Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia.

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

Resistance of selected saprobic and zoopathogenic fungi to cycloheximide.

Spore germination was used as an assay to measure the sensitivities of selected fungi (Achlya bisexualis, Cladosporium sp., Trichophyton mentagrophytes and Microsporum gypseum) to cyclohexamide and to determine their abilities to adapt to the drug. Two patterns of response were noted. The saprobes, A. bisexualis and Cladosporium sp., demonstrated acquired resistance. Spores from hyphae previously exposed to cycloheximide either germinated in the presence of concentrations of the drug that completely inhibited spores from unexposed hyphae (Achlya), or germinates with a shorter lag and to a greater extent in the presence of the antibiotic than did spores from unexposed hyphae (Cladosporium). Hyphae of Achlya adapted at concentrations of cycloheximide in which spores did not germinate and hyphae of Cladosporium adapted more rapidly than spores. Achlya adapted to only 12 muM-cycloheximide whereas Cladosporium acquired resistance to 18 mM-cycloheximide. These fungi lost this acquired resistance after a single transfer to media lacking cycloheximide. The zoopathogens, T. mentagrophytes and M. gypseum, had a contrasting response, indicating constitutive resistance. Conidia from unexposed hyphae showed 90 to 100% germination on media containing up to 18 mM-cycloheximide; prior exposure to the drug did not affect their response.

Inhibition of DNA synthesis by cycloheximide and blasticidin-S is independent of their effect on protein synthesis.

The effects of cycloheximide and related glutarimide antibiotics on DNA synthesis in Achlya bisexualis Coker and A. Couch were compared to those of other protein synthesis inhibitors, puromycin, p-fluorophenylalanine and blasticidin-S. The inhibitors had no significant effects on intrahyphal pool sizes of dTTP, dCTP, ATP, UTP and CTP, nor on the specific activity of the dTTP pool labelled by [3H] thymidine. DNA was the sole acid-insoluble product of [3H]-thymidine incorporation. Cycloheximide, isocycloheximide, streptimidone and blasticidin-S inhibited DNA synthesis rapidly and completely and anhydrocycloheximide was less effective. Cycloheximide acetate, puromycin and p-fluorophenylalanine did not inhibit DNA synthesis. It is concluded that the effects of the several glutarimide antibiotics and of blasticidin-S on DNA synthesis were independent of their effects on protein synthesis.