Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans.

Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Comparative transcript profiling of Candida albicans and Candida dubliniensis identifies SFL2, a C. albicans gene required for virulence in a reconstituted epithelial infection model.

Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. DeltaDeltasfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the DeltaDeltasfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, DeltaDeltasfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures.

Epichloë endophytes (holomorphic Epichloë spp. and anamorphic Neotyphodium spp.) are systemic, often heritable symbionts of cool-season grasses (subfamily Pooideae). Many epichloae provide protection to their hosts by producing anti-insect compounds. Among these are the loline alkaloids (LA), which are toxic and deterrent to a broad range of herbivorous insects but not to mammalian herbivores. LOL, a gene cluster containing nine genes, is associated with LA biosynthesis. We investigated coordinate regulation between LOL-gene expression and LA production in minimal medium (MM) cultures of Neotyphodium uncinatum. Expression of all LOL genes significantly fit temporal quadratic patterns during LA production. LOL-gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached at close to the time of most rapid LA accumulation, and gene expression declined to a very low level as amounts of LA plateaued. Temporal expression profiles of the nine LOL genes were tightly correlated with each other, but not as tightly correlated with proC and metE (genes for biosynthesis of precursor amino acids). Furthermore, the start days and peak days of expression significantly correlated with the order of the LOL-cluster genes in the genome. Hierarchical cluster analysis indicated three pairs of genes-lolA and lolC, lolO and lolD, and lolT and lolE-expression of which was especially tightly correlated. Of these, lolA and lolC tended to be expressed early, and lolT and lolE tended to be expressed late, in keeping with the putative roles of the respective gene products in the LA-biosynthesis pathway. Several common transcriptional binding sites were discovered in the LOL upstream regions. However, low expression of P(lolC2)uidA and P(lolA2)uidA in N. uncinatum transformants suggested induced expression of LOL genes might be subject to position effect at the LOL locus.

Symbioses of grasses with seedborne fungal endophytes.

Grasses (family Poaceae) and fungi of the family Clavicipitaceae have a long history of symbiosis ranging in a continuum from mutualisms to antagonisms. This continuum is particularly evident among symbioses involving the fungal genus Epichloe (asexual forms = Neotyphodium spp.). In the more mutualistic symbiota, the epichloe endophytes are vertically transmitted via host seeds, and in the more antagonistic symbiota they spread contagiously and suppress host seed set. The endophytes gain shelter, nutrition, and dissemination via host propagules, and can contribute an array of host fitness enhancements including protection against insect and vertebrate herbivores and root nematodes, enhancements of drought tolerance and nutrient status, and improved growth particularly of the root. In some systems, such as the tall fescue N. coenophialum symbioses, the plant may depend on the endophyte under many natural conditions. Recent advances in endophyte molecular biology promise to shed light on the mechanisms of the symbioses and host benefits.

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis.

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum.

Loline alkaloids are produced by mutualistic fungi symbiotic with grasses, and they protect the host plants from insects. Here we identify in the fungal symbiont, Neotyphodium uncinatum, two homologous gene clusters (LOL-1 and LOL-2) associated with loline-alkaloid production. Nine genes were identified in a 25-kb region of LOL-1 and designated (in order) lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, and lolE-1. LOL-2 contained the homologs lolC-2 through lolE-2 in the same order and orientation. Also identified was lolF-2, but its possible linkage with either cluster was undetermined. Most lol genes were regulated in N. uncinatum and N. coenophialum, and all were expressed concomitantly with loline-alkaloid biosynthesis. A lolC-2 RNA-interference (RNAi) construct was introduced into N. uncinatum, and in two independent transformants, RNAi significantly decreased lolC expression (P < 0.01) and loline-alkaloid accumulation in culture (P < 0.001) compared to vector-only controls, indicating involvement of lolC in biosynthesis of lolines. The predicted LolU protein has a DNA-binding site signature, and the relationships of other lol-gene products indicate that the pathway has evolved from various different primary and secondary biosynthesis pathways.

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants.

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.

Primer on the Immune System.

The human body regularly encounters and combats many pathogenic organisms and toxic molecules. Its ensuing responses to these disease-causing agents involve two interrelated systems: innate immunity and adaptive (or acquired) immunity. Innate immunity is active at several levels, both at potential points of entry and inside the body (see figure). For example, the skin represents a physical barrier preventing pathogens from invading internal tissues. Digestive enzymes destroy microbes that enter the stomach with food. Macrophages and lymphocytes, equipped with molecular detectors, such as Toll-like receptors (TLRs), which latch onto foreign structures and activate cellular defenses, patrol the inside of the body. These immune cells sense and devour microbes, damaged cells, and other foreign materials in the body. Certain proteins in the blood (such as proteins of the complement system and those released by natural killer cells, along with antimicrobial host-defense peptides) attach to foreign organisms and toxins to initiate their destruction.

Simplified extraction of ergovaline and peramine for analysis of tissue distribution in endophyte-infected grass tillers.

An improved extraction and cleanup procedure for quantitative analysis of ergovaline in Neotyphodium-infected grass tissues by high-performance liquid chromatography was developed, utilizing aqueous 2-propanol-lactic acid as extraction solvent. Losses of sample material and time requirements were significantly reduced, handling procedures simplified, and ergovaline and internal standard ergotamine recovered with similar efficiency from extracts. Analyses can be carried out on very small amounts (2-5 mg of dry weight) of samples and another endophyte-alkaloid, peramine, determined in the same extracts. Calibration curves with 2-propanol-lactic acid were linear over the range 0.004-0.938 microM ergovaline (= 2-500 ng/mL) in extracts, corresponding to 0.04-10 microg/g in samples. The distribution of ergovaline in the plant was extremely heterogeneous, indicating low in-planta mobility and strong regulation of accumulation by the internal plant environment. In contrast, peramine was much more uniformly distributed. These results clearly demonstrate very large differences in the tissue specificities of ergovaline and peramine.