A benzoate-activated promoter from Aspergillus niger and regulation of its activity.

The filamentous fungus Aspergillus niger is able to use benzoic acid as a sole carbon source by conversion to protocatechuic acid and subsequent metabolism. Synthesis of the first enzyme in this metabolic pathway, benzoate p-hydroxylase, is encoded by the bphA gene and positively regulated at the transcriptional level by benzoic acid. Methyl benzoate and para-aminobenzoate also act as inducers of the bphA gene. We show that bphA expression in A. niger in response to benzoate is confined to a 530-bp fragment from the bphA promoter region from -787 to -509 bp from the transcriptional start site. Electrophoretic mobility-shift assays show that a benzoate-response element, consisting of a single 6-bp sequence (5'-TAGTCA-3') within a 51-bp sequence in this region, is most likely to be involved in binding of one or more proteins that modulate the activity of the promoter in response to benzoic acid. We show through fusion of promoter fragments with the green fluorescent protein that the active sequences are located within a 200-bp sequence containing the TAGTCA benzoate-response element. Identification of the benzoate-response element in the bphA promoter region constitutes the first step in the development of a benzoate-inducible promoter system that could be used to control gene expression in fungi, and possibly in other organisms, such as plant and animal cells.

FLP recombinase-mediated site-specific recombination in rice.

The feasibility of using the FLP/FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene (neomycin phosphotransferase II, nptII) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA. Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA. While the hybrid progeny from various crosses exhibited beta-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/FRT system in rice genetic improvement.

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species.

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.