Progress 07/01/10 to 06/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The Project Director left the University. No progress to report.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Microalgae are a diverse set of photosynthetic prokaryotes and eukaryotes that use water, sunlight and carbon dioxide to produce biomass that includes oils appropriate for use as biodiesel. Different microalgae have unique oil compositions, meaning some varieties are less suitable for biodiesel production and, therefore, constitute undesirable contaminants of production systems. For the purpose of this proposal, desired high-oil-content algae are referred to as "elite" strains. Contamination of the algae growth system with unwanted algal competitors with low oil content, predators, or other pests will reduce nutrients available for elite algae strains, deplete elite algae populations through grazing, or otherwise reduce the productivity of the elite algae. In addition to biodiesel production, various private companies use microalgae for production of bioproducts such as food additives, vaccines, animal feed, probiotics and other products. Contamination of cultures is a potential limit to productivity in all these systems. Algae may be grown in closed or open systems; given the economic feasibility of open systems, this may become the dominant system for production. It is easy to understand the potential for contamination of this open system with unwanted organisms. Regardless, maintaining a production-scale monoculture even in a closed system is a significant challenge. To manage this threat, monitoring and remediation strategies must be developed. Toward this end, molecular diagnostic tools that faithfully identify elite algae strains, competitors, predators and pests are needed. The objectives of this project include: (1) develop quantitative molecular diagnostics for algae production systems, and (2) develop pest monitoring and management strategies for algae production systems. In 2012, my lab made progress toward both stated objectives. Toward objective 1, we developed our second generation of quantitative molecular diagnostics to detect elite algae and a known contaminant of production systems. These are DNA-based diagnostics that we tested on laboratory cultures to develop methodology and determine limits of detection, and then tested on production cultures from Solix BioSystems (Fort Collins, CO). Regarding objective 2, we monitored weedy species in lab and production samples using non-quantitative DNA-based diagnostics, quantitative DNA-based diagnostics, and flow cytometry. PARTICIPANTS: Stephen Chisholm is the Principle Investigator for this project and designed the quantitative diagnostics and helped develop the production sampling procedures. Scott Fulbright is a PhD student working on sample collection and analysis. Solix Biofuels (Fort Collins, CO) is a partner organization that provides algae production samples and access to equipment. TARGET AUDIENCES: The target audience for this project includes algae growers and researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcomes for 2012 relate to a change in knowledge. Having previously identified weeds commonly found in saline algae production systems, we developed quantitative DNA-based diagnostics to detect these organisms. The quantitative diagnostics we initially developed did not function as anticipated when used on production cultures. We developed alternate DNA-based quantitative diagnostics and demonstrated the effectiveness of these on both laboratory and production algae cultures. Furthermore, we demonstrated these diagnostics are far more sensitive and the data is more reproducible than methodologies currently in use by the algae industry (microscopy, flow cytometry, or a combination thereof). This knowledge will impact the available industry standards for culture monitoring. Throughout 2012, we also continued sampling of production cultures from Solix BioSystems. These samples were initially collected with the intent to monitor weedy species as described. Additionally, from a subset of samples we used metagenomics techniques to identify bacteria growing in production media with the desired algae. We found that any single production sample likely contains hundreds of different types of bacteria. The potential effects of these bacteria on promoting or hindering algae growth and fuel production remain unknown. Our initial characterization of bacteria populations in these cultures was not of a sufficient scale to correlate the presence of particular types of bacteria with impacts on algae growth. Therefore, during 2012 we altered our sample collection strategy to include collection of available data for various metrics of algae growth and overall health. In 2013, we will continue to use metagenomics to characterize bacteria in production cultures and endeavor to correlate the presence or absence of particular types of bacteria with the health of the algae culture. By cataloguing the sheer number of different types of bacteria in algae production cultures, our initial data set altered community perspectives regarding the likely importance of bacteria for culture health. Our forthcoming data correlating particular bacterial types with positive and negative impacts on algae growth or productivity will lay the groundwork for developing strategies to manage bacteria populations within algae cultures.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Microalgae are a diverse set of photosynthetic prokaryotes and eukaryotes that use water, sunlight and carbon dioxide to produce biomass that includes oils appropriate for use as biodiesel. Different microalgae have unique oil compositions, meaning some varieties are less suitable for biodiesel production and, therefore, constitute undesirable contaminants of production systems. For the purpose of this report, desired high-oil-content algae are referred to as "elite" strains. Contamination of the algae growth system with unwanted low-oil-content algal competitors, predators or other pests will reduce nutrients available for elite algae strains, deplete elite algae populations through grazing, or otherwise reduce the productivity of the elite algae. In addition to biodiesel production, various private companies use microalgae for production of bioproducts such as food additives, vaccines, animal feed, probiotics and other products. Contamination of cultures is a potential limit to productivity in all these systems. Algae may be grown in closed or open systems; given the economic feasibility of open systems, this may become the dominant system for production. It is easy to understand the potential for contamination of this open system with unwanted organisms. Regardless, maintaining a production-scale monoculture even in a closed system is a significant challenge. To manage this threat, monitoring and remediation strategies must be developed. Toward this end, molecular diagnostic tools that faithfully identify elite algae strains, competitors, predators and pests are needed. The objectives of this project include: (1) develop quantitative molecular diagnostics for algae production systems, and (2) develop pest monitoring and management strategies for algae production systems. In 2011, my lab made progress toward both objectives. Toward objective 1, we completed validation of several DNA-based quantitative diagnostics that specifically detect elite algae and the most common weedy algal contaminant of salt-water production systems. Regarding objective 2, throughout 2011 we collected weekly sample sets from the algae production systems at Solix BioSystems (Fort Collins, CO) that will allow us to monitor contamination of production cultures and maintain a biologically viable archive of cultures for future sampling. Though we do not characterize each sample, maintaining these weekly sample archives allows us to do forensic analyses following crashes of production cultures. The quantitative diagnostics mentioned above are effective at analyzing weedy low-oil-content algae competitors. In 2011, we also made significant advances toward developing procedures to determine the nature of bacterial populations within salt-water algae production systems. One goal is to identify bacterial pathogens of algae. Additionally, we hope to clarify what bacteria are beneficial for algae growth and develop inoculation procedures to ensue algae cultures contain appropriate quantities for these beneficial bacteria. PARTICIPANTS: Stephen Chisholm is the Principle Investigator for this project and designed the quantitative diagnostics and helped develop the production sampling procedures. Solix BioSystems (Fort Collins, CO) is a partner organization that provides algae production samples and access to equipment. TARGET AUDIENCES: The target audience for this work is the nascent algae biofuels and bioproducts industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcomes for 2011 relate to a change in knowledge. Having identified major eukaryotic contaminants of salt-water algae production systems in 2009 and 2010, in 2011 we tested multiple types of DNA-based quantitative diagnostics for performance on lab and production cultures, and compared our limits of detection (sensitivity of the diagnostics) to non-DNA-based methods for organism quantification, particularly flow cytometry. Broadly, we tested performance of quantitative PCR-based diagnostics that were designed to either: 1) amplify the same gene from all eukaryotes present in the production system and then detect organism-specific polymorphisms within these amplicons, or 2) amplify unique genomic regions for each organism known to be commonly found in salt-water algae production systems. For technical reasons related to amplification biases, the first strategy did not perform as well as the second. Our current diagnostics all use the second method involving amplifying unique gene regions for each different organism anticipated in the system. Also in 2011, we began monitoring bacteria within salt-water algae production systems. We expect that some bacterial pathogens may be present in these production systems. Additionally, other bacteria may perform functions necessary for efficient algae growth, such as making nutrients available for the elite algae or buffering the pH of the growth media. Prior to this work, it was unclear how complex bacterial communities would be in closed growth systems. We used a metagenomic community profiling approach to determine the species-level complexity of bacterial communities at different scales of production cultures, from small 200 ml flask cultures to large 400 liter production cultures. Our work determined that cultures have from approximately 150 to over 400 species of bacteria in them, with higher-volume cultures generally having more species of bacteria. Currently we are collecting additional samples that will allow us to identify potentially beneficial bacteria present in these cultures. This knowledge will allow development of probiotics (a cocktail of bacteria that are beneficial for algae growth) that can be co-inoculated with algae cultures to ensure robust growth of elite algae. A secondary outcome of our 2011 work is a change in action related to routine sampling of algae production cultures. We began developing sampling and archiving procedures in 2010, and in 2011 implemented a standard weekly sample collection and archiving protocol that allows us to examine any of over 20 points in the production system on a weekly basis. Additionally, we are working with colleagues in New Mexico to develop sampling kits and instructions for use by algae researchers at distant locations. Implementation of simple, standardized sampling strategies will allow us to request samples from algae production facilities throughout the US in a standardized format. These samples will allow us to characterize both the eukaryotes and prokaryotes present in the systems.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Microalgae are a diverse set of photosynthetic prokaryotes and eukaryotes that use water, sunlight and carbon dioxide to produce biomass that includes oils appropriate for use as biodiesel. Different microalgae have unique oil compositions, meaning some varieties are less suitable for biodiesel production and, therefore, constitute undesirable contaminants of production systems. For the purpose of this proposal, desired high-oil-content algae are referred to as "elite" strains. Contamination of the algae growth system with unwanted algal competitors with low oil content, predators, or other pests will reduce nutrients available for elite algae strains, deplete elite algae populations through grazing, or otherwise reduce the productivity of the elite algae. In addition to biodiesel production, various private companies use microalgae for production of bioproducts such as food additives, vaccines, animal feed, probiotics and other products. Contamination of cultures is a potential limit to productivity in all these systems. Algae may be grown in closed or open systems; given the economic feasibility of open systems, this may become the dominant system for production. It is easy to understand the potential for contamination of this open system with unwanted organisms. Regardless, maintaining a production-scale monoculture even in a closed system is a significant challenge. To manage this threat, monitoring and remediation strategies must be developed. Toward this end, molecular diagnostic tools that faithfully identify elite algae strains, competitors, predators and pests are needed. The objectives of this project include: (1) develop quantitative molecular diagnostics for algae production systems, and (2) develop pest monitoring and management strategies for algae production systems. In 2010, my lab made progress toward both stated objectives. Toward objective 1, we developed our first generation of quantitative molecular diagnostics to detect elite algae and a known contaminant of production systems. These are DNA-based diagnostics that we tested on laboratory cultures to develop methodology and determine limits of detection. Regarding objective 2, in November 2010, we implemented a routine sampling procedure at Solix Biofuels (Fort Collins, CO) that will allow us to monitor contamination of production cultures and maintain a biologically viable archive of cultures for future sampling. PARTICIPANTS: Stephen Chisholm is the Principle Investigator for this project and designed the quantitative diagnostics and helped develop the production sampling procedures. Solix Biofuels (Fort Collins, CO) is a partner organization that provides algae production samples and access to equipment. TARGET AUDIENCES: The target audience for this work is the nascent algae biofuels and bioproducts industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major outcomes for 2010 relate to a change in knowledge. Firstly, our monitoring of algae production cultures using non-quantitative diagnostics (developed in 2009, improved in 2010, and used to monitor cultures in 2010) identified additional contaminants of the production system. Future work will involve developing quantitative molecular diagnostics to identify such organisms. Secondly, we tested our first set of quantitative molecular diagnostics. In laboratory cultures, we determined the specificity and identified limits of detection for these diagnostics. We also began development of methodology for effective use of these diagnostics in a production setting; this aspect of the work is ongoing. Thirdly, we identified general technical limitations to these DNA-based diagnostics; this will lead us to develop alternate monitoring strategies and diagnostics in the coming year. A secondary outcome to our 2010 work is a change in action related to routine sampling of algae production cultures. Prior to our work, production samples were not regularly collected or maintained in a viable state. This meant that when an algae production culture failed, there was no historical archive of the culture to review for the possible sources of culture failure. In 2010 we developed production culture sampling and maintenance procedures for use in algae production. This strategy was implemented by Solix Biofuels in November 2010, and in 2011 we will continue to refine the routine based on feedback from Solix and based on our efforts to characterize stored cultures.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project will clarify the defense function of a family of proteins that are targeted by multiple bacterial effectors proteins, monitored by multiple host resistance (R) proteins and highly conserved in all land plants. If, as proposed, these proteins indeed function in immunity, then breeding and transgenic approaches may be used to introduce effector-resistant forms of these proteins into host cells in order to bolster the plant immune system. The objectives of the project include 1) generation of Arabidopsis plant lines in which expression of host targets of pathogen effectors are suppressed, and 2) functional characterization of these plant effector targets. In 2009, my laboratory made progress in several aspects of Objective 2, characterization of the plant effector targets. After we tried unsuccessfully to use a genetic approach to complete this aim, we developed tools for protein studies. Namely, we identified host proteins that interact with the host targets of effectors and began generating transgenic plant lines in which we will use fluorescence microscopy to study these proteins during interactions with microbes. Tools for analysis of protein interactors are presently limited to our characterization of Arabidopsis T-DNA insertion lines in which the gene encoding a potential interactor is disrupted (resulting in a likely null allele of the gene) as well as generating protein fusions that will be used in the coming year to directly study protein interactions in planta. Regarding generation of transgenic plants for fluorescence microscopy, we made 24 fusions between the green fluorescence protein (GFP) and plant proteins of interest (or mutated versions of plant proteins of interest). PARTICIPANTS: Work in this project was done entirely in the Chisholm tab at CSU. This includes training of two undergraduate honors researchers working on various aspects of the project. TARGET AUDIENCES: The target audience for this work currently includes other individuals or groups working on improving plant resistance to pathogens as well as those concerned with the evolution of pathogen virulence on plants. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The major 2009 outcomes from this work relate to a change in knowledge. Several plant proteins that interact with a specific host target of the bacterial effector protease AvrRpt2 were identified using a genetic screen. These putative interactors are members of two classes of host transcriptional regulators. If these interactions are confirmed in planta, this will represent a major advancement in understanding plant disease resistance signaling and bacterial pathogenesis. The proteins known to be targeted by AvrRpt2 are normally peripherally associated with the cytoplasmic face of the plant cell's plasma membrane; since these protein interactors are transcriptional regulators that function in the nucleus or chloroplasts, during interactions with microbes the targets of AvrRpt2 may move from the plasma membrane to the nucleus or chloroplast to stimulate or repress these transcriptional regulators. A second aspect of this project supports a model in which the targets of AvrRpt2 migrate from the plant cell plasma membrane to the nucleus following stimulation. Using fluorescence microscopy, translational fusions between GFP and targets of AvrRpt2 ("GFP-targets") were observed at the plasma membrane, as predicted. This localization is driven by a specific and reversible fatty acid modification. We prevented this modification using two strategies: 1) pharmacological inhibitors, and 2) protein mutagenesis. In both cases, prevention of this fatty acid modification resulted in accumulation of the GFP-target fusions in the nucleus (and, possibly, chloroplasts). Furthermore, we generated mutated versions of these GFP-target fusions that we believe mimic phosphorylation of these targets by other effector proteins, including AvrB. These "phosphorylation mimic" versions of the GFP-target fusions do not localize to the nucleus in the absence of fatty acid modification; this result suggests that the function of effector proteins is to prevent nuclear localization of these targets by either proteolytic cleavage (for example, by AvrRpt2) or phosphorylation (for example, by AvrB). By using enzymatic activities to prevent nuclear localization of targets, bacterial effector proteins may, therefore, prevent these targets from interacting with the nuclear-localized transcriptional regulators described in the preceding paragraph. Our 2009 data is being used as preliminary data for a 2010 resubmission of an NSF CAREER proposal.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project will clarify the defense function of a family of proteins that are targeted by multiple bacterial effectors proteins, monitored by multiple host resistance (R) proteins and highly conserved in all land plants. If, as proposed, these proteins indeed function in immunity, then breeding and transgenic approaches may be used to introduce effector-resistant forms of these proteins into host cells in order to bolster the plant immune system. Objective 1: Generation and genetic characterization of Arabidopsis lines in which expression of AvrRpt2 targets is suppressed Objective 2: Functional characterization of plant lines in which expression of AvrRpt2 targets is suppressed In 2008, my laboratory completed various aspects of Objective 1. Firstly, we obtained Arabidopsis T-DNA insertion lines with putative insertions in the promoters or coding regions of AvrRpt2 substrates The ABRC supplied several dozen seed for each requested T-DNA insertion line; typically these seed were a mixture of plants in which the T-DNA is segregating and may be absent, hemizygous or homozygous. Polymerase chain reaction (PCR)-based assays were used to identify plants homozygous for the T-DNA insertion in the gene of interest. We identified 15 homozygous lines corresponding to loci encoding 10 unique AvrRpt2 targets. Homozygous plants were selfed and the resulting progeny are being characterized as described in Objective 2. Secondly, we generated Arabidopsis and tobacco plants in which gene-silencing technologies are used to simultaneously suppress expression of AvrRPt2 targets. For the Arabidopsis plants, we generated over ten independent stable transgenic lines in which targets of AvrRpt2 are silenced by a hairpin construct; in tobacco plants, we are using transient virus-induced gene silencing to suppress expression of AvrRpt2 targets. PARTICIPANTS: Work in this project was done entirely in the Chisholm tab at CSU. This includes training of two undergraduate honors researchers working on various aspects of the project. TARGET AUDIENCES: The target audience for this work currently includes other individuals or groups working on improving plant resistance to pathogens as well as those concerned with the evolution of pathogen virulence on plants. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The major 2008 outcomes from this work relate to a change in knowledge. Using the Arabidopsis T-DNA insertion lines and silenced lines, we demonstrated that the AvrRpt2 targets are involved in defense against bacteria. Specifically, bacteria grow better on plants in which levels of a single or multiple AvrRpt2 are reduced. Surprisingly, some of our Arabidopsis T-DNA insertion lines show enhanced resistance to bacteria, indicating that some of the targets of AvrRpt2 may in fact be negative regulators of disease resistance. We are currently working to determine which targets are positive regulators of resistance and which are negative regulators. Furthermore, Arabidopsis lines in which multiple targets of AvrRpt2 are silenced show striking developmental phenotypes, in that they show a loss of apical dominance and produce a large number of rosette leaves. Tobacco plants in which AvrRpt2 targets are silenced display similar, but even more pronounced, phenotypes. Together, these results indicate that host proteins targeted by AvrRpt2 not only modulate host immune responses, but are involved in developmental regulation, possibly by regulating hormone signaling. A major focus of our 2009 work will be to determine the nature of these developmental phenotypes. Our 2008 data is being used as preliminary data for a 2009 USDA proposal in which we propose to extend our work to pathogen defense and plant development in rice plants. This data will also be included in a 2009 NSF CAREER proposal.
Publications
- No publications reported this period
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