Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

Abstract

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in bulbs in storage. In order to study the mechanisms of pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes EGFP (green), EYFP (yellow) or ECFP (cyan) using the AGL-1 strain of A. tumefaciens. Hygromycin B (100 μg/ml) resistant colonies were observed only when acetosyringone was added to the co-cultivation medium. Transformed colonies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N + membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, and they expressed green, yellow or cyano fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive for Hyg R lines but not for the untransformed isolate. The cyano fluorescence of the ECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying pathogenicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions.

Agrobacterium-mediated transformation of Fusarium proliferatum

“…GFP has long been used as a marker in the genetic transformation of fungi (Fang et al, 2006;Müller et al, 2006;Sebastianes et al, 2012;Rodrigues et al, 2013); however, other proteins can also be used with a similar efficiency. The DsRed protein, which showed similar expression to GFP, has been reported in other studies as a good transformant marker for host-pathogen interaction studies (Eckert et al, 2005;Helber and Requena, 2008;Lakshman et al, 2012).…”

“…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.…”

Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

“…and Fusarium sp., possess a large repertoire of lignocellulolytic enzymes due to their co-evolution with plants, and can convert released plant-derived sugars into ethanol [7–10]. In particular, the broad host range phytopathogen Fusarium oxysporum [11] can degrade and produce ethanol from various cellulosic substrates (e.g. untreated and pre-treated straw [12,13], brewer’s spent grain [14], potato waste [15]).…”

Rapid and Effective Oxidative Pretreatment of Woody Biomass at Mild Reaction Conditions and Low Oxidant Loadings

“…Microplate (96-well; Sarstedt, Germany) layout for three-tier screening. Fusarium oxysporum wild-type strain 11C (WT) and putative transformants were inoculated into columns (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and screened across five treatments: T1; no ethanol, T2; hygryomcinB (60 μg ml -1 ; Sigma, UK), T3; 0.5% (vv -1 ) ethanol (ethyl alcohol; Sigma, UK), T4; 6% (vv -1 ) ethanol T5; 10% (vv -1 ) ethanol with treatments positioned in rows (A-F) (Primary-tier) (A) or into rows (A-H) and then transferred and Generating and screening Fusarium oxysporum strain 11C transformants. Inoculation of Fusarium oxysporum strain 11C into Mung bean broth [32] for 5 days at 25°C and collection of conidia by filtration through sterile cheesecloth, washing twice with sterile distilled water and adjusting spore concentration to 10 4 conidia per ml (A) Inoculation of Agrobacterium tumefaciens strain AGL-1 to minimal medium (MM) [34] supplemented with Kanamycin (50 μg ml -1 ) for 2 days at 28°C (OD 600nm 0.4-0.6) followed by dilution to OD 600nm of 0.15 in induction medium (IM) [34] supplemented with 200 μM acetosyringone (AS) and incubation for 6 hours at 28°C (B) Co-cultivation of an equal volume of bacterial and fungal cells for 30 minutes at 28°C in liquid co-cultivation medium [34] followed by spreading 100 μl mix onto a UVsterilised cellulose filter membrane placed on solid cocultivation medium on large Petri plates (150x20mm) for 2 days at 25°C where if transformation is successful, T-DNA is transferred from AGL-1 to F. oxysporum strain 11C and randomly integration into the fungal genome results (C) Transfer of cellulose filter membrane to modified selection medium (SM) on large Petri plates (150x20mm) supplemented with 60 hygromycinB for 7-9 days at 25°C followed by isolation of hygromycinB (60 μg ml -1 ) resistant putatively transformed colonies into microtiter (96-well) plates with minimal medium [37] supplemented with hygromycinB (60 μg ml -1 ) for 3 days at 25°C (D) Transfer of putative transformants to microtiter (96-well) plate with minimal medium supplemented with ethanol and butanol selection for primary alcohol tolerance screening followed by purification and PCR prior to two additional rounds of screening (2° and 3°) and selection of candidates for future analysis (E).…”

“…and Fusarium sp., possess a large repertoire of lignocellulolytic enzymes due to their coevolution with plants, and can convert released plant-derived sugars into ethanol [7][8][9][10]. In particular, the broad host range phytopathogen Fusarium oxysporum [11] can degrade and produce ethanol from various cellulosic substrates (e.g. untreated and pre-treated straw [12,13], brewer's spent grain [14], potato waste [15]).…”