Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes

[EN] Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant ∆epl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonizationSIThis work was supported by the State of São Paulo Research Foundation (FAPESP) (Proc. 2012/16895-4, Proc. 2013/24452-8, and Proc. 2016/04274-6). Funding was also obtained from the Junta de Castilla y León, Spain (LE228U14) and the Spanish Government Grants from Ministerio de Economía y Competitividad (AGL2012-40041-C02-02 and AGL2015-70671-C2-2-R

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environmentSIFunding was obtained from the Junta de Castilla y Leon (SA260A11-2,LE125A12-2 and LE228U14) and Spanish Government grants MICINN-AGL2009-13431-C02-02, MINECO-AGL2012-40041-C02-01, AGL2012-40041-C02-02 and AGL2012-39798-C02-01. MGM and II-B were granted fellowships from the Spanish Ministry of Science and Innovation(AP2007-02835, BES-2013-063411

Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes

Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant Δepl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonization

STE20/PAKA Protein Kinase Gene Releases an Autoinhibitory Domain through Pre-mRNA Alternative Splicing in the Dermatophyte <i>Trichophyton rubrum</i>

Signaling pathways are highly diverse in filamentous fungi, allowing the cells to receive and process ambient information. Interaction of components from different pathways results in signaling networks. The mitogen-activated protein kinase (MAPK) pathway is dependent on phosphorylation that is accomplished by kinase proteins. Thus, the STE/PAK protein kinase family plays essential roles in MAPK signal transduction, regulating several cellular functions. The STE/PAK protein displays an autoinhibitory (Cdc42/Rac interactive binding&#8212;CRIB) domain on its N-terminal portion, which interacts with the C-terminal catalytic kinase domain. Based on current knowledge, for the STE/PAK kinase to be activated, molecular signals (e.g., interaction with the activated form of Rac1 and Cdc42 proteins) or proteolytic cleavage by caspase 3 is necessary. Both mechanisms release the kinase domain from the CRIB interaction. Here, we hypothesize a novel molecular mechanism for the activation of STE20/PAKA kinase in Trichophyton rubrum based on an alternative pre-mRNA splicing process. Our data suggest that, because of the retention of intron 1 of this gene, it is theoretically possible that the translation of STE20/PAKA kinase will be free of its autoinhibitory CRIB domain. These findings indicate a rapid response system to environmental changes. Furthermore, STE20/PAKA may be a potential T. rubrum virulence factor and an interesting target for new drugs against dermatophytes

<i>mus-52</i> disruption and metabolic regulation in <i>Neurospora crassa</i>: Transcriptional responses to extracellular phosphate availability

<div><p>Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (<i>mus</i>) genes in the model fungus <i>Neurospora crassa</i>, i.e. <i>mus-51</i>, <i>mus-52</i>, or <i>mus-53</i>, orthologous to the human genes <i>KU70</i>, <i>KU80</i>, and <i>LIG4</i>, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of <i>mus-52</i> deletion in <i>N</i>. <i>crassa</i> gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.</p></div

Gene Ontology (GO) enrichment analysis.

<p>(A) The 10 classes of genes in the <i>N</i>. <i>crassa</i> FGSC 2489 strain most affected by Pi availability. (B) The 10 classes of genes in the <i>N</i>. <i>crassa</i> FGSC 9568 strain most impacted by Pi availability. (C, D) The 10 classes of the most affected genes in <i>N</i>. <i>crassa</i> FGSC 9568 with respect to the wild-type strain in the low-Pi (C) and high-Pi (D) culture condition. Significantly enriched categories (P ≤ 0.05) are shown.</p

Dermatophyte Resistance to Antifungal Drugs: Mechanisms and Prospectus

Dermatophytes comprise pathogenic fungi that have a high affinity for the keratinized structures present in nails, skin, and hair, causing superficial infections known as dermatophytosis. A reasonable number of antifungal drugs currently exist on the pharmaceutical market to control mycoses; however, their cellular targets are restricted, and fungi may exhibit tolerance or resistance to these agents. For example, the stress caused by antifungal and cytotoxic drugs in sub-inhibitory concentrations promotes compensatory stress responses, with the over-expression of genes involved in cellular detoxification, drug efflux, and signaling pathways being among the various mechanisms that may contribute to drug tolerance. In addition, the ATP-binding cassette transporters in dermatophytes that are responsible for cellular efflux can act synergistically, allowing one to compensate for the absence of the other, revealing the complexity of drug tolerance phenomena. Moreover, mutations in genes coding for target enzymes could lead to substitutions in amino acids involved in the binding of antifungal agents, hindering their performance and leading to treatment failure. The relevance of each one of these mechanisms of resistance to fungal survival is hard to define, mainly because they can act simultaneously in the cell. However, an understanding of the molecular mechanisms involved in the resistance/tolerance processes, the identification of new antifungal targets, as well as the prospective of new antifungal compounds among natural or synthetic products, are expected to bring advances and new insights that facilitate the improvement or development of novel strategies for antifungal therapy

Kinase and kinase-related gene expression profile (heat map) of <i>N</i>. <i>crassa</i> FGSC 9568 mutant <i>vs</i> wild-type strains, grown in low- and high-Pi availability conditions.

<p>Expression scale is represented as log2-fold change.</p

Expression analysis of the <i>N</i>. <i>crassa</i> genes encoding TFs in wild-type FGSC 2489 and mutant FGSC 9568 strains grown in media with different Pi availability.

<p>(A, B) Fold change expression ratio of 9 genes encoding TFs in mutant FGSC 9568 <i>vs</i> FGSC 2489 grown in medium containing low-Pi (A) or high-Pi (B) availability. (C) Gene ID and description of the TFs analysed and their respective relative expression (log2) under conditions of low- and high-Pi availability.</p