An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae

Abstract

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.

Keywords: Agrobacterium tumefaciens-mediated transformation (ATMT); Verticillium dahliae; filamentous fungi; homologous recombination; protein labeling.

Figure 1

Overview of the in situ protein labeling methodology. (A) Construction of the tagging vectors utilized in this study. (B) Stepwise protocol for Agrobacterium tumefaciens-mediated transformation (ATMT). (C) Schematic of the design strategy for in situ tagging and the corresponding genomic integration resulting from the process. HPT BOX—hygromycin B resistance gene; HSVtk—herpes simplex virus thymidine kinase.

Figure 2

Construction of in situ GFP tagging vectors for VdCf2 and VdDMM2. (A) Schematic of primer design specific to the in situ GFP tagging approach. (B) Agarose gel electrophoresis confirmation of the PCR-amplified upstream and downstream flanking regions of insertions at VdCf2 and VdDMM2 genes loci. (C) Schematic of the in situ GFP tagging vector construction, including the primer sets within the HPT BOX, employed for validating the integration of the upstream and downstream flanking sequences into the pGKO-GFP-HPT vector.

Figure 3

The verification of in situ GFP tagging for VdCf2 and VdDMM2 in V. dahliae. (A) Transformants of VdCf2-GFP and VdDMM2-GFP grown on potato dextrose agar (PDA) plates with 5-fluoro-2′deoxyuridine (5FU) and hygromycin B. (B) Schematic of verification primer pairs specifically designed for the assessment of successful tagging. (C) PCR validation results for demonstrating the successful in situ GFP tagging of VdCf2 and VdDMM2, utilizing primer pairs VdCf2-V1/Hpt-R, VdCf2-V2/Hpt-F and VdDMM2-V1/Hpt-R, VdDMM2-V2/Hpt-F.

Figure 4

The subcellular localization for in situ GFP-tagged VdCf2 and VdDMM2 in V. dahliae cells. The fluorescence imaging of VdCf2-GFP and VdDMM2-GFP under confocal microscope (Leica TCS SP8). Scale bar: 15 μm.

Figure 5

The verification of in situ flag tagging for VdCf2 and VdDMM2 in V. dahliae. (A) Transformants of VdCf2-Flag and VdDMM2-Flag grown on PDA plates with 5FU and hygromycin B. (B) Schematic of verification primer pairs specifically designed for the assessment of successful tagging. (C) PCR validation results for demonstrating the successful in situ Flag tagging of VdCf2 and VdDMM2, utilizing primer pairs VdCf2-V1/Hpt-R, VdCf2-V2/Hpt-F, VdDMM2-V1/Hpt-R, and VdDMM2-V2/Hpt-F.

Figure 6

Western blot analysis of VdCf2 and VdDMM2 in situ Flag tagging in V. dahliae. (A) Western blot confirmation of VdCf2-Flag protein expression, with total protein loading visualized by Coomassie brilliant blue (CBB) staining. (B) Western blot confirmation of VdDMM2-Flag protein expression, with total protein loading visualized by CBB staining.