As described recently in the contribution by Peter Flegg, entitled: “The genetic transformation of mushrooms” (Mushrooms International Newsletter 86  3-5), there is a lot of discussion going on about the public and government acceptance of transgenic food crops in general. So, what is there to add for mushrooms?
Ever since the first breakthrough in Agaricus transformation a lot has changed affecting many scientists, especially in Europe. For some years all transformation work on Agaricus and many other food and even non-food crops has been questioned. People now believe that there should be benefits to consumers rather than exclusive benefits to spawn makers and growers. Moreover, transformation techniques should not be employed for solving problems (e.g. La France disease), that should preferably be solved using proper sanitary precautions (Wach, personal communication).
Flash back on initial transformation
In the 1970s, early 1980s it was generally acknowledged that the prospects of Biotechnology for a variety of organisms seemed almost unlimited. At that time several groups tried to achieve transformation of the white button mushroom using techniques that worked reasonably well in other filamentous fungi, like the industrially employed Aspergillus species. Reasons to start the transformation work, primarily inspired by the secondarily homothallic breeding system, ranged from pure scientific interest to direct commercial benefits. Problems associated with Agaricus cultivation, distribution and storage, like La France disease (dsRNA virus), other infections (e.g. Brown blotch disease with Pseudomonas tolaasii) or senescence-induced browning, were considered realistic targets for transformation.
There have been considerable world-wide, but unsuccessful efforts for about a decade, including those by the author at Jos Wessel’s lab. The author and his group now working at the Agrotechnological Research Institute in Wageningen (ATO B.V.; see Mushroom Personalities Harry Wichers and Hans Mooibroek, 1996) were the first to obtain a few stable Agaricus bisporus transformants by the end of 1993. The recipient strain used initially was the adenin-auxotrophic strain (ATCC 24663), which appeared to form protoplasts very efficiently and responded well to selection with hygromycin. Later on the protocol also appeared applicable to a derivative of commercial strain Horst U1 (ATCC 62462), be it less reliable. Fertile crosses between the transgenic mutant strain and a U1 homokaryon were easily obtainable. After filing a PCT-patent (Mooibroek et al., 1994) and a first communication at the Vancouver Congress in 1994 (Van de Rhee et al.), this work has been published as well as the results on highly efficient homologous, site-directed, integration of the transformation plasmid that contained the A. bisporus β-1,3-glucanase gene (Van de Rhee et al., 1996a; Van de Rhee et al., 1996b).
Our first target was to study mushroom browning, for which we isolated the A. bisporus tyrosinase genes and introduced one of them in the antisense orientation. Either approach using site-directed integration or antisense inhibition was supposed to provide straightforward tools for gene silencing in A. bisporus (see below). However, the multinuclear nature of fertile A. bisporus mycelia presented an additional problem. More details have been described by Stoop and Mooibroek, 1999. Another challenge was to isolate and identify the A. bisporus mannitol-dehydrogenase (MtDH) gene (Stoop and Mooibroek, 1998). With mannitol being the most abundant metabolite (up to about 50% DW and excluding water), white button mushrooms might as well be considered mannitol-rooms. In collaboration with a Swiss group (J. Sassoon and U. Baumann, University of Bern), the MtDH 3-dimensional structure has become available (Sassoon et al. 2001; Hörer et al., 2001), which now would allow, in principle, the generation of transgenic mushrooms containing altered mannitol profiles or altered MtDH enzymes and, thus, would allow the study of the function(s) of mannitol. Ultimately, such activities might also yield new commercial strains with for example a higher dry matter content or better pathogen resistance, if this is, at the end, what the consumer wants.
Other transformation protocols
More recently, a novel transformation protocol for different fungal species, including commercial heterokaryotic A. bisporus strain Horst U1, has been described (De Groot et al. 1998a), which is based on infection with the plant-pathogenic bacterium Agrobacterium tumefaciens and induction of its virulence (vir) gene with the plant hormone acetosyringone. Possible limitations of this Agrobacterium-mediated transformation system for A. bisporus may be that a priori it is not known which of the nuclei will have been transformed, that getting rid of the infectious Agrobacterium may be difficult and that co-transformation is much more difficult or impossible when more than two or three individual transgenes should be introduced. Perhaps most important, however, may be the necessity to include redundant DNA sequences in the transformation vector that have no function inAgaricus, but are needed for gene transfer only.
An alternative way to introduce donor DNA into a vast number of organisms, including many plant and fungal species, and even intact tissues, is particle bombardment. This technique is based on gas-driven bombardment with tungsten or gold particles coated with the donor DNA, thus penetrating the recipient tissue. A number of reports describe its use for transformation of intact plants and for a number of fungi. For A. bisporus, particle bombardment would have the advantage that the laborious and often problematic production and regeneration of protoplasts orAgrobacterium-mediated infection could be avoided. In our and other laboratories (Challen, personal communication), attempts have been undertaken to introduce hygromycin B resistance or other selectable markers into A. bisporus by particle bombardment. However, this technique has not yet resulted in the selection of stable Agaricus transformants or in a generally applicable system.
In a non-related ongoing project in our lab on the possible production of biodegradable bacterial polyesters (PHAs, polydroxyalkanoates) in potato, we have, in collaboration with the University of Wageningen Laboratory of Plant breeding, developed a reliable stable transformation system for potato using particle bombardment (Romano et al., 2001). The system allowed the simultaneous introduction of up to 4 different genes. Most important, however, is the observation that also expression cassettes (restriction fragments or PCR products) exclusively containing a promoter, a structural gene and a terminator, with no plasmid sequences, yielded hundreds of stable co-transformants. Further analysis of the organisation of the transgenes (Romano et al., in preparation) revealed the frequent integration of the selectable marker and PHA-synthetic genes into different chromosomal positions. This will allow the straightforward outcrossing of the selectable marker. If this system can also be adopted for Agaricustransformation, again another transformation tool will become available, the choice of which will depend on preference of the lab and on the final objective.
From a large number of organisms, including human, the nucleotide sequences of their entire genomes have been determined. This is the onset of the so-called Genomics-era, now with a combination of related disciplines and bio-informatics. For Agaricus, at this time there is only a joint initiative to start a genome sequencing project amongst a number of leading scientists, but no genuine funded project yet. Once this entire genome sequence will be available, the question is what it will be used for.
Now that a number of entire genomes have been sequenced, the number of surprises has decreased. It appears that in every organism the same types of basic genes are present and it would be a pity if such investment would only yield confirmatory results. A bit more surprising is the observation how sets of functionally related genes are organised and clustered in chromosomes and how this organisation is found in other related and non-related organisms (Comparative Genomics). Perhaps most surprising and most useful will be finding answers to the questions when and where a particular gene will become active (time-space distribution) and, ultimately, why. This information may also become available with the entire genome sequence once we will start to look at gene expression (e.g. micro-arrays), promoter sequences, transcriptional and other regulatory elements.
Also tools for systematic gene silencing (by high throughput RNAi, RNA-mediated interference) will be helpful in elucidating phenotypes associated with a particular gene function (Functional Genomics).
Coming back to the question in the intro: What is there to add for mushrooms? In my, modest, opinion at the present time there is no gene available for transformation that would only improve consumer appreciation without any benefits to spawn makers, growers or retailers. Properties like a higher dry matter content will not be the main interest of the consumer and mushrooms free of infections are what he expects and usually gets anyway. Also an extended shelf-life would probably mainly be exploited by the industry to improve the export window. For the promotion of other transgenic crops the industry has often used arguments other than the only honest one: more profits or less losses. The consumer will get confused if one group claims to produce healthier crops, whereas he knows very well that transgenic crops have a stamp of being unhealthy.
Therefore, let us use all the genetic tools available, and those to be generated, for applications such as strain typing, classical strain breeding and quality monitoring (Applied Genomics) and for pure scientific purposes in order to improve our understanding of mushroom biology. Maybe the industry will get return of investments from better understanding and as yet unexpected applications.
Hörer S, Stoop JMH, Mooibroek H, Baumann U, Sassoon J. The crystallographic structure of mannitol 2-dehydrogenase NADP+ binary complex fromAgaricus bisporus. J. Biol. Chem. 276 (2001) 27555-27561.
De Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen, AGM (1998a) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16: 839-842.
Mooibroek A, van de Rhee MD, Huizing HJ, Rats FH. Production and application of transgenic mushroom mycelium and fruitbodies. International PCT-patent application number PCT/NL93/00149, PCT/NL94/00164, WO95/02691.
Romano A, Raemakers K, Visser R, Mooibroek H. Transformation of Potato (Solanum tuberosum) using particle bombardment. Plant Cell Reports 20 (2001) 198-204, DOI 10.1007/s002990000314.
Romano A, Raemakers K, Bernardi J, Visser R, Mooibroek H. Organisation of transgenes in potato after particle bombardment-mediated co-transformation. In preparation.
Sassoon J, Hörer S, Stoop JMH, Mooibroek H, Baumann U. Crystallization and preliminary crystallographic analysis of mannitol dehydrogenase (MtDH) from the common mushroom Agaricus bisporus. Acta Crystallographica 57 (2001)> 711-713.
Stoop JMH and Mooibroek H. Cloning and characterization of NADP-dependent mannitol dehydrogenase from the button mushroom Agaricus bisporusand its expression in response to NaCl stress. Applied and Environmental Microbiology 64 (1998) 4689-4696.
Stoop JMH and Mooibroek H. Advances in genetic analysis and biotechnology of the cultivated button mushroom, Agaricus bisporus. Mini-review Applied Microbiology and Biotechnology 52 (1999) 474-483.
Van de Rhee MD, Graça PMA, Mooibroek H. Transformation of common mushroom, Agaricus bisporus. Abstract for presentation at the 5th International Mycological Congress, August 1994, Vancouver Canada.
Van de Rhee MD, Graça PMA, Huizing HJ, Mooibroek H. Transformation of common mushroom, Agaricus bisporus. Mol Gen Genet 250 (1996a) 252-258.
Van de Rhee MD, Mendes O, Werten MWT, Huizing HJ, Mooibroek H. Highly efficient homologous integration via tandem exo-ß1,3-glucanase genes in common mushroom, Agaricus bisporus. Curr Genet 30 (1996b) 166-173.
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