Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei. Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration. IMPORTANCEThe previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei.KEYWORDS Trichoderma reesei, filamentous fungi, gene silencing, industrial biotechnology, siRNA T richoderma reesei is a filamentous fungus that is used worldwide as a host for industrial enzyme production. The enzymes produced are used, for example, in pulp and paper production (1), in the food and feed industries (2), and in the textile industry (3, 4). T. reesei enzymes are also increasingly important because of their ability to turn lignocellulosic biomass into sugars that can be used to produce biofuels and chemicals (5, 6). The hydrolyzing enzymes (cellulase and hemicellulase) that are used to degrade lignocellulosic polysaccharides into fermentable sugars contribute substantially to the cost of bioethanol production (7). Systematic strain improvement through mutagenesis and screening has given rise to industrial strains producing over 100 g/liter extracellular proteins, with around one-half of the secreted proteins being the main cellulase, cellobiohydrolase I (8). Further improvements must be made, however, to make the production even more cost-effective and to improve the performance of the produced enzymes. Specific genetic manipulation of the enzyme production organism is a

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination 1,2 . Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) 3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion 3

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