The multifunctional beta-oxidation enzyme is required for full symptom development by the biotrophic maize pathogen Ustilago maydis

The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen Ustilago maydis is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in U. maydis and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the mfe2 gene encoding the multifunctional enzyme that catalyzes the second and third reactions in beta-oxidation of fatty acids in peroxisomes. The growth of the strains defective in mfe2 was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The mfe2 gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation, mfe2 mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the mfe2 gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of U. maydis.     

Complex mitonuclear interactions and metabolic costs of mating in male seed beetles

The lack of evolutionary response to selection on mitochondrial genes through males predicts the evolution of nuclear genetic influence on male‐specific mitochondrial function, for example by gene duplication and evolution of sex‐specific expression of paralogs involved in metabolic pathways. Intergenomic epistasis may therefore be a prevalent feature of the genetic architecture of male‐specific organismal function. Here, we assess the role of mitonuclear genetic variation for male metabolic phenotypes [metabolic rate and respiratory quotient (RQ)] associated with ejaculate renewal, in the seed beetle Callosobruchus maculatus, by assaying lines with crossed combinations of distinct mitochondrial haplotypes and nuclear lineages. We found a significant increase in metabolic rate following mating relative to virgin males. Moreover, processes associated with ejaculate renewal showed variation in metabolic rate that was affected by mitonuclear interactions. Mitochondrial haplotype influenced mating‐related changes in RQ, but this pattern varied over time. Mitonuclear genotype and the energy spent during ejaculate production affected the weight of the ejaculate, but the strength of this effect varied across mitochondrial haplotypes showing that the genetic architecture of male‐specific reproductive function is complex. Our findings unveil hitherto underappreciated metabolic costs of mating and ejaculate renewal, and provide the first empirical demonstration of mitonuclear epistasis on male reproductive metabolic processes.     

Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

An understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogen Ustilago maydis. However, mfe2 mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in the had1 gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog, had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of an mfe2Δ mutant. We also show that short-chain fatty acids induce cell death in U. maydis and that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms by U. maydis that includes potential metabolic contributions to proliferation in planta and an effect on virulence-related morphogenesis.     

Mitochondrial β‐oxidation regulates organellar integrity and is necessary for conidial germination and invasive growth in Magnaporthe oryzae

Fatty acids stored as triglycerides, an important source of cellular energy, are catabolized through β‐oxidation pathways predicted to occur both in peroxisomes and mitochondria in filamentous fungi. Here, we characterize the function of Enoyl‐CoA hydratase Ech1, a mitochondrial β‐oxidation enzyme, in the model phytopathogen Magnaporthe oryzae. Ech1 was found to be essential for conidial germination and viability of older hyphae. Unlike wild‐type Magnaporthe, the ech1Δ failed to utilize C14 fatty acid and was partially impeded in growth on C16 and C18 fatty acids. Surprisingly, loss of β‐oxidation led to significantly altered mitochondrial morphology and integrity with ech1Δ showing predominantly vesicular/punctate mitochondria in contrast to the fused tubular network in wild‐type Magnaporthe. The ech1Δ appressoria were aberrant and displayed reduced melanization. Importantly, we show that the significantly reduced ability of ech1Δ to penetrate the host and establish therein is a direct consequence of enhanced sensitivity of the mutant to oxidative stress, as the defects could be remarkably reversed through exogenous antioxidants. Overall, our comparative analyses reveal that peroxisomal lipid catabolism is essential for appressorial function of host penetration, whereas mitochondrial β‐oxidation primarily contributes to conidial viability and maintenance of redox homeostasis during host colonization by Magnaporthe.     

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.     

The relationship between respiration rate and peroxidase activities in maize root mitochondria

In the present study we provide the evidence of different respiration rates and peroxidase activities in maize (Zea mays L.) mitochondria isolated from germinated seeds and roots of 2-week-old seedlings. The negative relationships between mitochondrial respiration rate measured with NADH as substrate and activities of peroxidases that oxidized NADH in both oxidative and peroxidative cycles were found. The possible role of peroxidase in the regulation of reactive oxygen species metabolism in expense of NADH oxidation was hypothesized.     

Changes in glutathione-dependent redox status and mitochondrial energetic strategies are part of the adaptive response during the filamentation process in Candida albicans

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases called candidiasis. Its ability to grow in various morphological forms, such as unicellular budding yeast, filamentous pseudohyphae and hyphae, contributes to its survival in the diverse microenvironments it encounters in the host. During infection in vivo, C. albicans is faced with high levels of reactive oxygen species (ROS) generated by phagocytes, and the thiol-dependent redox status of the cells reflects their levels of oxidative stress. We investigated the role of glutathione during the transition between the yeast and hyphal forms of the pathogen, in relation to possible changes in mitochondrial bioenergetic pathways. Using various growth media and selective mutations affecting the filamentation process, we showed that C. albicans filamentation was always associated with a depletion of intracellular glutathione levels. Moreover, the induction of hypha formation resulted in general changes in thiol metabolism, including the oxidation of cell surface − SH groups and glutathione excretion. Metabolic adaptation involved tricarboxylic acid (TCA) cycle activation, acceleration of mitochondrial respiration and a redistribution of electron transfer pathways, with an increase in the contribution of the alternative oxidase and rotenone-insensitive dehydrogenase. Changes in redox status and apparent oxidative stress may be necessary to the shift to adaptive metabolic pathways, ensuring normal mitochondrial function and adenosine triphosphate (ATP) levels. The consumption of intracellular glutathione levels during the filamentation process may thus be the price paid by C. albicans for survival in the conditions encountered in the host.     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.