Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor.

In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases (lcc) sensu stricto (lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases (lcc10 and lcc11) that are not present in C. cinerea. The fet3-like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/sidA gene for siderophore biosynthesis. Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas (lcc3 and lcc8), in fruiting bodies (lcc7) or in the free-living mycelium grown on agar medium (lcc9 and lcc10), suggesting a specific function of these MCOs. The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi.

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis.

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

Sequence variation and evolution of Cryphonectria hypovirus 1 (CHV-1) in Europe.

Cryphonectria hypovirus 1 (CHV-1) acts as a naturally occurring biological control agent for chestnut blight, a destructive fungal disease of chestnut trees, which has been introduced into Europe in the 1930s. We have determined partial nucleotide and deduced amino acid sequences of the ORF A of 47 CHV-1 isolates collected in Europe over a period of 28 years. Phylogenetic analysis revealed the presence of four groups or single viruses, which showed sequence divergences ranging from 11 to 19%. These results confirm the previous subtype classification based on RFLP markers, with the exception of the two CHV-1 subtypes E and D, which appear to be related closer than anticipated previously. Dates of divergences between CHV-1 subtypes, calculated from nucleotide substitution rates, indicate that the CHV-1 subtypes diverged several hundreds years ago. Our results suggest that the genetic variation among CHV-1 subtypes did not evolve in Europe and support the hypothesis of multiple introductions of CHV-1 into Europe.

Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers.

A total of 72 hypovirus-infected isolates of the chestnut blight fungus Cryphonectria parasitica were sampled from nine European countries between 1975 and 1997. The double-stranded RNA of the Cryphonectria hypoviruses (CHV1) was isolated and reverse transcription (RT)-PCR products were obtained for two different regions of the viral genome (ORF A and ORF B) using primer sequences of the type species CHV1-EP713. Both PCR products of each viral isolate were digested with four restriction endonucleases recognizing sequences of four nucleotides. The restriction fragment length polymorphism (RFLP) analysis revealed 41 genetically distinct RFLP types of CHV1 with 10 types occurring more than once. Identical RFLP types were detected nine times among viruses collected in the same location. Cluster analysis based on the RFLP banding patterns separated the viral isolates into five CHV1 clusters or subtypes. Most viral isolates (64 out of 72) grouped into one large cluster which comprised all viruses from Italy (including CHV1-EP747), Switzerland, Crotia, Bosnia, Hungary, Greece, and the French island Corsica, as well as five out of 11 isolates from continental France. Two additional subtypes of CHV1 were found in France (one related to CHV1-EP713) and one each in Spain and Germany. The Swiss samples collected over a period of 20 years showed that very little RFLP variation has evolved during this time. The results of this study are consistent with the hypothesis of multiple introductions of CHV1 into Europe.