Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions

Tzima, Aliki; Paplomatas, Epaminondas J.; Schoina, Charikleia; Domazakis, Emmanouil; Kang, Seogchan

doi:10.1016/j.funbio.2014.04.009

Cited by 5 publications

(10 citation statements)

References 36 publications

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“…basicola and E . resinifera allows for easy identification of additional areas for optimization (Table 2; Loppnau et al, 2004; Tzima et al, 2014). Increasing the concentration of conidia and/or bacteria could raise transformation efficiency as this could improve the interactions between the two (Michielse, Hooykaas et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…Although it will be important to first improve the transformation efficiency of this protocol within these species, especially when using targeted mutation, the application of the current protocol to various species yielded GFP‐emitting transformants. The ability to detect GFP emissions in these few transformants means the infection pathway of these fungi within their hosts can be tracked (Maor et al, 1998; Moročko‐Bičevska & Fatehi, 2011; Tzima et al, 2014; Vu et al, 2018), an important aspect considering that most of the Ceratocystidaceae family are well‐known plant pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…However, care should be taken as increasing the bacteria too much could lead to less transformations as the fungus struggles to compete for nutrients and the bacteria become difficult to kill during subsequent selection, while too many fungal cells can result in false-positive colonies emerging. The co-incubation period on induction minimal media (IMM) agar could also be adjusted to the growth rates of each species, allowing the conidia to be at an optimum point of germination that makes the fungal cells more susceptible to Agrobacterium infection (Michielse, Hooykaas et al, 2005), and supplementing the induction and selection media with V8 juice (a commonly used supplement that is a blend of 8 vegetable juices) can increase their germination (Tzima et al, 2014). Although such optimizations were not attempted here, these could not only increase transformant yield, but might also prove crucial to inducing transformation in A. cleistominuta and E. polonica, the two species that could not be transformed using the current protocol.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Transferring an Agrobacterium‐mediated transformation protocol across eight genera in the Ceratocystidaceae

et al. 2021

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Establishing a transformation system is an essential first step for functional studies. Numerous transformation systems have been developed and optimized for filamentous ascomycetes, including Agrobacterium‐mediated transformation. Recently, such a system was developed for the African tree pathogen Ceratocystis albifundus. This fungus is part of the family Ceratocystidaceae, which includes many tree and plant pathogens of economic importance. Despite the many advantages associated with Agrobacterium‐mediated transformation, establishing and optimizing this system within a species are extremely arduous due to the many species‐specific parameters that must be optimized. This study aimed to apply the existing C. albifundus Agrobacterium‐mediated transformation protocol to eight diverse Ceratocystidaceae species from eight different genera. Minor adjustments were made to the already established protocol to increase transformant yield before it was used to successfully produce transformants for six of the eight targeted species without the need for any further species‐specific optimizations. Although the number of transformants obtained was less than for C. albifundus, this study proves that protocols for Agrobacterium‐mediated transformation might be more transferable than previously thought. It also provides a useful benchmark for future transformation studies in the Ceratocystidaceae and will make species‐specific optimization easier.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Transferring an Agrobacterium‐mediated transformation protocol across eight genera in the Ceratocystidaceae

et al. 2021

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“…Therefore, the ATMT system offers an efficient tool for random molecular manipulation. However, ATMT is time-consuming and prone to low transformation rate due to various factors, including acetosyringone concentration (Tzima et al, 2014), co-culturing time, co-culturing temperature and fungal inoculums, which were found to significantly impact the transformation frequency (Sorensen et al, 2014). The research about how the T-DNA is transferred into the yeast cells is on the way.…”

Section: Agrobacterium Tumefaciens-mediated Transformation (Atmt)mentioning

confidence: 99%

Genetic transformation of fungi

He¹,

Feng²,

Lu³

et al. 2017

Int. J. Dev. Biol.

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Transferring DNA into cells is an essential research method for molecular cloning and gene function studies. As molecular biology and materials physics develop, more and more new transformation methods have been applied to mammalian cells. Some techniques have been successfully developed for several types of fungi, but their efficiencies are extremely low. To better study the functional genes of fungi, and to improve the characteristics of fungi in an easy, safe and reliable way, many investigations have been conducted to effectively develop such technologies for a wide variety of species and to increase the efficiency and reproducibility of genetic transformation. The objective of this paper is to review the latest development of transformation methods used for the genetic transformation of fungi, including several promising transformation approaches, together with their advantages and drawbacks, which may open up novel methods for fungi research.

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“…These parameters include (i) fungal parameters such as the choice of the species and the initial material (e.g. spores, protoplasts, mycelium) (Michielse et al 2005), (ii) bacterial parameters such as the A. tumefaciens strain and its cell density (Flowers & Vaillancourt, 2005;Tzima et al 2014), and (iii) co-cultivation conditions such as the use of acetosyringone, incubation time, and temperature (Combier et al 2003;Tzima et al 2014). Among these factors, the addition of acetosyringone in the culture medium and the incubation time of the fungalbacterial co-cultivation are usually reported as important drivers of a successful ATMT.…”

Section: Introductionmentioning

confidence: 99%

Melanization and ageing are not drawbacks for successful agro-transformation of dark septate endophytes

et al. 2017

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Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and metabolism, and DSE genes have not been functionally characterized yet. Therefore developing molecular genetic tools to investigate the biological function of genes of interest in DSEs is of major significance. We investigated Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency in eight DSE strains belonging to Cadophora sp., Cadophora malorum, Leptodontidium sp., Phialophora mustea, and Cladosporium cladosporioides. ATMT efficiency was DSE-dependent and ranged from 0.6 to 125 %. We further focused on the effect of mycelium ageing on ATMT efficiency. Leptodontidium sp. Me07, Leptodontidium sp. Pr30, and C. cladosporioides CBS 101367 were significantly more transformed using 15-d-old mycelium (44.5, 6.9, and 1.1 %, respectively) as compared to 2-d-old mycelium (121, 28.7, and 25.1, respectively), whereas P. mustea Pr29 was more transformed using young mycelium (21.5 % compared to 5.3 % for the old mycelium). Finally, we focused on the effect of melanin content on ATMT efficiency. Melanin content in mycelium ranged from 0.9 to 3.2 mg g DW. Tricyclazole negatively modulated melanin content, while copper positively modulated it. However there was no correlation between hyphal melanin content and ATMT efficiency.

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Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions

Cited by 5 publications

References 36 publications

Transferring an Agrobacterium‐mediated transformation protocol across eight genera in the Ceratocystidaceae

Transferring an Agrobacterium‐mediated transformation protocol across eight genera in the Ceratocystidaceae

Genetic transformation of fungi

Melanization and ageing are not drawbacks for successful agro-transformation of dark septate endophytes

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