Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels.

Fungi lack the entire animal core apoptotic machinery. Nevertheless, regulated cell death with apoptotic markers occurs in multicellular as well as in unicellular fungi and is essential for proper fungal development and stress adaptation. The discrepancy between appearance of an apoptotic-like regulated cell death (RCD) in the absence of core apoptotic machinery is further complicated by the fact that heterologous expression of animal apoptotic genes in fungi affects fungal RCD. Here we describe the role of BcMcl, a methyl isocitrate lyase from the plant pathogenic fungus Botrytis cinerea, in succinate metabolism, and the connection of succinate with stress responses and cell death. Over expression of bcmcl resulted in elevated tolerance to oxidative stress and reduced levels of RCD, which were associated with accumulation of elevated levels of succinate. Deletion of bcmcl had almost no effect on fungal development or stress sensitivity, and succinate levels were unchanged in the deletion strain. Gene expression experiments showed co-regulation of bcmcl and bcicl (isocitrate lyase); expression of the bcicl gene was enhanced in bcmcl deletion and suppressed in bcmcl over expression strains. External addition of succinate reproduced the phenotypes of the bcmcl over expression strains, including developmental defects, reduced virulence, and improved oxidative stress tolerance. Collectively, our results implicate mitochondria metabolic pathways, and in particular succinate metabolism, in regulation of fungal stress tolerance, and highlight the role of this onco-metabolite as potential mediator of fungal RCD.

Production and Role of Hormones During Interaction of Fusarium Species With Maize (Zea mays L.) Seedlings.

It has long been known that hormones affect the interaction of a phytopathogen with its host plant. The pathogen can cause changes in plant hormone homeostasis directly by affecting biosynthesis or metabolism in the plant or by synthesizing and secreting the hormone itself. We previously demonstrated that pathogenic fungi of the Fusarium species complex are able to produce three major types of hormones: auxins, cytokinins, and gibberellins. In this work, we explore changes in the levels of these hormones in maize and mango plant tissues infected with Fusarium. The ability to produce individual phytohormones varies significantly across Fusarium species and such differences likely impact host specificity inducing the unique responses noted in planta during infection. For example, the production of gibberellins by F. fujikuroi leads to elongated rice stalks and the suppression of gibberellin biosynthesis in plant tissue. Although all Fusarium species are able to synthesize auxin, sometimes by multiple pathways, the ratio of its free form and conjugates in infected tissue is affected more than the total amount produced. The recently characterized unique pathway for cytokinin de novo synthesis in Fusarium appears silenced or non-functional in all studied species during plant infection. Despite this, a large increase in cytokinin levels was detected in F. mangiferae infected plants, caused likely by the up-regulation of plant genes responsible for their biosynthesis. Thus, the accumulation of active cytokinins may contribute to mango malformation of the reproductive organs upon infection of mango trees. Together, our findings provide insight into the complex role fungal and plant derived hormones play in the fungal-plant interactions.

Genetic alteration of UDP-rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP-KDG that adversely affects development and pathogenicity.

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs.

Comparative "Omics" of the Fusarium fujikuroi Species Complex Highlights Differences in Genetic Potential and Metabolite Synthesis.

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.

Translocation from nuclei to cytoplasm is necessary for anti A-PCD activity and turnover of the Type II IAP BcBir1.

Type II inhibitors of apoptosis (IAPs) belong to a subgroup of IAP-related proteins. While IAPs are restricted to animals, Type II IAPs are found in other phyla, including fungi. BcBir1, a Type II IAP from Botrytis cinerea has anti apoptotic-like programmed cell death (A-PCD) activity, which is important for pathogenicity of this fungus. Here we report on the role of sub-cellular localization of BcBir1 in protein turnover and anti A-PCD activity. Expression of BcBir1 in Saccharomyces cerevisiae had no effect on sensitivity of the yeast cells to A-PCD-inducing conditions, whereas expression of a truncated N' part reduced sensitivity of the cells to these conditions. The full-length BcBir1 protein was detected only in the yeast nucleus, whereas the N' part was observed both in the nucleus and cytoplasm. In B. cinerea, BcBir1 was mainly nuclear under optimal conditions, whereas under A-PCD-inducing conditions it shuttled to the cytoplasm and then it was completely degraded. Collectively, our results show that anti A-PCD activity of BcBir1 occurs in the cytoplasm, the C' end mediates regulation of steady state level of BcBir1 in the nucleus, and the N' end mediates anti A-PCD activity as well as fast degradation of BcBir1 in the cytoplasm.

Identification and functional characterization of indole-3-acetamide-mediated IAA biosynthesis in plant-associated Fusarium species.

The plant hormone indole-3-acetic acid (IAA) can be synthesized from tryptophan via the intermediate indole-3-acetamide (IAM). The two genes, IaaM (encoding tryptophan monooxygenase) and IaaH (encoding indole-3-acetamide hydrolase) that constitute the IAM pathway have been described in plant-associated bacteria. We have identified putative homologs of the bacterial IaaM and IaaH genes in four Fusarium species -Fusarium proliferatum, Fusarium verticillioides, Fusarium fujikuroi, and Fusarium oxysporum. In all four species the two genes are organized next to each other in a head to head orientation and are separated by a short non-coding region. However, the pathway is fully functional only in the orchid endophytic strain F. proliferatum ET1, which produces significant amounts of IAM and IAA. Minor amounts of IAM are produced by the corn pathogen F. verticillioides strain 149, while in the two other species, the rice pathogen F. fujikuroi strain m567 and the tomato pathogen F. oxysporum f. sp. lycopersici strain 42-87 the IAM pathway is inactive. Deletion of the entire gene locus in F. proliferatum ET1 resulted in drastic reduction of IAA production. Conversely, transgenic strains of F. fujikuroi over-expressing the F. proliferatum IAM genes produced elevated levels of both IAM and IAA. Analysis of the intergenic promoter region in F. proliferatum showed that transcriptional activation in direction of the IaaH gene is about 3-fold stronger than in direction of the IaaM gene. The regulation of the IAM genes and the limiting factors of IAA production via the IAM pathway are discussed.

Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling.

Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391-397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the 'substrate-trapping' mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.