Biolistic co-transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium

Abstract

Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG) 18 of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 Wg ml 31 glufosinate ammonium using an inverted microscope with 450^490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor. ß

PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola

“…This demonstrates the advantage of using Agrobacterium as a tool in fungal transformation. Overall the rate of PEG-mediated cotransformation is relatively low compared to fungi such as Coniothyrium minitans (Jones et al 1999) but is similar to ratios reported with Trichoderma harzianum (Bae & Knudsen 2000) and with biolistic co-transformation of Metarhizium anisopliae (Inglis et al 2000).…”

Basidiomycetes Telomeres – A Bioinformatics Approach

“…This sequence has been described in Aspergillus nidulans (Bhattacharyya & Blackburn, 1997), Beauveria bassiana (Padmavathi et al, 2003), Botrytis cinerea (Levis et al, 1997), Cladosporium fulvum (Coleman et al, 1993), Fusarium oxysporum (Inglis et al, 2000), Glomus intraradices (Hijri et al, 2007), Magnaporthe grisea (Gao et al, 2002), Metarrhizium anisopliae (Inglis et al, 2005), N. crassa (Wu et al, 2009), Pestalotiopsis microspora (Long et al, 1998), Pleurotus ostreatus (Perez et al, 2009), Pneumocystis carinii (Keely et al, 2001), and Ustilago maydis (Sanchez-Alonso & Guzman, 2008). However, variations of this sequence can be found in other fungi such as A. oryzae that has dodecanucleotide telomere repeats (Kusumoto et al, 2003) Incomplete and imperfect telomere units have been reported in: A. oryzae, Candida albicans, Kluyveromyces lactis, S. cerevisiae and S. pombe.…”

Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum

“…Nevertheless, improvements at the level of gene expression could be achieved by replacing the regulator signals of the genes of interest by sequences from highly expressed genes [29,39,41]. Gene expression controlled by Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthetase expression signals has been accomplished in Penicillium chrysogenum [22], Trichoderma reesei [30], Metarhizium anisopliae [16], Acremonium chrysogenum, and Sordaria macrospora [32] among other Wlamentous ascomycetes.…”