Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in ...which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars 1, 2. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor 3, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues 4–9 and thus can be considered a mutualism effector.
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► Identification of MiSSP7 as the first symbiotic effector protein critical to symbiosis ► MiSSP7 is actively taken up into plant cells without the need of fungal machinery ► MiSSP7 localizes to host nuclei upon cell entry where it alters host transcription ► Nuclear localization of MiSSP7 is necessary for its role in promoting symbiosis
For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic ...requirements of FP expression in the model ECM homobasidiomycete
Laccaria bicolor
and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in
Laccaria
. Nuclear FP localization was obtained via
in-frame
fusion of FPs between the intron-containing genomic gene sequences of
Laccaria
histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5′ fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in
Laccaria
and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an
Agrobacterium
-mediated transformation compatible plasmid set was designed for easy use of FPs in
Laccaria
. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal
in-frame
fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive
Agaricus bisporus gpdII
promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in
Laccaria
can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.
Summary
The ectomycorrhizal symbiosis is a predominant tree–microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long‐term and ...intimate relationship between the soil‐borne fungi and the roots of trees. Mycorrhiza‐induced Small‐Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8‐RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C‐terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small‐secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.
In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced ...by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1.
Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza.
We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle.
Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.
The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water‐transporting aquaporin in Laccaria ...bicolor. Picea glauca was inoculated with wild‐type (WT), mock transgenic or L. bicolor aquaporin JQ585595‐overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants.
Xerophilic fungal species of the genus Aspergillus are economically highly relevant due to their ability to grow on low water activity substrates causing spoilage of stored goods and animal feeds. ...These fungi can synthesize a variety of secondary metabolites, many of which show animal toxicity, creating a health risk for food production animals and to humans as final consumers, respectively. Animal feeds used for rabbit, chinchilla and rainbow trout production in Argentina were analysed for the presence of xerophilic Aspergillus section Aspergillus species. High isolation frequencies (>60%) were detected in all the studied rabbit and chinchilla feeds, while the rainbow trout feeds showed lower fungal charge (25%). These section Aspergillus contaminations comprised predominantly five taxa. Twenty isolates were subjected to taxonomic characterization using both ascospore SEM micromorphology and two independent DNA loci sequencing. The secondary metabolite profiles of the isolates were determined qualitatively by HPLC-MS. All the isolates produced neoechinulin A, 17 isolates were positive for cladosporin and echinulin, and 18 were positive for neoechinulin B. Physcion and preechinulin were detected in a minor proportion of the isolates. This is the first report describing the detailed species composition and the secondary metabolite profiles of Aspergillus section Aspergillus contaminating animal feeds.
Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for ...transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.
•Ectomycorrhizal research currently suffers from lack of molecular tools optimized for studies of the fungal partners•The pReNuK system presented here offers a nucleus-directed histone H2B-mCherry reporter optimized for studying promoter regulation in ECM model fungus Laccaria bicolor.•This fluorescent reporter is simple to detect, its expression is highly stable and non-toxic to fungal cells.•pReNuK is expected to be functional not only in Laccaria but also in other homobasidiomycetes
Summary
pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII ...promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.
Most boreal and temperate forest trees form a mutualistic symbiosis with soil borne fungi called ectomycorrhiza (ECM). In this association both partners benefit due to nutrient exchange at the ...symbiotic interface. Laccaria bicolor is the first mycorrhizal fungus with its genome sequenced thus making possible for the first time to analyze genome scale gene expression profiles of a mutualistic fungus. However, in order to be able to take full advantage of the genome sequence, reverse genetic tools are needed. Among them a high throughput transformation system is crucial. Herein we present a detailed protocol for genetic transformation of L. bicolor by means of Agrobacterium tumefaciens with emphasis on critical steps affecting the success and efficiency of the approach.
Summary
In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The ...expression profile of fHANT‐AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT‐AC as also N starvation and growth on urea and l‐asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT‐AC genes was detected in mycelia grown on variable concentrations of l‐glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT‐AC is not signalled via l‐glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA‐silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT‐AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT‐AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed.
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