Design rules for efficient transgene expression in plants.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons.

Diversity of sequences and expression patterns among alleles of a sugarcane loading stem gene.

Modern sugarcane cultivars are highly polyploid and aneuploid hybrids, which are propagated as clones. Their complex genome structure comprises 100-130 chromosomes and 10-13 hom(e)ologous copies of most loci. There is preliminary evidence of very high heterozygosity, with implications for genetic improvement approaches ranging from marker-assisted selection to transgenics.

Sugarcane Loading Stem Gene promoters drive transgene expression preferentially in the stem.

Promoter regions of six sugarcane Loading Stem Gene (ScLSG) alleles were analyzed using bioinformatic and transgenic approaches. Stable transgene expression analyses, on multiple independent lines per construct, revealed differences between ScLSG promoters in absolute levels and in tissue-selectivity of luciferase reporter activity. Four promoters drove peak expression in the sucrose-loading zone and maintained substantial expression throughout mature stems.

Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose.

Metabolic engineering of sugars and simple sugar derivatives in plants.

Carbon captured through photosynthesis is transported, and sometimes stored in plants, as sugar. All organic compounds in plants trace to carbon from sugars, so sugar metabolism is highly regulated and integrated with development. Sugars stored by plants are important to humans as foods and as renewable feedstocks for industrial conversion to biofuels and biomaterials.

Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants.

Transgene integration complexity in the recipient genome can be an important determinant of transgene expression and field performance in transgenic crops. We provide the first direct comparison of Agrobacterium-mediated transformation (AMT) and particle bombardment using whole plasmid (WP) and excised minimal cassettes (MC), for transformation efficiency, transgene integration complexity and transgene expression in plants. To enable direct comparison, a selectable marker and a luciferase reporter gene were linked in identical configurations in plasmids suitable for AMT or direct gene transfer into sugarcane.

Microbial sucrose isomerases: producing organisms, genes and enzymes.

Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production.

Isomaltulose is actively metabolized in plant cells.

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity.

Field performance of transgenic sugarcane expressing isomaltulose synthase.

Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3 years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217 mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars.

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars.

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars.

Genetic transformation of sweet sorghum.

Sweet sorghum has substantial potential as a biofuel feedstock, with advantages in some environments over alternatives such as sugarcane or maize. Gene technologies are likely to be important to achieve yields sufficient for food, fuel and fibre production from available global croplands, but sorghum has proven difficult to transform. Tissue culture recalcitrance and poor reproducibility of transformation protocols remain major challenges for grain sorghum, and there has been no reported success for sweet sorghum.

Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane.

Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole-targeted trehalulose synthase modified from the gene in 'Pseudomonas mesoacidophila MX-45' and controlled by the maize ubiquitin (Ubi-1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mM, with near-complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans.

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes.

Evaluation and application of a luciferase fusion system for rapid in vivo analysis of RNAi targets and constructs in plants.

The practical use of RNA-mediated approaches including antisense RNA, ribozymes and siRNAs for specific inhibition of gene expression is limited by lack of simple quantitative methods to rapidly test efficacy in vivo. There have been indications that cotransfer of target::reporter gene fusions with constructs designed against the target sequence, followed by quantification of transient reporter gene activity might be effective. Here, we report detailed testing of the approach in plants, using diverse target::luciferase fusions and antisense or ribozyme constructs.

Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing.

DNA gyrase from the albicidin producer Xanthomonas albilineans has multiple-antibiotic-resistance and unusual enzymatic properties.

The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species.

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip).

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer.

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades.

The phytotoxin albicidin is a novel inhibitor of DNA gyrase.

Xanthomonas albilineans produces a family of polyketide-peptide compounds called albicidins which are highly potent antibiotics and phytotoxins as a result of their inhibition of prokaryotic DNA replication. Here we show that albicidin is a potent inhibitor of the supercoiling activity of bacterial and plant DNA gyrases, with 50% inhibitory concentrations (40 to 50 nM) less than those of most coumarins and quinolones. Albicidin blocks the religation of the cleaved DNA intermediate during the gyrase catalytic sequence and also inhibits the relaxation of supercoiled DNA by gyrase and topoisomerase IV.

Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E.

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis.

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and "payload" proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics.

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors.

Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane.

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km.

Differentiation of Peanut Seedborne Potyviruses and Cucumoviruses by RT-PCR.

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively.

A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans.

Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted M:(r) 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS).