Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

Srinivasan, R.; Gothandam, Kodiveri Muthukaliannan

doi:10.1371/journal.pone.0158322

Cited by 27 publications

(16 citation statements)

References 46 publications

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“…For transformation and selection experiments solid and liquid De Walne's medium were employed, and adjusted to contain a final concentration of 0.5 M NaCl. For solid medium, 2.5% agar was added [43].…”

Section: Resultsmentioning

confidence: 99%

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Zhong

Cao

Zhang

et al. 2020

Biotechnol Biofuels

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Background: Dunaliella salina can produce glycerol under salt stress, and this production can quickly adapt to changes in external salt concentration. Notably, glycerol is an ideal energy source. In recent years, it has been reported that the mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is highly important to study the relationship between the MAPK cascade pathway and salt stress in D. salina and modify it to increase the production of glycerol. Results: In our study, we identified and analysed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1 and DsMEK1-X2 were both localized in the cytoplasm. qRT-PCR assays showed that DsMEK1-X2 was induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol production compared to the control and DsMEK1-X1-oe under salt stress. Under salt stress, the expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control. This finding indicated that DsMEK1-X2 was involved in the regulation of DsGPDH expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increased the proline content and reduced the MDA content under salt stress, and DsMEK1-X1 was able to regulate oxidative stress; thus, we hypothesized that DsMEK1-X1 could reduce oxidative damage under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 could interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1; however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines did not exhibit increased gene expression, except for DsMAPKKK9. Conclusion: Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can respond to salt stress by two different pathways. The DsMEK1-X1 response to salt stress reduces oxidative damage; however, the DsMAPKKK1/2/3/9/10/17-DsMEK1-X2-DsMAPK1 cascade is involved in the regulation of DsGPDH expression and thus glycerol synthesis under salt stress.

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Section: Resultsmentioning

confidence: 99%

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Zhong

Cao

Zhang

et al. 2020

Biotechnol Biofuels

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“…Glucose and other aldose monosaccharides activate the chromosomally encoded periplasmic protein (ChvE) which mediates a sugar-induced increase in Vir gene expression as well as Agrobacterium chemotaxis. In addition, glucose works synergistically with acetosyringone to enhance the transformation frequency [44]. Similar to transformation procedures for other monocots, acetosyringone (200 µM) was added to the co-culture media to induce A. tumefaciens to infect plants cells.…”

Section: Resultsmentioning

confidence: 99%

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

et al. 2019

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Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

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“…The effectiveness of Agrobacterium-mediated genetic transformation of microalgae is reported to be modulated by several factors. For instance, the Agrobacterium strain and its concentration (OD 600 ), the coculture time, infection medium, pH, temperature, and microalgae species have been suggested as key variables for the success of microalgae transformation (Cha et al, 2011;Úbeda-Mínguez et al, 2015;Srinivasan and Gothandam, 2016). Thus, it is of vital importance to find the most effective parameter conditions for a particular microalgae transformation.…”

Section: Introductionmentioning

confidence: 99%

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

Norzagaray-Valenzuela

Germán-Báez

Valdez-Flores

et al. 2018

Journal of Microbiological Methods

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Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

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Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

Cited by 27 publications

References 46 publications

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

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