The bZIP Protein MeaB Mediates Virulence Attributes in <i>Aspergillus flavus</i>

<div><p>LaeA is a fungal specific virulence factor of both plant and human pathogenic fungi. Transcriptional profiles of <i>laeA</i> mutants have been successfully exploited to identify regulatory mechanisms of secondary metabolism in fungi; here we use <i>laeA</i> mutants as tools to elucidate virulence attributes in <i>Aspergillus flavus</i>. Microarray expression profiles of Δ<i>laeA</i> and over-expression <i>laeA</i> (<i>OE::laeA</i>) were compared to wild type <i>A. flavus.</i> Strikingly, several nitrogen metabolism genes are oppositely mis-regulated in the Δ<i>laeA</i> and <i>OE::laeA</i> mutants. One of the nitrogen regulatory genes, the bZIP encoding <i>meaB,</i> is up-regulated in Δ<i>laeA</i>. Significantly, over-expression of <i>meaB</i> (<i>OE::meaB</i>) phenocopies the decreased virulence attributes of a Δ<i>laeA</i> phenotype including decreased colonization of host seed, reduced lipase activity and loss of aflatoxin B1 production in seed. However, a double knock-down of <i>laeA</i> and <i>meaB</i> (<i>KD::laeA,meaB</i>) demonstrated that <i>KD::laeA,meaB</i> closely resembled Δ<i>laeA</i> rather than wild type or Δ<i>meaB</i> in growth, aflatoxin biosynthesis and sclerotia production thus suggesting that <i>meaB</i> does not contribute to the Δ<i>laeA</i> phenotype. MeaB and LaeA appear to be part of regulatory networks that allow them to have both shared and distinct roles in fungal biology.</p></div

Northern analysis of <i>Aspergillus flavus</i> velvet complex mutants.

<p>VeA and LaeA mutants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074030#pone.0074030-Amaike2" target="_blank">[4]</a> grown in liquid GMM conditions under dark for 48 hours at 250 rpm. Note increase and decrease of <i>niaD</i> expression in the <i>laeA</i> deletion (Δ<i>laeA</i> TJW71.1) and over-expression (<i>OE::laeA</i> TJW79.13) respectively. Δ<i>veA</i> (TSA1.54) also shows decreased <i>niaD</i> expression. <i>OE::veA</i> = TSA2.46.</p

Fungal strains and plasmids used in this study.

<p><i>A. fum = A. fumigatus.</i></p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Aflatoxin production on different nitrogen sources.

<p>The indicated strains were grown in GMM lacking nitrogen, standard GMM (70.6 mM sodium nitrate), or GMM with 70.6 mM ammonium chloride for two days as described in Materials and Methods. All media was supplemented with uracil and uridine. 40% of extracted metabolites were loaded onto the TLC plates except in the cases of wild type on GMM with ammonium, as well as <i>OE::laeA</i> on GMM with nitrate and GMM with ammonium. For these three samples, 10% of extracted metabolites were loaded because of the high levels of AF biosynthesis. AF = aflatoxin standard.</p

Expression of nitrogen metabolism genes in comparisons of wild type to either Δ<i>laeA</i> or <i>OE::laeA</i> strains.

<p>Data summarized from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074030#pone.0074030-Georgianna1" target="_blank">[15]</a>.</p><p>− = decreased expression in <i>laeA</i> mutant versus wild type.</p><p>+ = increased expression in <i>laeA</i> mutant versus wild type.</p><p>NS = not significant difference in expression in <i>laeA</i> mutant versus wild type.</p><p><i>crnA</i>: nitrate transporter, <i>niiA</i>: nitrite reductase, <i>niaD</i>: nitrate reductase, <i>areA</i>: GATA transcriptional activator, <i>nmrA</i>: nitrogen metabolite repression regulator, <i>meaB</i>: methylammonium resistant B.</p

Sclerotia production of <i>meaB</i> mutants.

<p>(A) The strains listed were grown on GMM plus 2% sorbitol to induce sclerotia production. All media was supplemented with uracil and uridine. Asterisks indicate significant differences between each strain relative to the wild type as determined by a Student T test, with * = P<0.01 and ** = P<0.001. (B) One representative plate for each strain is shown here before removal of sclerotia. Despite less coverage on the plate, the <i>OE::laeA</i> sclerotia were of greater mass than the other strains.</p

Growth of <i>A. flavus meaB</i> mutants on different nitrogen sources.

<p>Plates containing GMM with no nitrogen, GMM with 10 mM sodium nitrate, GMM with 10 mM ammonium chloride, GMM with 10 mM ammonium chloride plus 30 mM sodium nitrite, GMM with 10 mM ammonium chloride plus 200 mM potassium chlorate, or GMM with 10 mM sodium nitrate plus 100 mM methylammonium chloride were inoculated with the indicated strains of <i>A. flavus</i> and grown for 3 days at 29°C. All media was supplemented with uracil and uridine.</p

Pathogenicity of <i>A. flavus meaB</i> mutants.

<p>(A) Growth of fungal colonies on living peanut cotyledons after 5 days of inoculation. (B) Conidia production on peanut cotyledons after 5 days of inoculation. Asterisk indicates statistical significance at P<0.05. (C) Lipase activity of <i>meaB</i> mutants. Asterisk indicates statistical significance at P<0.0001. (D) Thin layer chromotrography measurements of aflatoxin B1 extracted from seed in Panel A. AF = aflatoxin B1 standard.</p