Die Bedeutung des zentralen Blatt-Kohlenhydratstoffwechsels von Arabidopsis thaliana für die Interaktion mit Colletotrichum higginsianum

Hemibiotrophic pathogens interact with host plants in two consecutive phases. An initial biotrophic growth phase inside living host cells is followed by a necrotrophic phase, in which host tissue is sequentially killed. The hemibiotrophic fungus Colletotrichum higginsianum establishes itself through clearly defined infection steps on Arabidopsis thaliana in a compatible interaction. After breaching the host cell wall with an appressorium, the fungus forms intracellular biotrophic hyphae surrounded by the host plasma membrane. Following a short biotrophic phase, leaf tissue is colonized by secondary necrotrophic hyphae accompanied by extensive necrotic lesions on host leaves. In this study, it was shown that carbohydrate availability of Arabidopsis influences susceptibility towards C. higginsianum. Mutants with an impaired starch metabolism experienced faster fungal proliferation and showed increased disease symptoms. Accordingly, the reduced carbohydrate budget and not the duration of carbohydrate shortage increased susceptibility in this interaction. This observation was further supportet by a negative correlation between carbohydrate accumulation in the light and susceptibility towards C. higginsianum. By employing additional genotypes with nocturnal carbohydrate shortage, a clear coincidence between diurnal carbohydrate turnover and susceptibility was corroborated. Analyses of different defense responses revealed that the accumulation of secondary metabolites such as salicylic acid, camalexin and glucosinolates was limited due to reduced carbohydrate availability. Dark-induced carbohydrate shortage in different interaction phases indicated that carbohydrate supply by the host is dispensable during biotrophic growth of C. higginsianum. Furthermore, induced carbohydrate shortage affected susceptibility most during the necrotrophic interaction, indicating that affected defense increases susceptibility especially during necrotrophic colonization. An analysis of cell wall composition demonstrated that periodic carbohydrate shortage in different mutants suffering from nocturnal carbon starvation, caused reduced contents of the monosaccharides arabinose, galactose and galacturonic acid in pectic cell wall matrix polymers. Comparison to described cell wall mutants suggested that the observed cell wall defects reduce penetration resistance to C. higginsianum. In contrast to the increased susceptibility towards C. higginsianum, starchless mutants were more resistant towards the biotrophic powdery mildew fungus E. cruciferarum, which was not due to the observed changes in cell wall composition. Mutagenesis of starchless pgm mutants yielded a mutant population for forward genetic screens. By selecting suppressors of pgm hypersusceptibility, 63 mutants with reduced susceptibility towards C. higginsianum including 8 mutants with pgm-specific resistance could be identified. Selectivity of the screening was proven by the identification of a previously described resistant mutant. Moreover, a mutant showing pgm-specific resistance could be mapped to a 800 kb genomic region on chromosome 3 containing interesting candidate genes for further analyses.Hemibiotrophe Pathogene interagieren mit ihren Wirtspflanzen in zwei zeitlich aufeinanderfolgenden Phasen. In einer initialen biotrophen Phase wachsen die Pathogene innerhalb lebendender Wirtszellen, während das Wirtsgewebe in der nachfolgenden nekrotrophen Phase sequentiell abgetötet wird. Colletotrichum higginsianum ist ein hemibiotropher Pilz, der eine kompatible Interaktion mit Arabidopsis thaliana eingeht und dabei verschiedene klar definierte Infektionsschritte durchläuft. Nachdem die Zellwand des Wirtes mit einem Appressorium durchstoßen wurde, bildet der Pilz intrazelluläre biotrophe Hyphen die von der Plasmamembran der Wirtszelle umgeben sind. Nach einer kurzen biotrophen Phase wird das Blattgewebe von sekundären nekrotrophen Hyphen besiedelt, woraufhin großflächige nekrotische Läsionen auf Blättern der Wirtspflanze entstehen. In dieser Arbeit konnte gezeigt werden, dass die Kohlenhydratverfügbarkeit von Arabidopsis die Suszeptibilität gegenüber C. higginsianum beeinflusst. Mutanten mit einem beeinträchtigten Stärkestoffwechsel wurden schneller durch den Pilz besiedelt und wiesen nach Etablierung der Interaktion stärkere Krankheitssymptome auf. Quantitative Analysen des intrazellulären Pilzwachstums zeigten, dass diese Mutanten unabhängig von der Dauer der Lichtphase eine erhöhte Suszeptibilität gegenüber C. higginsianum aufwiesen. Daraus konnte geschlossen werden, dass nicht die Dauer des Zuckermangels, sondern die Reduktion des Kohlenhydratbudgets die erhöhte Suszeptibilität vermittelt. Dieser Zusammenhang wurde auch durch eine negative Korrelation zwischen der Kohlenhydratakkumulation während der Lichtphase und der Suszeptibilität gegenüber C. higginsianum unterstützt. Durch die Untersuchung weiterer Genotypen mit einem nächtlichen Kohlenstoffmangel konnte eine klare Koinzidenz zwischen dem diurnalen Kohlenhydratumsatz und der Suszeptibilität weiter bekräftigt werden. Analysen verschiedener induzierter Abwehrreaktionen zeigten, dass die Kapazität zur Bildung von Sekundärmetaboliten wie Salicylsäure, Camalexin und Glucosinolaten in stärkefreien Mutanten aufgrund einer reduzierten Kohlenhydratverfügbarkeit vermindert war. Durch Dunkel-induzierten Kohlenhydratmangel während verschiedener Phasen der Interaktion zwischen Arabidopsis und C. higginsianum konnte gezeigt werden, dass der Pilz während des biotrophen Wachstums nicht auf die Versorgung mit Kohlenhydraten durch den Wirt angewiesen ist. Außerdem wirkte sich ein induzierter Kohlenhydratmangel während der nekrotrophen Interaktionsphase besonders stark auf die Suszeptibilität für den Pilz aus. Beeinträchtigungen induzierter Abwehrreaktionen könnten sich folglich besonders während des nekrotrophen Wachstums des Pilzes auf die Suszeptibilität auswirken. Analysen der Zellwandzusammensetzung verschiedener Mutanten mit einem nächtlichen Kohlenhydratmangel zeigten, dass der regelmäßig wiederkehrende Kohlenhydratmangel zu verringerten Gehalten der Monosaccharide Arabinose, Galactose und Galacturonsäure im Pektinanteil der Zellwandmatrix führte. Vergleichende Analysen mit beschriebenen Zellwandmutanten lieferten klare Hinweise, dass dieser Zellwanddefekt die Penetrationsresistenz von Arabidopsis gegen C. higginsianum reduziert. Im Gegensatz zu der erhöhten Suszeptibilität für C. higginsianum hatten stärkefreie Mutanten eine erhöhte Resistenz gegen den obligat biotrophen Mehltaupilz Erysiphe cruciferarum, die nicht durch die beobachteten Zellwandveränderungen begründet war. Durch eine Mutagenese stärkefreier pgm Pflanzen konnte eine Population von Mutanten für vorwärtsgenetische Durchmusterungen erzeugt werden. Durch die Selektion auf Suppressor-Phänotypen der Hypersuszeptibilität von pgm konnten 63 Mutanten mit einer reduzierten Suszeptibilität gegenüber C. higginsianum identifiziert werden, von denen 8 Mutanten eine pgm-spezifische Resistenz zeigten. Durch die Identifizierung einer Mutante, die bereits als resistent beschrieben war, konnte die Selektivität der Durchmusterung belegt werden. Zudem konnte eine Mutante mit pgm-spezifischer Resistenz auf einen 800 kb umfassenden Genombereich von Chromosom 3 kartiert werden, der interessante Kandidatengene für weiterführende Analysen beinhaltet

Loss of the arabidopsis protein kinases ANPs affects root cell wall composition, and triggers the cell wall damage syndrome

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional β-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance

Deficiencies in the Mitochondrial Electron Transport Chain Affect Redox Poise and Resistance Toward Colletotrichum higginsianum

To investigate if and how the integrity of the mitochondrial electron transport chain (mETC) influences susceptibility of Arabidopsis toward Colletotrichum higginsianum, we have selected previously characterized mutants with defects at different stages of the mETC, namely, the complex I mutant ndufs4, the complex II mutant sdh2-1, the complex III mutant ucr8-1, and a mutant of the uncoupling protein ucp1-2. Relative to wild type, the selected complex I, II, and III mutants showed decreased total respiration, increased alternative respiration, as well as increased redox charge of the NADP(H) pool and decreased redox charge of the NAD(H) pool in the dark. In the light, mETC mutants accumulated free amino acids, albeit to varying degrees. Glycine and serine, which are involved in carbon recycling from photorespiration, and N-rich amino acids were predominantly increased in mETC mutants compared to the wild type. Taking together the physiological phenotypes of all examined mutants, our results suggest a connection between the limitation in the re-oxidation of reducing equivalents in the mitochondrial matrix and the induction of nitrate assimilation into free amino acids in the cytosol, which seems to be engaged as an additional sink for reducing power. The sdh2-1 mutant was less susceptible to C. higginsianum and did not show hampered salicylic acid (SA) accumulation as previously reported for SDH1 knock-down plants. The ROS burst remained unaffected in sdh2-1, emonstrating that subunit SDH2 is not involved in the control of ROS production and SA signaling by complex II. Moreover, the ndufs4 mutant showed only 20% of C. higginsianum colonization compared to wild type, with the ROS burst and the production of callose papillae being significantly increased compared to wild type. This indicates that a restriction of respiratory metabolism can positively affect pre-penetration resistance of Arabidopsis. Taking metabolite profiling data from all investigated mETC mutants, a strong positive correlation of resistance toward C. higginsianum with NADPH pool size, pyruvate contents, and other metabolites associated with redox poise and energy charge was evident, which fosters the hypothesis that limitations in the mETC can support resistance at post-penetration stages by improving the availability of metabolic power

Corrigendum: Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional β-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance.Highlights: The loss of ANP function affects cell wall composition and leads to typical cell wall damage-induced phenotypes, such as ectopic lignification and jasmonic acid accumulation

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues

Perception of a divergent family of phytocytokines by the Arabidopsis receptor kinase MIK2

Plant genomes encode hundreds of receptor kinases and peptides, but the number of known plant receptor-ligand pairs is limited. We report that the Arabidopsis leucine-rich repeat receptor kinase LRR-RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) is the receptor for the SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) phytocytokines. MIK2 is necessary and sufficient for immune responses triggered by multiple SCOOP peptides, suggesting that MIK2 is the receptor for this divergent family of peptides. Accordingly, the SCOOP12 peptide directly binds MIK2 and triggers complex formation between MIK2 and the BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) co-receptor. MIK2 is required for resistance to the important root pathogen Fusarium oxysporum. Notably, we reveal that Fusarium proteomes encode SCOOP-like sequences, and corresponding synthetic peptides induce MIK2-dependent immune responses. These results suggest that MIK2 may recognise Fusarium-derived SCOOP-like sequences to induce immunity against Fusarium. The definition of SCOOPs as MIK2 ligands will help to unravel the multiple roles played by MIK2 during plant growth, development and stress responses

Reduced carbohydrate availability enhances the susceptibility of Arabidopsis toward Colletotrichum higginsianum

Colletotrichum higginsianum is a hemibiotrophic ascomycete fungus that is adapted to Arabidopsis (Arabidopsis thaliana). After breaching the host surface, the fungus establishes an initial biotrophic phase in the penetrated epidermis cell, before necrotrophic growth is initiated upon further host colonization. We observed that partitioning of major leaf carbohydrates was shifted in favor of sucrose and at the expense of starch during necrotrophic fungal growth. Arabidopsis mutants with impaired starch turnover were more susceptible toward C. higginsianum infection, exhibiting a strong negative correlation between diurnal carbohydrate accumulation and fungal proliferation for the tested genotypes. By altering the length of the light phase and employing additional genotypes impaired in nocturnal carbon mobilization, we revealed that reduced availability of carbon enhances susceptibility in the investigated pathosystem. Systematic starvation experiments resulted in two important findings. First, we showed that carbohydrate supply by the host is dispensable during biotrophic growth of C. higginsianum, while carbon deficiency was most harmful to the host during the necrotrophic colonization phase. Compared with the wild type, the increases in the total salicylic acid pool and camalexin accumulation were reduced in starch-free mutants at late interaction stages, while an increased ratio of free to total salicylic acid did not convey elevated pathogenesis-related gene expression in starch-free mutants. These observations suggest that reduced carbon availability dampens induced defense responses. In contrast, starch-free mutants were more resistant toward the fungal biotroph Erysiphe cruciferarum, indicating that reduced carbohydrate availability influences susceptibility differently in the interaction with the investigated hemibiotrophic and biotrophic fungal pathogens