Dr. Pal Maliga

News

February 2, 2012
Plants swap chloroplasts via grafts

November 27, 2011
2011 NJ Inventor of the Year

Research overview

Plastids are semi-autonomous organelles with a relatively small (120-180 kb), highly polyploid genome present in 1,000 to 10,000 copies per cell. The best-known plastids, chloroplasts, convert sunlight into chemical energy. Plastid engineering, in contrast to nuclear engineering, offers higher protein yields, the opportunity to express several genes controlling complex traits, and natural containment that prevents transgene flow via pollen. During the past twenty years we developed protocols for transformation of the tobacco (Nicotiana tabacum) plastid genome, efficient post-transformation excision of the marker genes, and high-level expression of recombinant proteins. Patents based on research from our laboratory cover all aspects of the genetic manipulation of plastid genomes.

Currently, we pursue research in the following areas:

(1) Plastid marker excision using phage recombinases. Marker gene excision is typically accomplished by integration of a recombinase gene in the plant nucleus. The objective of research supported by the National Institute of Food and Agriculture (NIFA) USDA Biotechnology Risk Assessment Grants (BRAG) Program is marker gene excision by recombinases directly exported from Agrobacterium into chloroplasts by Type IV secretion.

(2) Genetic control of plastid inheritance in Medicago truncatula. Plastids in most plants are inherited by the maternal parent only. The genus Medicago is exceptional among flowering plants because some of its species inherit plastids maternally, paternally or biparentally. Currently we are conducting a mode of plastid inheritance survey supported by the NIFA USDA BRAG Program in Medicago truncatula, a diploid species, with the intent to identify the genes that determine maternal, paternal or biparental modes of plastid inheritance. The ultimate goal is to experimentally modify the mode of plastid inheritance to prevent gene flow via pollen. Currently we use plastid DNA polymorphic markers to follow plastid inheritance. Our ongoing efforts on plastid transformation in Medicago truncatula aim at developing a visual marker system to replace PCR-based tracking of plastid DNA in crosses. Plastid transformation in Medicago (alfalfa) will also be useful to provide a vehicle for oral delivery of vaccines.

(3) Plastid engineering in cereal crops. Plastid transformation is routine in tobacco, tomato, potato, soybean and lettuce, but is not reproducible in any of the cereal crops. The objective of this new program is to develop a marker system that enables routine plastome engineering in cereals, particularly maize. In support of this goal we are developing new marker and vector systems, sequence plastid genomes using next generation technologies, study plastid inheritance, and intend to determine the level of containment achievable by plastid localization of transgenes. The latter is particularly important in the wind-pollinated cereal crops, in which pollen is an efficient vehicle for transgene dissemination.

(4) Inter-cellular movements of DNA-containing organelles in plants. Land plants developed highly sophisticated intercellular channels or plasmodesmata, which mediate cell-to-cell movement of nutrients, hormones and information macromolecules. Functional equivalents of plasmodesmata in animal cells are the tunneling nanotubes, which were shown to mediate intercellular trafficking of organelles, including mitochondria. Our objective is to test whether or not organelles move between cells in plants. Our experimental approach is grafting different species of tobacco with plastid, mitochondrial and nuclear markers and selecting for new combinations of the markers to recover rare events.

Recent Publications

Thyssen, G, Svab Z, Maliga P. 2012. Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny. Plant J.. 72:84-8. AbstractWebsite

Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

Thyssen, G, Svab Z, Maliga P. 2012. Cell-to-cell movement of plastids in plants. Proc. Natl. Acad. Sci. U.S.A.. 109:2439-43. AbstractWebsite

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Tungsuchat-Huang, T, Maliga P. 2012. Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. Plant J.. 70:717-25. AbstractWebsite

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only 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 to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) 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 that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-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.

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3rd International Symposium on Chloroplast Genomics and Genetic Engineering [3rd ISCGGE]

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