Molecular tools for breeding basidiomycetes

The industrial production of edible basidiomycetes is increasing every year as a response to the increasing public demand of them because of their nutritional properties. About a dozen of fungal species can be currently produced for food with sound industrial and economic bases. Notwithstanding, this production is threatened by biotic and abiotic factors that make it necessary to improve the fungal strains currently used in industry. Breeding of edible basidiomycetes, however, has been mainly empirical and slow since the genetic tools useful in the selection of the new genetic material to be introduced in the commercial strains have not been developed for these fungi as it was for other organisms. In this review we will discuss the main genetic factors that should be considered to develop breeding approaches and tools for higher basidiomycetes. These factors are (i) the genetic system controlling fungal mating; (ii) the genomic structure and organisation of these fungi; and (iii) the identification of genes involved in the control of quantitative traits. We will discuss the weight of these factors using the oyster mushroom Pleurotus ostreatus as a model organism for most of the edible fungi cultivated industrially

Identification of new antifungal metabolites produced by the yeast Metschnikowia pulcherrima involved in the biocontrol of postharvest plant pathogenic fungi

Several strains of the yeast Metschnikowia pulcherrima exhibit strong antagonistic activity against postharvest pathogens and may have broad biotechnological potential as biocontrol agents. However, the nature and interplay of the mechanisms contributing to this antifungal activity are still largely unknown. This study characterizes the antifungal compounds present in the exometabolome of two yeast strains that previously showed an efficient inhibition of Botrytis cinerea infection. We show that a yeast-fungus co-culture assay is a good system to examine the antagonistic interaction and elucidate the nature of the produced yeast metabolites. As a result, our UPLC-MS/MS analysis identified a total of 35 differentially secreted metabolites, potentially involved in the biocontrol of gray mold. Subsequent in vitro analysis and in vivo tomato, grape and apple fruit protection assays with such metabolites allowed us to identify several new antifungal compounds, with 3-amino-5-methylhexanoic acid, biphenyl-2,3-diol and sinapaldehyde being the most active (with up to 90–100% reduction in the infection of tomato and apple with B. cinerea). In addition, the first two metabolites protected tomatoes against Alternaria alternata infection. It was observed that these metabolites negatively affected the cell membrane integrity and mycelial morphology of B. cinerea and increased the intracellular level of ROS. Furthermore, other unexpected metabolites with interesting biotechnological applications were identified for the first time as being secreted by yeast cells, such as piperideine and protoemetine (alkaloids), p-coumaroyl quinic acid (phenylpropanoid), β-rhodomycin (antibiotic), hexadecanedioic acid (long chain fatty acid) or taurocholic acid (bile acid). This fact highlights that the antifungal activity of M. pulcherrima may result from synergistic action of several active molecules

Towards understanding of fungal biocontrol mechanisms of different yeasts antagonistic to Botrytis cinerea through exometabolomic analysis

There is increased interest in research on yeasts as potential phytopathogen biocontrol agents due to increasing restrictions in the use of chemical pesticides. Yeast strains from a range of genera and species have been reported to inhibit postharvest decay in different fruits. However, the mechanisms behind these yeast biocontrol capacities have not been completely deciphered because they are complex and act synergistically. In this study, we performed a thorough untargeted analysis of the exometabolome generated in a co-culture of the fungal plant pathogen Botrytis cinerea with four antagonistic yeast strains: Pichia fermentans (two strains), Issatchenkia terricola and Wickerhamomyces anomalus. As a result, general and strain-specific antifungal mechanisms and molecules were identified. The P. fermentans strains secreted the highest number of differential metabolites to the extracellular medium when co-cultured with B. cinerea. In vitro antagonistic and in vivo pathogen protection assays were performed with the selected metabolites. Among a plethora of 46 differentially secreted metabolites related to yeast-fungus competitive interaction, the phenylpropanoid trans-cinnamic acid and the alkaloid indole-3-carboxaldehyde were identified as the best antagonistic metabolites against gray mold infection under in vivo protection assays. Both metabolites caused damage to the fungal membrane and increased ROS generation in spores of B. cinerea. In addition, enhanced yeast secretion to the extracellular medium of oxylipins, dipeptides, alkaloids or antibiotics deserve to be further investigated as signaling or antagonistic molecules. This study opens the door to future investigations of roles of these molecules in yeast metabolism and application of this knowledge for biotechnological purposes.This work was financed by the Departamento de Desarrollo Económico y Empresarial from the Gobierno de Navarra (Spain): grants 0011-1365-2021-000079 and 0011-1411-2019-000009. Open Access funding provided by Universidad Pública de Navarra

Innervation of the Human Cavum Conchae and Auditory Canal: Anatomical Basis for Transcutaneous Auricular Nerve Stimulation

The innocuous transcutaneous stimulation of nerves supplying the outer ear has been demonstrated to be as effective as the invasive direct stimulation of the vagus nerve for the treatment of some neurological and nonneurological disturbances. Thus, the precise knowledge of external ear innervation is of maximal interest for the design of transcutaneous auricular nerve stimulation devices. We analyzed eleven outer ears, and the innervation was assessed by Masson’s trichrome staining, immunohistochemistry, or immunofluorescence (neurofilaments, S100 protein, and myelin-basic protein). In both the cavum conchae and the auditory canal, nerve profiles were identified between the cartilage and the skin and out of the cartilage. The density of nerves and of myelinated nerve fibers was higher out of the cartilage and in the auditory canal with respect to the cavum conchae. Moreover, the nerves were more numerous in the superior and posterior-inferior than in the anterior-inferior segments of the auditory canal. The present study established a precise nerve map of the human cavum conchae and the cartilaginous segment of the auditory canal demonstrating regional differences in the pattern of innervation of the human outer ear. These results may provide additional neuroanatomical basis for the accurate design of auricular transcutaneous nerve stimulation devices

Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts

Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.info:eu-repo/semantics/publishedVersio