‘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.     

Evaluation of reconstitution of immunodeficient mice by partially purified thymic extracts.

Partially purified thymus products were used to evaluate the maturation of T lymphocytes in immunodeficient mice. Three different bovine thymic extracts, designated thymic extracts A, B, and C, were successful in increasing the longevity of conventionally raised nude mice. Daily injection of bovine thymic extracts A, B, and C and mouse thymus extracts failed to mature a population of T lymphocytes and restore the capacity to reject heart allografts. Preincubation of normal syngeneic bone marrow with thymic extract B in vitro before injection into nude mice also failed to reconstitute the host's ability to reject heart grafts. The number of antibody plaque-forming cells of sheep red blood cells in lethally irradiated, bone marrow-reconstituted mice could be increased by preincubating the bone marrow cells with bovine thymic extract fraction B before injection followed by daily injections. A similar but less marked increase in plaque-forming cells was obtained by the daily injection of bovine thymic extract fraction C. Complete functional maturation of T lymphocytes in immunodeficient animals appears to require more than thymic extract stimulation of bone marrow cells or pre-T lymphocytes.     

Identification of immunoglobulins associated with complement fixation, agglutination, and low pH buffered antigen tests for brucellosis.

Serums from infected cattle, cattle with persistent postvaccinal antibody, and serologically "positive" noninfected cattle were fractionated into major immunoglobulin classes by diethylaminoethyl (DEAE)-cellulose chromatography and by sucrose density gradient centrifugation. Each fraction was assayed for anti-Brucella activity by standard tube-agglutination test (STT), buffered tube-agglutination test (BTT), and complement-fixation test (CF). In the serums from experimentally infected cattle, anti-Brucella antibody could be found by all tests in 6 DEAE fractions and in slow, fast, and sediment regions of the density gradient. Serums from cattle with persistent postvaccinal titers had STT activity in all 6 DEAE fractions, BTT activity in 5 fractions, and CF activity in only 1 fraction. The STT and BTT activities were found in the slow and the sediment regions of the gradient, whereas the CF activity was found only in the slow region. Serums from a chronically infected animal had STT and BTT activities in 2 DEAE fractions and CF activity in only 1. The STT, BTT, and CF activities were found in the slow and the sediment regions of the gradient. The principal antibody in serums from noninfected cattle was immunoglobulin M, which had all of the CF activity and most of the STT and BTT activities. Low levels of STT and BTT activities were found in 3 other DEAE fractions. Only STT and BTT activities were found in the fast and the sediment regions of the gradient.     

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     

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.