Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species)

Prasad, Binod; Vadakedath, Nithya; Jeong, Hyun-Jeong; General, Thiyam; Cho, Man-Gi; Lein, Wolfgang

doi:10.1007/s00253-014-5900-7

“…Several microalgal species were transformed by Agrobacterium , including Chlamydomonas (Kumar et al, 2004 ; Pratheesh et al, 2014 ), as well as other algae of biotechnological value, such as H. pluvialis (Kathiresan et al, 2015 ), Schizochytrium (Cheng et al, 2012 ), Isochrysis galbana , and Isochrysis sp. (Prasad et al, 2014 ).…”

Section: Nuclear Transformationmentioning

confidence: 99%

Transgene Expression in Microalgae—From Tools to Applications

Doron

¹

,

Segal

²

,

Shapira

³

2016

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Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while C. reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.

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“…Agrobacterium has been used extensively to 315 transfer DNA to eukaryotic algae in the green algal lineage with subsequent chromosomal integration (29)(30)(31)(32). In contrast, to our knowledge, the only other example beyond our results of using A. tumefaciens to deliver DNA maintained as an episome in the recipient was with yeast (14).…”

Section: Discussionmentioning

confidence: 91%

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

Kang

¹

,

Bielinski

²

,

Bolt

³

et al. 2017

Preprint

0

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Bacterial conjugation utilizes a type IV secretion system and a DNA transfer mechanism to deliver DNA from one cell to another. Conjugative partners are conventionally confined to the prokaryotic domain. In a prominent exception, Agrobacterium tumefaciens type IV secretion-mediated transfer of DNA to plant cells can result in subsequent chromosomal integration. Recently, we demonstrated interdomain conjugation from Escherichia coli to the diatom Phaeodactylum tricornutum with the subsequent maintenance of an episome at chromosomal copy numbers if it contains diatom centromeres or centromere-like elements. The genes involved in the conjugation process can be separated into those encoding the type IV secretion system, also called the mating pair formation (MPF) genes, and genes involved in DNA processing called the mobilization (MOB) genes. Various protein families compose each class of conjugation genes, including common MOB types F, P, and Q and MPF types F, P, and T. The conjugative transfer from E. coli to P. tricornutum was demonstrated with a vector expressing MOBP and MTFP. Here we show that the MOBPsystem can be deleted and complemented with a MOBQ system in E.coli-diatom conjugations with subsequent episomal maintenaince. Utilization of both MOBP and MOBQ systems results in substantially higher efficiencies in E. coli-diatom conjugation. Finally, we demonstrate conjugative gene transfer between P. tricornutum and A. tumefaciens expressing a MPFT, the first demonstration of this system in diatoms,resulting in episomal maintainance or chromosomal integration, depending on the ex-conjugant. The promiscuity of MOB and MTF systems permitting prokaryote to diatom conjugative DNA transfer suggest major environmental and evolutionary importance of this process. The increased efficiency of dual MOB systems immediately improves genetic engineering in diatoms and has interesting basic cellular biology implications.

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“…We also observed that A. tumefaciens could perform interdomain conjugation with P. tricornutum resulting in establishment of episomes in the recipient when the cargo plasmid contained a centromeric maintenance sequence. Agrobacterium has been used extensively to 315 transfer DNA to eukaryotic algae in the green algal lineage with subsequent chromosomal integration (29)(30)(31)(32). In contrast, to our knowledge, the only other example beyond our results of using A. tumefaciens to deliver DNA maintained as an episome in the recipient was with yeast (14).…”

Section: Discussionmentioning

confidence: 91%

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

Kang

¹

,

Bielinski

²

,

Bolt

³

et al. 2017

Preprint

0

View full text Add to dashboard Cite

Bacterial conjugation utilizes a type IV secretion system and a DNA transfer mechanism to deliver DNA from one cell to another. Conjugative partners are conventionally confined to the prokaryotic domain. In a prominent exception, Agrobacterium tumefaciens type IV secretion-mediated transfer of DNA to plant cells can result in subsequent chromosomal integration. Recently, we demonstrated interdomain conjugation from Escherichia coli to the diatom Phaeodactylum tricornutum with the subsequent maintenance of an episome at chromosomal copy numbers if it contains diatom centromeres or centromere-like elements. The genes involved in the conjugation process can be separated into those encoding the type IV secretion system, also called the mating pair formation (MPF) genes, and genes involved in DNA processing called the mobilization (MOB) genes. Various protein families compose each class of conjugation genes, including common MOB types F, P, and Q and MPF types F, P, and T. The conjugative transfer from E. coli to P. tricornutum was demonstrated with a vector expressing MOBP and MTFP. Here we show that the MOBPsystem can be deleted and complemented with a MOBQ system in E.coli-diatom conjugations with subsequent episomal maintenaince. Utilization of both MOBP and MOBQ systems results in substantially higher efficiencies in E. coli-diatom conjugation. Finally, we demonstrate conjugative gene transfer between P. tricornutum and A. tumefaciens expressing a MPFT, the first demonstration of this system in diatoms,resulting in episomal maintainance or chromosomal integration, depending on the ex-conjugant. The promiscuity of MOB and MTF systems permitting prokaryote to diatom conjugative DNA transfer suggest major environmental and evolutionary importance of this process. The increased efficiency of dual MOB systems immediately improves genetic engineering in diatoms and has interesting basic cellular biology implications.

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Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species)

Cited by 35 publications

References 40 publications

Transgene Expression in Microalgae—From Tools to Applications

Transgene Expression in Microalgae—From Tools to Applications

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

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