Composite poplars: a novel tool for ectomycorrhizal research

Dimitri Neb,Arpita Das,Uwe Nehls,Annette Hintelmann

doi:10.1007/s00299-017-2212-2

Abstract

Key MessageComposite poplars were used for ectomycorrhiza formation. Structurally normal mycorrhizas of transgenic roots revealed better fungal sugar support. Targeting fluorescent proteins to peroxisomes allowed easy in planta visualization of successful transformation.A bottle neck in ectomycorrhizal research is the time demand for generation of transgenic plants. An alternative strategy for such root-centered research might be the formation of the so-called composite plants, where transgenic roots are formed by non-transgenic shoots. We have developed an Agrobacterium rhizogenes-mediated root transformation protocol using axenic Populus tremula × tremuloides and P. tremula × alba cuttings. When comparing four different bacterial strains, A. rhizogenes K599 turned out to be the most suitable for poplar transformation. Transgenic roots revealed only minor hairy root phenotype when plants were grown on agar plates with synthetic growth medium in the absence of a sugar source. When using different ectomycorrhizal fungi, formation of ectomycorrhizas by transgenic roots of composite poplars was not affected and mycorrhizas were anatomically indistinguishable from mycorrhizas of non-transgenic roots. Elevated trehalose content and marker gene expression, however, pointed towards somewhat better fungal carbon nutrition in ectomycorrhizas of transgenic compared to non-transgenic roots. Cell wall autofluorescence of poplar fine roots is an issue that can limit the use of fluorescent proteins as visual markers for in planta analysis, especially after ectomycorrhiza formation. By targeting marker proteins to peroxisomes, sensitive fluorescence detection, easily distinguishable from cell wall autofluorescence, was obtained for both poplar fine roots and ectomycorrhizas.

Highlights

Due to a frequently nutrient limited environment combined with extended seasonality, boreal and temperate forests dominate large parts of the northern hemisphere
To elucidate the potential of composite plants in ectomycorrhizal research and to speed up molecular and functional research of this type of symbiosis, we have developed an A. rhizogenes-based protocol for a fast generation of composite poplars and investigated the ability of transgenic roots to form functional ectomycorrhizas
Except for the agropine A. rhizogenes strain 15834 that revealed a kinetic similar to that of the water control, root formation was retarded when plants were inoculated with agrobacteria (Fig. 1)

Summary

Introduction

Due to a frequently nutrient limited environment combined with extended seasonality, boreal and temperate forests dominate large parts of the northern hemisphere. To guarantee optimal function of these ecosystems, the mutualistic interaction of tree roots with ectomycorrhizal (ECM) fungi is essential (Smith and Read 2008). In this type of symbiosis, the fungal partner provides soil-based water and mineral nutrients, while the plant serves the basic carbohydrates from photosynthesis. With respect to ectomycorrhizal plant colonization, a (limited) number of laboratory- and field-based (e.g., Stefani et al 2009; Danielsen et al 2013) studies revealed no general impact of transgenic properties on symbiosis. Due to the time request for generation and propagation of transgenic plants, which usually lasts for more than a year (Alpizar et al 2006; Hampp et al. Vol.:(0123456789)

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Conclusion