Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Abstract

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.

Agrobacterium tumefaciens-Mediated Transformation for Investigating Pathogenicity Genes of the Phytopathogenic Fungus Colletotrichum sansevieriae

“…Agrobacterium tumefaciens-mediated transformation (AtMT) used to be restricted to plant DNA transformation [9] and initially, AtMT was performed for the transformation of the yeast Saccharomyces cerevisiae [1], and then, this technique has been exploited for a wide variety of different fungal species over the past 12 years [5,7,12,16,27,30,34,38]. Good results were also obtained with the AtMT of Colletotrichum spp.…”

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

“…A. tumefaciens-T-DNA transfer has been extensively proven to be an efficient transformation system for filamentous fungi (Nizam et al, 2010;Vieira and Camilo, 2011;Maruthachalam et al, 2012). The binary vector pPm43GW-GFP-HPH, containing cassette with E. coli hph-resistance gene under the regulation of the Aspergillus nidulans trpC promoter and the PtGFP cassette containing the promoter toxA-5'-UTR from Pyrenophora tritici-repentis, was successfully employed.…”

“…Such advantages include a high efficiency in transformation, a high percentage of single T-DNA insertion (Blaise et al, 2007), and the possibility to operate on distinct types of cells, spores, hyphae, or fructifications (Michielse et al, 2005). ATMT has been long used to transfer genes to a wide variety of fungi, including plant pathogenic or symbiotic fungi, mushrooms, industrial fungi, and biological control fungi (Ando et al, 2009;Sharma and Kuhad, 2010;Vieira and Camilo, 2011). A. tumefaciens has the natural ability to transfer a segment of its Ti plasmid, known as 'T-DNA' to plant or fungal cells, becoming randomly integrated into nuclear chromosomes.…”

INTRODUKSI GEN METALLOTHIONEIN TIPE II KE DALAM RUMPUT LAUT Kappaphycus alvarezii MENGGUNAKAN Agrobacterium tumefaciens

“…Peningkatan mutu genetik dapat dilakukan dengan kultur jaringan, fusi protoplas, dan transformasi genetik pada rumput laut Porphyra yezoensis (Cheney, 2000). Metode transformasi genetik menggunakan Agrobacterium tumefaciens umum digunakan pada tanaman tingkat tinggi, fungi, khamir, dan sel manusia (Vieira & Camilo, 2011 …”