Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large‐scale transposon tagging

Abstract

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia(+) revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016, displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans. The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.

Plant defense response against Fusarium oxysporum and strategies to develop tolerant genotypes in banana

“…Recently homologs of three Foc pathogenicity genes, SIX genes called SIX1, SIX7 and SIX8 were identified by Meldrum et al (2012). Insertional mutant banks of Fol generated by inserting the novel modified impala element (fungal transposon) can be used for further investigation of pathogenicity genes (Lopez-Berges et al 2009). …”

A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB

“…Generation and molecular characterization of the F. oxysporum Dfmk1, Dste12, and DmeaB mutants were described previously (Di Pietro et al, 2001;Ló pez-Berges et al, 2009;Rispail and Di Pietro, 2009). Fusarium graminearum isolate PH-1 and Magnaporthe oryzae isolate Guy-11 were used as indicated.…”

“…Targeted replacement of the F. oxysporum MeaB gene in the Dfmk1 background was performed using a fusion PCR construct and protocol described previously (Ló pez-Berges et al, 2009). Targeted replacement of the entire coding region of the F. oxysporum AreA gene with the hygromycin resistance cassette under control of the A. nidulans GpdA promoter (PgpdA) and TrpC terminator (Ttrpc) was performed using the split-marker method (Catlett et al, 2003) following the protocol previously described (Rispail and Di Pietro, 2009) (see Supplemental Figure 7 online).…”

The bZIP Transcription Factor MeaB Mediates Nitrogen Metabolite Repression at Specific Loci

“…For cDNA synthesis, 1 g total RNA was reverse transcribed using the oligo(dT) [12][13][14][15][16][17][18] primer and SuperScript II (Invitrogen) according to the manufacturer's instructions. PCR mixtures contained 25 ng DNA, 5 pmol of each primer, 200 nM deoxynucleotide triphosphates, and 1 unit BioTherm DNA polymerase (GeneCraft GmbH, Lüdinghausen, Germany).…”

“…Recently, it has been reported that under conditions of nitrogen sufficiency MeaB activates nmrA expression by binding to a conserved sequence in the nmrA promoter, thus indirectly modulating AreA activity (43). MeaB orthologues have not yet been characterized in any other fungus, although recently meaB was identified by transposon tagging as a virulence factor in the plant pathogen Fusarium oxysporum (17), and in F. fujikuroi meaB was identified by microarray analysis as a gene whose transcription is regulated in response to nitrogen availability (32).…”