‘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.     

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     

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H₂O₂. A small amount of non-specific generation of H₂O₂ was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H₂O₂ at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H₂O₂ at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H₂O₂ generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane. Received 21 September 2001/ Accepted in revised form 25 October 2001     

钙素对叶用莴苣营养吸收和生长发育的影响

 采用NFT 栽培, 研究了叶用莴苣钙素与主要无机成分间的相互关系, 以及对叶用莴苣生长发育的影响。结果表明, 缺钙胁迫引起叶绿素含量降低, 缘腐病发病率上升, 叶片干、鲜样质量降低。生长中后期, 特别是新生内叶易发生钙素营养失调症。营养液中钙浓度过高会降低钾、镁的吸收, 而对氮、磷吸收影响不大。提高营养液中NH₄⁺ -N 比例会抑制钙吸收, 降低其有效性。     

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     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

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.