‘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.     

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     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002     

Estimation of Genetic Variation Among Verticillium Isolates using AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis was used to study genetic variation among 76 isolates of Verticillium. A dendrogram based on the AFLP data revealed three main groups. One group consisted of 35 European isolates derived from Brassica napus together with five Californian isolates taken from B. oleracea. This group displayed a high degree of genetic similarity and included three isolates earlier classified as Verticillium longisporum, indicating that all isolates in this group probably should be regarded as members of V. longisporum. V. dahliae isolates constituted the second group while the third group contained four V. albo-atrum isolates. In addition to these three groups, a cluster of six V. nigrescens isolates was observed. However, the genetic distances between the isolates of V. nigrescens were much higher than those between members in the other groups and the bootstrap value for the V. nigrescens cluster was subsequently low. Four isolates classified as V. tricorpus were highly diverse and did not cluster together. Analysis of molecular variance revealed that the isolates of V. longisporum were separated into four subgroups, based on geographic origin. The study furthermore shows that AFLP is a suitable method for studying population structure in Verticillium.     

Inconspicuous restraint of rice seedling growth by root-infecting fungi in soil of a rice paddy field

 Rice seedling growth, estimated by plant height and root development and discoloration, was better in pasteurized soil than in unpasteurized soil obtained from a flooded rice field. Rice seedlings also grew better in sterilized soil modified by adding roots harvested from the pasteurized soil than in soil modified by adding roots harvested from the unpasteurized soil. The results demonstrate that seedling growth in the rice field soil was inhibited by soil microorganisms, even though no typical symptoms such as seedling blight or damping-off appeared. Pythium aristosporum is suggested to be involved in the inhibition. Thus, it appears that inconspicuous restraint of rice seedling growth could occur in soils of rice paddy fields. Received: May 20, 2002 / Accepted: October 16, 2002 Acknowledgments The authors thank Dr. T. Ichitani, former professor at Osaka Prefectural University, for providing an isolate of Pythium aristosporum for comparison, and Mr. Mitsuaki Sato of Akita Prefectural College of Agriculture for technical assistance.     

微管、微丝特异性抑制剂处理对水稻抗病性的影响

 微管特异性抑制剂oryzalin、微丝特异性抑制剂细胞松弛素A (cytochalasin A,CA)和细胞松弛素D (cytochalasin D,CD)的试验表明:oryzalin在5~50μmol/L、CA在0.5~1.0μg/mL、CD在1~20μg/mL的浓度范围内,对稻瘟病菌孢子的萌发和附着胞的形成基本上没有影响。采用以上几种细胞骨架特异性抑制剂处理水稻叶鞘都可以不同程度地抑制寄主细胞抗病菌扩展的能力。在抑制剂处理的水稻叶鞘细胞中,病菌扩展的速度加快。进一步的观察发现,抑制剂处理抑制水稻细胞抗病菌的扩展能力与水稻的抗病防卫反应如原生质颗粒化、多酚类物质的积累和HR发生的延迟是相关的。     

Functional heterogeneity in resources within landscapes and herbivore population dynamics

Large mammalian herbivores are notorious for their propensity towards population irruptions and crashes, yet many herbivore populations remain relatively stable. I explore how resource heterogeneity within landscapes dampens population instability, using a metaphysiological modelling approach considering patch state distributions. Resource heterogeneity is functionally stabilizing through spreading consumption away from preferred resources before these become critically depleted. Lower-quality resources act as a buffer against starvation during critical periods of the seasonal cycle. Enriching resource quality is destabilizing, even if patch diversity is maintained, because food quantity then becomes the limitation. The potential consequences of landscape fragmentation are explored using the Serengeti ecosystem, characterised by broadscale resource gradients, as a hypothetical example. Further insights provided by the model are illustrated with specific examples concerning the effects of patch scales and waterpoint distribution. A metaphysiological modelling approach enables the basic consequences of landscape heterogeneity to be distinguished from further effects that may arise from specific patch scales and configurations, without the distracting detail of spatially explicit models.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.