Abstract
Arginase (coded by agaA) provides the exclusive catabolic route for arginine utilization in the fungi studied so far. A mutant/disruptant of agaA is unable to grow on l-arginine as the sole nitrogen source. Transforming such a strain with an agaA expression construct allows it to utilize l-arginine. The agaA − recipient host along with an agaA expression construct describes arginase as a conditional nutritional marker. A protocol to use this arginine selection for A. niger transformation is demonstrated.
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- 1.
The agaA selection requires a recipient strain that has arginase-less phenotype. This could be achieved by disruption or mutation in the host agaA gene. The agaA mutants of fungal recipient strains can be isolated and are reported for Neurospora crassa (Morgan 1970) as well as A. nidulans (Bartnik et al. 1977). However, a recipient strain with agaA deletion or disruption is desirable; this circumvents complications due to spontaneous reversion possible with point mutations.
- 2.
Sterilize all growth media and transformation reagents by autoclaving. Store the transformation reagents at 4 °C. Each Petri plate should be dispensed with 20 mL of uniformly spread agar medium.
- 3.
Sterilize l-arginine solution separately by filtration and then add it to the autoclaved MM. Ensure that the final pH is between 5.5 and 6.0.
- 4.
Prepare PEG(t) solution freshly before transformation.
- 5.
Using agaA as a nutritional selection marker in A. niger was demonstrated with two different promoters namely, the citrate synthase promoter (PcitA) of A. niger and a truncated tryptophan synthase promoter (PtrpC) of A. nidulans (Dave et al. 2012). The promoter of agaA itself is under nitrogen metabolite regulation and has regulatory elements that respond to l-arginine induction. Therefore, it is desirable to construct a marker that expresses agaA cDNA from a constitutive promoter. Heterologous expression of A. niger agaA cDNA could complement a car1 (yeast agaA homolog) deletion in S. cerevisiae (Jayashri 2006). A constitutive yeast promoter (Pgpd) was used for this purpose. Any functional combination of a promoter and arginase cDNA would work well as arginase marker for selection.
- 6.
Carry out all the steps for the preparation and transformation of protoplasts under aseptic conditions.
- 7.
Maintain the fungal stock cultures as PDA slants. Inoculate the spores from the stock cultures on PDA plates to generate seed cultures. Allow fungal growth on these plates at 37 °C for initiation of conidiation (3–4 days) and then transfer them to room temperature for the spores to mature (10–12 days). Harvest spores from fresh seed plates (12–15 days old) to use as inoculums. Prepare spore suspensions for inoculation in DDW with 0.005 % Tween 20. Count the spores using Neubauer counting chamber and inoculate with approximately 108 spores per 100 mL of medium. For preparing protoplasts, inoculate A. niger spores into four 1 L flasks (each containing 400 mL of MM).
- 8.
Pre-cool DDW and OM at 4 °C for washing mycelia. Wash the harvested mycelia with OM till it looks shiny/slimy.
- 9.
The amount of lysing enzyme used for making protoplasts needs standardization depending upon its strength/efficiency. (You may need only 10 mg/mL if fresh enzymes are used).
- 10.
Pre-dissolve BSA powder in a small volume of OM. Ensure that the solution does not froth while dissolving BSA.
- 11.
Visible growth can be observed after 48 h of incubation on arginine selection plates. True transformants grow better/faster than others upon prolonged incubation (colony size increases). The false positives, if any, do not grow further and die out. Passage the putative transformants on arginine selection plates for five generations. Culture these transformants on MM (i.e. without selection pressure) for the sixth generation and subsequently transfer them back on MM + Arg plates to ensure their genetic stability. Single spore these transformants on MM + Arg plates to obtain a genetically homogenous culture.
- 12.
All filamentous fungal genomes sequenced so far contain an agaA gene.
References
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Acknowledgments
This research was funded by the New Millennium Indian Technology Leadership Initiative of Council of Scientific and Industrial Research (NMITLI-CSIR), India. This work was also supported in part by research grant from Board of Research in Nuclear Science-Department of Atomic Energy (BRNS-DAE) and research fellowship from University Grants Commission, UGC, Council of Scientific and Industrial Research, CSIR and Department of Science and Technology Women Scientist Scheme, DST-WOS-A.
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Dave, K., Ahuja, M., Jayashri, T.N., Sirola, R.B., Dave, K., Punekar, N.S. (2015). Arginase (agaA) as a Fungal Transformation Marker. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 2. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10503-1_12
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