Agrobacterium rhizogenes transformed calli of the holoparasitic plant Phelipanche ramosa maintain parasitic competence

Libiaková, Dagmara; Ruyter-Spira, Carolien; Bouwmeester, Harro J.; Matúšová, Radoslava

doi:10.1007/s11240-018-1466-x

Cited by 11 publications

(6 citation statements)

References 35 publications

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“…As our experiments show, the protocol works with both A. rhizogenes and A. tumefaciens, and allows the expression of reporter proteins from different binary vectors and under the control of different promoters. The T‐DNA of pRedRoot, a binary vector developed for A. rhizogenes , contains the dsRed protein under control of the Ubi10 promoter (Libiakova et al, 2018), while the A. tumefaciens line used by Bobik et al (2019) contains a GFP gene controlled by the 35S promoter. Both promoters allow strong constitutive expression of transgenes.…”

Section: Discussionmentioning

confidence: 99%

“…Cuscuta reflexa , Cuscuta campestris, and Cuscuta platyloba were grown in a greenhouse on the host Pelargonium zonale under continuous illumination and a constant temperature of 21°C (Förste et al, 2020). The bacteria and binary T‐DNA‐containing vectors pRedRoot and XM82, respectively, were kindly contributed by Prof. Harro Bouwmeester (University of Amsterdam, Netherlands) ( A. rhizogenes ) and Prof. Tessa Burch‐Smith (University of Tennessee, USA) ( A. tumefaciens ) and are described in more detail in other studies (Limpens et al, 2004; Libiakova et al, 2018; Bobik et al, 2019). Cultures of A. rhizogenes MSU440 and A. tumefaciens GV3101 without binary plasmids were grown on LB medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L, agar 7,5 g/L) supplemented with 100 mg/L Spectinomycin or 50 mg/L Rifampicin plus 50 mg/L Gentamycin, respectively.…”

Section: Methodsmentioning

confidence: 99%

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A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

2020

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The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium‐mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T‐DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T‐DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

2020

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show abstract

“…As our experiments show, the protocol works with both, A. rhizogenes and A. tumefaciens and allows the expression of reporter proteins from different binary vectors and under the control of different promoters. The T-DNA of pRedRoot, a binary vector developed for A. rhizogenes , contains the dsRed protein under control of the Ubi10 promoter (Libiakova et al, 2018), while the A. tumefaciens line used by Bobik et al (2019) contains a GFP gene controlled by the 35S promoter. Both promoters allow strong constitutive expression of transgenes.…”

Section: Discussionmentioning

confidence: 99%

“…Cuscuta reflexa, Cuscuta campestris and Cuscuta platyloba were grown in a greenhouse on the host Pelargonium zonale under continuous illumination and a constant temperature of 21 °C (Förste et al, 2019). The bacteria and binary T-DNA-containing vectors pRedRoot and XM82, respectively, were kindly contributed by Prof. Harro Bouwmeester (University of Amsterdam, Netherlands) ( A. rhizogenes ) and Prof. Tessa Burch-Smith (University of Tennessee, USA) ( A. tumefaciens ) and are described in more detail in other studies (Limpens et al, 2004; Libiakova et al, 2018; Bobik et al, 2019). Cultures of A. rhizogenes MSU440 and A. tumefaciens GV3101 without binary plasmids were grown on LB medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L, agar 7,5 g/L) supplemented with 100 mg/L Spectinomycin or 50 mg/L Rifampicin plus 50 mg/L Gentamycin, respectively.…”

Section: Methodsmentioning

confidence: 99%

A highly efficient protocol for transformingCuscuta reflexabased on artificially induced infection sites

Lachner

Galstyan

Krause

2020

Preprint

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A protocol that yields high numbers of transformed cells in the adhesive disks of Cuscuta reflexa 27 upon exposure to agrobacteria brings closer the vision of generating genetically modified 28 Cuscuta. 29 30 ABSTRACT 31 A current bottleneck in the functional analysis of the emerging parasitic model plant Cuscuta and 32 the exploitation of its recently sequenced genomes is the lack of efficient transformation tools. 33 Here, we describe the development of a novel highly efficient Agrobacterium-mediated 34 transformation protocol for Cuscuta reflexa based on the parasitic structure referred to as 35 adhesive disk. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary 36 transformation vectors with reporter fluorochromes yielded high numbers of transformation 37 events. An overwhelming majority of transformed cells were observed in the cell layer below the 38 adhesive disk's epidermis, suggesting that these cells are particularly susceptible to infection. Co-39 transformation of these cells happens frequently when Agrobacterium strains carrying different 40 constructs are applied together. Explants containing transformed tissue expressed the fluorescent 41 markers in in vitro culture for several weeks, offering a possibility for development of 42 transformed cells into callus. 43 44 45 3 46 48 worldwide and infects a broad range of host plants. The parasite starts an attack by winding 49around the host stem. A multicellular feeding structure termed the haustorium, which can reach 50 2-3 mm in size in some species and originates from a secondary meristem in the cortex close to 51 the vascular bundles, develops within a few days and breaches the host tissue integrity using 52 mechanical pressure and enzymatic digestion of cell walls (Nagar et al., 1984; Johnsen et al., 53 2015; Kaiser et al., 2015). To make the penetration possible, a sucker-shaped attachment organ 54 provides the necessary counterforce. The development of this organ, termed "adhesive disk" or 55

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“…Answers to these questions must await further effective and relevant functional validation tools in root parasitic plants. To date, very few studies have been devoted to the transformation of these parasitic organisms: Triphysaria versicolor (Tomilov et al , 2007), Striga hermonthica (D. Njora, unpublished), Phelipanche aegyptiaca (Fernández‐Aparicio et al , 2011) and P. ramosa (Libiaková et al , 2018). This reflects the complexity of implementing, particularly in the obligatory species, such a protocol that is time‐consuming and presents one major bottleneck: the impossibility to date to generate transgenic seeds.…”

mentioning

confidence: 99%

Are root parasitic plants like any other plant pathogens?

Delavault

2020

New Phytologist

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Agrobacterium rhizogenes transformed calli of the holoparasitic plant Phelipanche ramosa maintain parasitic competence

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Cited by 11 publications

References 35 publications

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

A highly efficient protocol for transformingCuscuta reflexabased on artificially induced infection sites

Are root parasitic plants like any other plant pathogens?

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