RNA Silencing in Plants

RNA silencing-related mechanisms have been documented in almost all living organisms and RNA silencing is now used as board term to describe the vast array of related processes involving RNA–RNA, RNA–DNA, RNA–protein or protein–protein interactions that ultimately result in the repression of gene expression. In plants, the parallel RNA silencing pathways have evolved to extraordinary levels of complexity and diversity, playing crucial roles in providing protection against invading nucleic acids derived from viruses or replicating transposons, controlling chromatin modifications as well as regulating endogenous gene expression to ensure normal plant growth and development. The aims of this chapter are (1) to provide an overview of the initial curious observations of RNA silencing-related phenomena in plants, (2) to outline the parallel gene silencing pathways of plants, and (3) to discuss current applications of RNA silencing technologies to not only study but also modify plant development

High-throughput vectors for efficient gene silencing in plants

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs, pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Construct design for efficient, effective and high-throughput gene silencing in plants

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes

A very simple leaf assay is described that rapidly and reliably identifies transgenic plants expressing the hygromycin resistance gene, hph or the phosphinothricin resistance gene, bar. Leaf tips were cut from plants propagated either in the glasshouse or in tissue culture and the cut surface embedded in solid medium containing the appropriate selective agent. Non-transgenic barley or rice leaf tips had noticeable symptoms of either bleaching or necrosis after three days on the medium and were completely bleached or necrotic after one week. Transgenic leaf tips remained green and healthy over this period. This gave unambiguous discrimination between transgenic and non-transgenic plants. The leaf assay was also effective for dicot plants tested (tobacco and peas).

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken: sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Firmicutes dominate the bacterial taxa within sugar-cane processing plants

Sugar cane processing sites are characterised by high sugar/hemicellulose levels, available moisture and warm conditions, and are relatively unexplored unique microbial environments. The PhyloChip microarray was used to investigate bacterial diversity and community composition in three Australian sugar cane processing plants. These ecosystems were highly complex and dominated by four main Phyla, Firmicutes (the most dominant), followed by Proteobacteria, Bacteroidetes, and Chloroflexi. Significant variation (p , 0.05) in community structure occurred between samples collected from ‘floor dump sediment’, ‘cooling tower water’, and ‘bagasse leachate’. Many bacterial Classes contributed to these differences, however most were of low numerical abundance. Separation in community composition was also linked to Classes of Firmicutes, particularly Bacillales, Lactobacillales and Clostridiales, whose dominance is likely to be linked to their physiology as ‘lactic acid bacteria’, capable of fermenting the sugars present. This process may help displace other bacterial taxa, providing a competitive advantage for Firmicutes bacteria.

Model-predictive control and closed-loop stability considerations for nonlinear plants described by local ARX-type models

In this paper, a model-predictive control (MPC) method is detailed for the control of nonlinear systems with stability considerations. It will be assumed that the plant is described by a local input/output ARX-type model, with the control potentially included in the premise variables, which enables the control of systems that are nonlinear in both the state and control input. Additionally, for the case of set point regulation, a suboptimal controller is derived which has the dual purpose of ensuring stability and enabling finite-iteration termination of the iterative procedure used to solve the nonlinear optimization problem that is used to determine the control signal.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Mini-scale method for nuclear run-on transcription assay in plants

We present a mini-scale method for nuclear run-on transcription assay. In our method, all the centrifuge steps can be carried out by using micro-tubes for short time (5 min each) throughout the process, including isolation of transcriptionally active nuclei and purification of labeled RNA after synthesis of RNA in isolated nuclei. The assay can be performed using a small amount of plant tissue, which enables analysis of developmental changes in transcriptional status of given genes in a single individual plant. Successful results were obtained using the tissues of flower and leaf of petunia and embryo of pea, suggesting that the method is potentially applicable to a variety of plant tissues.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Ecological risk analysis of transgenic plants

Public concern about the safety of many forms of industrial technology are known to be linked to a range of factors including a perceived lack of confidence in regulatory decision making.1 The use of transgenic plants in agriculture may be seen as an issue that could generate similar concern. Criticism has been made about the completeness of knowledge on the potential for aberrant behaviour of genetically manipulated organisms (GMO's) in release environments, and the adequacy of existing pre­‐release screening and assessment methodologies (Goldberg & Tjaden, 1990). Such comments are important because any perceived shortcomings in the pre-release assessment of GMO safety may lead to decreased public support of the technology -­‐and the industry itself...

Control of solar powered micro-grids using electric vehicles

Large scale solar plants are gaining recognition as potential energy sources for future. In this paper, the feasibility of using electric vehicles (EVs) to control a solar powered micro-grid is investigated in detail. The paper presents a PSCAD/EMTDC based model for the solar powered micro-grid with EVs. EVs are expected to have both the vehicle-to-grid (V2G) and grid-to-vehicle (G2V) capability, through which energy can either be injected into or extracted from the solar powered micro-grid to control its energy imbalance. Using the model, the behaviour of the micro-grid is investigated under a given load profile, and the results indicate that a minimum number of EVs are required to meet the energy imbalance and it is time dependent and influenced by various factors such as depth of charge, commuting profiles, reliability etc...

Pasture Degradation and Recovery in Australia's Rangelands : Learning from History

"The extended drought periods in each degradation episode have provided a test of the capacity of grazing systems (i.e. land, plants, animals, humans and social structure) to handle stress. Evidence that degradation was already occurring was identified prior to the extended drought sequences. The sequence of dry years, ranging from two to eight years, exposed and/or amplified the degradation processes. The unequivocal evidence was provided by: (a) the physical 'horror' of bare landscapes, erosion scalds and gullies and dust storms; (b) the biological devastation of woody weeds and animal suffering/deaths or forced sales, and; (c) the financial and emotional plight of graziers and their families due to reduced production in some cases leading to abandonment of properties or, sadly, deaths (e.g. McDonald 1991, Ker Conway 1989)."--Publisher website

Recovering Functional Integrity in Pastures Dominated by an Invasive Grass Species

Exotic grasses have been introduced in countries worldwide for pasture improvement, soil stabilisation and ornamental purposes. Some of these introductions have proven successful, but many have not (Cook & Dias 2006). In Australia, the Commonwealth Plant Introduction Scheme was initiated in 1929, and over-time introduced more than 5000 species of grasses, legumes and other forage and browse plants (Cook & Dias 2006). Lonsdale (1994) suggested that, in tropical Australia, 13% of introductions have become a problem, with only 5% being considered useful for agriculture. Low (1997) suggested that 5 out of 18 of Australia's worst tropical environmental weeds were intentionally introduced as pasture grasses. The spread and dominance of invasive grass species that degrade the quality of pastures for production can impact significantly on the livelihoods of small proprietors. Although Livestock grazing contributes only a small percentage to the world's GDP (1.5%), maintaining the long-term stability of this industry is crucial because of the high social and environmental consequence of a collapse. One billion of the world's poor are dependent on livestock grazing for food and income with this industry occupying more than 25% of the world's land base (Steinfeld et al. 2006). The ling-term sustainability of livestock grazing is also crucial for the environment. A recent FAO report attributed livestock production as a major cause of five of the most serious environmental problems: global warming, land degredation, air and water pollution, and the loss of biodiversity (Steinfeld et al. 2006). For these reasons, finding more effective approaches that guide the sustainable management of pastures is urgently needed. In Australia more than 55% of land use is for livestock grazing by sheelp and/or cattle. This land use dominate in the semi-arid and arid regions where rainfall and soil conditions are marginal for production (Commonwealth of Australia 2004). Although the level of agriculture production by conglomerates is increasing, the majority of livestock grazing within Australia remains family owned and operated (Commonwealth of Australia 2004). The sustainability of production from a grazed pasture is dependent on its botanical composition (Kemp & Dowling 1991, Kemp et al. 1996). In a grazed pasture, the dominance of an invasive grass species can impact on the functional integrity of the ecosystem, including production and nutrient cycling; wwhich will in turn, affect the income of proprietors and the ability of the system to recover from disturbance and environmental change. In Australia, $0.3 billion is spent on weed control in livestock production, but despite this substantial investment $1.9 billion is still lost in yield as a result of weeds (Sinden et al. 2004). In this paper, we adaprt a framework proposed for the restoration of degraded rainforest communities (Lamb & Gilmour 2003, Lamb et al. 2005) to compare and contrast options for recovering function integrity (i.e. a diverse set of desirable plant species that maintain key ecological processes necessary for sustainable production and nutrient cycling) within pasture communities dominated by an invasive grass species. To do this, we uase a case-study of the invasion of Eragrostis curvula (Africal lovegrss; hereafter, Lovegrass), a serious concern in Australian agricultural communities (Parsons and Cuthbertson 1992). The spread and dominance of Lovegrass is a problem because its low palatability, low nutritional content and competitiveness affect the livelihood of graziers by reducing the diversity of other plant species. We conclude by suggesting modifications to this framework for pasture ecosystems to help increase the effiency of strategies to protect functional integrity and balance social/economic and biodiversity values.

Priority threat management of invasive plant species in the Lake Eyre Basin

This project is led by scientists in conservation decision appraisal and brings together a group of experts working across the Lake Eyre Basin (LEB). The LEB covers a sixth of Australia, with an array of globally significant natural values that are threatened by invasive plants, among other things. Managers at various levels are investing in attempts to control, contain and eradicate these invasive plant species, under severe time and resources limitations. To date there has been no basin-wide assessment of which weed management strategies and locations provide the best investments for maximising outcomes for biodiversity per unit cost. Further, there has been no assessment of the extent of ecosystem intactness that may be lost without effective invasive plant species management strategies. Given that there are insufficient resources to manage all invasive plant species everywhere, this information has the potential to improve current investment decisions. Here, we provide a prioritisation of invasive plant management strategies in the LEB. Prioritisation was based on cost-effectiveness for biodiversity benefits. We identify the key invasive plant species to target to protect ecosystem intactness across the bioregions of the LEB, the level of investment required and the likely reduction in invasive species dominance gained per dollar spent on each strategy. Our focus is on strategies that are technically and socially feasible and reduce the likelihood that high impact invasive plant species will dominate native ecosystems, and therefore change their form and function. The outputs of this work are designed to help guide decision-making and further planning and investment in weed management for the Basin. Experts in weed management, policy-making, community engagement, biodiversity and natural values of the Basin, attended a workshop and agreed upon 12 strategies to manage invasive plants. The strategies focused primarily on 10 weeds which were considered to have a high potential for broad, significant impacts on natural ecosystems in the next 50 years and for which feasible management strategies could be defined. Each strategy consisted of one or more supporting actions, many of which were spatially linked to IBRA (Interim Biogeographical Regionalisation of Australia) bioregions. The first strategy was an over-arching recommendation for improved mapping, information sharing, education and extension efforts in order to facilitate the more specific weed management strategies. The 10 more specific weed management strategies targeted the control and/or eradication of the following high-impact exotic plants: mesquite, parkinsonia, rubber vine, bellyache bush, cacti, mother of millions, chinee apple, athel pine and prickly acacia, as well as a separate strategy for eradicating all invasive plants from one key threatened ecological community, the GAB (Great Artesian Basin dependant) mound springs. Experts estimated the expected biodiversity benefit of each strategy as the reduction in area that an invasive plant species is likely to dominate in over a 50-year period, where dominance was defined as more than 30% coverage at a site. Costs were estimated in present day terms over 50 years largely during follow up discussions post workshop. Cost-effectiveness was then calculated for each strategy in each bioregion by dividing the average expected benefit by the average annual costs. Overall, the total cost of managing 12 invasive plant strategies over the next 50 years was estimated at $1.7 billion. It was estimated that implementation of these strategies would result in a reduction of invasive plant dominance by 17 million ha (a potential 32% reduction), roughly 14% of the LEB. If only targeting Weeds of National Significance (WONS), the total cost was estimated to be $113 million over the next 50 years. Over the next 50 years, $2.3 million was estimated to eradicate all invasive plant species from the Great Artesian Basin Mound Springs threatened ecological community. Prevention and awareness programs were another key strategy targeted across the Basin and estimated at $17.5 million in total over 50 years. The cost of controlling, eradicating and containing buffel grass were the most expensive, over $1.5 billion over 50 years; this strategy was estimated to result in a reduction in buffel grass dominance of a million ha in areas where this species is identified as an environmental problem. Buffel grass has been deliberately planted across the Basin for pasture production and is by far the most widely distributed exotic species. Its management is contentious, having economic value to many graziers while posing serious threats to biodiversity and sites of high cultural and conservation interest. The strategy for containing and locally eradicating buffel grass was a challenge to cost based on expert knowledge, possibly because of the dual nature of this species as a valued pastoral grass and environmental weed. Based on our conversations with experts, it appears that control and eradication programs for this species, in conservation areas, are growing rapidly and that information on the most cost-effective strategies for this species will continue to develop over time. The top five most cost-effective strategies for the entire LEB were for the management of: 1) parkinsonia, 2) chinee apple, 3) mesquite, 4) rubber vine and 5) bellyache bush. Chinee apple and mother of millions are not WONS and have comparatively small populations within the semi-arid bioregions of Queensland. Experts felt that there was an opportunity to eradicate these species before they had the chance to develop into high-impact species within the LEB. Prickly acacia was estimated to have one of the highest benefits, but the costs of this strategy were high, therefore it was ranked 7th overall. The buffel grass strategy was ranked the lowest (10th) in terms of cost effectiveness. The top five most cost-effective strategies within and across the bioregions were the management of: 1) parkinsonia in the Channel Country, 2) parkinsonia in the Desert Uplands, 3) mesquite in the Mitchell Grass Downs, 4) parkinsonia in the Mitchell Grass Downs, and 5) mother of millions in the Desert Uplands. Although actions for several invasive plant species like parkinsonia and prickly acacia were concentrated in the Queensland part of the LEB, the actions involved investing in containment zones to prevent the spread of these species into other states. In the NT and SA bioregions of the LEB, the management of athel pine, parkinsonia and cacti were the main strategies. While outside the scientific research goals of study, this work highlighted a number of important incidental findings that led us to make the following recommendations for future research and implementation of weed management in the Basin: • Ongoing stakeholder engagement, extension and participation is required to ensure this prioritisation effort has a positive impact in affecting on-ground decision making and planning. • Short term funding for weed management was identified as a major reason for failure of current efforts, hence future funding needs to be secure and ongoing. • Improved mapping and information sharing is essential to implement effective weed management. • Due to uncertainties in the outcomes and impacts of management options, strategies should be implemented as part of an adaptive management program. The information provided in this report can be used to guide investment for controlling high-impact invasive plant species for the benefits of biodiversity conservation. We do not present a final prioritisation of invasive plant strategies for the LEB, and we have not addressed the cultural, socio-economic or spatial components necessary for an implementation plan. Cost-effectiveness depends on the objectives used; in our case we used the intactness of ecosystems as a surrogate for expected biodiversity benefits, measured by the extent that each invasive plant species is likely to dominate in a bioregion. When other relevant factors for implementation are considered the priorities may change and some actions may not be appropriate in some locations. We present the costs, ecological benefits and cost-effectiveness of preventing, containing, reducing and eradicating the dominance of high impact invasive plants through realistic management actions over the next 50 years. In doing so, we are able to estimate the size of the weed management problem in the LEB and provide expert-based estimates of the likely outcomes and benefits of implementing weed management strategies. The priorities resulting from this work provide a prospectus for guiding further investment in management and in improving information availability.