In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N 2 . Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.

The codon-modified cryV-Bt gene (cryV-Bt) from Bacillus thuringiensis subsp. kurstaki Berliner, which is specifically toxic to Lepidoptera and Coleoptera insects, and a potato virus Yo coat protein gene (PVYocp), in which the aphid transmission site was inactivated, were cotransformed into potato (Solanum tuberosum L.) `Spunta' via Agrobacterium tumefaciens Conn. We demonstrated the integration and expression of both genes by molecular analysis and bioassays. All cryV-Bt/PVYocp-transgenic lines were more resistant to potato tuber moth (Phthorimaea operculella Zeller) and PVYo infection than nontransgenic `Spunta'. Four cryV-Bt/PVYocp transgenic lines were equal in potato tuber moth mortality to a cryV-Bt transgenic line, but of these four only two lines were equivalent in PVYo titer levels to a PVYocp-transgenic line. We identified two transgenic lines, 6a-3 and 6a-5, which showed greater resistance to potato tuber moth and PVYo than the other cryV-Bt/PVYocp transgenic lines. This study indicated that multiple genes, conferring insect pest resistance and virus resistance, could be engineered into and expressed simultaneously in a potato cultivar.

Late blight of potato (Solanum tuberosum L.), incited by Phytophthora infestans (Mont.) de Bary, is a devastating disease affecting tuber yield and storage. Recent work has isolated a resistance gene, RB, from the wild species Solanum bulbocastanum Dun. Earlier work in Toluca, Mexico, observed significant levels of field resistance under intense disease pressure in a somatic hybrid containing RB. In this study, five transgenic RB lines were recovered from the late blight susceptible line MSE149-5Y, from the Michigan State University (MSU) potato breeding program. Transgenic lines were molecularly characterized for the RB transgene, RB transcript, and insertion number of the kanamycin resistance gene NPTII. Transgenic lines and the parent line were evaluated for resistance in field and laboratory tests. Molecular characterization alone did not predict which lines were resistant. Three of the RB transformed MSE149-5Y lines showed increased resistance under field conditions at MSU and increased resistance in detached leaf evaluations using multiple isolates individually (US-1, US-1.7, US-8, US-10, and US-14). Transfer of RB into late blight susceptible and resistant lines could provide increased protection to potato late blight. The use of the RB gene for transformation in this way creates a partially cisgenic event in potato because the gene's native promoter and terminator are used. This type of transformation provides a chance to generate greater public acceptance of engineered approaches to trait introgression in food crops.

The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L.) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic 'Spunta' clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to 'Spunta'. These Bt-cry5 transgenic potato plants with high potato tuber moth resistance have value in integrated pest management programs.

Potato tuber moth (Phthorimaea operculella Zeller) is a highly destructive pest of the cultivated potato (Solanum tuberosum L.) in the tropics and subtropics and causes significant damage to both leaves and tubers. Development of host plant resistance is a central component of an integrated pest management (IPM) program for potato tuber moth. The purpose of this research was to augment natural resistance by transforming potato with a codon-modified CryV-Bt gene using Agrobacterium-mediated techniques. `Lemhi Russet' potato and two clones with different host plant resistance mechanisms, USDA8380-1 (leaf leptines) and L235-4 (glandular trichomes), were transformed with the CryV-Bt gene. Gene integration of regenerated plants was confirmed by polymerase chain reactions and Southern analyses; gene transcription was evaluated by northern analyses. Detached leaf bioassays showed that high levels of Bt expression occurred in the CryV-Bt transgenic lines (`Lemhi Russet' and L235-4), providing up to 96% control of potato tuber moth larvae, compared with 3% and 54% control in L235-4 and USDA8380-1, respectively. These transgenic lines can be used for breeding purposes to develop cultivars for (and eventual introduction into) IPM systems.