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Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

Agrobacterium‐mediated transfer from a binary vector was used to produce transgenic Nicotiana tabacum plants that expressed coat protein of the plant virus, alfalfa mosaic virus (AMV). Expression levels of the chimeric gene, which was under the control of the cauliflower mosaic virus 19S promoter, were determined in primary transformed plants, in the progeny from self‐fertilization and in the progeny from crosses to normal tobacco. RNA transcripts that were of the expected size as well as a protein of the Mr and antigenicity of AMV coat protein accumulated in the transgenic plants. Plants that expressed the highest levels of coat protein developed fewer primary infections following inoculation with two strains of AMV and developed systemic infection slower than did plants that did not express coat protein. Resistance was specifically against virions of the AMV strains. AMV RNA and the unrelated virus, tobacco mosaic virus, were as infectious on progeny that expressed coat protein as they were on progeny that did not. The relationship between the virus resistance expressed by these transgenic plants and that observed in virus cross‐protection is discussed.

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.Spinosyns, the active ingredients in Dow AgroSciences' new Naturalyte line of insect control products, are produced by fermentation of the actinomycete Saccharopolyspora spinosa. Spinosyns are macrolides ( Fig. 1) consisting of a 21-carbon tetracyclic lactone to which are attached two deoxysugars: tri-O-methylated rhamnose and forosamine (6). The most active components of the spinosyn family of compounds are spinosyns A and D, which differ from each other by a single methyl substituent at position 6 of the polyketide. Other factors in this family have different levels of methylation and are significantly less active. Both the rhamnose and forosamine moieties are essential for the insecticidal activity of spinosyns (2). Spinosad is highly effective against target insects and has an excellent environmental and mammalian toxicological profile (2,13,14).Spinosyn biosynthesis occurs via the nonglycosylated intermediate, the aglycone (AGL). Rhamnose is the first sugar attached and is tri-O-methylated to yield the intermediate pseudoaglycone.Only after the rhamnose is attached can the forosamine sugar be incorporated (M. C. Broughton, M. L. B. Huber, L. C. Creemer, H. A. Kirst, and J. R. Turner, Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991, abstr. K-58, p. 224, 1991. Both trimethyl rhamnose and forosamine are believed to be synthesized from glucose-1-phosphate via the common intermediate 4-keto-6-deoxy-D-glucose (Fig. 2). The biosynthetic pathway for rhamnose (Fig. 2) has been elucidated in enteric bacteria, where the deoxysugar is an element of surface antigens (8, 18). The first step, activation of glucose by addition of a nucleotidyl diphosphate (NDP), is catalyzed by an NDP-glucose synthase (the gtt gene product). The second step, dehydration to NDP-4-keto-6-deoxyglucose, is catalyzed by glucose dehydratase (the gdh gene product). 4-Keto-6-deoxy-D-glucose is the common intermediate to many deoxysugar biosynthetic pathways, and the enzymes encoded by the gtt and gdh genes may supply the precursors for all of them. Rhamnose synthesis requires two additional enzymes, a 3Ј5Ј epimerase (encoded by epi) and a 4Ј ketoreductase (encoded by kre), that are unique to the pathway. They convert the NDP-4-keto-6-deoxyglucose to NDP-L-rhamnose, the activated sugar that is the substrate of th...

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The tcdA gene of Photorhabdus luminescens encodes a 283-kDa protein, toxin A, that is highly toxic to a variety of insects, including some agriculturally important pests. We tested the efficacy of transgenic toxin A in Arabidopsis thaliana for control of feeding insects. Plants with toxin A expression above about 700 ng/mg of extractable protein were highly toxic to tobacco hornworm (Manduca sexta). Toxin A isolated from transgenic plants also strongly inhibited growth of the southern corn rootworm (Diabrotica undecimpunctata howardi). Addition of 5' and 3' untranslated regions of a tobacco osmotin gene (osm) increased toxin A production 10-fold and recovery of insect-resistant lines 12-fold. In the best line, high toxin A expression and insect resistance were maintained for at least five generations in all progeny. The intact tcdA mRNA represents the largest effective transgenic transcript produced in plants to date. These results may open a new route to transgenic pest control in agriculture.