Novel sul I binary vectors enable an inexpensive foliar selection method in Arabidopsis

Thomson, James G.; Cook, Meridith A.; Guttman, Mara; Smith, Jamison; Thilmony, Roger

doi:10.1186/1756-0500-4-44

Cited by 19 publications

(20 citation statements)

References 28 publications

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“…Therefore, in all transformation vectors, the sul coding sequence was fused to the transit peptide from the Rubisco small subunit of pea ( Pisum sativum ) to target the Sul protein (i.e., the sulfonamide‐resistant DHPS) to the chloroplast. The successful generation of transgenic plants with these constructs in a number of plant species (Guerineau et al ., ; Wallis et al ., ; Hadi et al ., ; Thomson et al ., ) seemed to confirm the chloroplast localization of DHPS and the entire folate pathway. However, subsequently obtained evidence for a mitochondrial localization of DHPS and the downstream enzymes of the pathway has cast considerable doubt on the chloroplast location of the folate pathway in plants (Rébeillé et al ., ; Hanson and Gregory, , ).…”

Section: Introductionmentioning

confidence: 67%

“…For initial transformation experiments, vector p35S sul was used. It contains the sul cassette (driven by the CaMV 35S promoter and the RbcS transit peptide) that was used in all previous transformation studies that employed sulfadiazine selection (Guerineau et al ., ; Wallis et al ., ; Hadi et al ., ; Thomson et al ., ; Dal Bosco et al ., ). These experiments confirmed that transgenic plants can be obtained by selection for 20 or 25 mg/L sulfadiazine.…”

Section: Resultsmentioning

confidence: 98%

“…Due to the essentiality of the folate pathway in bacteria and plants, sulfonamides are extensively used as antibiotics to control bacterial infections and as herbicides for weed control. In addition, sulfonamides can be used as selection agents in plant transformation (Guerineau et al ., ; Wallis et al ., ; Hadi et al ., ; Thomson et al ., ). Resistance to sulfonamides is conferred by DHPS variants that are insensitive to the drug.…”

Section: Introductionmentioning

confidence: 97%

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A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Tabatabaei

Bosco

Bednarska

et al. 2018

Plant Biotechnology Journal

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The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide-insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low-level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co-transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high-throughput genetic transformation of plants and algae.

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Section: Introductionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 97%

See 1 more Smart Citation

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Tabatabaei

Bosco

Bednarska

et al. 2018

Plant Biotechnology Journal

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“…Approximately 100,000 T1 seeds from each of the three transformations were sown on flats of soil for approximately 10 d, then heavily misted with a solution of 500 mg L 21 sulfadiazine containing 0.03% (w/v) of the surfactant Silwet L-77, as described by Thomson et al (2011). Plants that survived the sulfadiazine application and displayed red fluorescence were transferred to pots of untreated soil and grown to maturity.…”

Section: Mutant Complementationmentioning

confidence: 99%

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis

et al. 2015

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The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.

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“…A fragment including 35S promoter, gene, and terminator was then extracted from these plasmids using SacI and ApaI and introduced into the multicloning site of pSUN (Thomson et al, 2011). After floral dipping, seedlings were selected using sulfadiazine.…”

Section: Plant Transformationmentioning

confidence: 99%

Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone

et al. 2016

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ORCID IDs: 0000-0002-6534-5347 (J.S.); 0000-0001-6773-0583 (E.R.V.); 0000-0001-9053-4134 (N.I.); 0000-0002-2150-8538 (G.R.); 0000-0001-7203-0688 (I.Z.).In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked b-D-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) b-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for b-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membraneanchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3. In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1. We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound b-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer.

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Novel sul I binary vectors enable an inexpensive foliar selection method in Arabidopsis

Cited by 19 publications

References 28 publications

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis

Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone

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