Identification of plant genes putatively involved in the perception of fungal ergosterol‐squalene

[EN] Trichoderma biocontrol strains establish a complex network of interactions with plants, in which diverse fungal molecules are involved in the recognition of these fungi as nonpathogenic organisms. These molecules act as microbial-associated molecular patterns that trigger plant responses. Previous studies have reported the importance of ergosterol produced by Trichoderma spp. for the ability of these fungi to induce plant growth and defenses. In addition, squalene, a sterol biosynthetic intermediate, seems to play an important role in these interactions. Here, we analyzed the effect of different concentrations of ergosterol and squalene on tomato (Solanum lycopersicum) growth and on the transcription level of defense- and growth-related genes. We used an RNA-seq strategy to identify several tomato genes encoding predicted pattern recognition receptor proteins or WRKY transcription factors, both of which are putatively involved in the perception and response to ergosterol and squalene. Finally, an analysis of Arabidopsis thaliana mutants lacking the genes homologous to these tomato candidates led to the identification of a WRKY40 transcription factor that negatively regulates salicylic acid-related genes and positively regulates ethylene- and jasmonate-related genes in the presence of ergosterol and squaleneSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO-AGL2015–70671-C2-2-R and MICINN-RTI2018–099600-B-I00 to S.G.), and also by the Junta de Castilla y León (Spain) (LE251P18). L. Lindo was granted a fellowship by the University of León (Spain

Requirement of Two Acyltransferases for 4-O-Acylation during Biosynthesis of Harzianum A, an Antifungal Trichothecene Produced by Trichoderma arundinaceum

Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4-O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungiSIThe Spanish Ministry of Economy and Competitiveness supported this work (MINECO-AGL2015-70671-C2-2-R to S.G.), and the University of León granted L.L. a fellowshi

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

[EN] Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungiSISG received funding from the Spanish Ministry of Economy and Competitiveness (grant number MINECO-AGL2015-70671-C2-2-R). TL received funding from the National Institute of Agricultural Sciences, Rural Development Administration (grant number PJ00843203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Effect of Farnesol in Trichoderma Physiology and in Fungal–Plant Interaction

[EN] Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1, encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1-overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactionsSIThis research was funded by the Spanish I+D+i Grants AGL2012-40041-C02-02, AGL2015-70671-C2-2-R, RTI2018-099600-B-I00 and PID2021-123874OB-I00, financed by the MCIN/ AEI/10.13039/501100011033. GC-H was awarded with a Grant from the Ministry of Education, Culture, and Sport (Spain) (Grant number FPU15/04681). NM-R was awarded with a Grant from the Junta de Castilla y León (Spain) (ORDEN EDU/875/2021, 13 July 2021

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma

[EN] Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.S

Nuevas terapias dirigidas para el tratamiento del cáncer

El cáncer es el término que se utiliza para englobar un conjunto de enfermedades que se caracterizan por el crecimiento descontrolado de células alteradas molecularmente por mutaciones o modificaciones epigenéticas.En la presente revisión describimos algunas terapias dirigidas que se están utilizando actualmente en clínic

Caracterización evolutiva y molecular de la biosíntesis de trichotecenos y análisis del papel de los esteroles de membrana de trichoderma en la interacción con plantas = Evolutionary and molecular characterization of the trichothecen biosynthesis and analysis of the role of Trichoderma membrane sterols in the interaction with plants

364Los trichotecenos son una familia de toxinas derivadas de la vía de los terpenos que son producidos por múltiples géneros fúngicos. Durante este trabajo se ha llevado a cabo un análisis filogenético de estos hongos productores de trichotecenos y de los genes involucrados en esta ruta biosintética, así como estudios químicos y de biología molecular que permiten aclarar la función de cada gen. De esta forma, se ha visto que la diversidad estructural existente entre los más de 150 trichotecenos detectados hasta el momento se debe, desde el punto de vista evolutivo, a una ganancia o pérdida de genes tri y a los eventos que conducen a la variación de determinados genes para la adquisición de nuevas funciones. También se ha visto que la producción de diversidad de trichotecenos se debe, en parte, a fenómenos de transferencia horizontal de genes debido a que la evolución de las especies no está siempre relacionada con la evolución independiente de los clústeres de genes tri o de los genes individuales. Durante este trabajo se han obtenido mutantes de Trichoderma arundinaceum con los genes tri3, tri17, tri6, tri10 y tri18 delecionados; los cuales han permitido el estudio de la función de cada gen en la producción de trichotecenos (harzianum A en este caso). Así, se ha visto que tri6 y tri10 son genes reguladores de la producción de trichotecenos. TRI6 codifica para una proteína de dedos de zinc Cys2Hys2. Su eliminación a nivel genómico compromete totalmente la producción de harzianum A (HA) y reduce la expresión de los genes tri, resultando por tanto ser un regulador positivo de los genes tri. Tiene además funciones como regulador global afectando a genes presentes en 10 de los 49 clústeres de biosíntesis de metabolitos secundarios detectados en esta especie. tri10, sin embargo, no es esencial, pero la deleción del gen tri10 reduce en más del 80% la producción de harzianum A. La proteína TRI10 también ejerce una regulación positiva de los genes tri en las primeras horas de crecimiento, tiempo en el que los genes reguladores tri6 y tri10 se expresan más. Se definió la función de los genes tri3, tri17 y tri18. Las funciones de TRI17 y TRI18 fueron descritas por primera vez. TRI17 es la encargada de sintetizar la cadena lateral poliquetídica del HA: 8-carboxi-2,4,6-trieneoil-CoA. Tri18 es una acetiltransferasa necesaria junto a TRI3 para la conversión de trichodermol a harzianum A. Se ha demostrado que TRI3 cataliza la acetilación en C4 del trichodermol para formar trichodermina, mientras TRI18 cataliza la sustitución del grupo acetilo en C4 de la trichodermina por la cadena lateral sintetizada por el gen TRI17. Mientras TRI17 y TRI18 son esenciales para la producción de harzianum A, la eliminación del TRI3 conserva aproximadamente un 10% de la producción de este trichoteceno. Esto puede ser debido a que TRI18 conserva parte de la funcionalidad asignada al TRI3 o a la acción de alguna acetiltransferasa inespecífica existente en T. arundinaceum. Por otra parte, la reducción de producción de HA no refleja una reducción en la actividad antimicrobiana de la especie. Esto ha sido atribuido, al menos en parte, a un aumento en la producción de aspinolidas, compuestos poliquetídicos con actividad antibiótica. Este hecho, sumado al aumento de ergosterol que acompaña la reducción de producción de trichotecenos permite deducir que la producción de trichotecenos está siendo utilizada por el hongo para regular la biodisponibilidad de farnesil difosfato, un intermediario clave en la vía de los terpenos del metabolismo primario. Sumado a lo anterior, la secuenciación de los genomas de los mutantes y el estudio de RNAseq llevado a cabo con la cepa silvestre y el mutante delecionado en tri6 han permitido hacer un análisis bioinformático exhaustivo de la cepa que ha llevado a la detección de genes y análisis de clústeres. Esto es especialmente importante para detectar el potencial biosintético de la cepa y dejar abierta la puerta a futuros estudios. Finalmente, y continuando con la relación del ergosterol con la biosíntesis de trichotecenos y la compleja red de interacciones que establece Trichoderma con la planta se procedió a hacer un estudio del efecto de ergosterol y de escualeno en plantas. A lo largo de este trabajo se ha analizado el efecto de ergosterol/escualeno en el crecimiento y transcripción de genes de plantas de tomate mediante un estudio de RNAseq comparativo de dos condiciones: con y sin los citados compuestos. Así se han detectado dos receptores putativos de estos compuestos y un factor de transcripción WRKY presunta-mente involucrado en la respuesta desencadenada en la planta por estos compuestos. Mutantes de Arabidopsis thaliana delecionados en un gen homólogo a este factor de transcripción revelaron su importancia como regulador negativo de la vía del salicilato y positivo de las vías del etileno y del jasmonato en presencia de ergosterol/escualeno