TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes

Abstract

Species of the genus <i>Trichoderma</i> are ubiquitous in the environment and are widely used in agriculture, as biopesticides, and in the industry for the production of plant cell wall-degrading enzymes. <i>Trichoderma</i> represents an important genus of endophytes, and several <i>Trichoderma</i> species have become excellent models for the study of fungal biology and plant-microbe interactions; moreover, are exceptional biotechnological factories for the production of bioactive molecules useful in agriculture and medicine. Next-generation sequencing technology coupled with systematic construction of recombinant DNA molecules provides powerful tools that contribute to the functional analysis of <i>Trichoderma</i> genetics, thus allowing for a better understanding of the underlying factors determining its biology. Here, we present the creation of diverse vectors containing (i) promoter-specific vectors for <i>Trichoderma</i>, (ii) gene deletions (using hygromycin phosphotransferase as selection marker), (iii) protein localization (mCherry and eGFP, which were codon-optimized for <i>Trichoderma</i>), (iv) gene complementation (neomycin phosphotransferase) and (v) overexpression of encoding gene proteins fused to fluorescent markers, by using the Golden Gate cloning technology. Furthermore, we present the design and implementation of a binary vector for <i>Agrobacterium</i>-mediated transformation in <i>Trichoderma</i> to increase the homologous recombination rate and the generation of a novel selection marker based on carboxin resistance.