Progress 12/15/04 to 12/14/08
Outputs
OUTPUTS: SUMMARY OF FINAL OUTPUTS FOR THE WHOLE PROJECT Activities of project personnel: Dr. Quan Zhang is the post-doctoral fellow who worked on this project and was responsible for the cloning of STR. Over the course of the project, he learned a broad range of genetic, molecular biology and cell biology skills. He presented his research data at the 2008 Annual Meeting of American Society of Plant Biologists at Merida, Mexico. The title was "Positional cloning of STR, a gene required for arbuscular mycorrhizal symbiosis in Medicago truncatula". In addition, part of the results has been presented at the seminar at Boyce Thompson Institute for Plant Research (BTI) and as a poster at BTI annual retreat. Over the course of the project, Dr. Harrison attended the annual USDA-NRI Plant-Microbe Awardees meetings and in each case, the str project was described on a poster. Dr. Harrison also gave several seminars which included discussion of the str mutant. These included: Department of Plant and Soil Science, University of Delaware (Graduate student enrichment speaker), March 8-9th, 2007. Department of Biology Seminar Series, Clemson University, South Carolina, February 23, 2007. Department of Plant Biology seminar series, Michigan State University, East Lansing, Michigan, September 17, 2007. As a result of this project, we established collaboration and connections with scientists in the USDA-ARS at Minnesota, with scientists at U.C. Davis, and with a research group at the University of Lausanne, Switzerland. As detailed in the annual reports and summarized in the Outcome/Impact section, the major output from this research project was the cloning of the STR gene. PARTICIPANTS: Dr. Quan Zhang (post-doc) and Dr. Maria J. Harrison (PI) worked on this project. The main experiments were performed by Dr. Zhang. Experimental planning, analysis of results and preparation of manuscripts was carried out by Dr. Zhang and Dr. Harrison TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
FINAL PROJECT OUTCOME This project led to a detailed characterization of the first M. truncatula mutant, str, impaired only in AM symbiosis. The str mutant shows wildtype interactions with rhizobia and with various biotrophic fungal and oomycete pathogens. STR was cloned by positional cloning. This was a particularly challenging process as STR resides at the extreme end of M. truncatula chromosome 4 and recombination in this region of the genome is severely repressed. As a consequence of the positional cloning, several new markers were developed that will be useful to the M. truncatula community. STR encodes a integral membrane protein and based on conserved features it is a novel transporter. The sequence does not provide clues about the transported substrate, but it is possibly a secondary metabolite or a lipid molecule. STR is expressed constitutively in root vascular tissue and induced in root cortical cells with arbuscules during symbiosis. Transcripts were not detected in any aerial tissues of the plant. Phylogenetic profiles show that STR protein sequence is well conserved in plant species that form arbuscular mycorrhizal symbiosis, but is absent from the Arabidopsis thaliana genome. Arabidopsis is not able to form AM symbiosis. FINAL PROJECT IMPACT Vascular plants and arbuscular mycorrhizal (AM) fungi have been associated since the Devonian age and today, they are found in a huge array of plant species in ecosystems throughout the world. The effects of a mycorrhizal P supply on plant growth are well documented. In addition to enhancing phosphorus acquisition, there are equally significant impacts to soil quality and soil aggregate stability. Through these and other mechanisms, the AM symbiosis influences plant biodiversity and ecosystem productivity. Although these associations are formed by a huge array of species and influence plant growth in environments throughout the world, we know relatively little of the mechanisms underlying their development. An improved understanding of the formation and functioning of the symbiosis is necessary to enable the rational management and/or exploitation of the AM symbiosis in agriculture and land management practices. The work supported by this award led to the cloning of the first plant gene (STR) required specifically for AM symbiosis. STR is a transporter and plays an essential role in development of the arbuscule-cortical cell interface. This is the major nutrient exchange interface in the AM symbiosis and is central to the functioning of the symbiosis. Previously in legumes, genes required for both nodulation and AM symbiosis have been identified, but STR is unique in that it is required exclusively for AM symbiosis. As such, it provides the first information about processes specific for AM symbiosis and in particular, the first information about processes essential for development of the biotrophic interface, where mineral nutrient transport to the plant occurs. STR, along with other transporters, such as MtPT4, clearly impact symbiotic function. Consequently, they are targets for breeding crop varieties that have the genetic potential to benefit maximally from symbioses with AM fungi.
Publications
- A manuscript describing the str mutant, the positional cloning of STR and analysis of the STR gene was submitted in January 2009.
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Progress 12/15/06 to 12/14/07
Outputs
Outputs. Activities: Dr. Quan Zhang is the post-doctoral fellow working on this project. Over the past year, his major activities have been to continue the mapping and complete the positional cloning of a gene required for AM symbiosis. Details of the cloning are outlined below. Dr. Harrison attended the "Genes to Products - Agricultural Plant, Microbe, and Biobased Product Research," USDA-NRI Plant-Microbe Awardees meeting, March 12-14, 2007, Bethesda, Maryland. The str project was described on a poster. Dr. Harrison also gave three seminars which included discussion of the str mutant. These included:Department of Plant and Soil Science, University of Delaware (Graduate student enrichment speaker), March 8-9th, 2007. Department of Biology Seminar Series, Clemson University, South Carolina, February 23, 2007. Department of Plant Biology seminar series, Michigan State University, East Lansing, Michigan, September 17, 2007 Dr. Harrison serves as a member of the Board of
Directors of the International Society for Molecular Plant Microbe Interactions.
Impacts
Outcome: Over the past year, we finally finished the cloning of str, as summarized below. Using the sequence information obtained from the newly sequenced BAC 222J12, we developed additional markers to assist with the mapping of str. We then further increased the mapping population to 8712 individuals. This is an extremely large mapping population but was necessary because recombination is suppressed in the region containing str. From this population, we obtained three additional recombinants that allowed us to position str within a 230kb region. Sequence prediction programs predict 65 genes in this region including a significant proportion of genes associated with transposable elements. We then initiated two approaches to find str. Using long-range, high fidelity, PCR, we amplified this 230 Kb region from str in 10 kb overlapping segments. We intended to sequence the region from str using one of the next generation sequencing technologies such as 454. Simultaneously,
we began analysis of some of the candidate genes in the regions. We designed primers to analyze transcripts of genes in this contig and monitored expression patterns to determine which genes were expressed in roots. We then sequenced the most promising candidates. One candidate, gene 23 contained a single C to T transition that created a premature stop codon in the protein and a truncated, non functional protein. A wildtype copy of the STR gene was then used to complement the str mutant. This demonstrated that the gene in question was indeed str. This is the first M. truncatula mutant required specifically for AM symbiosis to be cloned by a positional cloning approach. The data are being prepared for publication. Impact: Vascular plants and arbuscular mycorrhizal (AM) fungi have been associated since the Devonian age and today, they are found in a huge array of plant species in ecosystems throughout the world. The effects of a mycorrhizal P supply on plant growth are well documented,
but in addition to enhancing phosphorus acquisition, there are equally significant impacts to soil quality and soil aggregate stability. Through these and other mechanisms, the AM symbiosis influences plant biodiversity and ecosystem productivity. Although these associations are formed by a huge array of species and influence plant growth in environments throughout the planet, we know relatively little of the mechanisms underlying their development. The work supported by this award is providing insights into plant components required for development of the arbuscule-cortical cell interface. This is the major nutrient exchange interface in the symbiosis and central to the functioning of the symbiosis. Currently, str is the only mutant known to impact arbuscule formation specifically and thus the cloning of str has the potential to provide the first insights into the molecular events that control the development of the arbuscule. An improved understanding of the formation and
functioning of the symbiosis is necessary to enable the rational management and/or exploitation of the AM symbiosis in agriculture and land management practices.
Publications
- No publications reported this period
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Progress 12/15/05 to 12/15/06
Outputs
Plants have evolved the ability to cope with many different environmental stresses and in some cases, the strategy involves enlisting the help of rhizosphere microbes. One widespread example is the interaction between arbuscular mycorrhizal fungi (AMF) and vascular plants, which occurs widely in ecosystems throughout the world. AMF help plants acquire mineral nutrients, including phosphorus and nitrogen, from soil. The AM symbiosis is a highly co-evolved partnership. The AMF live within the cortical cells where they form highly branched hyphae called arbuscules. These are the major site of nutrient exchange between the symbionts. The goals of this project are to contribute to an understanding of the molecular basis of development and regulation of the AM symbiosis by cloning a gene, required for arbuscule development. The mutant, named str (stunted arbuscule), is unable to support arbuscule development and thus the symbiosis arrests in this line. In the first year of
this project, we developed protocols for large scale mapping, mapped str to the south arm of chromosome4, and increased the mapping population to 3903 plants. In this past year, we continued to progress towards the cloning of str, as summarized below. 1) While continuing to improve the mapping protocol, we increased the mapping population to 4818 individuals (F2 and F3 plants). 2) Several additional markers on the south end of chromosome 4 were used to map the whole population. One marker, namely 53O24, still shows complete linkage to str. Breaks have been confirmed between this marker and previous makers closest to str. 3) Using a probe made from a DNA sequence close to the last available genetic marker, we identified additional BACs that are located further south of the chromosome 4. One of the BAC is currently being sequenced by Oklahoma University Genome Center. 4) We designed new SSR markers in the chromosome region where str is located. These markers maybe used for future fine
mapping. In addition to progress towards the cloning of str, we further characterized the str phenotype. Under extreme low phosphorus conditions, wild type mycorrhizal plants grow at a relatively normal level, by acquiring phosphorus from soil through AMF. In contrast, growth of mycorrhizal str is comparable to nonmycorrhizal wild type plants, and they show signs of phosphorus deficiency. This indicates that the str interaction is not functional for phosphate transfer. In addition, we compared the transcript profiles of str relative to wild type, under both mycorrhizal and nonmycorrhizal conditions. A set of genes down-regulated in mycorrhizal str roots have been identified and are under further investigation.
Impacts
Vascular plants and arbuscular mycorrhizal (AM) fungi have been associated since the Devonian age and today, they are found in a huge array of plant species in ecosystems throughout the world. The effects of a mycorrhizal P supply on plant growth are well documented, but in addition to enhancing phosphorus acquisition, there are equally significant impacts to soil quality and soil aggregate stability. Through these and other mechanisms, the AM symbiosis influences plant biodiversity and ecosystem productivity. Although these associations are formed by a huge array of species and influence plant growth in environments throughout the planet, we know relatively little of the mechanisms underlying their development. The work supported by this award is providing insights into plant components required for development of the arbuscule-cortical cell interface. This is the major nutrient exchange interface in the symbiosis and central to the functioning of the symbiosis.
Currently, str is the only mutant known to impact arbuscule formation specifically and thus the cloning of str has the potential to provide the first insights into the molecular events that control the development of the arbuscule. An improved understanding of the formation and functioning of the symbiosis is necessary to enable the rational management and/or exploitation of the AM symbiosis in agriculture and land management practices.
Publications
- No publications reported this period
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Progress 12/15/04 to 12/14/05
Outputs
The majority of the vascular flowering plants, including most crop species, are able to develop symbiotic associations with arbuscular mycorrhizal (AM) fungi. The associations develop in the roots where the AM fungi colonize the root cortex to access carbon provided by the plant, while assisting the plant with the acquisition of mineral nutrients, particularly phosphorus (P), from the soil. The AM symbiosis is a highly co-evolved partnership, in which the coordinate differentiation of both partners results in a stable, symbiotic state that enables the bi-directional exchange of nutrients between symbionts. The goals of the project are to contribute to an understanding of the molecular basis of development and regulation of the AM symbiosis by cloning a gene, required for arbuscule development. A novel Medicago truncatula mutant that is unable to form a complete AM symbioses has been identified. The mutant, named str (stunted arbuscule), is unable to support arbuscule
development and thus the symbiosis arrests in this line. The objectives of work in the first year included further characterization of the str phenotype, the initial mapping of the str locus, and development of a large mapping population to enable fine mapping of str. We have undertaken additional phenotypic characterization of str and shown that the mutation impacts the interaction with three different AM fungi. We are now preparing to assess the transcript profiles in str relative to wildtype and 4 biological replicate sets of material have been created. Further assessment of the suitability of this material for use on arrays is in progress. The str mutant was crossed with ecotype A20 and the F1 was allowed to self giving rise to an F2 population. 126 F2 individuals were planted and inoculated with G. versiforme and the phenotypes scored. 28 individuals showed the str mutant phenotype, while the remaining 98 individuals showed a wildtype phenotype. This population was used for the
initial mapping of the str locus. Two simple sequence repeat (SSR) markers, evenly distributed on each of the 8 chromosomes, were chosen for initial mapping. In this F2 population, the frequency of recombination between str and markers on chromosome 4 is significantly lower than 50%. No recombination was detected between str and markers south of marker 005H08. Additional markers selected from linkage group 4 and used to evaluate the population. Based on these data, it is concluded that str is located on south arm of chromosome 4. Prior to increasing the mapping population, we developed protocols that enabled us to scale up our genotyping and phenotyping activities. In particular, we developed a DNA preparation system that allows the preparation of 400 DNA preps in a single day. To do this, we use a 96-well format genomic DNA isolation protocol (adapted from the CTAB protocol in Arabidopsis- A Laboratory Manual). This permits the isolation of DNA of a quality suitable for use with SSR
and CAPs markers. The mapping population has now been increased to 3903 individuals (a mixture of F2 and F3 plants) and additional mapping, using markers from the south arm of linkage group 4, is in progress.
Impacts
Vascular plants and arbuscular mycorrhizal (AM) fungi have been associated since the Devonian age and today, they are found in a huge array of plant species in ecosystems throughout the world. The effects of a mycorrhizal P supply on plant growth are well documented, but in addition to enhancing phosphorus acquisition, there are equally significant impacts to soil quality and soil aggregate stability. Through these and other mechanisms, the AM symbiosis influences plant biodiversity and ecosystem productivity. Although these associations are formed by a huge array of species and influence plant growth in environments throughout the planet, we know relatively little of the mechanisms underlying their development. The work supported by this award is providing insights into plant components required for development of the arbuscule-cortical cell interface. This is the major nutrient exchange interface in the symbiosis and central to the functioning of the symbiosis.
Currently, str is the only mutant known to impact arbuscule formation specifically and thus the cloning of str has the potential to provide the first insights into the molecular events that control the development of the arbuscule. An improved understanding of the formation and functioning of the symbiosis is necessary to enable the rational management and/or exploitation of the AM symbiosis in agriculture and land management practices.
Publications
- No publications reported this period
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