{"doi":"10.1101/2025.11.30.691285","title":"Construction of a randomly barcoded insertional mutant library in the filamentous fungus <i>Trichoderma atroviride</i>","abstract":"Abstract Filamentous fungi play key roles in ecosystems, agriculture, biotechnology, symbiosis, and disease, yet the large-scale characterization of gene function in these organisms remains limited by low transformation efficiencies and their multinucleate, syncytial cells, which complicate high-throughput screening strategies. To address the challenge of high-throughput screening in filamentous fungi, we developed methods to construct a genome-wide barcoded insertional mutant library in Trichoderma atroviride , a filamentous fungus widely used as a biocontrol agent against bacterial and fungal plant pathogens. Our strategy leveraged randomly barcoded transfer DNA insertions from plasmid libraries containing hundreds of millions of unique DNA barcodes and a broad host-range drug resistance marker delivered via Agrobacterium tumefaciens into T. atroviride . By optimizing transformation conditions, we achieved up to 600 independent transformants per infection event, resulting in a library of over 31,000 mapped insertions disrupting 7,104 of the 11,863 predicted genes in the T. atroviride genome. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling both fundamental investigations of fungal biology and engineering approaches toward improved medical applications, biotechnology, and sustainable agriculture.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":559623,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9502,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1460903,"name":"José Manuel Villalobos‐Escobedo","orcid":"0000-0002-8412-6748","position":1,"is_corresponding":false},{"id":356772,"name":"Jeffrey M. Skerker","orcid":"0000-0003-2653-1566","position":2,"is_corresponding":false},{"id":1296960,"name":"Ran Shi","orcid":"0009-0004-2436-2900","position":3,"is_corresponding":false},{"id":906027,"name":"Adriana M. Rico-Ramírez","orcid":"0000-0002-4196-8427","position":4,"is_corresponding":false},{"id":1460904,"name":"Catharine A. Adams","orcid":"0000-0002-0914-0806","position":5,"is_corresponding":false},{"id":2171,"name":"Adam P. Arkin","orcid":"0000-0002-4999-2931","position":6,"is_corresponding":false},{"id":336360,"name":"Adam M. Deutschbauer","orcid":"0000-0003-2728-7622","position":7,"is_corresponding":false},{"id":292006,"name":"N. Louise Glass","orcid":"0000-0002-4844-2890","position":8,"is_corresponding":false},{"id":689491,"name":"Lori B. Huberman","orcid":"0000-0003-2638-8528","position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":null,"created_at":"2026-07-19T02:55:34.849815Z","pmid":"41377505","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}