Jan Schmid

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

A simple method for extraction of fungal genomic DNA

T. H. AL‐SAMARRAI and J. SCHMID.2000.We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze‐dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8–32 μg of high molecular weight DNA per 30 mg of freeze‐dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow‐growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min−1 in the presence of CsCl. The modified method yielded 1·5–2 μg of high molecular weight DNA per colony.

Growth of Epichloe/Neotyphodium and p-endophytes in leaves of Lolium and Festuca grasses

Epichloe spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloe and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloe/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloe/Neotyphodium and host cells in natural associations were observed.

Dual roles for calcium ions in apical growth of Neurospora crassa

We report initial attempts to define the role of Ca2+ in the polarized extension of Neurospora crassa. Growth of the organism was diminished in media containing less than 1 mM-Ca2+; extension was more severely impaired than biomass synthesis, resulting in the formation of stubby, bulbous hyphae, even of spherical cells. Reduced extension and abnormal morphology were correlated with the loss of surface-bound Ca2+, probably associated with the cell wall. Intracellular Ca2+ may be represented by material that fluoresces brightly in the presence of chlortetracycline. Punctate fluorescent bodies and diffuse fluorescence were both arrayed in a longitudinal gradient, maximum apically. Addition of the calcium ionophore A23187 induced dissipation of the fluorescence; concurrently, the hyphae lost as much as one half of their Ca2+ content. Extension continued almost unabated, but multiple branches quickly emerged from the apex. The observations suggest that a cytoplasmic Ca2+ gradient is not required for polarized extension, but may play a role in ensuring the dominance of the apex.

Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection

Epidemiological studies, using the probe Ca3, have shown that in a given patient population a single cluster of genetically related Candida albicans isolates usually predominates. The authors have investigated whether these local clusters are part of a single group, geographically widespread and highly prevalent as an aetiological agent of various types of candidiasis. An unrooted neighbour-joining tree of 266 infection-causing C. albicans isolates (each from a different individual) from 12 geographical regions in 6 countries was created, based on genetic distances generated by Ca3 fingerprinting. Thirty-seven per cent of all isolates formed a single genetically homogeneous cluster (cluster A). The remainder of isolates were genetically diverse. Using the maximum branch length within cluster A as a cut-off, they could be divided into 37 groups, whose prevalence ranged between 0.3% and 9%. Strains from cluster A were highly prevalent in all but one geographical region, with a mean prevalence across all regions of 41%. When isolates were separated into groups based on patient characteristics or type of infection, strains from cluster A had a prevalence exceeding 27% in each group, and their mean prevalence was 43% across all patient characteristics. These data provide evidence that cluster A constitutes a general-purpose genotype, which is geographically widespread and acts as a predominant aetiological agent of all forms of candidiasis in all categories of patients surveyed.

Transcellular ion currents and extension ofNeurospora crassa hyphae

SummaryHyphae ofNeurospora crassa, like many other tipgrowing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. In order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. Omission of K+ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. Omission of glucose markedly reduced both inward and outward currents; addition of sodium azide virtually abolished the flow of electric current. Growing hyphae also generate a longitudinal pH gradient: the medium surrounding the apex is slightly more alkaline than the bulk phase, while medium adjacent to the trunk turns acid. The results suggest thatNeurospora hyphae generate a proton current; protons are expelled distally by the H+-ATPase and return into the apical region by a number of pathways, including the symport of protons with phosphate and potassium ions. Calcium influx may also contribute to the electric current that enters the apical region. There seems to be no simple obligatory linkage between the intensity of the transcellular electric current and the rate of hyphal extension. Calcium ions, however, are required in micromolar concentrations for extensions and morphogenesis of hyphal tips.

In planta regulation of extension of an endophytic fungus and maintenance of high metabolic rates in its mycelium in the absence of apical extension.

ABSTRACT The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfectedL. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the myceliums metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the myceliums metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non‐synonymous single nucleotide polymorphisms (NS‐SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site‐directed mutagenesis showed that the TM‐12 NS‐SNPs, and principally the G1473A NS‐SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele‐specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy–Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne.

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.

Genetic Relationship between Human and Animal Isolates of Candida albicans

ABSTRACT Analyzing Candida albicans isolates from different human and animal individuals by Ca3 fingerprinting, we obtained no evidence for host-specific genotypes and for the existence of species-specific lineages, even though a certain degree of separation between human and animal isolates was found. Therefore, animals could potentially serve as reservoirs for human Candida infection.