Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor.

Cysteine-rich peptides such as metallothioneins (MTs) are involved in metal homeostasis and detoxification in many eukaryotes. We report the characterization and expression of two MT genes, LbMT1 and LbMT2 from the ectomycorrhizal fungus Laccaria bicolor under metal stress conditions. LbMT1 and LbMT2 differ with respect to the length of the encoded peptides (58 versus 37 aa, respectively) and also by their expression patterns in response to metals. The expression levels of both LbMT1 and LbMT2 increased as a function of increased external Cu concentration, the expression levels for LbMT2 were always significantly higher compared with those of LbMT1. Only LbMT1, but not LbMT2, responded to Cd supply in the range of 25-100 µM while Zn did not affect the transcription of either LbMT1 or LbMT2. Both genes also responded to oxidative stress, but to a lesser extent compared to their responses to either Cu or Cd stress. Heterologous complementation assays in metal-sensitive yeast mutants indicated that both LbMT1 and LbMT2 encode peptides capable of conferring higher tolerance to both Cu and Cd. The present study identified LbMTs as potential determinants of the response of this mycorrhizal fungus to Cu and Cd stress.

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.

Hebeloma cylindrosporum- a model species to study ectomycorrhizal symbiosis from gene to ecosystem.

The basidiomycete Hebeloma cylindrosporum has been extensively studied with respect to mycorrhiza differentiation and metabolism and also to population dynamics. Its life cycle can be reproduced in vitro and it can be genetically transformed. Combined biochemical, cytological, genetical and molecular approaches led to the characterisation of mutant strains affected in mycorrhiza formation. These studies demonstrated the role of fungal auxin as a signal molecule in mycorrhiza formation and should allow the characterisation of essential fungal genes necessary to achieve a compatible symbiotic interaction. Random sequencing of cDNAs has identified numerous key functional genes which allowed dissection of essential nitrogen assimilation pathways. H. cylindrosporum also proved to be a remarkable model species to uncover the dynamics of natural populations of ectomycorrhizal fungi and the way in which they respond and adapt to anthropogenic disturbance of the forest ecosystem. Although studies on mycorrhiza differentiation and functioning and those on the population dynamics of H. cylindrosporum have been carried out independently, they are likely to converge in a renewed molecular ecophysiology which will envisage how ectomycorrhizal symbiosis functions under varying field conditions. Contents Summary 481 I. Introduction 482 II. Taxonomy, distribution, autecology, and host range of H. cylindrosporum 482 III. The Hebeloma cylindrosporum toolbox 483 IV. Mycorrhiza differentiation 486 V. Nutritional interactions 488 VI. Genetic diversity and dynamics of H. cylindrosporum populations in P. pinaster forest ecosystems 491 VII. Future directions 494 Acknowledgements 494 References 494.

Isolation of UV-induced mutations in the areA nitrogen regulatory gene of Aspergillus niger, and construction of a disruption mutant.

The areA gene of filamentous fungi encodes a positive-acting transcriptional factor required for the expression of genes involved in the utilisation of nitrogen sources other than ammonium and glutamine. In Aspergillus niger we have isolated three UV-induced areA mutants and constructed a well-defined disruption allele of the areA gene. The areA gene was genetically localised on Linkage Group III, 3.6 map units (m.u.) from bioA1 and 4.5 m.u. from lysA7. Analysis of the expression of the nitrate reductase encoding gene and of nitrate reductase activities show that the mutated areA strains behave as loss-of-function mutants and can be classified as areAr type. In addition, growth tests were performed using several nitrogen sources in combination with glucose. The results suggest that, unlike the case in A. nidulans, in A. niger the AreA protein also plays a role in the presence of ammonium. Furthermore, the spectrum of protease activities secreted by A. niger differs from that produced by A. nidulans, as only A. niger is able to degrade elastin.

Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid.

Two tobacco genes (TOGT) with homology to glucosyltransferase genes known to be induced by salicylic acid (SA) also responded rapidly to a fungal elicitor or to an avirulent pathogen. SA, although an efficient inducer, was shown not to be essential in the signal transduction pathway regulating TOGT gene expression during the resistance response. Recombinant TOGT proteins produced in Escherichia coli exhibited low, but significant, glucosyltransferase activity towards SA, but very high activity towards hydroxycoumarins and hydroxycinnamic acids, with glucose esters being the predominant products. These results point to a possible important function in defense of these glucosyltransferases in conjugating aromatic metabolites prior to their transport and cross-linking to the cell wall.

Aspergillus as a host for heterologous protein production: the problem of proteases.

Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected.

Production of the homologous pectin lyase B protein in six genetically defined protease-deficient Aspergillus niger mutant strains.

An expression cassette has been transformed into six protease-deficient (prt) mutant strains of Aspergillus niger. Transformants were tested for improved production of the proteolytically susceptible PELB tester protein. In four complementation groups (prtA, B, D and F) distinct improvement of PELB yield was observed. These in vivo experiments in single prt mutants confirmed earlier in vitro PELB degradation data and demonstrated how the use of protease-deficient mutants can significantly improve protein production in A. niger. The strong effects of several prt alleles on the stability of the PELB tester protein have initiated a more detailed genetical and molecular characterization of the prt mutations. Mapping of the cloned protease genes pepA [I], B [II], C [IV], D [I], E [IV] and F [IV] indicated that none of the prt mutations represent alleles of the presently cloned protease (pep) genes from A. niger. Analysis of the expression of the pep genes in prt strains demonstrated that the strongly reduced protease activities observed in several prt mutants are not reflected by reduced transcription levels for a number of extracellular proteases. These results indicate that the mode of action of the prt genes constitute an interesting group of new genetic functions which severely affect protease production, and as such improve protein production, in A. niger.

Expression of the Sclerotinia sclerotiorum polygalacturonase pg1 gene: possible involvement of CREA in glucose catabolite repression.

Northern-blot analysis of RNA isolated from Sclerotinia sclerotiorum grown on either glucose or polygalacturonate as the sole carbon source showed that pg1, encoding a neutral polygalacturonase, was not expressed during growth in both media. In contrast, transcripts of this gene were detected during infection of sunflower germlings. Analysis of the promoter sequence revealed a number of cis-acting sequences known to regulate the expression of many fungal promoters. Protein-DNA-binding experiments showed that proteins extracted from mycelia grown on polygalacturonate or glucose interacted with different regions of the promoter. The GST-CREA fusion protein, containing the two zinc fingers of the Aspergillus nidulans repressor CREA involved in carbon catabolite repression, forms several complexes with DNA fragments carrying the consensus 5'-SYGGRG-3'. These results suggest that a CREA homolog may be involved in the regulation of pg1.

Regulation by Galacturonic Acid of Pectinolytic Enzyme Production by Sclerotinia sclerotiorum

Production of polygalacturonases and pectinases from Sclerotinia sclerotiorum was induced in vitro by galacturonic acid. The inductive effect of galacturonic acid was abolished by the presence of glucose, leading to a basal enzyme production. Zymograms of extracellular enzymes showed that galacturonic acid induced the synthesis of six polygalacturonase and one pectin-methylesterase isoforms. Immunoblotting revealed that an exo-polygalacturonase and an exo-polymethylgalacturonase were secreted in all conditions. They are not glucose repressed and not regulated by galacturonic acid. These constitutive enzymes provide the pathogen with the inherent ability to release galacturonic acid from plant cell walls and to trigger inducible enzyme synthesis.

Molecular karyotype of the phytopathogenic fungus Sclerotinia sclerotiorum.

Molecular techniques have been used to characterize different field isolates of Sclerotinia sclerotiorum, an ubiquitous phytopathogen. Chromosomal DNA resolved by pulsed-field gel electrophoresis (PFGE) revealed that S. sclerotiorum contains at least 16 chromosomes ranging from 1.5 Mb to 4.0 Mb. The size of the haploid genome was estimated to be 43.5 Mb. Six field isolates with different levels of virulence on sunflower germlings or green beans were differentiated by random amplification of polymorphic DNA (RAPD), and analysed by clamped homogeneous electric field electrophoresis. This analysis revealed few chromosome-length polymorphisms among these strains. Chromosomal DNA hybridization indicated that the endopolygalacturonase-encoding pg1 gene is localized on the smallest chromosome of all the strains, whereas the ribosomal DNA mapped to different-sized chromosomes. The less-aggressive strain was characterized by the presence of a supernumary small band, presumably consisting of dsRNA. In contrast to numerous other phytopathogenic fungi, this study reveals a strong karyotypic stability among the strains of S. sclerotiorum which may be preserved by the sexual mode of reproduction of this species

Characterization of a multigene family encoding an endopolygalacturonase in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum produces several polygalacturonases which together with other pectinolytic enzymes are involved in the degradation of pectin. A number of different genomic clones were isolated by screening a genomic DNA library in phage EMBL3. Southern-blot and restriction mapping indicate that seven genes constitute two subfamilies of a multigene family encoding endopolygalactutonase. Using pulsed-field gel electrophoresis to separate S. sclerotiorum chromosomes each subfamily was found to hybridize to a different chromosome. A comparison of the nucleotide sequence for the coding region of three members of the gene family reveals surprisingly few base substitutions suggesting that this gene family arose from recent multiple duplication events.