-2 ¹⁰⁰, Ldh-2 ¹¹⁴, Idh-1 ¹⁰⁰, Idh-2 ¹⁰⁰, and Mdh-2 ¹¹¹) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow.