The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1--LaeA--is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.This is the publisher’s final pdf. The published article is copyrighted by Wiley-Blackwell and can be found at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-2958/issue

Functional Analyses of Trichoderma reesei LAE1 Reveal Conserved and Contrasting Roles of This Regulator

The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.Keywords: Flavus, Secondary metabolism, Aspergillus nidulans, Gene expression, Hypocrea jecorina, DNA methylation, Histone H3, Protein kinase, Neurospora crassa, Penicillin biosynthesi

The role of methyltransferase LAE1 and related proteins for Trichoderma interaction with its environment

Zsfassung in dt. SpracheDas zuerst in Aspergillus spp. gefundene lae1 -Gen kodiert für eine putative Methyltransferase, die in einem Komplex mit VeA,(VeA und VELB )als Master-Regulator des Sekundärstoffwechsels, der Virulenz sowie der Entwicklung wirkt. LaeA reguliert auch die Biosynthese von Sekundärmetaboliten in dem Penicillin-Produzenten Penicillium chrysogenum und den phytopathogenen Pilzen Fusarium fujikuroi (zB Gibberelline) und Cochliobolus heterostrophus. In allen diesen Prozessen wirkt LaeA auf Ebene der Chromatin Modifikation, und ermöglicht dadurch die koordinierte Transkruiption von Genclustern. Berichte über die Beteiligung von LaeA in anderen zellulären Prozessen sind nicht verfügbar.Trichoderma ist eine PIlzgattung mit breiten Anwendungen. T. reesei ist bekannt für seine Produktion von Enzymen für die Hydrolyse pflanzlicher Biomasse, während T. atroviride sich durch einen ausgeprägten Mykoparasitismus aufweist und daher industriell als Biokontrollmittel angewandt wird. Die Gene, die Cellulasen kodieren, befinden sich in Clustern in Genom und werden koordiniert exprimiert. Ein genomisches Clustering von unter mykoparasitischen Bedingungen exprimierten Genen ist auch in T. atroviride beobachtet worden. Um herauszufinden, ob LAE1 von T. reesei die Expression von Cellulasen reguliert verwendete ich einen reversen genetischen Ansatz: in einer lae1 Deletionsmutante beobachtete ich einen vollständigen Verlust der Expression aller sieben Cellulasen, Hilfs-Faktoren für die Celluloseabbau, [beta]-Glucosidasen und Xylanasen. Eine deutlich erhöhte Cellulasetranskription ergab sich in Mutanten mit erhöhter Expression von LAE1. Chromatinimmunpräzipitation, verbunden mit Hochdurchsatz-Sequenzierung ("ChIP-seq") zeigte jedoch, dass offensichtlich die LAE1 Expression nicht mit H3K4 di oder Trimethylierung (indikativ aktiver Transkription) oder H3K9 Trimethylierung (typisch für Heterochromatin Regionen) in CAZyme kodierenden Regionen korreliert. Genom-weite ChIP-Sequenzierung entdeckte 4089 Gene, die eine oder mehrere dieser Methylierung Marken ertragen, von denen 75 zeigten eine Korrelation zwischen beiden H3K4me2 oder H3K4me3 und Regulierung durch LAE1, was darauf hindeutet, dass LAE1 nicht direkt H3K4 oder H3K9 Methylierung moduliert.Eine genomweite Genexpressionsanalyse zeigte ferner, dass LAE1 positiv die Transkription von 7 der 17 Polyketid- oder nicht-ribosomalen Peptidsynthetasen reguliert. Außerdem wurden Gene, Proteine mit Ankyrin-Domainen, Eisenaufnahme, Heterokaryon Inkompatibilitäts-Proteine, PTH11-Rezeptoren und verschiedene Oxidasen / Monoxygenasen kodieren, durch LAE1 reguliert. Um zu untersuchen, ob das Velvet A Protein aus T. reesei für die Regulierung der Bildung von Cellulase durch LAE1 erforderlich ist, wurde das T. reesei Ortholog von vel1 charakterisiert. Deletion von vel1 beeinflusste nicht die Cellulase Genexpression in T. reesei, aber vel1 Überexpression hat sie stark verbessert. Ähnliche Ergebnisse wurden auch für die Bildung von Xylanase und ß-Xylosidase Enzymaktivitäten erhalten. Die Stimulierung der Cellulase Genexpression durch Überexpression von vel1 war abhängig von einer funktionellen LAE1 Allel und damit wurde ein vel1-unabhängigen Modus der Aktivierung durch LAE1 in T. reesei angedeutet.In T. atroviride führte der Verlust der Funktion von LAE1 zu einer stark verringerten mykoparasitischen Aktivität und einer signifikanten Unterexpression von mehreren Genen, die früher als unter mykoparasitischer Interaktion hochreguliert gezeigt wurden, wie z. B Proteasen, GH16 ß-Glucanasen, Polyketidsynthasen und kleine Cystein-reiche sekretierte Proteine. Dies korrelierte auch mit der teilweisen Verringerung der Bildung von fungiziden wasserlöslichen Metaboliten und flüchtigen Metaboliten. Zusammenfassend deuten meine Daten auf neue Rollen von LAE1 in der Physiologie von Trichoderma hin.LAE1 scheint ein Regulator des Fitness von Pilzen in ihrem spezifischen Habitat zu sein.In Aspergillus spp. the laeA (loss of aflR expression) gene encodes a putative methyltransferase that acts in a complex with VeA, i.e., Velvet A, and VelB i.e., Velvet-like B, as master regulator of secondary metabolism, virulence as well as development. LaeA is also required for the biosynthesis of secondary metabolites in the penicillin producer Penicillium chrysogenum and the phytopathogenic fungi Fusarium fujikuroi (e.g., gibberellins) and Cochliobolus heterostrophus respectively- In all these processes, LaeA acts at the level of chromatin modification thereby rendering gene clusters to be coordinately transcribed. Reports about the involvment of LaeA in other cellular processes is not available. Trichoderma is a genus of filamentous fungi with broad range of applications. T. reesei is well known for its production of enzymes used for plant biomass hydrolysis whereas T. atroviride is conspicuous for its vigorous mycoparasitism and is therefore industrially applied as a biocontrol agent. The genes encoding cellulases are found in clusters in genome and expressed coordinately. Similarly, genomic clustering of mycoparasitic genes have been also observed in T. atroviride. In order to learn whether LAE1 of T. reesei is involved in the expression of cellulase genes I used reverse genetics to show that is indeed the case: in a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, [beta]-glucosidases and xylanases were no longer expressed.Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Interestingly, chromatin immunoprecipitation followed by highthroughput sequencing ("ChIP-seq") showed that lae1 expression was not obviously correlated with H3K4 di or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions. Genome-wide ChIP sequencing detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1, suggesting that LAE1 does not directly modulate H3K4 or H3K9 methylation.Genome-wide gene expression analysis showed that lae1 positively regulates 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-containing proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. To investigate whether the Velvet A protein would be required for the regulation of cellulase formation by LAE1, I characterized the T. reesei orthologue vel1.Deletion of vel1 did not affect cellulase gene expression in T. reesei, but vel1 overexpression strongly enhanced it. Similar findings were also obtained for the formation of xylanase and ß-xylosidase enzyme activities. The stimulation of cellulase gene expression by overexpressing vel1 was dependent on a functional lae1 allele, suggesting the operation of a vel1-independent pathway of activation by LAE1 in T. reesei.In T. atroviride, loss of function of lae1 results in a loss of mycoparasitic activity correlated with a significant underexpression of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which was also reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Summarizing, my data point to new roles of LAE1 in the physiology of Trichoderma. LAE1 appears to be a regulator of the fungus fitness in its specialized habitat.<br /

The Putative Protein Methyltransferase LAE1 of <i>Trichoderma atroviride</i> Is a Key Regulator of Asexual Development and Mycoparasitism

<div><p>In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus <i>Trichoderma atroviride</i> is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of <i>lae1</i> gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a <b>Δ</b><i>lae1</i> and 30–50% increased in <i>lae1</i>-overexpressing (OE<i>lae1</i>) strains. In darkness, <b>Δ</b><i>lae1</i> strains did not sporulate, and the OE<i>lae1</i> strains produced as much spores as the parent strain. Loss-of-function of <i>lae1</i> also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of <i>lae1</i> also increased the sensitivity of <i>T. atroviride</i> to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the <i>OElae1</i> strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the <b>Δ</b><i>lae1</i> strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows <i>T. atroviride</i> LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.</p></div

Asexual sporulation of <i>T. atroviride</i>.

<p>(A) Quantitation of conidiation of the parent (P1), <b>Δ</b><i>lae1-1</i> and <i>OElae1</i> strains on PDA in light (white bars) and in darkness (full bars). Values are means of at least three independent biological experiments. Similar investigations with strain <b>Δ</b><i>lae1-2</i> yielded values within ±8% of those of <b>Δ</b><i>lae1-1.</i> All values are statistically different by the students t-test (p<0.05).</p

Effect of LAE1, and the blue light receptors BLR1 and BLR2 on each others expression: (A) Expression of <i>blr1</i> (dark grey) and <i>blr2</i> (light grey) in Δ<i>lae1-1</i> and <i>OElae1</i> in light (L) and darkness (D); (B) expression of <i>lae1</i> in Δ<i>blr1</i> and Δ<i>blr2</i> in light (L) and darkness (D).

<p>Vertical bars indicate the standard deviation (N ≥3). Expression of <i>lae1, blr1</i> and <i>blr2</i> was normalized to the expression of <i>tef1</i>. Relative gene expression is calculated as the ratio of the normalized expression in the mutant in –fold of that of the parent strain P1. None of the difference was found to be statistically relevant by students t-test (p>0.15).</p

Phenotype of confrontation of <i>T. atroviride</i> P1 and the<i>lae1</i> mutants <i>OElae1</i> and Δ<i>lae1</i> (all T) against <i>B. cinerea</i> (B), <i>A. alternata</i> (A) and <i>R. solani</i> (R) after termination of growth of the latter three fungi.

<p>Left plates are photographed from the backside, right plates are photographed from top. (B) Test for production of WSC: <i>T. atroviride</i> parent strain, and the <b>Δ</b><i>lae1-1</i> and <i>OElae1</i> mutants were grown on PDA agar covered by cellophane, and then removed and <i>Alternaria alternata</i> (<i>Aa</i>), <i>Rhizoctonia solani</i> (<i>Rs</i>) and <i>Sclerotinia sclerotiorum</i> (<i>Ss</i>) placed on these plates. The plates were photographed after 7 days.</p