Papers by Stevens Brumbley
Plant Biotechnology Journal, Mar 1, 2007
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Australasian Plant Pathology, 2006
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Metarhizium anisopliae is a naturally occurring biological control of many insects including the ... more Metarhizium anisopliae is a naturally occurring biological control of many insects including the greyback canegrub (Dermolepida albohirtum), a sugarcane pest in Australia. While there have been some gene-based approaches into identifying determinants for biological control and developing improved strains, our study provides a comparative proteomics approach into identifying key proteins produced by Metarhizium anisopliae during infection of greyback canegrubs. At the same time, we have developed a proteomic map for the greyback canegrub larvae responsible for significant losses of the crop. Pathogenicity related proteins were identified by both a liquid culture and solid culture approach. Solid culture approach is thought to give a more realistic view of infection process compared to liquid culture. In order to identify novel target proteins, differential displays of proteomic maps of healthy/infected cuticle (HC/IC) as well as healthy/infected whole grub (HWG/IWG) were produced and analysed using Progenesis image analysis software. Proteomic map of healthy fungus (Metarhizium anisopliae) (MY) was also generated successfully. A total of 156 protein spots on HWG, 88 unique spots on IWG, 17 spots from HC and 14 spots from MY were analysed by mass spectrometry. Of these, 61 protein spots from HWG, 40 protein spots from IWG, 15 protein spots from HC and 9 protein spots from MY were confirmed by identification using mass spectrometry. Among the identified proteins were different forms of actin and tropomyosin, an ATP binding protein, arginine kinase, formate dehydrogenalase, enolase, tara like protein isoform and heat shock proteins. Further identification for most of the protein spots has been hindered due to the limited number of suitable/accessible databases. Metarhizium anisopliae has been successfully transformed to benomyl resistance using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans using particle bombardment as a preparation for introducing genes encoding the identified pathogenesis factors into Metarhizium.1 page(s
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Applied and Environmental Microbiology, Feb 1, 2008
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Current synthetic & systems biology, Oct 26, 2015
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Journal of Plant Physiology, Mar 1, 2010
Metabolic engineering of plant peroxisomes for biotechnological purposes typically requires effic... more Metabolic engineering of plant peroxisomes for biotechnological purposes typically requires efficient peroxisomal targeting of heterologous proteins. Type I peroxisomal targeting signals (PTS1) consist of three uncleaved amino acids (SKL or a conserved variant) at the carboxyl terminus and direct nuclear-encoded proteins into the peroxisomes of eukaryotic cells. PTS1 fusion with a heterologous protein results in peroxisomal targeting of that protein, but the minimal length of PTS1 required for efficient targeting in plants is vague. Here, we determine short effective PTS1 sequences derived from plant peroxisomal proteins to target four heterologous proteins, namely the green fluorescent protein (GFP) and the three enzymes required for polyhydroxybutyrate (PHB) production, PhaA, PhaB and PhaC, each fused to the C-terminus of GFP. Transient expression analysis in leaf cells of Saccharum sp. (sugarcane interspecific hybrids) indicated that a three amino acid (ARL) PTS1 effectively targeted only GFP and PhaB to peroxisomes. The same signal was not sufficient to target PhaA and only inefficiently targeted PhaC. An alternative, prototypic three amino acid (SKL) PTS1 was also insufficient to target PhaA and inefficient in targeting PhaC, whilst a six amino acid (RAVARL) PTS1 efficiently targeted both of these enzymes. This study highlights the need for more than a three amino acid PTS1 to target some heterologous proteins to plant peroxisomes.
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Springer eBooks, Oct 28, 2009
Synthesis of polyhydroxyalkanoates (PHAs) in crop plants is viewed as an attractive approach for ... more Synthesis of polyhydroxyalkanoates (PHAs) in crop plants is viewed as an attractive approach for the production of this family of biodegradable plastics in large quantities and at low costs. Synthesis of PHAs containing various monomers has so far been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHAs in crop plants remains a challenging project. PHA synthesis at a high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth in some plants. The challenges for the future are to succeed in the synthesis of PHA copolymer with a narrow range of monomer composition, at levels that do not compromise plant productivity, and in creating methods for efficient and economical extraction of polymer from plants. These goals will undoubtedly require a deeper understanding of plant biochemical pathways as well as advances in biorefinery.
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Australasian Plant Pathology, 2003
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BMC Biotechnology, Sep 10, 2014
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Current Microbiology, Aug 18, 2011
Yeasts associate with numerous insects, and they can assist the metabolic processes within their ... more Yeasts associate with numerous insects, and they can assist the metabolic processes within their hosts. Two distinct yeasts were identified by PCR within the planthopper Perkinsiella saccharicida, the vector of Fiji disease virus to sugarcane. The utility of both microbes for potential paratransgenic approaches to control Fiji leaf gall (FLG) was assessed. Phylogenetic analysis showed one of the microbes is related to yeast-like symbionts from the planthoppers: Laodelphax striatellus, Nilaparvata lugens, and Sogetella furcifera. The second yeast was a member of the Candida genus, a group that has been identified in beetles and recently described in planthoppers. Microscopy revealed the presence of yeast in the fat body of P. saccharicida. The Candida yeast was cultured, and transformation was accomplished by electroporation of Candida albicans codon optimized plasmids, designed to integrate into the genome via homologous recombination. Transgenic lines conferred resistance to the antibiotic nourseothricin and expression of green fluorescent protein was observed in a proportion of the yeast cells. Stably transformed yeast lines could not be isolated as the integrative plasmids presumably replicated within the yeast without integration into the genome. If stable transformation can be achieved, then this yeast may be useful as an agent for a paratransgenic control of FLG.
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Australasian Plant Pathology, 2006
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Macromolecular Symposia, Jul 1, 2016
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Plant Physiology, Dec 31, 2009
Genome-scale metabolic network models have been successfully used to describe metabolism in a var... more Genome-scale metabolic network models have been successfully used to describe metabolism in a variety of microbial organisms as well as specific mammalian cell types and organelles. This systems-based framework enables the exploration of global phenotypic effects of gene knockouts, gene insertion, and up-regulation of gene expression. We have developed a genome-scale metabolic network model (AraGEM) covering primary metabolism for a compartmentalized plant cell based on the Arabidopsis (Arabidopsis thaliana) genome. AraGEM is a comprehensive literature-based, genome-scale metabolic reconstruction that accounts for the functions of 1,419 unique open reading frames, 1,748 metabolites, 5,253 gene-enzyme reaction-association entries, and 1,567 unique reactions compartmentalized into the cytoplasm, mitochondrion, plastid, peroxisome, and vacuole. The curation process identified 75 essential reactions with respective enzyme associations not assigned to any particular gene in the Kyoto Encyclopedia of Genes and Genomes or AraCyc. With the addition of these reactions, AraGEM describes a functional primary metabolism of Arabidopsis. The reconstructed network was transformed into an in silico metabolic flux model of plant metabolism and validated through the simulation of plant metabolic functions inferred from the literature. Using efficient resource utilization as the optimality criterion, AraGEM predicted the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. AraGEM is a viable framework for in silico functional analysis and can be used to derive new, nontrivial hypotheses for exploring plant metabolism.
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Plant Biotechnology Journal, Feb 28, 2012
Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemi... more Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.
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Plant Biotechnology Journal, Sep 21, 2004
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Papers by Stevens Brumbley