Nicolas Rouhier

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 ∼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.

Obligate biotrophy features unraveled by the genomic analysis of rust fungi

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation.

Glutathione, a tripeptide with the sequence gamma-Glu-Cys-Gly, exists either in a reduced form with a free thiol group or in an oxidized form with a disulfide between two identical molecules. We describe here briefly the pathways involved in the synthesis, reduction, polymerization, and degradation of glutathione, as well as its distribution throughout the plant and its redox buffering capacities. The function of glutathione in xenobiotic and heavy metal detoxification, plant development, and plant-pathogen interactions is also briefly discussed. Several lines of evidence indicate that glutathione and glutaredoxins (GRXs) are implicated in the response to oxidative stress through the regeneration of enzymes involved in peroxide and methionine sulfoxide reduction. Finally, emerging functions for plant GRXs and glutathione concern the regulation of protein activity via glutathionylation and the capacity of some GRXs to bind iron sulfur centers and for some of them to transfer FeS clusters into apoproteins.

Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Plant Glutathione Peroxidases Are Functional Peroxiredoxins Distributed in Several Subcellular Compartments and Regulated during Biotic and Abiotic Stresses

We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of −295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.

The plant thioredoxin system

Abstract.Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cell. In plants, the thioredoxin system is particularly complex since at least 20 thioredoxin isoforms are found in the plant model Arabidopsis thaliana. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. Different pathways allowing thioredoxin reduction also coexist in the plant involving ferredoxin-thioredoxin reductase, thioredoxin reductases and the glutathione/glutaredoxin system. This review discusses the literature of plant thioredoxins with emphasis on recent findings in the field.

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters.

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein‐glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe–2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase‐mediated assembly of an identical [2Fe–2S] cluster in apo‐GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe–S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe–2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe–2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe–S clusters or in the regulation of the chloroplastic Fe–S cluster assembly machinery.

Plant glutaredoxins: still mysterious reducing systems

Glutaredoxins are ubiquitous oxidoreductases which are similar to thioredoxins and possess a typical glutathione-reducible CxxC or CxxS active site. We present here the current knowledge about these proteins in plants. At least 31 glutaredoxin genes are present in Arabidopsis thaliana, a value close to the thioredoxin gene number. Based essentially on active site sequences, a classification of these multiple genes is proposed. The specificity of the various apparently redundant forms within the glutaredoxin group or between glutaredoxin and thioredoxin can be analysed in terms of differential spatiotemporal expression of the genes, specificity vs. target proteins and mode of catalysis (glutathiolation/ deglutathiolation processes appear to be a specific function of glutaredoxin). Additional putative functions are proposed for plant glutaredoxins based on their targets in other organisms and in the light of the existence of hybrid proteins containing glutaredoxin modules in their N- or C-terminal part.

Poplar Peroxiredoxin Q. A Thioredoxin-Linked Chloroplast Antioxidant Functional in Pathogen Defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.

Glutaredoxins: roles in iron homeostasis

Glutaredoxins, proteins traditionally involved in redox reactions, are also required for iron-sulfur cluster assembly and haem biosynthesis. These new roles are probably related to the ability of some glutaredoxins to bind labile [2Fe-2S] clusters and to transfer them rapidly and efficiently to acceptor proteins. Recent results point to putative roles for glutaredoxins in the sensing of cellular iron and in iron-sulfur cluster biogenesis, either as scaffold proteins for the de novo synthesis of iron-sulfur clusters or as carrier proteins for the transfer of preformed iron-sulfur clusters. Based on prokaryote genome analysis and in vivo studies of iron regulation in yeast, we propose putative new roles and binding partners for glutaredoxins in the assembly of metalloproteins.