Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: ACTIVITIES: Marker genes are essential for uniform transformation of the polyploid plastid genomes of plant cells. Funded by the USDA BRAG Program, we previously developed efficient systems for plastid marker excision. Excision was accomplished by Agrobacterium-mediated transformation of the plant nucleus with plastid-targeted phage recombinases, which efficiently excised the target-site flanked marker gene in plastids. The recombinase gene was subsequently segregated away in the seed progeny. The objective of Project NJ12928 was: (1) to achieve marker excision with recombinases exported from Agrobacterium to plastids and (2) to carry out the marker excision in plants rather than in tissue culture cells. Experiments to achieve Objective 1 are still in progress. (a) To detect recombinase delivery, we developed inactive (spectinomycin resistance) marker genes, which could be activated by changes effected by Agrobacterium-delivered recombinase. Two generations of marker genes performed well in E. coli (cells carrying the inactive aadA were sensitive, the activated aadA resistant to spectinomycin), but the inactive form always conferred resistance to plastids. (b) To enable recombinase targeting from Agrobacterium to plastids, the Cre recombinase was fused with a plastid-targeting signal at its N-terminus and a Type IV protein secretion signal at it C -terminus. The constructs tested thus far were either imported into plastids but rapidly degraded in Agrobacterium, or were stable in Agrobacterium but failed to be imported into plastids. Further testing of improved versions of these systems is ongoing. Objective 2 has been accomplished. The target site-flanked plastid marker has been excised by recombinase expressed from T-DNA of Agrobacterium injected into plants, yielding marker-free seed progeny. A novel visual marker, the aurea-aadA gene, which yielded a visual pigment phenotype in leaves, enabled the recovery of plastid marker-free plants. EVENTS: PI in 2012 gave lectures and disseminated information at the following events and locations: (1) Plant and Animal Genome Conference XX, Plant Organellar Genetics Workshop, San Diego, CA, January 14-18, 2012; speaker and organizer (2) 7th Annual Tripartite Meeting of the Americas between the University of Sao Paulo, Rutgers University, and The Ohio State University, Columbus, Ohio, May 31 - June 2, 2012; speaker (3) Annual Project Director's Meeting for Biotechnology Risk Assessment Grants (BRAG) Program, Riverdale, MD, June 5-6, 2012; speaker PARTICIPANTS: PI/PD Prof. Pal Maliga directed research. Dr. Tarinee Tungsuchat-Huang, a Research Associate, was the lead contributor to the project. Ms. Sugey Sinagawa Garcia tested the stability of transplastomes carrying target-site flanked marker genes. These plants were injected with Agrobacterium to detect marker excision in plants. Ms. Sinagawa Garcia defended her Ph.D. thesis at Cinvestav, Irapuato, Mexico, based on research at Rutgers. Ms. Kristina Slivinski constructed the aurea spectinomycin resistance genes that formed the basis of the research thesis submitted in partial fulfillment of the requirements of the Bachelor of Arts Degree in Genetics. TARGET AUDIENCES: (1) Plant biologists interested in engineering the photosynthetic machinery; biotechnologists interested in the expression of recombinant proteins in chloroplasts and engineering transgenic crops. (2) Regulatory agencies, who should be aware of the technical feasibility of plastid marker excision. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) Successful manipulation of the plastid genome (ptDNA) has been carried out thus far in tissue culture cells. Plastid marker gene excision directly in plants by injected Agrobacterium cells is a major step forward. Because Cre is expressed from a non-integrated T-DNA copy, in many cases there is no T-DNA to segregate away. In planta plastid marker excision is enabled by the aurea-aadA a visual-selectable marker gene. The gene confers spectinomycin resistance in culture and a visual phenotype in leaves. This visual marker will be particularly useful in vegetatively propagated, heterozygous crops such as potato and fruit trees, in which elimination of the marker through segregation is incompatible with variety preservation. This marker will be a valuable tool for breeders as they generate commercially viable transplastomic crops. (2) Expression of marker genes in plastids, that are silent in E. coli, point to principal differences in the translation machinery of the two prokaryotic-type systems. This information will be published in the scientific literature. The results of our research will aid biotechnologists in designing regulated transgene expression systems. (3) Our third-generation marker genes are designed to be sufficiently sensitive to detect the import of a single Cre enzyme molecule from Agrobacterium into the chloroplast. Although the engineered Cre does not accumulate in Agrobacterium at high levels, relatively low levels of the enzyme, coupled with a sensitive detection system, may be sufficient to confirm Cre import from Agrobacterium into chloroplasts. If this occurs, we will attempt to re-engineer Agrobacterium to deliver the T-DNA to plastids.
Publications
- Maliga, P. (2012) Plastid transformation in flowering plants. In Genomics of Chloroplasts and Mitchondria (Bock, R. and Knoop, V. eds). Springer, pp. 393-414 .
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: (1) Engineering a silent spectinomycin resistance (aadA) marker gene that is inducible by Cre. According to the design, floxed stop codons disrupt the aadA coding region. CRE expression excises the stop codons, thereby reconstituting a functional aadA reading frame. We tested expression of our constructs in the absence of CRE in E. coli and plastids. We found that, as expected, the construct did not confer spectinomycin resistance to E. coli. However, partial spectinomycin resistance was detected when the same constructs were introduced into the plastid genome. We are currently testing additional designs to overcome the problem of leakiness. (2) Protein fusion to direct CRE recombinase from Agrobacterium to plastids. We constructed CRE fusion proteins which, when expressed in the plant nucleus, were translated on cytoplasmic ribosomes, imported into chloroplasts and efficiently excised the target sequences from the plastid genome. However, the fusion proteins were unstable in Agrobacterium. We are now constructing new fusion proteins and testing them for stability in Agrobacterium and targeting to chloroplasts. (3) We reported in 2009 that plastid markers in planta could be excised with recombinases delivered on the T-DNA of Agrobacterium injected into plants. These data have now been submitted for publication. The significance of the new system is discussed in the Outcomes/Impacts section. EVENTS: PI gave lectures and disseminated information at the following events and locations: (1) Plant and Animal Genome Conference XIX, San Diego, CA, January 14-19, 2011. Somatic Cell Genetics Workshop, Invited Speaker; (2) International Conferenc on Plant Transformation Technologies II, Vienna, Austria, February 19-22, 2011. Invited Speaker; (3) Plant-Based Vaccines & Antibodies, 8-10 June 2011, Porto, Portugal, Invited Speaker. PARTICIPANTS: During the past year Dr. Tarinee Tungsuchat Huang, a Research Associate, carried out the project. TARGET AUDIENCES: The target audience is plant biotechnologists constructing transgenic crops and needing the technical tools to remove undesirable marker genes. PROJECT MODIFICATIONS: The experiments were carried out as planned. However, we found that the original CRE design was unstable in Agrobacterium. We have corrected the design, but require additional time to complete testing plastid marker excision by Cre delivered via the Agrobacterium Type IV protein secretion machinery. The original grant expired in September 2011. An attempt was made to request additional funding for two more years, but PIs application was turned down. Grant was renewed for one year as no-cost extension.
Impacts
Successful manipulation of the plastid genome (ptDNA) so far has been carried out in tissue culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants yielding genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (barau) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex differentiating at the injection site contained marker-free plastids in 30% to 40% of the injected plants, of which 7% transmitted the marker-free plastids to the seed progeny. The success of obtaining seed with marker-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.
Publications
- (1) Maliga, P. and Bock, R. (2011) Plastid biotechnology: food, fuel and medicine for the 21st century. Plant Physiol. 155: 1501-1510.
- (2) Tungsuchat-Huang, T. and Maliga, P (2011) Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. submitted
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Research during 2009-2010 was conducted along two parallel tracks denoted by (1) and (2). (1) Detecting low-level Cre recombinase activity delivered by Type IV secretion. (1a) Detecting Cre delivery to chloroplasts by activation of a silent spectinomycin resistance (aadA; encoding AAD protein) gene. We have constructed two generations of silent aadA genes and tested activation achieved by excision of a blocking fragment. We verified a dramatic increase from undetectable to significant AAD accumulation as the result of gene activation. However, undetectable AAD accumulation from the inactive gene was sufficient to confer low-level spectinomycin resistance. Testing 3rd generation constructs is in progress. (1b) Development of novel aadA marker genes enabling detection of marker excision in plants by restoration of leaf color. We completed testing the aurea aadA genes and now have an operational system to test plastid marker excision in plants by injected Agrobacterium. (2) Engineering CRE for export from Agrobacterium to chloroplasts. We tested 1st generation CRE enzymes designed to target the CRE recombinase from Agrobacterium to chloroplasts. The fusion protein carries a plastid-targeting signal at its N-terminus and a TypeIV secretion signal at its C-terminus. When expressed from a plant nuclear gene, CRE was imported into chloroplasts and efficiently excised the loxP-flanked target sequence. Thus, the TypeIV secretion signal does not interfere with chloroplast import. However, when the same protein was expressed in Agrobacterium, the protein was degraded. We determined that the cause of protein degradation in Agrobacterium was the plastid targeting sequence. We are now testing alternative plastid targeting sequences in 2nd generation CRE constructs. EVENTS: PI gave lectures and disseminated information generated through the project at the following events and locations: (1) 5th Solanaceae Genome Workshop, Cologne, Germany, October 15, 2008. (2) Department of Biology, Syracuse University, Syracuse, NY, October 30/October 31, 2008. (3) International Center for genetic Engineering and Biotechnology, New Delhi, India, December 8, 2008. (4) Global Potato Conference 2008, New Delhi, India, December 9-12, 2008. (5) Department of Plant Molecular Biology, University of New Delhi, South Campus, New Delhi, India, December 11, 2008. (6) 2009 Society of In Vitro Biology Conference, Charleston, SC, June 10, 2009. (7) Molecular Genetics of Chloroplasts and Mitochondria, Joint Symposium of German Academy of Sciences Leopoldina, SFB 429 and SFB-TR1, Berlin, Germany September 20-23, 2009. (8) 6th Tripartite Workshop of the Americas in Biotechnology and Bioenergy, University of Sao Paulo, Brazil, December 2-5th, 2009. (9) Plant and Animal Genome Conference XVIII, San Diego, CA, January 9-13, 2010. Workshop Speaker & Organizer. (10) 2nd International Symposium on Chloroplast Genomics and Genetic Engineering, Maynooth, Ireland, June 20-23, 2010. Invited Speaker and Session Chair. (11) 56th Brazilian Congress of Genetics, Sociedade Brasileira de Genetica (SBG), Guaruja - SP, Brazil, 14-17 September 2010. PARTICIPANTS: PI/PD Prof. Pal Maliga directs research. During the first phase of the project Ms. Sinagawa Garcia tested the stability of transplastomes carrying target-site flanked marker genes. These plants were injected with Agrobacterium to detect marker excision in plants. Ms. Sugey Sinagawa Garcia was a Ph.D. student at Cinvestav, Irapuato, Mexico, who defended her Ph.D. thesis in 2009 based on research at Rutgers. Dr. Tarinee Tungsuchat Huang, a Postdoctoral Researcher, is the lead contributor to the project. Ms Kristina Slivinski is currently employed as a full time technician working on the project. Ms. Kristina Slivinsiki just graduated with a Bachelor of Arts Degree in Genetics. Construction of aurea spectinomycin resistance genes formed the basis of the research thesis submitted in partial fulfillment of the requirements of the Bachelor of Arts Degree. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the aurea bar gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aurea aadA genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.
Publications
- Maliga, P. and Svab, Z. (2010) Engineering the plastid genome of Nicotiana sylvestris, a diploid model species for plastid genetics. In: Plant Chromosome Engineering: Methods and Protocols, James J. Birchler, ed., Humana Press, 701: 37-50.
- Tungsuchat-Huang, T., Slivinski, K.M., Sugey Ramona Sinagawa-Garcia, S.R. and Maliga, P. (2011) Visual spectinomycin resistance (aadAau) gene for facile identification of transplastomic sectors in tobacco leaves. Plant Molecular Biology, In press.
- Tungsuchat-Huang, T., Sinagawa-Garcia, S.R., Paredes-Lopez, O.P. and Maliga, P. (2010) Study of plastid genome stability in tobacco reveals that the loss of marker gene is more likely by gene conversion than by recombination between 34-bp loxP repeats. Plant Physiol. 153: 252-259.
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