Kirstin Feussner

Metabolic priming by a secreted fungal effector

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves of Arabidopsis during Extended Darkness

All photosynthetic organisms face the difficulty of maintaining cellular metabolism in the absence of photosynthetic active radiation during the night. Although many consuming metabolic pathways (e.g., fatty acid synthesis) are only active in the light, plant cells still require basic levels of metabolic energy and reductive power during the night for sustained growth and development.Fatty acid β-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core β-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal β-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly α-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable α-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of α-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and β-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal β-oxidation plays a major role in dark-treated plants after depletion of starch reserves.

A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize

The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin–proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses. DOI: http://dx.doi.org/10.7554/eLife.01355.001

Verticillium longisporum infection affects the leaf apoplastic proteome, metabolome, and cell wall properties in Arabidopsis thaliana.

Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes.

The COP9 signalosome mediates transcriptional and metabolic response to hormones, oxidative stress protection and cell wall rearrangement during fungal development.

The COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.

Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana

The bioactive lipid ceramide is produced by the enzyme ceramide synthase, which exists in several isoforms in most eukaryotic organisms. Here, we investigated functional differences between the three ceramide synthase isoforms in Arabidopsis thaliana. The biochemical properties of the three ceramide synthases were investigated by comparing lipid profiles of yeast strains expressing LOH1, LOH2 or LOH3 with those of wild-type and loh1, loh2 and loh3 knockout plants. Expression profiles of the ceramide synthases and of the pathogenesis-related gene PR-1 were investigated by real-time PCR. Each ceramide synthase isoform showed a characteristic preference regarding acyl-CoA chain length as well as sphingoid base hydroxylation, which matches the pattern of ceramide and glucosylceramide species found in leaves. After extended culture under short-day conditions, loh1 plants showed spontaneous cell death accompanied by enhanced expression of PR-1. The levels of free trihydroxy sphingoid bases as well as ceramide and glucosylceramide species with C(16) fatty acid were significantly elevated while species with C(20) -C(28) fatty acids were reduced. These data suggest that spontaneous cell death in the loh1 line is triggered either by the accumulation of free trihydroxy sphingoid bases or ceramide species with C(16) fatty acid.

The Alphabet of Galactolipids in Arabidopsis thaliana.

Galactolipids constitute the major lipid class in plants. In recent years oxygenated derivatives of galactolipids have been detected. They are discussed as signal molecules during leaf damage, since they accumulate in wounded leaves in high levels. Using different analytical methods such as nuclear magnetic resonance, infra-red spectroscopy, and high performance liquid chromatography/mass spectrometry (HPLC/MS) earlier reports focused on the analysis of either oxidized or non-oxidized species and needed high levels of analytes. Here, we report on the analysis of the galactolipid subfraction of the Arabidopsis leaf lipidome by an improved HPLC/MS2-based method that is fast, robust, and comparatively simple in its performance. Due to a combination of phase partitioning, solid phase fractionation, liquid chromatography, and MS2 experiments this method has high detection sensitivity and requires only low amounts of plant material. With this method 167 galactolipid species were detected in leaves of Arabidopsis thaliana. Out of these 79 being newly described species. From all species the head group and acyl side chains were identified via MS2 experiments. Moreover, the structural identification was supported by HPLC/time-of-flight (TOF)-MS and gas chromatography (GC)/MS analysis. The quantification of different galactolipid species that accumulated 30 min after a mechanical wounding in A. thaliana leaves showed that the oxidized acyl side chains in galactolipids are divided into 65% cyclopentenones, 27% methyl-branched ketols, 3.8% hydroperoxides/straight-chain ketols, 2.0% hydroxides, and 2.6% phytoprostanes. In comparison to the free cyclopentenone derivatives, the esterified forms occur in a 149-fold excess supporting the hypothesis that galactolipids might function as storage compounds for cyclopentenones. Additional analysis of the ratio of non-oxidized to oxidized galactolipid species in leaves of wounded plants was performed resulting in a ratio of 2.0 in case of monogalactosyl diacylglycerol (MGD), 8.1 in digalactosyl diacylglycerol (DGD), and 0.6 in the acylated MGD. This indicates that galactolipid oxidation is a major and rapid metabolic process that occurs class specific.

Phosphoenolpyruvate Provision to Plastids Is Essential for Gametophyte and Sporophyte Development in Arabidopsis thaliana

This work examines double mutants defective in providing phosphoenolpyruvate (PEP) to the plastids and finds that PEP is essential for multiple steps in plant development, reflecting the central importance of PEP in multiple plant metabolic pathways, including synthesis of aromatic amino acids, lipids, flavonoids, and lignin. Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/−)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/−) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/−) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

Two Pathways of Sphingolipid Biosynthesis Are Separated in the Yeast Pichia pastoris

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.

Phloem-Specific Expression of Yang Cycle Genes and Identification of Novel Yang Cycle Enzymes in Plantago and Arabidopsis

This work reports on the identification and characterization of enzymes of the Yang cycle, which recycle 5-methylthioadenosine to Met. It shows that the genes for all Yang cycle enzymes are expressed primarily in the phloem. One of the identified enzymes is a trimeric protein composed of subunits encoded by up to three separate genes in other organisms. The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis.