A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors

Rice production is known to be severely affected by virus transmitting rice pests, brown planthopper (BPH) and green leafhopper (GLH) of the order hemiptera, feeding by phloem abstraction. ASAL, a novel lectin from leaves of garlic (Allium sativum) was previously demonstrated to be toxic towards hemipteran pests when administered in artificial diet as well as in ASAL expressing transgenic plants. In this report ASAL was targeted under the control of phloem-specific Agrobacterium rolC and rice sucrose synthase-1 (RSs1) promoters at the insect feeding site into popular rice cultivar, susceptible to hemipteran pests. PCR, Southern blot and C-PRINS analyses of transgenic plants have confirmed stable T-DNA integration and the transgenes were co-segregated among self-fertilized progenies. The T₀ and T₁ plants, harbouring single copy of intact T-DNA expression cassette, exhibit stable expression of ASAL in northern and western blot analyses. ELISA showed that the level of expressed ASAL was as high as 1.01% of total soluble protein. Immunohistofluorescence localization of ASAL depicted the expected expression patterns regulated by each promoter type. In-planta bioassay studies revealed that transgenic ASAL adversely affect survival, growth and population of BPH and GLH. GLH resistant T₁ plants were further evaluated for the incidence of tungro disease, caused by co-infection of GLH vectored Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), which appeared to be dramatically reduced. The result presented here is the first report of such GLH mediated resistance to infection by RTBV/RTSV in ASAL expressing transgenic rice plant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.