Establishment Of Ectomycorrhizal Symbiosis Research Articles

Colonization by the Mycorrhizal Helper Bacillus pumilus HR10 Is Enhanced During the Establishment of Ectomycorrhizal Symbiosis Between Hymenochaete sp. Rl and Pinus thunbergii.

There are complex interactions between mycorrhizal helper bacteria (MHBs) and ectomycorrhizal (ECM) fungi, with MHBs promoting mycorrhizal synthesis and ECM fungi regulating plant rhizobacterial colonization, diversity, and function. In this study, to investigate whether the ECM fungus Hymenochaete sp. Rl affects the survival and colonization of the MHB strain Bacillus pumilus HR10 in the rhizosphere, the biomass of B. pumilus HR10 was measured in the rhizosphere and mycorrhizosphere. In addition, extracts of Hymenochaete sp. Rl and Pinus thunbergii were evaluated for their effect on B. pumilus HR10 colonization (growth, sporulation, biofilm formation, extracellular polysaccharide and extracellular protein contents, flagellar motility, and expression of colonization-related genes). The results showed that inoculation of Hymenochaete sp. Rl significantly increased the biomass of B. pumilus HR10 in the rhizosphere; however, while extracts of Hymenochaete sp. Rl and P. thunbergii did not affect the biomass or spore formation of HR10, they did affect its biofilm formation, extracellular polysaccharide and extracellular protein production, and flagellar motility. Furthermore, the addition of symbiont extracts affected the expression of chemotaxis-related genes in HR10. When the extracts were added separately, the expression of srf genes in HR10 increased; when the extracts were added simultaneously, the expression of the flagellin gene fliG in HR10 increased, but there was no significant effect on the expression of srf genes, consistent with the results on biofilm production. Thus, Hymenochaete sp. Rl and P. thunbergii roots had a positive effect on colonization by B. pumilus HR10 at the rhizosphere level through their secretions.

The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis.

To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.

Arthrinium phaeospermum isolated from Tuber borchii ascomata: the first evidence for a “Mycorrhization Helper Fungus”?

A filamentous fungus was isolated from Tuber borchii Vitt. fruiting bodies, and it was identified as an Arthrinium phaeospermum (Corda) M.B. Ellis strain, an “endophyte” that forms various associations with healthy leaves, stems, and roots of plants. Molecular analysis confirmed the association of this filamentous fungus with the ascocarps of all collection sites in Salento, Apulia (South Italy). An in vitro symbiosis system between Cistus creticus L. and T. borchii was set up; A. phaeospermum appears to be able to promote mycorrhiza formation in Cistus seedlings, inducing primary root shortening and an increase of secondary roots, similar to the effect of Mycorrhization Helper Bacteria (MHB). Compartmented and uncompartmented bioassays were carried out to investigate the effects of exudates/volatiles released by the truffle-hosted fungus on root architecture; the results showed root shortening in compartmented bioassay suggesting that volatiles released by the fungus alone are sufficient to alter root morphology in early phase of interaction before the mycorrhiza formation. The first evidence for an influence of a truffle-hosted fungus on ectomycorrhizal symbiosis establishment is reported.

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling.

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the “symbiosis toolkits” and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.

Mycorrhizal induction of phenolic compounds and antioxidant properties of fungi and seedlings during the early steps of symbiosis

Ectomycorrhizal (ECM) symbiosis plays a major role in nutrient cycling and the functioning of forest ecosystems. Trees with well-developed ectomycorrhizas are more resistant to environmental stresses such as drought and to biotic stresses such as root pathogens. The establishment of ECM symbiosis is triggered by signals produced by both partners. These signals lead to morphological changes and a complex development of specific structures in both the plant root and the fungus. The development of the ECM fungi, Paxillus involutus and Pisolithus arhizus, in the presence and the absence of the symbiont—Pinus pinaster—was evaluated as well as their antioxidant properties and phenolic compounds composition in response to the symbiotic association. ECM fungi grew less in the presence of P. pinaster, with P. arhizus being less affected in growth and thus being more adapted to this association. Protocatechuic acid was found only in P. involutus, while P. pinaster roots, both in association and isolated, proved to have other phenolic acids, such as p-hydroxybenzoic and p-coumaric acids. The symbiosis between P. involutus and P. pinaster had no major effects on the symbionts, while the association between P. arhizus and P. pinaster seems to generally decrease the antioxidant effects of both symbionts, despite the increase in p-coumaric and cinnamic acids in the ECM fungi.

The auxin‐inducible GH3 homologue Pp‐GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.

Comparative effects of auxin transport inhibitors on rhizogenesis and mycorrhizal establishment of spruce seedlings inoculated with Laccaria bicolor.

We compared the effects of two auxin transport inhibitors (2,3,5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA)) on rhizogenesis and mycorrhizal establishment of Picea abies L. (Karst.) seedlings inoculated with Laccaria bicolor S238N (Maire) Orton. Inoculation of seedlings with L. bicolor under in vitro conditions strongly increased host root and shoot growth. Although TIBA had no effect on taproot growth, NPA decreased taproot growth and deformed the root apex into a globular shape in both non-inoculated seedlings and seedlings inoculated with L. bicolor. Inoculation with L. bicolor strongly increased lateral rhizogenesis of the seedlings, and application of 100 microM indole-3-acetic acid (IAA) partially reproduced this effect. Although TIBA completely inhibited the stimulatory effect of L. bicolor on lateral root formation, NPA inhibited it only partially. Both TIBA and NPA counteracted the effect of exogenous IAA on lateral rhizogenesis. Inoculation with L. bicolor significantly increased shoot growth and seedling dry biomass, whereas application of exogenous IAA had no effect on either parameter. There was no effect of NPA on shoot growth and biomass production. The presence of TIBA completely prevented the development of ectomycorrhizal structures (mantle and Hartig net). In the presence of NPA, the number of seedlings colonized by the fungus was reduced and the degree of development of ectomycorrhizal structures was variable, but not completely prevented. In medium lacking tryptophan, neither TIBA nor NPA inhibited the release of IAA produced by L. bicolor in pure culture. When 100 microM tryptophan was added to the medium, TIBA significantly increased the amount of IAA released by the fungus, whereas NPA had no significant effect. We conclude that fungal IAA plays an important role in plant rhizogenesis and in the establishment of ectomycorrhizal symbiosis.

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor.

Norway spruce (Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap-root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μm) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.

Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir

The mycorrhiza helper bacterium Pseudomonas fluorescens BBc6, isolated from a Laccaria bicolor sporocarp, consistently promotes L. bicolor-Douglas fir (Pseudotsuga menziesii) ectomycorrhizal formation, even with low doses of bacterial inoculum. In order to describe this phenomenon more accurately, we have looked at the location and survival of the introduced bacterial strain in the soil and in the rhizosphere during the establishment of mycorrhizal symbiosis in glasshouse and nursery experiments. Bacterial populations were quantified with a spontaneous, stable, rifampin-resistant mutant, BBc6R8, which phenotypically conformed to the parental strain. BBc6R8 populations declined rapidly, reaching the detection limit after 19 weeks, and did not increase either when L. bicolor sporocarps were forming in autumn or when Douglas fir roots resumed growing in spring. BBc6R8 was neither an endophyte nor a rhizobacterium. Furthermore, it was not particularly associated with either mycorrhizas of Douglas fir-L. bicolor or L. bicolor sporocarps. Surprisingly, a significant mycorrhiza helper effect was observed when the inoculated BBc6R8 population had dropped as low as 30 CFU g of dry matter(sup-1) in the soil. This study raises questions concerning the bacterial concentration in the soil which is effective for promotion of mycorrhizal establishment and the timing of the bacterial effect. It allows us to develop working hypotheses, which can be tested experimentally, to identify the mechanisms of the mycorrhiza helper effect.

Auxin overproducer mutants of Hebeloma cylindrosporum Romagnesi have increased mycorrhizal activity

summaryThe ectomycorrhizal activity (expressed as the number of mycorrhizas per plant) of indole‐3‐acetic acid (IAA) overproducer mutants of Hebeloma cylindruspurum Rumagnesi and of their mono‐ and dikaryotic progenies has been studied in an attempt to clarify the role of fungal IAA in the establishment of ectomycorrhizal symbiosis, The original mutants obtained from the hi monokaryon produced a higher number of mycorrhizas than did the wild type. The study of the mycorrhizal activity of synthesized dikaryons heterozygous for the FIR (fluoroindoleresistant) mutations, which are responsible for IAA overproduction, showed that these mutations are recessive.The vitro fruiting of a dikaryon heterozygous for a FIR mutation allowed the isolation of IAA‐overproducing sib‐monokaryons. Their average mycorrhizal activity was higher than that of non‐overproducers. indicating that, although the mycorrhizal activity of sib‐monokaryons is under polygenic control, high IAA production gives an advantage in mycorrhiza formation. This was confirmed by the higher number of mycorrhizas formed by synthesized dikaryons homozygous for a FIR mutation than by wild type mycelia.The mycorrhizas formed by the mono‐ or dikaryotic wild types were characterized by a uniseriate Hartig net, whereas it was pluriseriate in the case of mycorrhizas formed by IAA overproducer mutants. The mutants stimulated the growth of the host plants to the same extent as did the wild types, indicating that growth stimulation is not a direct consequence of fungal auxin production. The usefulness of these mutants as a model to study the role of fungal IAA in mycorrhiza formation is discussed.