Biolistic transformation of conidia of Botryotinia fuckeliana

Abstract

Botryotinia fuckeliana, the causal agent of grey mould, was biolistically transformed to hygromycin B resistance using a plasmid (pOHT) containing a bacterial hygromycin phosphotransferase gene fused to regulatory sequences from Aspergillus nidulans. Multiple copies of the plasmid, precipitated onto tungsten particles, were delivered into the conidia by a helium-driven gene gun. Southern analysis showed that the plasmid was integrated into the fungal genome at one single locus. After five subsequent transfers on selective medium, all transformants were mitotically stable. When propagated on non-selective medium, four out of eight transformants retained their resistance to hygromycin B. Southern analysis of the fifth generation of transformants showed that no genetic rearrangements occurred during vegetative growth of stable transformants.

Biological applications of gold nanoparticles

“…The ballistic acceleration of the gene-loaded micro-or nanoparticles is realized by different means like macroscopic bullets, gas pressure or electric discharges 61 and some types of guns are commercially available. Traditionally, gene guns have been used for the introduction of plasmid DNA into plant cells, 62,63 which results in expression of the corresponding proteins inside the cells. In this case ballistic introduction with massive particles is advantageous as it allows for traversing the rigid cell walls which surround the membranes of plant cells.…”

A Major Effect Gene Controlling Development and Pathogenicity in Botrytis cinerea Identified Through Genetic Analysis of Natural Mycelial Non-pathogenic Isolates

“…Cloning of genomic DNA fragments in plasmid pWAM6 were carried out using the Gateway BP Clonase II Enzyme Mix according to manufacturer's indications (Thermofisher). Plasmid pWAM6 was derived from plasmid pNR4 (Zhang et al, 2011) by replacing the 1,9 kb XbaI-HindIII fragment containing the nourseothricin resistance cassette with a 2,6 kb XbaI-HindIII fragment containing the hygromycin resistance cassette from pOHT (Hilber et al, 1994).…”

Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein

“…When needed, PCR amplified DNA fragments were cloned in the pGEM-T vector (Promega, Madison, WI, USA). Cloning and subcloning strategies made use of plasmids pBluescript II SK + (Stratagene, La Jolla, CA, USA) as the backbone of derived constructs and of plasmid pOHT ( Hilber et al, 1994 ) as the source of a hygromycin B resistance cassette.…”