Identificación de genes inducidos en la cepa de biocontrol Trichoderma harzianum cect 2413 durante la interacción con plantas de tomate. Caracterización biológico-funcional de los genes qid74 y asp1

[ES]Tradicionalmente, las especies del género Trichoderma eran consideradas como hongos saprófitos del suelo, de vida libre, cuya habilidad para parasitar a hongos fitopatógenos dio lugar a su uso como agentes de control biológico. Posteriormente, han sido definidas como simbiontes oportunistas avirulentos de plantas por su capacidad de asociarse de manera íntima con las raíces vegetales y, como resultado, promover el crecimiento de los cultivos y estimular sus respuestas de defensa frente al estrés biótico generado tras el ataque de organismos fitopatógenos y plagas. Hoy en día, se han redefinido como simbiontes multifuncionales antiestrés debido a los múltiples efectos beneficiosos que le brindan a los cultivos. Sin embargo, apenas se conoce como se relacionan los mecanismos moleculares que subyacen tras la interacción Trichoderma-planta. Entender a nivel molecular que factores intervienen en dicha interacción es crucial para explotar las propiedades beneficiosas de ciertas especies de Trichoderma a nivel agrícola. Con el propósito de profundizar en los mecanismos moleculares implicados en la interacción Trichoderma-planta, se diseñó un microarray de alta densidad de oligonucleótidos para especies del género Trichoderma y se utilizó para analizar la respuesta transcriptómica de T. harzianum CECT 2413 durante la interacción con plantas de tomate. Mediante esta aproximación se identificaron diversos genes fúngicos potencialmente implicados en el establecimiento de la asociación simbiótica con la planta, muchos de los cuales no habían sido previamente relacionados con dicho proceso, incluyendo genes implicados en la biosíntesis de óxido nítrico, serotonina y melatonina, desintoxicación de compuestos tóxicos y xenobióticos, actividades micoparasíticas, desarrollo del micelio o aquellos asociados con la formación de estructuras de infección del tejido vegetal. Adicionalmente, se llevó a cabo una caracterización biológico-funcional de dos de estos genes (qid74 y asp1) mediante estrategias de sobreexpresión, silenciamiento y/o disrupción génica. El gen qid74 codifica para una proteína de pared celular rica en cisteína y se había relacionado previamente con la protección celular del micelio de T. harzianum CECT 2413 y con su adherencia a superficies hidrofóbicas. En el presente estudio se encontró que la expresión de qid74 durante las primeras horas de interacción con plantas de tomate es inducida por la pared celular vegetal. Además, se demostró que qid74 está implicado en la modificación del sistema radical vegetal y, consecuentemente, en el efecto biofertilizador de T. harzianum. El gen asp1 codifica para una aspartil-peptidasa extracelular. En el presente estudio se encontró que asp1 cumple un papel esencial en la respuesta antagonista que despliega T. harzianum CECT 2413 frente al hongo fitopatógeno R. solani y que su expresión durante las primeras horas de interacción con plantas de tomate es inducida como consecuencia del reconocimiento por parte del hongo de un sustrato potencialmente colonizable. Además, nuestros resultados sugirieron que la proteína ASP1 secretada por el hongo provoca en la planta respuestas de defensa a nivel local dirigidas a limitar la colonización del tejido vegetal durante el establecimiento de la asociación simbiótica

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray

<p>Abstract</p> <p>Background</p> <p>It has recently been shown that the <it>Trichoderma </it>fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of <it>Trichoderma </it>to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight <it>Trichoderma </it>spp. and 9,121 genome-derived transcripts of <it>T. reesei</it>, and we have used this microarray to examine the gene expression of <it>T. harzianum </it>either alone or in the presence of tomato plants, chitin, or glucose.</p> <p>Results</p> <p>Global microarray analysis revealed 1,617 probe sets showing differential expression in <it>T. harzianum </it>mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to <it>Trichoderma</it>-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues.</p> <p>Conclusion</p> <p>The effectiveness of the <it>Trichoderma </it>HDO microarray to detect different gene responses under different growth conditions in the fungus <it>T. harzianum </it>strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other <it>Trichoderma </it>spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of <it>T. harzianum </it>within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the <it>Trichoderma</it>-plant interaction.</p

Un método simple y preciso para la cuantificación específica de biomasa en cultivos mixtos de hongos filamentosos por PCR cuantitativa

Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.La producción de enzimas lignocelulolíticas por hongos filamentosos tiene un gran potencial a nivel industrial debido a sus diversas aplicaciones. Los cultivos fúngicos mixtos y particularmente las biopelículas fúngicas mixtas constituyen un sistema de fermentación prometedor para una mayor producción enzimática. Sin embargo, no se ha abordado cuánto de esta mejora depende de la proporción de biomasa mixta. En este sentido, el objetivo de este estudio fue desarrollar un método para cuantificar de forma específica y precisa la biomasa fúngica mixta. Para este propósito, se recolectaron cultivos mixtos de biopelículas de 48 a 120 h de crecimiento compuestos por Aspergillus niger y Trichoderma reesei, dos hongos filamentosos utilizados industrialmente para la producción de celulasas; el micelio se pulverizó y el ADN se extrajo para ensayos de qPCR con cebadores específicos para cada hongo. Los cebadores se diseñaron a partir de regiones no conservadas de las secuencias de los genes de actina y β-tubulina de A. niger y T. reesei. La especificidad de estos cebadores se probó in silico y experimentalmente. Se obtuvo una correlación estadísticamente significativa entre la biomasa calculada mediante qPCR y los datos de biomasa en peso seco. Mediante este método, fue posible detectar cambios en las proporciones de los micelios en las biopelículas a lo largo del tiempo, lo que sugiere una interacción competitiva entre estos dos hongos. En conclusión, este método permite una cuantificación específica y precisa de la biomasa fúngica mixta y también podría aplicarse a diferentes sistemas de cultivo mixto para estudiar interacciones microbianas

Primary alveolar hypoventilation and XXXXY chromosopathy

The association of primary alveolar hypoventilation (PAH) and chromosomic diseases has not been described previously. A 19 year-old man with Fraccaro’s syndrome (XXXXY karyotype) was admitted to evaluate chronic hypercapnic respiratory failure, pulmonary arterial hypertension and cor pulmonale. PAH was diagnosed. As effective treatment, such as non-invasive positive pressure ventilation (NIPPV), is available for this disorder we should intensify the search for PAH in patients with chromosome disease

Carbon dioxide rebreathing in non-invasive ventilation. Analysis of masks, expiratory ports and ventilatory modes

Background and Aim. Carbon dioxide (CO2) rebreathing is a complication of non-invasive ventilation (NIV). Our objectives were to evaluate the ability of masks with exhaust vents (EV) to avoid rebreathing while using positive pressure (PP) NIV with different levels of expiratory pressure (EPAP). Concerning volume-cycled NIV, we aimed to determine whether cylindrical spacers located in the circuit generate rebreathing. Materials and methods. 5 healthy volunteers were evaluated. Bi-level PP was used with 3 nasal and 2 facial masks with and without EV. Spacers of increasing volume attached to nasal hermetic masks were evaluated with volume NIV. Inspired CO2 fraction was analyzed. Results. Rebreathing was zero with all nasal masks and EPAP levels. Using facial masks 1 volunteer showed rebreathing. There was no rebreathing while using all the spacers. Conclusions. In healthy volunteers, nasal and facial masks with EV prevent rebreathing. In addition, the use of spacers did not generate this undesirable phenomenon

Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications

RNA-seq analysis in plant–fungus interactions

Many fungi are pathogens that infect important food and plantation crops, reducing both yield and quality of food products. Understanding plant–fungus interactions is crucial as knowledge in this area is required to formulate sustainable strategies to improve plant health and crop productivity. High-throughput RNA-sequencing (RNA-seq) enables researchers to gain insights of the mixed and multispecies transcriptomes in plant–fungus interactions. Interpretation of huge data generated by RNA-seq has led to new insights in this area, facilitating a system approach in unraveling interactions between plant hosts and fungal pathogens. In this review, the application and challenges of RNA-seq analysis in plant–fungus interactions will be discussed

Gene Expression Analysis of Non-Clinical Strain of Aspergillus fumigatus (LMB-35Aa): Does Biofilm Affect Virulence?

Aspergillus fumigatus LMB-35Aa, a saprophytic fungus, was used for cellulase production through biofilms cultures. Since biofilms usually favor virulence in clinical strains, the expression of the related genes of the LMB 35-Aa strain was analyzed by qPCR from the biomass of planktonic cultures and biofilms developed on polyester cloth and polystyrene microplates. For this, virulence-related genes reported for the clinical strain Af293 were searched in A. fumigatus LMB 35-Aa genome, and 15 genes were identified including those for the synthesis of cell wall components, hydrophobins, invasins, efflux transporters, mycotoxins and regulators. When compared with planktonic cultures at 37 &deg;C, invasin gene calA was upregulated in both types of biofilm and efflux transporter genes mdr4 and atrF were predominantly upregulated in biofilms on polystyrene, while aspHs and ftmA were upregulated only in biofilms formed on polyester. Regarding the transcription regulators, laeA was downregulated in biofilms, and medA did not show a significant change. The effect of temperature was also evaluated by comparing the biofilms grown on polyester at 37 vs. 28 &deg;C. Non-significant changes at the expression level were found for most genes evaluated, except for atrF, gliZ and medA, which were significantly downregulated at 37 &deg;C. According to these results, virulence appears to depend on the interaction of several factors in addition to biofilms and growth temperature