Fabiano Sillo

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship

AbstractMain conclusionOrchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.

Genome-wide analysis of cell wall-related genes in Tuber melanosporum

A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components.

Comparative Genomics of Sibling Fungal Pathogenic Taxa Identifies Adaptive Evolution without Divergence in Pathogenicity Genes or Genomic Structure

It has been estimated that the sister plant pathogenic fungal species Heterobasidion irregulare and Heterobasidion annosum may have been allopatrically isolated for 34–41 Myr. They are now sympatric due to the introduction of the first species from North America into Italy, where they freely hybridize. We used a comparative genomic approach to 1) confirm that the two species are distinct at the genomic level; 2) determine which gene groups have diverged the most and the least between species; 3) show that their overall genomic structures are similar, as predicted by the viability of hybrids, and identify genomic regions that instead are incongruent; and 4) test the previously formulated hypothesis that genes involved in pathogenicity may be less divergent between the two species than genes involved in saprobic decay and sporulation. Results based on the sequencing of three genomes per species identified a high level of interspecific similarity, but clearly confirmed the status of the two as distinct taxa. Genes involved in pathogenicity were more conserved between species than genes involved in saprobic growth and sporulation, corroborating at the genomic level that invasiveness may be determined by the two latter traits, as documented by field and inoculation studies. Additionally, the majority of genes under positive selection and the majority of genes bearing interspecific structural variations were involved either in transcriptional or in mitochondrial functions. This study provides genomic-level evidence that invasiveness of pathogenic microbes can be attained without the high levels of pathogenicity presumed to exist for pathogens challenging naïve hosts.

Expression and phylogenetic analyses of the Gel/Gas proteins of Tuber melanosporum provide insights into the function and evolution of glucan remodeling enzymes in fungi

The β(1,3)-glucanosyltransferases of the GH72 family are redundant enzymes that are essential for the formation and dynamic remodeling of the fungal wall during different stages of the life cycle. Four putative genes encoding glycosylphosphatidylinositol (GPI)-anchored β(1,3)-glucanosyltransferases, designated TmelGEL1, TmelGEL2, TmelGEL4 and TmelGAS4, have been annotated in the genome of Tuber melanosporum, an ectomycorrhizal fungus that also produces a hypogeous fruiting body (FB) of great commercial value (black truffle). This work focuses on the characterization and expression of this multigene family by taking advantage of a laser microdissection (LMD) technology that has been used to separate two distinct compartments in the FB, the hyphae and the asci containing the ascospores. Of the four genes, TmelGEL1 was the most up-regulated in the FB compared to the free-living mycelium. Inside the FB, the expression of TmelGEL1 was restricted to the hyphal compartment. A phylogenetic analysis of the Gel/Gas protein family of T. melanosporum was also carried out. A total of 237 GH72 proteins from 51 Ascomycotina and 3 Basidiomycota (outgroup) species were analyzed. The resulting tree provides insight into the evolution of the T. melanosporum proteins and identifies new GH72 paralogs/subfamilies. Moreover, it represents a starting point to formulate new hypotheses on the significance of the striking GH72 gene redundancy in fungal biology.

Identification of genes differentially expressed during the interaction between the plant symbiont Suillus luteus and two plant pathogenic allopatric Heterobasidion species

The effects of biological invasions by non-native species have been widely studied in terms of environmental, economic, and human health impacts. However, little is known about the consequences that non-native plant pathogens may determine on host plant symbionts, such as ectomycorrhizal (ECM) fungi. In this study, interactions between Suillus luteus, an ECM fungus of pine trees, and the allopatrically differentiated fungal pathogens of pines Heterobasidion irregulare and H. annosum were investigated in dual culture by morphological and gene expression analyses. Growth of S. luteus was inhibited by both Heterobasidion species, but based on statistical analysis, growth inhibition was due to the isolate rather than to the species. The expression analysis on genes related to cell wall hydrolytic enzymes and hydrophobins, putatively involved in the fungus–fungus interaction, allowed to identify significantly up- and down-regulated genes both in the symbiont and in the pathogens. Based on the transcript analysis, it was not possible to distinguish the impact of the two pathogenic species on the ECM fungus. The only exception was a S. luteus gene coding for a putative chitinase (SlGH18_8356) that was found to be differentially regulated during interaction with H. irregulare compared to H. annosum.

Selection processes in simple sequence repeats suggest a correlation with their genomic location: insights from a fungal model system

BackgroundAdaptive processes shape the evolution of genomes and the diverse functions of different genomic regions are likely to have an impact on the trajectory and outcome of this evolution. The main underlying hypothesis of this study is that the evolution of Simple Sequence Repeats (SSRs) is correlated with the evolution of the genomic region in which they are located, resulting in differences of motif size, number of repeats, and levels of polymorphisms. These differences should be clearly detectable when analyzing the frequency and type of SSRs within the genome of a species, when studying populations within a species, and when comparing closely related sister taxa. By coupling a genome-wide SSR survey in the genome of the plant pathogenic fungus Heterobasidion irregulare with an analysis of intra- and interspecific variability of 39 SSR markers in five populations of the two sibling species H. irregulare and H. annosum, we investigated mechanisms of evolution of SSRs.ResultsResults showed a clear dominance of trirepeats and a selection against other repeat number, i.e. di- and tetranucleotides, both in regions inside Open Reading Frames (ORFs) and upstream 5’ untranslated region (5’UTR). Locus per locus AMOVA showed SSRs both inside ORFs and upstream 5’UTR were more conserved within species compared to SSRs in other genomic regions, suggesting their evolution is constrained by the functions of the regions they are in. Principal coordinates analysis (PCoA) indicated that even if SSRs inside ORFs were less polymorphic than those in intergenic regions, they were more powerful in differentiating species. These findings indicate SSRs evolution undergoes a directional selection pressure comparable to that of the ORFs they interrupt and to that of regions involved in regulatory functions.ConclusionsOur work linked the variation and the type of SSRs with regions upstream 5’UTR, putatively harbouring regulatory elements, and shows that the evolution of SSRs might be affected by their location in the genome. Additionally, this study provides a first glimpse on a possible molecular basis for fast adaptation to the environment mediated by SSRs.

HRM analysis provides insights on the reproduction mode and the population structure of Gnomoniopsis castaneae in Europe

Gnomoniopsis castaneae is an emergent nut rot agent of chestnut in southern Europe. To elucidate its population genetics, three simple sequence repeat (SSR) and two hypervariable markers were developed and assessed through highresolution melting (HRM) analysis on 132 isolates collected from 10 sites in Italy, France and Switzerland. High allele diversity (ranging from 0.23 to 0.40 depending on site) and number of haplotypes (49) were observed. More than 70% of the molecular variance could be accounted among isolates within sites. Multilocus analysis showed absence of linkage disequilibrium, suggesting a predominant role played by sexual reproduction and random mating. Data analyses indicated the presence of at least two putative distinct subpopulations and this was confirmed by several approaches, including analysis of shared haplotypes, multivariate and Bayesian analyses. Based on data of allelic diversity, the possibility that the pathogen could have been introduced is discussed. This work assessed the genetic variability and the sexual strategies of G. castaneae in Europe, adding useful information on the epidemiology of this fungal plant pathogen.

Understanding plant cell-wall remodelling during the symbiotic interaction between Tuber melanosporum and Corylus avellana using a carbohydrate microarray

AbstractMain conclusionA combined approach, using a carbohydrate microarray as a support for genomic data, has revealed subtle plant cell-wall remodelling duringTuber melanosporumandCorylus avellanainteraction. Cell walls are involved, to a great extent, in mediating plant–microbe interactions. An important feature of these interactions concerns changes in the cell-wall composition during interaction with other organisms. In ectomycorrhizae, plant and fungal cell walls come into direct contact, and represent the interface between the two partners. However, very little information is available on the re-arrangement that could occur within the plant and fungal cell walls during ectomycorrhizal symbiosis. Taking advantage of the Comprehensive Microarray Polymer Profiling (CoMPP) technology, the current study has had the aim of monitoring the changes that take place in the plant cell wall in Corylus avellana roots during colonization by the ascomycetous ectomycorrhizal fungus T. melanosporum. Additionally, genes encoding putative plant cell-wall degrading enzymes (PCWDEs) have been identified in the T. melanosporum genome, and RT-qPCRs have been performed to verify the expression of selected genes in fully developed C. avellana/T. melanosporum ectomycorrhizae. A localized degradation of pectin seems to occur during fungal colonization, in agreement with the growth of the ectomycorrhizal fungus through the middle lamella and with the fungal gene expression of genes acting on these polysaccharides.

Plant and fungal gene expression in mycorrhizal protocorms of the orchid Serapias vomeracea colonized by Tulasnella calospora

Little is known on the molecular bases of plant–fungal interactions in orchid mycorrhiza. We developed a model system to investigate gene expression in mycorrhizal protocorms of Serapias vomeracea colonised by Tulasnella calospora. Our recent results with a small panel of genes as indicators of plant response to mycorrhizal colonization indicate that genes related with plant defense were not significantly up-regulated in mycorrhizal tissues. Here, we used laser microdissection to investigate whether expression of some orchid genes was restricted to specific cell types. Results showed that SvNod1, a S. vomeracea nodulin-like protein containing a plastocyanin-like domain, is expressed only in protocorm cells containing intracellular fungal hyphae. In addition, we investigated a family of fungal zinc metallopeptidases (M36). This gene family has expanded in the T. calospora genome and RNA-Seq experiments indicate that some members of the M36 metallopeptidases family are differentially regulated in orchid mycorrhizal protocorms.

ANALYSIS OF GENOTYPIC DIVERSITY PROVIDES A FIRST GLIMPSE ON THE PATTERNS OF SPREAD OF THE WOOD DECAY FUNGUS PERENNIPORIA FRAXINEA IN AN URBAN PARK IN NORTHERN ITALY

Perenniporia fraxinea is a fungal pathogen causing wood decay in roots and bole of a wide variety of broadleaf tree species. Despite its ecological importance, little is know about the infection biology of this fungus and in particular of its ability to infect trees through the mycelia growth through root contacts. To clarify its spreading mechanisms, a genetic analysis of 20 P. fraxinea isolates obtained from basidiomata collected from closely located Robinia pseudoacacia and Quercus robur trees in the Vernavola Urban Park (Pavia, Italy) and in surrounding areas was performed. Random Amplified Microsatellites (RAMs) fingerprinting was conducted allowing to distinguish 19 different haplotypes. High intrapopulation diversity was confirmed by somatic incompatibility tests (SITs), which were performed by dual- culturing isolates in vitro in all possible combinations, resulting in detection of 16 compatibility groups. These results, together with Non-metric MultiDimensional Scaling (NMDS) analysis on genetic data, suggest that spread through root contacts is unlikely for P. fraxinea. In addition, a significant negative correlation between spatial distribution and kinship coefficients was observed in isolates from the Vernavola Urban Park, suggesting a limited dispersal potential of P. fraxinea basidiospores. This report provides a first glimpse of the primary mechanisms of spread of P. fraxinea.