Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

Abstract

BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.

Vegetative compatibility groups explain variation in the virulence ofVerticillium dahliaeon strawberry

“…A cumulative logit model is regression model for a cumulative logit [53]: where x k represents the k th explanatory variate, the k effect of x k , and Table 1). The VdAve1 random insertion vector was constructed using USER-Brick vector system, [54]. Core USER Bricks, as well as the constitutive fungal promoter PgpdA and the selective marker Hygromycin, were amplified from the plasmid pRF-HU2-F/R and pRF-HUE-F/R using the primers specified in [54]).…”

“…The VdAve1 random insertion vector was constructed using USER-Brick vector system, [54]. Core USER Bricks, as well as the constitutive fungal promoter PgpdA and the selective marker Hygromycin, were amplified from the plasmid pRF-HU2-F/R and pRF-HUE-F/R using the primers specified in [54]). Two different insertion vectors were assembled; one containing the Ave1 native promoter, and another using the constitutive promoter PgpdA.…”

“…The known avirulence gene gene VdAve1 and its native promoter (called PAve1) were cloned from the 'race 1' isolate 12067 using the primer pair VdAve1C-F/R and NPAve1-F/R, respectively (Supplementary Table 1). The VdAve1 random insertion vector was constructed using USER-Brick vector system, 42 . Core USER Bricks, as well as the constitutive fungal promoter PgpdA and the selective marker Hygromycin, were amplified from the plasmid pRF-HU2-F/R and pRF-HUE-F/R using the primers specified in 42 ).…”

Agrobacterium-mediated transformation of Fusarium proliferatum

“…Although the literature contains no report of the transformation of F. proliferatum, other species of the genus Fusarium have been transformed via A. tumefaciens, such as F. circinatum (Covert et al, 2001), F. graminearum (Lysøe et al, 2006), F. oxysporum Lakshman et al, 2012), F. culmorum (Michielse et al, 2005), F. solani Bao, 2009), F. equiseti (Maciá-Vicente et al, 2009), F. virguliforme (Pudake et al, 2013) and F. avenaceum (Sørensen et al, 2014). In these studies, the presence of acetosyringone led to a more efficient transformation.…”

Agrobacterium tumefaciens-Mediated Transformation

“…This technique has primarily become possible due to the development of proofreading DNA polymerases that do not stall at the 2-deoxyuridines in the primers, which has signifi cantly reduced the risk of PCR introduced mutations (Nørholm 2010 ). The USER Fusion technique has allowed my lab to develop a new cloning strategy (USERBricks) for fast and effi cient construction of vectors intended for performing targeted genome modifi cations (Sørensen et al 2014 ). The USERBrick system consists of a set of standardized building blocks, e.g., vector backbones, selection markers, and promoters, that can be combined to generate vectors intended for gene replacement, in locus overexpression, GFP-tagging, and ectopic expression.…”