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

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

Comparison of Pigment Production by Filamentous Fungal Strains under Submerged (SmF) and Surface Adhesion Fermentation (SAF)

Although synthetic colorants are widely used in many industries due to their high stability at different conditions in industrial processes, evidence of its negative impact on health and the environment is undeniable. Filamentous fungi are well known for their use as alternative sources to produce natural pigments. However, an adequate comparison of the productivity parameters between the fermentation systems could be limited to their heterogeneous conditions. Even though Solid-State Fermentations (SSF) on natural substrates are widely used for pigments production, complex media, and non-controlled variables (T, pH, medium composition), these systems could not only hamper the finding of accurate productivity parameters, but also mathematical modeling and genomics-based optimization. In this context, the present study screened five pigment-producing fungi by comparing Submerged (SmF) and Surface Adhesion Fermentation [biofilm (BF) and Solid-State (SSF)] with defined media and controlled variables. For this purpose, we used the same defined media with sucrose as the carbon source for pigment production on SmF, BF, and SSF, and BF and SSF were carried out on inert supports. Five molecularly identified Penicillium and Talaromyces strains isolated from the Peruvian rainforest were selected for their ability to produce yellowish-orange colorants. Highest productivities were obtained from T. brunneus LMB-HP43 in SmF (0.18 AU/L/h) and SSF (0.17 AU/L/h), and P. mallochii LMB-HP37 in SSF (0.18 AU/L/h). Both strains also exhibited the highest yields (AU/g biomass) in the three fermentation systems, reaching values greater than 18-folds in SSF compared to the other strains. Conversely, T. wortmannii LMB-HP14 and P. maximae LMB-HP33 showed no ability to produce pigments in the SSF system. The performed experiments accurately compared the effect of the fermentation system on yield and productivity. From this, further genomics approaches can be considered for an extensive analysis of pigment synthesis pathways and a genomics-driven optimization in the best fermentation system

SÍNTESIS DE NANOCELULOSA COMO REFUERZO MECÁNICO DE ALMIDÓN TERMOPLÁSTICO

Nanocellulose was successfully synthetized from microcrystalline cellulose by an acid hydrolysis process. The sample characterization was performed employing X-ray diffraction, zeta potential and confocal Raman microscopy. Nanocellulose-reinforced thermoplastic starch (TPS) composites were prepared by solution casting method, in which a small concentration of nanocellulose improved the elastic modulus of TPS. This property was calculated using the atomic force microscopy nanoindentation method. We conclude that nanocellulose is a good mechanical reinforcement for composites from commercial sources as starch.Se sintetizó exitosamente nanocelulosa a partir de celulosa microcristalina mediante un proceso de hidrólisis ácida. La caracterización de la muestra se realizó mediante difracción de rayos X, potencial zeta y microscopía Raman confocal. Se prepararon compuestos de almidón termoplástico (TPS, por sus siglas en inglés) reforzados con nanocelulosa mediante el método “casting solution”, en el que una pequeña concentración de nanocelulosa mejoró el módulo de elasticidad del TPS. Esta propiedad se calculó mediante el método de nanoindentación por microscopía de fuerza atómica. Llegamos a la conclusión de que la nanocelulosa es un buen refuerzo mecánico para los compuestos de fuentes comerciales como el almidón