A novel strategy for obtaining kanamycin resistance in <i>Arabidopsis thaliana</i> by silencing an endogenous gene encoding a putative chloroplast transporter

Aufsatz, Werner; Nehlin, Lilian; Voronin, Viktor; Schmidt, Agnes; Matzke, A. J. M.; Matzke, Marjori

doi:10.1002/biot.200800156

Cited by 15 publications

(20 citation statements)

References 13 publications

(22 reference statements)

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“…A recent article describes independent mutations of the MAR1 locus (At5g26820) that are sufficient to achieve kanamycin resistance in Arabidopsis (Aufsatz et al, 2009). These findings agree with our data; however, Aufsatz et al report that resistance is kanamycin specific and does not carry over to gentamicin or hygromycin.…”

Section: Discussionsupporting

confidence: 92%

“…More recently, it has been described as RTS3, and two mutations in the gene (rts3-1 and rts3-2; Fig. 2, A and B) were shown to confer kanamycin resistance at 40 mg/L (Aufsatz et al, 2009). Native expression of At5g26820 was able to complement the mutant mar1-1 (data not shown).…”

Section: Isolation and Map-based Cloning Of The Multiplementioning

confidence: 88%

“…Interestingly, the RTS3 putative transport protein is predicted to be chloroplast localized, indicating that it may be acting as an entry point for antibiotics into this subcellular compartment. However, no experimental evidence has yet been provided to support this prediction (Aufsatz et al, 2009). We have isolated three independent rts3 mutants that also confer resistance to kanamycin, and we have found that this resistance extends to other aminoglycoside antibiotics as well.…”

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confidence: 91%

“…Our lab has found that overexpression of the Arabidopsis multidrug resistance1 confers resistance to multiple herbicides and a single antibiotic, cycloheximide (Thomas et al, 2000;Windsor et al, 2003). The most recent report reveals that mutations in and RNA interference-based down-regulation of the putative transporter gene RTS3 (for RNAmediated Transcriptional gene Silencing) confer resistance to kanamycin (Aufsatz et al, 2009).…”

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confidence: 99%

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Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

et al. 2009

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Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics; however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here, we report that mutations in the Arabidopsis (Arabidopsis thaliana) locus Multiple Antibiotic Resistance1 (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Additionally, yeast expressing MAR1 are hypersensitive to the aminoglycoside G418. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:yellow fluorescent protein fusion localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside gentamicin, while mar1-1 mutant chloroplasts accumulate less than the wild type. MAR1 overexpression lines are slightly chlorotic, and chlorosis is rescued by exogenous iron. MAR1 expression is also down-regulated by low iron. These data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis and allows opportunistic entry of multiple antibiotics into the chloroplast.

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Section: Discussionsupporting

confidence: 92%

Section: Isolation and Map-based Cloning Of The Multiplementioning

confidence: 88%

mentioning

confidence: 91%

mentioning

confidence: 99%

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Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

et al. 2009

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“…It might contain a mutation conferring kanamycin resistance independent of transgene-based NPTII expression, e.g. by affecting a gene required for kanamycin uptake, possibly similar to a chloroplast transporter that has been described by Aufsatz et al (2009).…”

Section: Discussionmentioning

confidence: 99%

Genetic analysis of RNA-mediated gene silencing in Arabidopsis thaliana

2012

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In plants, a particular class of small noncoding RNAs, short interfering RNAs, can serve as a signal to induce cytosine methylation at homologous genomic DNA regions in the nucleus. If the targeted DNA regions have promoter function, this RNAdirected DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing of transgenes provides a versatile system for the study of epigenetic gene regulation in plants. In our experimental setup in Arabidopsis thaliana, transcription of a promoterinverted repeat provides a RNA signal that triggers de novo cytosine methylation and TGS of a homologous nopaline synthase promoter (proNOS) in trans. Utilising this two component transgene system in a forward-genetic screen for ''suppressor of silencing'' mutations, we were able to identify new candidates for factors involved in RdDM of transgenic as well as endogenous target regions.

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Selectable marker genes from plants: reliability and potential

Rosellini

2011

In Vitro Cell.Dev.Biol.-Plant

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Selectable marker genes (SMGs) are still useful to efficiently obtain transgenic plants, although marker-free techniques are available, but with limitations. The presence of SMGs, especially bacterial antibiotic resistance genes, in transgenic crops is criticized. Fortunately, several genes isolated from plants are available that can serve as SMGs.Here, I review the plant genes reported to have been used as SMGs. Some are wild-type genes that, when overexpressed, confer a selective advantage during in vitro plant regeneration, whereas some are mutated genes encoding enzymes resistant to inhibitory chemicals. Most of the genes have not yet been tested in a significant number of species. The effect of SMGs expression on the phenotype has often been superficially examined and should be better characterized. The sequence conservation of some SMGs could allow derivation of a SMGs from any plant species, if an intragenic or cisgenic approach to genetic engineering is preferred. I conclude that several promising SMGs have been isolated from plants, allowing avoidance of bacterial genes for transformation, transgene stacking, and intragenic or cisgenic engineering approaches. Nonetheless, further testing in more plant species would be useful to fully assess phenotypic neutrality, efficiency, and versatility. Patent rights restrict the immediate use of most plant SMGs for commercial applications, but freely available marker systems do exist.

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A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter

Cited by 15 publications

References 13 publications

Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

Genetic analysis of RNA-mediated gene silencing in Arabidopsis thaliana

Selectable marker genes from plants: reliability and potential

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