Auxins are hormones that regulate growth and development in plants. Besides plants, various microorganisms also produce auxins. Here we investigate whether and how the phytopathogenic fungus Leptosphaeria maculans biosynthesizes auxins. We characterized the auxin profile of in vitro grown L. maculans. The culture was further supplied with the auxin biosynthetic-precursors tryptophan and tryptamine and gene expression and phytohormone content was analyzed. L. maculans in vitro produced IAA (indole-3-acetic acid) as the predominant auxin metabolite. IAA production could be further stimulated by supplying precursors. Expression of indole-3-pyruvate decarboxylase LmIPDC2, tryptophan aminotransferase LmTAM1 and nitrilase LmNIT1 genes was mainly upregulated after adding tryptophan and correlated with IAA production, suggesting that these genes are the key components of auxin biosynthesis in L. maculans. Tryptamine acted as a potent inducer of IAA production, though a pathway independent of LmIPDC2/LmTAM1 may be involved. Despite L. maculans being a rich source of bioactive IAA, the auxin metabolic profile of host plant Brassica napus was not altered upon infection. Exogenous IAA inhibited the growth of L. maculans in vitro when supplied in high concentration. Altogether, we showed that L. maculans is capable of IAA production and we have identified biosynthetic genes that were responsive to tryptophan treatment.
- MeSH
- aminohydrolasy genetika MeSH
- biosyntetické dráhy MeSH
- Brassica napus mikrobiologie MeSH
- fylogeneze MeSH
- genetická transkripce MeSH
- houby klasifikace genetika metabolismus MeSH
- karboxylyasy genetika metabolismus MeSH
- kyseliny indoloctové metabolismus farmakologie MeSH
- Leptosphaeria enzymologie genetika růst a vývoj metabolismus MeSH
- regulace genové exprese u hub MeSH
- regulátory růstu rostlin metabolismus MeSH
- tryptaminy metabolismus farmakologie MeSH
- tryptofan metabolismus farmakologie MeSH
- tryptofantransaminasa genetika metabolismus MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. METHODS: To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. KEY RESULTS: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. CONCLUSIONS: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.
The Dothideomycete Leptosphaeria maculans, a worldwide fungal pathogen of oilseed rape (Brassica napus), secretes a broad spectrum of molecules into the cultivation medium during growth in vitro. Here, candidate elicitor molecules, which induce resistance in B. napus to L. maculans, were identified in the cultivation medium. The elicitation activity was indicated by increased transcription of pathogenesis-related gene 1 (PR1) and enhanced resistance of B. napus plants to the invasion of L. maculans. The elicitation activity was significantly lowered when the cultivation medium was heated to 80°C. Active components were further characterized by specific cleavage with the proteolytic enzymes trypsin and proteinase K and with glycosidases α-amylase and β-glucanase. The elicitor activity was eliminated by proteolytic digestion while glycosidases had no effect. The filtered medium was fractionated by either ion-exchange chromatography or isoelectric focusing. Mass spectrometry analysis of the most active fractions obtained by both separation procedures revealed predominantly enzymes that can be involved in the degradation of plant cell wall polysaccharides. This is the first study searching for L. maculans-specific secreted elicitors with a potential to be used as defense-activating agents in the protection of B. napus against L. maculans in agriculture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:918-928, 2016.
- MeSH
- antifungální látky analýza metabolismus farmakologie MeSH
- Ascomycota účinky léků metabolismus MeSH
- Brassica napus chemie metabolismus mikrobiologie MeSH
- chromatografie iontoměničová MeSH
- fungální proteiny analýza metabolismus MeSH
- hmotnostní spektrometrie MeSH
- kultivační média chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.
- MeSH
- alely MeSH
- antioxidancia farmakologie MeSH
- Ascomycota účinky léků izolace a purifikace metabolismus MeSH
- Brassica napus účinky léků růst a vývoj metabolismus mikrobiologie MeSH
- chromatografie kapalinová MeSH
- cyklopentany metabolismus MeSH
- ethyleny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- hmotnostní spektrometrie MeSH
- interakce hostitele a patogenu účinky léků MeSH
- kotyledon účinky léků metabolismus mikrobiologie MeSH
- kyselina abscisová metabolismus MeSH
- kyselina askorbová farmakologie MeSH
- kyselina salicylová metabolismus MeSH
- oxylipiny metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulátory růstu rostlin metabolismus MeSH
- signální transdukce * účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
Growth is the best visible sign of plant comfort. If plants are under stress, a difference in growth with control conditions can indicate that something is going wrong (or better). Phytohormones such as auxin, cytokinins, brassinosteroids or giberellins, are important growth regulators and their role in plant growth was extensively studied. On the other hand the role of salicylic acid (SA), a phytohormone commonly connected with plant defense responses, in plant growth is under-rated. However, studies with SA-overaccumulating mutants directly showed an influence of SA on plant growth. Recently we characterized an Arabidopsis SA-overaccumulating mutant impaired in phosphatidylinositol-4-kinases (pi4kIIIβ1β2). This mutant is dwarf. The crossing with mutants impaired in SA signaling revealed that pi4kIIIβ1β2 stunted rosette is due to high SA, while the short root length is not. This brings into evidence that upper and lower parts of the plants, even though they may share common phenotypes, are differently regulated.
- MeSH
- 1-fosfatidylinositol-4-kinasa genetika MeSH
- Arabidopsis anatomie a histologie enzymologie MeSH
- kořeny rostlin anatomie a histologie MeSH
- kyselina salicylová metabolismus MeSH
- listy rostlin anatomie a histologie MeSH
- mutace genetika MeSH
- proteiny huseníčku genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIIIβ1β2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIIIβ1β2 phenotype. The pi4kIIIβ1β2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIIIβ1β2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIIIβ1β2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIIIβ1β2, sid2 pi4kIIIβ1β2 and NahG pi4kIIIβ1β2 had similar amounts of SA as the wild-type (WT), npr1pi4kIIIβ1β2 had more SA than pi4kIIIβ1β2 despite being less dwarfed. This indicates that PI4KIIIβ1 and PI4KIIIβ2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIIIβ1β2 was not suppressed in any of the triple mutants. The pi4kIIIβ1β2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.
- MeSH
- 1-fosfatidylinositol-4-kinasa genetika metabolismus MeSH
- Arabidopsis anatomie a histologie enzymologie genetika růst a vývoj MeSH
- down regulace genetika MeSH
- fenotyp MeSH
- genom rostlinný MeSH
- genotyp MeSH
- kinetika MeSH
- kořeny rostlin anatomie a histologie růst a vývoj MeSH
- kyselina salicylová metabolismus MeSH
- listy rostlin genetika růst a vývoj MeSH
- metabolismus lipidů genetika MeSH
- modely genetické MeSH
- mutace genetika MeSH
- nemoci rostlin genetika imunologie mikrobiologie MeSH
- odolnost vůči nemocem genetika imunologie MeSH
- proteiny huseníčku genetika metabolismus MeSH
- Pseudomonas fyziologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- signální transdukce MeSH
- upregulace genetika MeSH
- výhonky rostlin růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Preparations with elicitation activity were obtained from the mycelium of Leptosphaeria maculans , a fungal pathogen of oilseed rape (Brassica napus). Crude delipidated and deproteinized extract from fungal cell walls induced expression of pathogenesis related gene 1 (PR1), hydrogen peroxide accumulation, and enhanced resistance of B. napus plants toward infection by L. maculans. Elicitation activity significantly decreased after treatment of a crude extract with α- or β-glucanase. Monosaccharide composition analysis of a crude extract purified by ion-exchange chromatography revealed glucose (∼58 mol %), mannose (∼22 mol %), and galactose (∼18 mol %) as the major sugars. FT-IR and NMR spectra confirmed the presence of both carbohydrate and polypeptide components in the purified product. Correlation NMR experiments defined trisaccharide bound to O-3 of serine residue α-D-Glcp-(1→2)-β-D-Galf-(1→6)-α-D-Manp-(1→3)-L-Ser. Terminal α-D-Glcp and (1→6)-β-D-glucan were also detected. The obtained results strongly support the conclusion that these carbohydrates induce defense response in B. napus plants.
- MeSH
- Ascomycota chemie růst a vývoj imunologie MeSH
- Aspergillus niger enzymologie MeSH
- Brassica napus účinky léků imunologie metabolismus mikrobiologie MeSH
- buněčná stěna chemie MeSH
- buněčné extrakty chemie izolace a purifikace farmakologie MeSH
- chemie zemědělská metody MeSH
- down regulace MeSH
- fungální proteiny analýza chemie metabolismus MeSH
- fungicidy průmyslové chemie izolace a purifikace farmakologie MeSH
- glykosidhydrolasy biosyntéza genetika metabolismus MeSH
- glykosidy analýza chemie metabolismus MeSH
- hydrolýza MeSH
- mycelium chemie růst a vývoj imunologie MeSH
- odolnost vůči nemocem účinky léků MeSH
- oligosacharidy analýza chemie metabolismus MeSH
- peptidové fragmenty analýza chemie metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- rostlinné proteiny biosyntéza genetika metabolismus MeSH
- semenáček účinky léků imunologie metabolismus mikrobiologie MeSH
- upregulace účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interaction of a plant with a fungal pathogen is an encounter with hundreds of molecules. In contrast to this, a single molecule often decides between the disease and resistance. In the present article, we describe the defense responses triggered by AvrLm1, an avirulence gene from a hemibiotrophic ascomycete, Leptosphaeria maculans, responsible for an incompatible interaction with Brassica napus. Using multiple hormone quantification and expression analysis of defense-related genes, we investigated signaling events in Rlm1 plants infected with two sister isolates of L. maculans differentiated by the presence or absence of AvrLm1. Infection with the isolate carrying AvrLm1 increased the biosynthesis of salicylic acid (SA) and induced expression of the SA-associated genes ICS1, WRKY70, and PR-1, a feature characteristic of responses to biotrophic pathogens and resistance gene-mediated resistance. In addition to SA-signaling elements, we also observed the induction of ASC2a, HEL, and CHI genes associated with ethylene (ET) signaling. Pharmacological experiments confirmed the positive roles of SA and ET in mediating resistance to L. maculans. The unusual cooperation of SA and ET signaling might be a response to the hemibiotrophic nature of L. maculans. Our results also demonstrate the profound difference between the natural host B. napus and the model plant Arabidopsis in their response to L. maculans infection.
- MeSH
- Ascomycota metabolismus MeSH
- Brassica napus účinky léků metabolismus mikrobiologie MeSH
- časové faktory MeSH
- ethyleny metabolismus MeSH
- fungální proteiny metabolismus farmakologie MeSH
- kyselina salicylová metabolismus MeSH
- listy rostlin účinky léků metabolismus mikrobiologie MeSH
- nemoci rostlin MeSH
- regulace genové exprese u hub MeSH
- signální transdukce fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH
