New Tools for Engineering the Arabidopsis Plastid Genome

Yu, Qiguo; LaManna, Lisa M; Kelly, Megan E.; Lutz, Kerry Ann; Maliga, Pál

doi:10.1104/pp.19.00761

Cited by 13 publications

(5 citation statements)

References 16 publications

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“…Chloroplast localization of GFP in the fluorescent sectors was confirmed by laser scanning confocal microscopy (Figure 2b). GFP fluorescence as a reliable indicator of transplastomic state has been documented in Arabidopsis (Yu et al ., 2019b).…”

Section: Resultsmentioning

confidence: 99%

“…Vectors pMRR13, pMRR18 and pMRR15 yielded 6.6%, 2.9% and 0.5% AAD, while the second ORF yielded 42.2%, 12.7% and 29.4% GFP (Figure 4a and Table 2). The mRNA levels are the highest from the pMRR13 construct, with high‐level GFP accumulation from the second ORF, which is why we used this construct for the early identification of transplastomic clones in Arabidopsis (Yu et al ., 2017; Yu et al ., 2019b).…”

Section: Resultsmentioning

confidence: 99%

“…Because polycistronic mRNAs are not expressed in the nucleus (Kozak, 2005), we hypothesized that selection for a marker gene encoded in the first ORF, and GFP accumulation from the second ORF should indicate plastid transformation. This hypothesis was confirmed by the finding that bombardment followed with spectinomycin selection yielded GFP‐expressing Arabidopsis clones, which were confirmed to be transplastomic by DNA gel blot analyses (Yu et al ., 2019b). Here we report a similar finding, plastid transformation confirmed by DNA gel blot analyses in tobacco, when the clones are selected for kanamycin resistance and are expressing GFP.…”

Section: Discussionmentioning

confidence: 99%

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Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression

Tungsuchat-Huang

Verma

et al. 2020

The Plant Journal

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SUMMARY 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression

Tungsuchat-Huang

Verma

et al. 2020

The Plant Journal

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“…In plastid engineering via homologous recombination (HR) between the transformed vector's targeting regions and the wild-type plastid DNA, foreign DNA is integrated into the plastid genome at a predefined and desired position [42]. Thus, HR enables the insertion of transgene sequences responsible for position effects in nuclear transformation studies [36].…”

Section: Deployment Of Homology-based Recombination In Chloroplastsmentioning

confidence: 99%

Chloroplast Genome Engineering: A Plausible Approach to Combat Chili Thrips and Other Agronomic Insect Pests of Crops

Bulle,

Sheri,

Aileni

et al. 2023

Plants

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The world population’s growing demand for food is expected to increase dramatically by 2050. The agronomic productivity for food is severely affected due to biotic and abiotic constraints. At a global level, insect pests alone account for ~20% loss in crop yield every year. Deployment of noxious chemical pesticides to control insect pests always has a threatening effect on human health and environmental sustainability. Consequently, this necessitates for the establishment of innovative, environmentally friendly, cost-effective, and alternative means to mitigate insect pest management strategies. According to a recent study, using chloroplasts engineered with double-strand RNA (dsRNA) is novel successful combinatorial strategy deployed to effectively control the most vexing pest, the western flower thrips (WFT: Frankliniella occidentalis). Such biotechnological avenues allowed us to recapitulate the recent progress of research methods, such as RNAi, CRISPR/Cas, mini chromosomes, and RNA-binding proteins with plastid engineering for a plausible approach to effectively mitigate agronomic insect pests. We further discussed the significance of the maternal inheritance of the chloroplast, which is the major advantage of chloroplast genome engineering.

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“…High-frequency plastid transformation in Arabidopsis was achieved by choosing plants lacking a duplicated acetyl-CoA-carboxylase enzyme that makes plants hyper-sensitive to spectinomycin, the selective agent used in chloroplast transformation (Yu et al, 2017; Ruf et al, 2019; Yu et al, 2019). Inherent polyploidy of leaf cells and the tendency for somaclonal variation make difficult to regenerate fertile plants from transplastomic tissue culture cells (Galbraith et al, 1991; Melaragno et al, 1993; Bairu et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Split GFP assay to show chloroplast targeting ofAgrobacteriumVirD2 protein

Matsuoka,

Ferranti,

Maliga

2023

Preprint

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Regenerating fertileArabidopsis thalianaplants from tissue culture cells with transformed plastid genomes is difficult, because of somaclonal variation in tissue culture cells. For nuclear gene transformation, tissue culture limitations were overcome in Arabidopsis by direct transformation of the female gametes using the floral dip protocol and identification of transgenic events in the seed progeny. DuringAgrobacteriumtransformation the VirD2 protein guides the T-complex, consisting of single stranded transferred-DNA (T-DNA) coated with VirE2 proteins, to the plant nucleus. To enable floral dip transformation of the plastid DNA, we retargeted VirD2 to chloroplasts. We show plastid targeting of VirD2 in a split GFP assay, where VirD2-GFP11complements GFP1-10in chloroplasts. Floral dip transformation of plastids will avoid tissue culture altogether, making plastid transformation readily available for the research community.

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New Tools for Engineering the Arabidopsis Plastid Genome

Cited by 13 publications

References 16 publications

Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression

Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression

Chloroplast Genome Engineering: A Plausible Approach to Combat Chili Thrips and Other Agronomic Insect Pests of Crops

Split GFP assay to show chloroplast targeting ofAgrobacteriumVirD2 protein

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