Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

Handbook of British fungi, with full descriptions of all the species, and illustrations of the genera. By M. C. Cooke, M.A.

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