RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

MAIN CONCLUSION: Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Novel plant transformation vectors containing the superpromoter.

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2' (mas2') transcriptional activating element plus minimal promoter. We tested a superpromoter-beta-glucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2', and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.

Maize (Zea mays L.).

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts.

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.

Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.