Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

Abstract

BackgroundLarge efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation.ResultsHigh-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs—19-nt duplex with 2-nt 3′ overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)—into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis.ConclusionOur quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0287-4) contains supplementary material, which is available to authorized users.

An efficient gene disruption method for the woody plant pathogen Botryosphaeria dothidea

“…The results of this study showed that when using the same molar mass of GHR cassette and plasmid DNA in the transformation system, the GHR cassette gave much higher GD efficiency than the plasmid. This result agrees with findings in V. dahliae [26], Lecanicillium lecanii [27], Fusarium graminearum [28], Candida albicans [29] and Acremonium implicatum [30]. Moreover, although the GD efficiency increased when the GHR cassette quantity was increased from 5 to 25 μg per 10 6 protoplasts, multiple insertions occurred when the GHR cassette quantity was more than 15 μg per 10 6 protoplasts.…”

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach

“…Rehman et al (2016) optimized the protoplast isolation, regeneration, and transformation efficiency of Verticillium dahlia , the causal agent of Verticillium wilt. The maximum release of protoplasts was obtained using 200 mg driselase in 10 ml NaCl (0.7 M) at 2.5 h after incubation.…”

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies

“…Transient assays in grasses mostly rely on protoplasts[12-14]. However, expression of a gene in protoplasts may not always mimic in planta native state and, also experience inconsistent efficiency due to variability in quality of protoplasts and size of vector transformed[15]. Here, we have established a simplified transient assay with Agrobacterium , also known as agroinfiltration, for transient transformation of sorghum and demonstrated its application by confirming gene editing in sorghum leaves using GFP as a marker.…”