Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One‐step PCR‐based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination‐mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination‐mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one‐step PCR‐based and fusion PCR‐based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high‐throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

Accurate determination of protein localization, levels, or protein−protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.

Histone modifications and chromatin‐binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation‐Sequencing (ChIP‐Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation‐Sequencing (fCUT&Tag‐Seq) protocol specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein‐DNA binding efficiency. We then successfully applied fCUT&Tag‐Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae , Neurospora crassa, Fusarium graminearum , and Sporisorium scitamineum , showing good signal‐to‐noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin‐binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag‐Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.

Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used ChIP-Seq method. Here, we present an optimized CUT&Tag-Seq protocol (fCUT&Tag-Seq) specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae , Neurospora crassa , Fusarium graminearum , and Sporisorium scitamineum , showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity. Impact Statement: We developed a faster, lowLJinput method to study how genes are turned on and off in fungi, even in tough species that are difficult to analyze with standard methods. Our new approach, named fCUT&Tag-Seq, requires only 10,000 cells and can be completed in just two days. It delivers clearer, more reliable results and has already been successfully applied to multiple fungi, including crop pathogens. By revealing the molecular “control switches” that govern fungal development and virulence, we expect it will accelerate basic research and help identify new targets for controlling destructive plant diseases.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step-PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increases the efficiency of recombination-mediated genetic targeting. Here we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step-PCR-based and fusion-PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomics assays at multi-population and multi-species levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems. Keypoints Developed the web tool GetPrimers to assist automatic primer design for PCR-based genetic targeting. It runs for any given locus on any genetic background of any given species with full genome scalability. Experimental validations have been performed across multiple organism systems and experimental setups.

Accurate determination of protein localization, levels, or protein-protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex-situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.