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

Abstract

Species of the genus Trichoderma 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. Trichoderma represents an important genus of endophytes, and several Trichoderma 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 Trichoderma 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 Trichoderma, (ii) gene deletions (using hygromycin phosphotransferase as selection marker), (iii) protein localization (mCherry and eGFP, which were codon-optimized for Trichoderma), (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 Agrobacterium-mediated transformation in Trichoderma to increase the homologous recombination rate and the generation of a novel selection marker based on carboxin resistance.

Unveiling a Microexon Switch: Novel Regulation of the Activities of Sugar Assimilation and Plant-Cell-Wall-Degrading Xylanases and Cellulases by Xlr2 in Trichoderma virens

“…The OEXlr2-α (long version) and OEXlr2-β (short version) vectors were used to create the overexpressing vectors. The TrichoGate system was used as a Golden Gate subcloning strategy adapted for Trichoderma and other filamentous fungi [105]. Different TrichoGate vectors were used, including (1) pTrichoGate-3, containing the constitutive promoter from the translation elongation factor 1 alpha (tef1α); (2) pTrichoGate-16, containing the terminator of the nopaline synthase (T-nos); and (3) pTrichoGate-20, containing the neomycin phosphotransferase II (nptII), which confers resistance to the antibiotic Geneticin.…”

Designing artificial synthetic promoters for accurate, smart, and versatile gene expression in plants

“…Functional testing of modules driven by synthetic promoters (MSPs) generally involves two steps: MSP assembly and expression pattern analysis. Numerous popular tools (e.g., Gibson, Golden Gate, COMPASS, MoClo, GoldenBraid, MODAL, and PaperClip) have been developed for assembly of reusable DNA parts into working modules, thereby accelerating MSP assembly ( Sarrion-Perdigones et al., 2013 ; Coll et al., 2016 ; Naseri et al., 2019 ; Nogueira-López et al., 2019 ; Lukan et al., 2022 ). Multiple synthetic parts with different universal adaptors are ligated simultaneously to generate anticipated modules, facilitating assembly work flow ( Sarrion-Perdigones et al., 2013 ; Engler et al., 2014 ).…”

A User’s Guide to Golden Gate Cloning Methods and Standards

“…The most common example of this is the assembly of a genetic circuit from individual transcriptional units: for practical reasons, it is often advantageous to assemble each transcriptional unit separately (stage 1 of the assembly) and then combine the separate transcriptional units into a single construct (stage 2 of the assembly). Another example is when a transcriptional unit must first be assembled individually, and then combined with additional modules such as helper genes, , selection markers, ,,− replication origins for different hosts, , centromeres, , targeting sequences for genomic integration, ,,,,,, or origins of transfer ( oriT ).…”