Disruption of A. fumigatus gliZ resulted in a mutant isolate unable
to produce gliotoxin [10]. RNAi-mediated silencing of sirZ in L. maculans revealed that sirZ is essential for the transcription of sirodesmin biosynthetic genes and consequently production of sirodesmin PL [11]. In this paper we describe the identification of three genes that regulate sirodesmin PL and are unlinked to the sirodesmin gene cluster. One of these genes is denoted as cpcA (cross pathway control A), www.selleckchem.com/products/elacridar-gf120918.html and is involved in regulation of amino acid biosynthesis in fungi such as Saccharomyces cerevisiae, Aspergillus nidulans, and A. fumigatus [12–14]. This pathway acts as a metabolic switch to enable the fungus to synthesize amino acids during periods of amino acid limitation. In this paper we describe the effect of starvation on the expression of sirodesmin biosynthetic genes and sirodesmin PL production in L. maculans wild type and cpcA-silenced isolates. Results Identification of genes flanked by T-DNA insertions in sirodesmin-deficient mutants of L. maculans To generate sirodesmin-deficient mutants of L. maculans, wild type isolate IBCN 18 was transformed with plasmid pGTII, which contains T-DNA with a selectable marker (hygromycin-resistance) thus generating random insertional mutants [15]. Two hundred such mutants were then screened using a bioassay that
exploits the antibacterial properties of sirodesmin PL [2]. Six-day-old cultures of the mutants grown on 10% Campbell’s V8 juice agar were overlaid with a suspension of Bacillus subtilis. The presence or absence of zones of clearing Selleckchem Tariquidar of the bacterial lawn around the fungal colony 16 h later reflected the presence or absence, respectively, of sirodesmin PL. Three mutants, as well as a previously characterized mutant in the peptide Arachidonate 15-lipoxygenase learn more synthetase gene (ΔsirP) in the sirodesmin biosynthetic pathway [6], did not clear the B. subtilis lawn. Sirodesmin-deficiency of these mutants was confirmed by HPLC analysis of filtrate of six-day-old cultures grown on 10% Campbell’s V8 juice, whereby a peak eluting at 18.2 min in the wild type and co-incident with that of sirodesmin PL, was absent from profiles
of the three mutants (data not shown). Quantitative RT-PCR showed extremely low levels of transcripts of the sirodesmin pathway-specific transcription factor, sirZ, in the three T-DNA mutants compared to the wild type strain (Figure 1). In these mutants a single copy of T-DNA had inserted in either the 5′ or 3′ untranslated regions of predicted genes (Table 1). Figure 1 Quantitative Reverse Transcription PCR analysis of the sirodesmin pathway-specific transcription factor, sirZ . in Leptosphaeria maculans wild type (IBCN 18) and sirodesmin-deficient mutants GTA6, GTA7 and GTA9. Cultures were grown for six days in 10% V8 juice. Gene expression level is normalised to that of actin. Values are means ± SE of triplicate reactions of three independent biological samples.