By José Manuel Villalobos-Escobedo
Filamentous fungi are involved in processes ranging from nutrient cycling and material decomposition to agriculture and human health. Some are allies of plants, helping control pests and pathogens; others can cause disease, harm amphibian and bat populations, and contribute to crop losses of up to 30% worldwide each year (1).
Understanding what each of their genes does could help explain these capabilities, but the functions of most fungal genes remain unknown. Studying them one at a time is also slow and costly. Now, a new tool makes it possible to analyze thousands of genes simultaneously and determine which ones are essential for fungal growth under different conditions.
The study, Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus, presents a barcoded mutant library that allows researchers to study thousands of fungal genes at once and uncover their functions.
The strategy involves disrupting different genes and assigning each mutant a unique genetic tag. Researchers can then track which mutants become more or less abundant as the fungus grows under different conditions, revealing which genes are required for survival and growth in each environment.
Unlocking the hidden genes of a “mold”
The researchers created a library containing more than 83,000 mutants of Trichoderma atroviride, each identified by a unique DNA barcode. This filamentous fungus occurs naturally in soil and is used in agriculture as a biological control agent to help protect plants from microorganisms that can harm them.
The researchers also adapted a strategy previously used in yeasts and bacteria, known as RB-TDNAseq, for use in this multicellular, multinucleate organism, T. atroviride.
The method uses the bacterium Agrobacterium tumefaciens to insert DNA fragments carrying a unique tag, or “barcode,” at different locations in the fungus’s genome.
Each insertion disrupts a different gene. Because every mutant carries its own barcode, thousands of mutants can be pooled into a single population, allowing researchers to track the behavior of each one using a high-throughput sequencing technique called BarSeq.
Using this approach, the researchers built a library of 83,311 insertions that disrupted 5,331 of the 11,863 predicted genes in the T. atroviride genome.
They then exposed this mixed population of mutants to different growth conditions—including media lacking specific amino acids and media in which fructose or xylose was the only carbon source—and tracked which mutants became less abundant under each condition. A decline indicated that the disrupted gene was required for growth in that environment.
The analysis identified genes already known to be involved in the synthesis of sulfur-containing amino acids, confirming that the method was working as expected.
It also uncovered genes involved in fructose metabolism, including a hexokinase required to process the sugar, as well as genes essential for using xylose, a major component of plant cell walls. These included a transcriptional regulator homologous to xlnR and a xylitol dehydrogenase.
To validate these findings, the team turned to deletion mutants of the model fungus Neurospora crassa. When the corresponding genes were missing, the mutants were unable to grow with xylose or xylan as a carbon source, just as the analysis predicted.
Beyond these specific examples, the study’s most significant contribution is methodological. The plasmid library, containing nearly 290 million unique DNA barcodes, together with the optimized transformation protocol, could be adapted to other species of filamentous fungi—not just members of the genus Trichoderma.
The same strategy has already been used to generate a comparable library in a different thermophilic fungus, providing evidence that the approach can be transferred across a broad range of fungal species.
This platform provides a foundation for large-scale studies of gene function in fungi with medical, agricultural, and biotechnological applications.
In the long term, the approach could help identify targets for new antifungal treatments for humans, animals, and plants; improve biological control agents such as Trichoderma; and support the development of fungal strains optimized to produce enzymes or other compounds of industrial interest.
References
- Huberman LB, Villalobos-Escobedo JM, Skerker JM, Shi R, Rico-Ramírez AM, Adams CA, Arkin AP, Deutschbauer AM, Glass NL. Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus. PNAS. 2026;123(35):e2616888123.
Author
José Manuel Villalobos-Escobedo. Research professor at the Institute for Obesity Research at Tecnológico de Monterrey, where he leads the Functional Genomics for Bioprospecting Laboratory. His research combines functional genomics, metagenomics, and metabolic modeling to study fungi and bacteria with applications in biotechnology.
