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 tool to prevent transgene flow via pollen. We have 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.
Our current research interests are in the development of efficient plastid transformation protocols in the model plant Arabidopsis thaliana and the related oilseed crop Brassica napus. As an alternative to biolistic DNA delivery, we are working on chloroplast transformation by Agrobacterium. Additionally, we are interested in building synthetic chloroplast operons and the expression of recombinant proteins for therapeutic applications.
Engineering the plastid and mitochondrial genomes of flowering plants
Nature Plants (2022) 8, 996-1006, https://doi.org/10.1038/s41477-022-01227-6
Engineering the plastid genome based on homologous recombination is well developed in a few model species. Homologous recombination is also the rule in mitochondria, but transformation of the mitochondrial genome has not been realized in the absence of selective markers. The application of transcription activator-like (TAL) effector-based tools brought about a dramatic change because they can be deployed from nuclear genes and targeted to plastids or mitochondria by an N-terminal targeting sequence. Recognition of the target site in the organellar genomes is ensured by the modular assembly of TALE repeats. In this paper, I review the applications of TAL effector nucleases and TAL effector cytidine deaminases for gene deletion, base editing and mutagenesis in plastids and mitochondria. I also review emerging technologies such as post-transcriptional RNmodification to regulate gene expression, Agrobacterium- and nanoparticle-mediated organellar genome transformation, and self-replicating organellar vectors as production platforms.
Multiple sgRNAs for one-step inactivation of the duplicated acetyl-coenzyme A carboxylase 2 (ACC2) genes in Brassica napus
LaManna, L.M., Parulekar, M.S. & Maliga, P. (2022) Plant Physiol. 189: 178-187
Efficient plastid transformation in Arabidopsis thaliana requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast ACCase encoded in the ACC2 nuclear gene. To obtain plastid transformation-competent Brassica napus, we decided to inactivate all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. B. napus (2n= 38, AACC) is a recent interspecific hybrid of B. rapa (2n=20, AA) and B. oleracea (2n=18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric EC1.2p promoter known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologues in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type copy remained. The B. napus T2 seedlings lacking wild-type ACC2 gene copies exhibit a spectinomycin hypersensitive phenotype suggesting that they will be a useful resource for chloroplast genome transformation.
Prospects for reengineering Agrobacterium tumefaciens for T-DNA delivery to chloroplasts
Aki Matsuoka and Pal Maliga (2021) Plant Physiol. 186: 215-220. DOI:10.1093/plphys/kiab081
Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression.
Yu Q, Tungsuchat-Huang T, Verma K, Radler MR, Maliga P (2020) Plant J. 103, 2318-2329. doi: 10.1111/tpj.14864
We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.
New Tools for Engineering the Arabidopsis Plastid Genome.
Yu, Q., Lamanna, L.M., Kelly, M.E., Lutz, K.A. and Maliga, P. (2019) Plant Physiol. 181: 394-398. DOI:10.1104/pp.19.00761
Engineered RNA-binding protein for transgene activation in non-green plastids.
Yu Q, Barkan A, Maliga P. (2019) Nat Plants. 5:486-490.
Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes.
Rojas M, Yu Q, Williams-Carrier R, Maliga P, Barkan A. (2019) Nat Plants. 5:505-511.
Efficient plastid transformation in Arabidopsis
Yu, Q., Lutz, K.A. and Maliga, P. (2017) Plant Physiol. 175: 186-193.
Cell-to cell movement of mitochondria in plants
Gurdon, C., Svab, Z., Feng, Y. Kumar, D., and P. Maliga, P. (2016) Proc. Natl. Acad. Sci. USA 113: 3395-400.
Cell-to-cell movement of plastids in plants
Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants
Tungsuchat-Huang, T. & Maliga, P. (2012) Plant J. 70: 717-725.
Plastid biotechnology: food, fuel and medicine for the 21st century
Maliga, P. and Bock, R. (2011) Plant Physiol. 155: 1501-1510.
Pal Maliga is Distinguished Professor of Plant Biology at Rutgers University. He obtained an MS degree at Eötvös Loránd University (ELTE) in Budapest and a PhD at József Attila University (JATE) in Szeged, Hungary. Since at Rutgers his research group developed methods for the stable transformation of land plant chloroplast genomes. Chloroplast genome engineering in higher plants has led to an explosion of research concerning the chloroplast genome’s role in photosynthesis, functional analysis of plastid genes by reverse genetics, and mechanisms of plastid gene regulation.
His current research interests are development of reproducible protocols for plastid transformation in Arabidopsis thaliana, including Agrobacterium-mediated transformation of the chloroplast genome, and expression of recombinant proteins in tobacco chloroplasts.
Former Graduate Students
- Lisa Lamanna
- Qiguo Yu
Former Undergraduate Students
- Amanda Chen
- Mugdha Parulekar
- Alifya Quresh