Multiple recent horizontal transfers of a large genomic region in cheese making fungi.

While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti--called Wallaby--present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes.

Partition of the Botrytis cinerea complex in France using multiple gene genealogies.

In micro-organisms biodiversity is often underestimated because relevant criteria for recognition of distinct evolutionary units are lacking. Phylogenetic approaches have been proved the most useful in fungi to address this issue. Botrytis cinerea, a generalist fungus causing gray mold, illustrates this problem. It long has been thought to be a single variable species. Recent population genetics studies have shown that B. cinerea is a species complex. However conflicting partitions were proposed. To identify the most relevant partitions within the B. cinerea complex we used a multiple-gene genealogies approach. We sequenced portions of four nuclear genes, of which genealogies congruently clustered into two well supported groups corresponding to Groups I and II previously described, indicating that they represent phylogenetic species. Estimates of migration rates and genetic differentiation showed that these groups had been isolated for a long time, without detectable gene flow. This was confirmed by the high number of polymorphic sites fixed within each group. The genetic diversity was lower within Group I, as revealed by DNA polymorphism and vegetative incompatibility tests. Groups I and II exhibited phenotypic differences in their phenology, host range, size of asexual spores and vegetative compatibility. All these morphological and molecular aspects suggest that B. cinerea Groups I and II may be different cryptic species, isolated for a long time. Phylogenies and molecular analyzes of variance revealed no genetic structure according to the other suggested partitions for the B. cinerea complex (i.e., among host plants, between strains with and without transposable elements, nor between strains responsible for noble rot and gray mold. This suggests that recombination regularly occurs, or occurred until recently, within B. cinerea Group II. This also was supported by recombination rates at each locus. Multiple-gene genealogies showed their utility by providing a relevant partition criterion for the B. cinerea complex.

Ascomycete diversity in soil-feeding termite nests and soils from a tropical rainforest.

Molecular microbial ecology has revealed remarkable biodiversity - prokaryotic and eukaryotic - in numerous soil environments. However, no culture-independent surveys of the termitosphere exists, although termites dominate tropical rainforests. Here, we focused on soil feeders, building nests with their soil-born faeces, enriched with clay-organic complexes, thus contributing to the improvement of soil fertility. In order to assess the fungal community composition of these termitaries compared with soils not foraged by termites, samples of the two types were collected in the Lopé rainforest, Gabon, and processed for generation of fungal internal transcribed spacer (ITS) clone libraries. Although primers were universal, most of the recovered sequences represented Ascomycete that were previously uncharacterized and the proportions of which reached 72.5% in soils and 80% in termitaries. Their affiliation with identified fungi was analysed in performing a phylogenetic tree based on 5.8S rDNA. Furthermore, the ascomycete communities of soil-feeding termitaries and soils shared only 6.3% of sequences. This discrepancy of composition between soil and nest may result from the building behaviour of termites, as the organic matter in the nest is chemically modified, and some vacant ecological microniches are available for more specialized fungi.

Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea.

Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent (CND) genes among 2839 B. cinerea genes. Among the co-regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug.

The endopolygalacturonase 1 from Botrytis cinerea activates grapevine defense reactions unrelated to its enzymatic activity.

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassahet-c vegetative incompatibility locus, and its use as a population marker.

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.