Hevea genetic transformation using a single-chain variable fragment (scFv) specific to cassiicolin, the causal agent of Corynespora leaf disease

The pathogenic fungus Corynespora cassiicola a serious threat to rubber trees (Hevea brasiliensis); the infected leaves develop necrotic lesions and abscise, leaving the tree unproductive. The destructiveness of C. cassiicola has been largely attributed to cassiicolin, a secreted protein toxin of the fungus. Recombinant antibody technology coupled to genetic transformation offer hope to curtail the disease in Hevea. Single chain variable fragment (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the functional antibody gene. A scFv phage library was constructed from cassiicolin immunized Balb/C mice spleen IgG heavy and light variable chains. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. Nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. The hemagglutinin (HA) tagged scFv expressed in Escherichia coli is discerned as a band at circa 30 kDa on SDS-PAGE. The corresponding band was detected by anti-HA IgG on a Western immunoblot of the gel. Homology-based modelling was employed to create three-dimensional structures of the scFv antibody and cassiicolin, and binding (docking) of the antibody to the toxin antigen was simulated. Furthermore, deactivation of cassiicolin by the scFv was demonstrated by detached leaf bio-assay on selected susceptible Hevea clones. Efforts are underway to genetically transform Hevea clones with the cassiicolin-specific scFv gene to enhance resistance to Corynespora leaf disease. (Texte intégral

Isolation and characterisation of latex cyanogenic glucosidase in Hevea brasiliensis

Latex from the rubber tree (Hevea brasiliensis) is separated into the rubber cream, the clear aqueous phase (C-serum), and the sediment, which consists mainly of vacuole-like organelles called lutoids by high-speed centrifugation. The fluid content of the latter, known as B-serum has drawn attention for many years, owing to its hydrolytic enzymes and pathogenesis-related proteins. A number of these proteins were also pursued in latex allergy studies. [bêta]-glucosidase is among the first hydrolases detected in the H. brasiliensis latex B-serum. It is in the leaf tissue, however, that a [bêta]-glucosidase was first isolated and purified. Besides [bêta]-monoglucosides, this enzyme from Hevea leaves cleaves a wide range of indigenous heteroglycosides including coniferin and cyanoglucosides (linamarin and lataustralin) indicating a possible role in lignification and cyanogenesis. Accordingly it is given the name cyanogenic glycosidase (linamarase). In cassava, hydrolysis of cyanoglucosides by linamarase produces cyanohydrins and glucose. Subsequent breakdown of cyanohydrins either spontaneous or by an á-hydroxynitrile lyase releases a ketone and hydrogen cyanide (HCN), which among others has a role in plant defense. Likewise, the H. brasiliensis linamarase has been implicated in the resistance against South American Leaf Blight, a devastating disease caused by the pathogenic fungi Microcyclus ulei. The present study is aimed at isolating, identifying and characterising a cyanogenic [bêta]-glucosidase in the B-serum, as well as cloning and sequencing of its transcript in H. brasiliensis latex. (Résumé d'auteur

Hevea genetic transformation for enhanced recombinant pharmaceutical production by the use of hevein promoter

Pharmaceuticals produced in the latex cytosol of genetically transformed Hevea can be harvested nondestructively by conventional tapping. This study evaluates the promoter activity of the 5’-untranslated upstream regulating region of hevein gene, which encodes the most abundant soluble protein in Hevea latex. Constructs were prepared with the test hevein promoter fragments Hev P1 (0.35kb), Hev P2 (0.45kb) and Hev P3 (0.73kb) that were inserted 5’ to the pharmaceutical genes i.e. human protamine 1 (HP1) and human atrial natriur tic factor (HANF), in pGPTV-Kan expression vector. The expression vectors containing HP1 and HANF were electroporated into Agrobacterium tumefaciens GV2260 containing supervirulent plasmid pToK47, which were then used to infect Hevea anther callus. The growth of the putative transformed Hevea callus was monitored on kanamycin selection media. The presence of the pharmaceutical genes (HP1 and HANF), the hevein promoter fragments, and nptII selection marker were verified by PCR on sampled putative transformed callus. The remaining callus tissues will be sub-cultured and transferred into differentiation media for embryoids formation and plantlet regeneration

Analysis of latex expressed sequence tags (ESTs) in Hevea brasiliensis

Natural rubber from Hevea brasiliensis latex consists of a high molecular weight polymer composed of isopentenyl diphosphate (IPP) molecules. Latex is the cytoplasmic fluid contained in the vessel tubes or laticifers which are located in the bark of the Hevea tree. As cytoplasm, latex would be expected to contain general cellular processes carried out by eukaryotic cells and more specifically, rubber biosynthesis. In this project, we are interested in building a profile of genes expressed in latex. These expressed genes or Expressed Sequence Tags (ESTs) are obtained by sequencing random cDNA clones which represent the genes transcribed in latex. Our objective is to generate a pool of Hevea ESTs which would serve as a molecular genetics resource to facilitate various aspects of rubber research. Furthermore, an EST library will also create the way for microarray-based studies of Hevea gene expression. In this study, we report the current status of EST sequencing of clones from a latex cDNA library. (Résumé d'auteur

ScFv antibody specific to Cassiicolin, a host-selective toxin of Corynespora cassiicola

Corynespora cassiicola leaf disease is a serious threat to rubber trees; the fungus infected leaves develop necrotic lesions and abscind, leaving the tree unproductive. The destructiveness of C. cassiicola has been largely attributed to cassiicolin, a host-selective protein toxin secreted by the fungus. Recombinant antibody technology offers hope to curtail the disease; single chain variable fragment (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the antibody gene. Anti-cassiicolin scFv phage library was constructed from cassiicolin immunized Balb/C mice spleen IgG heavy and light variable chains. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. The nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. The hemagglutinin (HA) tagged scFv expressed in Escherichia coli is discerned as a band at circa 30 kDa on SDS-PAGE. The corresponding band was detected by anti-HA IgG on a Western immunoblot of the gel. Deactivation of cassiicolin by the scFv was demonstrated by leaf bio-assay on PB 260, a susceptible clone. Leaf bioassay is in progress to evaluate deactivation of cassiicolin on the other susceptible clones that are also amenable for genetic modification i.e. GL 1, RRIM 600 and BRIM 2020

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Abstract. -Objectives: Hevea brasiliensis extract could potentially be employed as a relatively low cost resource for various anti-fungal activities due to the simplicity of latex preparation and the abundance of latex that can be obtained in rubber producing regions. The present study was aimed at examining the species specific anti-fungal property of H. brasilensis latex C-serum against Aspergillus niger. Results: The results showed that the latex Cserum exerted a specific antifungal activity against Aspergillus niger, but not Candida albicans. Low toxicity of the C-serum was demonstrated in Brine Shrimp Lethality Test (BSLT) with an LC 50 value of 98.4 mg/ml. Conclusions: Pending further more elaborated investigations, H. brasiliensis latex C-serum, with its species specific anti-fungal and cancer-origin cell line specific anti-proliferation properties, would probably contribute in healthcare in addition to its traditional role in polymer industry