Reprogramming of the apoplast metabolome of Lolium perenne upon infection with the mutualistic symbiont Epichloë festucae

SummaryEpichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

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Chitin Deacetylases are Required for Epichloë festucae Endophytic Cell Wall Remodelling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-20-0347-r ◽

2021 ◽

Author(s):

Nazanin Noorifar ◽

Matthew Savoian ◽

Arvina Ram ◽

Yonathan Lukito ◽

Berit Hassing ◽

...

Keyword(s):

Cell Wall ◽

Lolium Perenne ◽

Molecular Probes ◽

Symbiotic Association ◽

In Planta ◽

Intercellular Spaces ◽

Free Living ◽

Chitin Deacetylase ◽

Symbiotic Interaction ◽

Epichloë Festucae

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network, and also grows as an epiphyte. However, little is known about the cell wall remodelling mechanisms required to avoid host defence and maintain intercalary growth within the host. Here we use a suite of molecular probes to show that the E. festucae cell wall is remodelled by conversion of chitin to chitosan during infection of L. perenne seedlings as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth the cell wall is remodelled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalysed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.

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VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae

Eukaryotic Cell ◽

10.1128/ec.00034-14 ◽

2014 ◽

Vol 14 (1) ◽

pp. 13-24 ◽

Cited By ~ 14

Author(s):

Jennifer T. Niones ◽

Daigo Takemoto

Keyword(s):

Transcription Factor ◽

Antifungal Activity ◽

Critical Role ◽

Antifungal Compound ◽

Symbiotic Association ◽

Wild Type ◽

Exogenous Dna ◽

Content Type ◽

Type Isolate ◽

Epichloë Festucae

ABSTRACT Symbiotic association of epichloae endophytes ( Epichloë/Neotyphodium species) with cool-season grasses of the subfamily Pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. While the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little is known about the antibiosis mechanism against grass pathogens by the mutualistic endophyte. In this study, an Epichloë festucae mutant defective in antimicrobial substance production was isolated by a mutagenesis approach. In an isolated mutant that had lost antifungal activity, the exogenous DNA fragment was integrated into the promoter region of the vibA gene, encoding a homologue of the transcription factor VIB-1. VIB-1 in Neurospora crassa is a regulator of genes essential in vegetative incompatibility and promotion of cell death. Here we show that deletion of the vibA gene severely affected the antifungal activity of the mutant against the test pathogen Drechslera erythrospila . Further analyses showed that overexpressing vibA enhanced the antifungal activity of the wild-type isolate against test pathogens. Transformants overexpressing vibA showed an inhibitory activity on test pathogens that the wild-type isolate could not. Moreover, overexpressing vibA in a nonantifungal E. festucae wild-type Fl1 isolate enabled the transformant to inhibit the mycelial and spore germination of D. erythrospila . These results demonstrate that enhanced expression of vibA is sufficient for a nonantifungal isolate to obtain antifungal activity, implicating the critical role of VibA in antifungal compound production by epichloae endophytes.

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Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504154112 ◽

2015 ◽

Vol 112 (17) ◽

pp. 5533-5538 ◽

Cited By ~ 81

Author(s):

Manuel Benedetti ◽

Daniela Pontiggia ◽

Sara Raggi ◽

Zhenyu Cheng ◽

Flavio Scaloni ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Inducible Promoter ◽

Spectrometric Analysis ◽

In Planta ◽

Gene Encoding ◽

Polygalacturonase Inhibiting Protein ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP–PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

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Expression of Erwinia chrysanthemi Pectinase Genes pelI, pelL, and pelZ During Infection of Potato Tubers

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.10.845 ◽

1999 ◽

Vol 12 (10) ◽

pp. 845-851 ◽

Cited By ~ 12

Author(s):

Sylwia Jafra ◽

Izabela Figura ◽

Nicole Hugouvieux-Cotte-Pattat ◽

Ewa Lojkowska

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Pectate Lyase ◽

Soft Rot ◽

Erwinia Chrysanthemi ◽

Potato Tubers ◽

Wild Type ◽

In Planta ◽

Gene Encoding ◽

Glucuronidase Activity

Erwinia chrysanthemi mutants, containing transcriptional fusions of one of the minor pectate lyase genes (pelI, pelL, pelZ) with the reporter gene encoding β-glucuronidase activity, were studied for their ability to cause disease symptoms and to synthesize pectinases after inoculation of potato tubers. The strains affected in pelI and pelL genes displayed reduced virulence on potato tubers, demonstrating the important role of these isoenzymes in soft rot disease. Inactivation of the pelZ gene slightly influences the ability to macerate. Analysis of the bacterial population showed rapid multiplication of bacteria during infection. Similar kinetics of growth were observed for all mutants and for the wild-type strain. Comparison of the mutants and the wild-type strain showed that the pelI, pelL, and pelZ mutants synthesized reduced levels of Pels. The expression of pelZ is fivefold higher in planta than in bacterial cultures. In contrast, both pelI and pelL are highly (10-fold factor) induced in planta, which is characteristic of the plant-inducible pectate lyases.

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Systems level modelling of metabolism in fungal endophytes - implications for the symbiosis with ryegrass

NZGA: Research and Practice Series ◽

10.33584/rps.13.2006.3049 ◽

2007 ◽

Vol 13 ◽

pp. 203-206

Author(s):

Rajiv Chaturvedi ◽

Tanya Soboleva ◽

Linda Johnson ◽

Anthony Parsons ◽

Susanne Rasmussen

Keyword(s):

Amino Acids ◽

Metabolic Network ◽

Lolium Perenne ◽

Fungal Endophytes ◽

Gene Replacement ◽

Symbiotic Association ◽

Enzymatic Reactions ◽

In Planta ◽

Network Modelling ◽

A Genome

We used constraint based stoichiometric modelling of metabolic fluxes in EpichloÃ‘' festucae (FL1), a targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae, and the symbiotic association of these endophytic fungi and their host Lolium perenne. SidF encodes an excreted ironchelating siderophore and the sidF knockouts (KO) are impaired in their ability to take up iron. After constructing the metabolic network at a genome scale, we applied constraints on enzymatic reactions that require iron as co-factor to study the variations in metabolic network capabilities of the siderophore mutant versus wildtype, in culture and in planta. We compared fluxes calculated for the production of amino acids with observed concentrations of these amino acids in planta. We report a counter-intuitive result from considering metabolism on a systems level in our models. Keywords: stoichiometric metabolic network modelling, flux balance analysis, symbiosis, Neotyphodium lolii, Lolium perenne, EpichloÃ‘' festucae.

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Redox Regulation of an AP-1-Like Transcription Factor, YapA, in the Fungal Symbiont Epichloë festucae

Eukaryotic Cell ◽

10.1128/ec.00129-13 ◽

2013 ◽

Vol 12 (10) ◽

pp. 1335-1348 ◽

Cited By ~ 17

Author(s):

Gemma M. Cartwright ◽

Barry Scott

Keyword(s):

Oxidative Stress ◽

Transcription Factor ◽

Redox Regulation ◽

Bzip Transcription Factor ◽

Wild Type ◽

In Planta ◽

Content Type ◽

Epichloë Festucae ◽

Whole Plant ◽

Cellular Phenotypes

ABSTRACTOne of the central regulators of oxidative stress inSaccharomyces cerevisiaeis Yap1, a bZIP transcription factor of the AP-1 family. In unstressed cells, Yap1 is reduced and cytoplasmic, but in response to oxidative stress, it becomes oxidized and accumulates in the nucleus. To date, there have been no reports on the role of AP-1-like transcription factors in symbiotic fungi. An ortholog of Yap1, named YapA, was identified in the genome of the grass symbiontEpichloë festucaeand shown to complement anS. cerevisiaeΔyap1mutant. Hyphae of theE. festucaeΔyapAstrain were sensitive to menadione and diamide but resistant to H2O2, KO2, andtert-butyl hydroperoxide (t-BOOH). In contrast, conidia of the ΔyapAstrain were very sensitive to H2O2and failed to germinate. Using a PcatA-eGFPdegron-tagged reporter, YapA was shown to be required for expression of a spore-specific catalase gene,catA. Although YapA-EGFP localized to the nucleus in response to host reactive oxygen species during seedling infection, there was no difference in whole-plant and cellular phenotypes of plants infected with the ΔyapAstrain compared to the wild-type strain. Homologs of theS. cerevisiaeandSchizosaccharomyces pomberedox-sensing proteins (Gpx3 and Tpx1, respectively) did not act as redox sensors for YapA inE. festucae. In response to oxidative stress, YapA-EGFP localized to the nuclei ofE. festucaeΔgpxC, ΔtpxA, and ΔgpxCΔtpxAcells to the same degree as that in wild-type cells. These results show thatE. festucaehas a robust system for countering oxidative stress in culture andin plantabut that Gpx3- or Tpx1-like thiol peroxidases are dispensable for activation of YapA.

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Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum

Molecular Biology of the Cell ◽

10.1091/mbc.e18-02-0132 ◽

2019 ◽

Vol 30 (4) ◽

pp. 453-466 ◽

Cited By ~ 7

Author(s):

Petros Batsios ◽

Hellen C. Ishikawa-Ankerhold ◽

Heike Roth ◽

Michael Schleicher ◽

Catherine C. L. Wong ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Three Dimensional ◽

Model Organism ◽

Live Cell ◽

Actin Binding ◽

Spectrometric Analysis ◽

Wild Type ◽

Gene Encoding ◽

Adhesion Sites

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.

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Tolerance to, and Uptake and Degradation of 2,4,6-Trinitrotoluene (TNT) are Enhanced by the Expression of a Bacterial Nitroreductase Gene in Arabidopsis thaliana

Zeitschrift für Naturforschung C ◽

10.1515/znc-2005-3-412 ◽

2005 ◽

Vol 60 (3-4) ◽

pp. 272-278 ◽

Cited By ~ 34

Author(s):

Mami Kurumata ◽

Misa Takahashi ◽

Atsushi Sakamoto ◽

Juan L. Ramos ◽

Ales Nepovim ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Transgenic Plants ◽

Degradation Products ◽

Plant Genome ◽

Pcr Analysis ◽

Wild Type ◽

In Planta ◽

Gene Encoding ◽

Plant Body ◽

Wide Range

Abstract Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

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Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽

10.21273/hortsci.33.3.519d ◽

1998 ◽

Vol 33 (3) ◽

pp. 519d-519 ◽

Cited By ~ 4

Author(s):

Kenneth R. Schroeder ◽

Dennis P. Stimart

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Height ◽

Dry Weight ◽

Nicotiana Alata ◽

Wild Type ◽

Gene Encoding ◽

Delayed Senescence ◽

Gene Construct ◽

Shoot Dry Weight ◽

Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

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Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

Life ◽

10.3390/life11070595 ◽

2021 ◽

Vol 11 (7) ◽

pp. 595

Author(s):

Stephen Gargan ◽

Paul Dowling ◽

Margit Zweyer ◽

Jens Reimann ◽

Michael Henry ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Biochemical Properties ◽

Mass Spectrometric ◽

Cellular Stress Response ◽

Diaphragm Muscle ◽

Spectrometric Analysis ◽

Mitochondrial Enzymes ◽

Wild Type ◽

Potential Marker

Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy. The proteomic survey of wild type mice and the dystrophic mdx-4cv model revealed a broad spectrum of sarcomere-associated proteoforms, including components of the thick filament, thin filament, M-band and Z-disk, as well as a variety of muscle-specific markers. Interestingly, the mass spectrometric analysis revealed unusual expression levels of contractile proteins, especially isoforms of myosin heavy chain. As compared to diaphragm muscle, both proteomics and immunoblotting established isoform MyHC14 as a new potential marker in wild type EOMs, in addition to the previously identified isoforms MyHC13 and MyHC15. Comparative proteomics was employed to establish alterations in the protein expression profile between normal EOMs and dystrophin-lacking EOMs. The analysis of mdx-4cv EOMs identified elevated levels of glycolytic enzymes and molecular chaperones, as well as decreases in mitochondrial enzymes. These findings suggest a process of adaptation in dystrophin-deficient EOMs via a bioenergetic shift to more glycolytic metabolism, as well as an efficient cellular stress response in EOMs in dystrophinopathy.

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