Procedures and Prospects for DNAMediated Transformation of Ectomycorrhizal Fungi

Department of Botany and Microbiology, Auburn University, Auburn, AL 36849

Environmental Research Laboratory, Environmental Protection Agency, Corvallis, OR 97333, USA

I. Background considerations 281

A. Limitations to conventional genetic study 282

B. Fungal transformation systems 282

C. Prospects for molecular genetic manipulation 284

II. Protoplasts and vectors for transformation 285

A. Protoplast formation 286

B. Representative vectors 286

III. Transformation protocols 287

A. Polyethylene glycol-calcium chloride treatment 288

B. Other procedures 290

IV. Concluding remarks 291

References 292

I. Background considerations

Mycorrhizal fungi represent an integral component of the root-soil ecosystem or rhizosphere (Harley and Smith, 1983; Curl and Truelove, 1986) and, if genetically altered to further benefit the plant, offer potential to improve the establishment and yield of many crop plants. Certainly, genetic manipulation of mycorrhizal fungi, through studies involving genes for agronomically important traits or for improved

METHODS IN MICROBIOLOGY VOLUME 23 ISBN 0-12-521523-1

Copyright © 1991 by Academic Press Limited AH rights of reproduction in any form reserved symbiosis, has the potential to improve agricultural and forest production world-wide. Despite their ubiquity in the rhizosphere and their importance to plants, mycorrhizal fungi have unfortunately not been amenable to detailed genetic study. Only more recently, owing to the development of procedures for DNA-mediated transformation of filamentous fungi, has the prospect for research with mycorrhizal fungi been extended to include molecular genetic manipulations.

A. Limitations to conventional genetic study

The extent of mutualistic interdependence between a mycorrhizal fungus and a plant root system, while of overall benefit to both partners, limits the ability to study the fungus as an experimental or genetic system. The endomycorrhiza or vesicular-arbuscular mycorrhiza involve a limited number of species of zygomycetous fungi (Schneck, 1982). These fungi do sporulate during vegetative or mitotic growth, often in profusion, but they are developmentally dependent biotrophs with no known sexual or meiotic spore states (Harley and Smith, 1983). The vesicular-arbuscular mycorrhizal fungi, as obligate symbionts with no known sexual phase, thus offer little or no opportunity for conventional or other genetic investigation. By contrast, the ectomycorrhiza collectively involve more than 1000 species of mainly basidiomycetous and ascomycetous fungi (Trappe, 1962; Zak, 1973), most with an ability to grow saprophytically. Many ectomycorrhizal fungi thus can grow vegetatively, often quite well, in pure culture, but in general they do not sporulate in the vegetative state (Hutchinson, 1989) and are rarely able to complete a sexual cycle under standard laboratory conditions (Kropp, 1988). Their sexual or meiotic spores, when produced, germinate asynchronously and often only at low frequency (Fries, 1983; Kropp, 1988). Such intrinsic limitations for sexual competence are the principal reason why ectomycorrhizal fungi have not been examined extensively through conventional genetic methods. To date, only very few ectomycorrhizal basidio-mycetes, species principally from the genera Laccaria and Hebeloma, have been examined critically through breeding experiments.

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