Agrobacterium tumefaciens-Mediated Transformation of Tomato

Eck, Joyce Van; Keen, Patricia; Tjahjadi, Michelle

doi:10.1007/978-1-4939-8778-8_16

Cited by 94 publications

(58 citation statements)

References 10 publications

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“…To induce mutations in both Fls3 and Fls2.1/2.2 in the same plant (ΔFls2.1/2.2/3), the constructs used to induce the individual mutations were transformed into the Agrobacterium tumefaciens strain LBA4404 and the cultures were mixed 1:1 prior to tomato transformation. Tomato transformations were performed at the Boyce Thompson Institute transformation facility (Gupta and Van Eck, 2016;Van Eck et al, 2019). More information is available in Supplemental Figure S1, Supplemental Tables S1, S4, and S5, and Supplemental Methods, as well as on the Plant CRISPR database at plantcrispr.org.…”

Section: Generation Of Crispr/cas9-mediated Knockout Linesmentioning

confidence: 99%

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

et al. 2020

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Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

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Section: Generation Of Crispr/cas9-mediated Knockout Linesmentioning

confidence: 99%

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

et al. 2020

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“…As with tomato transformation methodology and reports on transformation of P. philadelphica, segments from cotyledons are the explant that works best for A. tumefaciensmediated transformation (Simpson et al 1995;Van Eck et al 2019). However, when we investigated plant regeneration from cotyledon and hypocotyl explants we found that only hypocotyl explants regenerated shoots.…”

Section: Discussionmentioning

confidence: 81%

“…Development of the method reported here was based on our experience with transformation of other Solanaceae family members because although there are reports of plant regeneration and transformation of other Physalis species, we were unsuccessful with application of these methods to P. pruinosa (Otroshy et al 2013;Simpson et al 1995;Van Eck et al 2019;Wang et al 2011). Therefore, we chose to investigate the applicability of our tomato regeneration and transformation approaches and found these methods to be very effective for P. pruinosa (Van Eck et al 2019).…”

Section: Discussionmentioning

confidence: 99%

Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

Swartwood

Eck

2019

Plant Cell Tiss Organ Cult

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Physalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production of P. pruinosa fruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration and Agrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing of P. pruinosa. Cotyledon and hypocotyl explants from 7 to 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected with Agrobacterium tumefaciens strain AGL1 containing the pJL33 binary vector that has the green fluorescent protein reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on PCR analysis for the presence of the nptII gene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement of P. pruinosa. Key message Methodology for regeneration and transformation of Physalis pruinosa is a key component for the genetic improvement of this underutilized fruit crop for future agricultural production. Keywords AGL1 • Green fluorescent protein • Groundcherry • Kanamycin • Solanaceae Abbreviations GFP Green fluorescent protein GUS Beta-glucuronidase MS Murashige and Skoog nptII Neomycin phosphotranferase II

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“…To induce mutations in both Fls3 and Fls2.1/2.2 in the same plant 20 (∆Fls2.1/2.2/3), the constructs used to induce the individual mutations were transformed into the Agrobacterium tumefaciens strain LBA4404 and the cultures were mixed 1:1 prior to tomato transformation. Tomato transformations were performed at the Boyce Thompson Institute transformation facility Van Eck et al, 2019). More information may be found in the Supplemental Methods, Table S1, Table S4-S5, Fig.…”

Section: Generation Of Crispr Cas9-mediated Knockout Linesmentioning

confidence: 99%

Molecular characterization of differences between the tomato immune receptors Fls3 and Fls2

Roberts

Liu

Wan

et al. 2020

Preprint

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Plants mount defense responses by recognizing indications of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) contains two MAMPs, flg22 and flgII-28, that are recognized by tomato receptors Flagellin sensing 2 (Fls2) and Flagellin sensing 3 (Fls3), respectively. It is unknown to what degree each receptor contributes to immunity and if they promote immune responses using the same molecular mechanisms. Characterization of CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants revealed that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, striking differences were observed in certain host responses mediated by the two receptors.Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species (ROS) and an increase in transcript abundance of 44 tomato genes, with two genes serving as reporters for Fls3. Fls3 had greater in vitro kinase activity and interacted differently with the Pst effector AvrPtoB as compared to Fls2. Using chimeric Fls2/Fls3 proteins, we found that no receptor domain was solely responsible for the Fls3 sustained ROS, suggesting involvement of multiple structural features. This work reveals differences in the immunity outputs between Fls2 and Fls3, suggesting they use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

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Agrobacterium tumefaciens-Mediated Transformation of Tomato

Cited by 94 publications

References 10 publications

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2

Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

Molecular characterization of differences between the tomato immune receptors Fls3 and Fls2

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