Sergio Casas-Flores

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Colonization of Arabidopsis roots by Trichoderma atroviride promotes growth and enhances systemic disease resistance through jasmonic acid/ethylene and salicylic acid pathways

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

SUMMARY The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

The Epl1 and Sm1 proteins from Trichoderma atroviride and Trichoderma virens differentially modulate systemic disease resistance against different life style pathogens in Solanum lycopersicum.

Fungi belonging to the genus Trichoderma, commonly found in soil or colonizing plant roots, exert beneficial effects on plants, including the promotion of growth and the induction of resistance to disease. T. virens and T. atroviride secrete the proteins Sm1 and Epl1, respectively, which elicit local and systemic disease resistance in plants. In this work, we show that these fungi promote growth in tomato (Solanum lycopersicum) plants. T. virens was more effective than T. atroviride in promoting biomass gain, and both fungi were capable of inducing systemic protection in tomato against Alternaria solani, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst DC3000). Deletion (KO) of epl1 in T. atroviride resulted in diminished systemic protection against A. solani and B. cinerea, whereas the T. virens sm1 KO strain was less effective in protecting tomato against Pst DC3000 and B. cinerea. Importantly, overexpression (OE) of epl1 and sm1 led to an increase in disease resistance against all tested pathogens. Although the Trichoderma WT strains induced both systemic acquired resistance (SAR)- and induced systemic resistance (ISR)-related genes in tomato, inoculation of plants with OE and KO strains revealed that Epl1 and Sm1 play a minor role in the induction of these genes. However, we found that Epl1 and Sm1 induce the expression of a peroxidase and an α-dioxygenase encoding genes, respectively, which could be important for tomato protection by Trichoderma spp. Altogether, these observations indicate that colonization by beneficial and/or infection by pathogenic microorganisms dictates many of the outcomes in plants, which are more complex than previously thought.

Mango (Mangifera indica L.) cv. Kent fruit mesocarp de novo transcriptome assembly identifies gene families important for ripening.

Fruit ripening is a physiological and biochemical process genetically programmed to regulate fruit quality parameters like firmness, flavor, odor and color, as well as production of ethylene in climacteric fruit. In this study, a transcriptomic analysis of mango (Mangifera indica L.) mesocarp cv. “Kent” was done to identify key genes associated with fruit ripening. Using the Illumina sequencing platform, 67,682,269 clean reads were obtained and a transcriptome of 4.8 Gb. A total of 33,142 coding sequences were predicted and after functional annotation, 25,154 protein sequences were assigned with a product according to Swiss-Prot database and 32,560 according to non-redundant database. Differential expression analysis identified 2,306 genes with significant differences in expression between mature-green and ripe mango [1,178 up-regulated and 1,128 down-regulated (FDR ≤ 0.05)]. The expression of 10 genes evaluated by both qRT-PCR and RNA-seq data was highly correlated (R = 0.97), validating the differential expression data from RNA-seq alone. Gene Ontology enrichment analysis, showed significantly represented terms associated to fruit ripening like “cell wall,” “carbohydrate catabolic process” and “starch and sucrose metabolic process” among others. Mango genes were assigned to 327 metabolic pathways according to Kyoto Encyclopedia of Genes and Genomes database, among them those involved in fruit ripening such as plant hormone signal transduction, starch and sucrose metabolism, galactose metabolism, terpenoid backbone, and carotenoid biosynthesis. This study provides a mango transcriptome that will be very helpful to identify genes for expression studies in early and late flowering mangos during fruit ripening.

Performance of innovative PU-foam and natural fiber-based composites for the biofiltration of a mixture of volatile organic compounds by a fungal biofilm.

The performance of perlite and two innovative carriers that consist of polyurethane (PU) chemically modified with starch; and polypropylene reinforced with agave fibers was evaluated in the biofiltration of a mixture of VOCs composed of hexane, toluene and methyl-ethyl-ketone. At a total organic loading rate of 145 gCm(-3)h(-1) the elimination capacities (ECs) obtained were 145, 24 and 96 gCm(-3)h(-1) for the biofilters packed with the PU, the reinforced polypropylene, and perlite, respectively. Specific maximum biodegradation rates of the mixture, in the biofilters, were 416 mgCg(protein)(-1)  h(-1) for the PU and 63 mgCg(protein)(-1) h(-1) for perlite, which confirms the highest performance of the PU-composite. 18S rDNA analysis from the PU-biofilter revealed the presence of Fusarium solani in its sexual and asexual states, respectively. The modified PU carrier significantly reduced the start-up period of the biofilter and enhanced the EC of the VOCs. Thus, this study gives new alternatives in the field of packing materials synthesis, promoting the addition of easily biodegradable sources to enhance the performance of biofilters.

Differential distribution of transcripts from genes involved in polyamine biosynthesis in bean plants

Partial cDNAs sequences for arginine decarboxylase (Pvadc), S-adenosylmethionine decarboxylase (Pvsamdc) and spermidine synthase (Pvspds) were isolated from the bean Phaseolus vulgaris using primers designed from conserved regions of enzymes belonging to plant species. Sequence analysis showed that the Pvadc, Pvsamdc and Pvspds genes were most closely related to the orthologous genes from Glycine max, Phaseolus lunatus and Pisum sativum, respectively. The expression patterns of the genes, together with that of ornithine decarboxylase (Pvodc), were analysed in young and mature leaves, stems, roots, root tips, petals, stigma, ovaries, filaments and anthers of bean plants. Pvsamdc was found to be expressed at similar levels in all tissues. The other transcripts showed tissue specific expression. Pvadc was barely expressed in petals and not at all in roots tips, Pvspds was mainly expressed in roots, stigma and filaments, and Pvodc was detected only in roots.

3 The Bright and Dark Sides of Fungal Life

Sunlight is one of the most important cues for living organisms on earth. Fungi are a wide group of microorganisms, which life is importantly impacted by light, even though they typically grow in damp, dark habitats. In these organisms light regulates several physiological and developmental processes, including phototropism, synthesis of pigments, circadian rhythms, sexual and asexual development, and primary and secondary metabolism, among other processes. Such responses are initiated by sensing light, which is perceived through a protein chromophore complex to transduce the signal downstream. These protein/chromophore complexes are called photoreceptors and are classified based on the chromophore they bind and what part of the visible light spectrum they perceive. Fungi are considered as some of the most important producers of secondary metabolites that are used for anthropocentric purposes, including antibiotics, enzymes used in industry, pigments, aromas, flavors, etc. Since the production of several of these compounds is stimulated by light, it could be used as a gratuitous inducer. Intriguingly, recent evidences point to a role of light in fungal virulence of human and plant pathogens. In this chapter we discuss the impact of light on the physiology and development in fungi.

Histone Deacetylase HDA-2 Regulates Trichoderma atroviride Growth, Conidiation, Blue Light Perception, and Oxidative Stress Responses

ABSTRACT Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichodermaatroviride, the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and Δhda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δhda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1, indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δhda-2 mutants display high sensitivity whereas Δblr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δblr strains but misregulation in Δhda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δblr and Δhda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS. IMPORTANCE Trichoderma atroviride is a free-living fungus commonly found in soil or colonizing plant roots and is widely used as an agent in biocontrol as it parasitizes other fungi, stimulates plant growth, and induces the plant defense system. To survive in various environments, fungi constantly sense and respond to potentially threatening external factors, such as light. In particular, UV light can damage biomolecules by producing free-radical reactions, in most cases involving reactive oxygen species (ROS). In T. atroviride, conidiation is essential for its survival, which is induced by light and mechanical injury. Notably, conidia are typically used as the inoculum in the field during biocontrol. Therefore, understanding the linkages between responses to light and exposure to ROS in T. atroviride is of major basic and practical relevance. Here, the histone deacetylase-encoding gene hda-2 is induced by light and ROS, and its product regulates growth, conidiation, blue light perception, and oxidative stress responses.

The Trichoderma atroviride photolyase-encoding gene is transcriptionally regulated by non-canonical light response elements.

The BLR‐1 and BLR‐2 proteins of Trichoderma atroviride are the Neurospora crassa homologs of white collar‐1 and ‐2, two transcription factors involved in the regulation of genes by blue light. BLR‐1 and BLR‐2 are essential for photoinduction of phr‐1, a photolyase‐encoding gene whose promoter exhibits sequences similar to well‐characterized light regulatory elements of Neurospora, including the albino proximal element and the light response element (LRE). However, despite the fact that this gene has been extensively used as a blue light induction marker in Trichoderma, the function of these putative regulatory elements has not been proved. The described LRE core in N. crassa comprises two close but variably spaced GATA boxes to which a WC‐1/‐2 complex binds transiently upon application of a light stimulus. Using 5′ serial deletions of the phr‐1 promoter, as well as point mutations of putative LREs, we were able to delimit an ~ 50 bp long region mediating the transcriptional response to blue light. The identified light‐responsive region contained five CGATB motifs, three of them displaying opposite polarity to canonical WCC binding sites. Chromatin immunoprecipitation experiments showed that the BLR‐2 protein binds along the phr‐1 promoter in darkness, whereas the application of a blue light pulse results in decreased BLR‐2 binding to the promoter. Our results suggest that BLR‐2 and probably BLR‐1 are located on the phr‐1 promoter in darkness ready to perform their function as transcriptional complex in response to light.