Functional Analysis of the Nitrogen Metabolite Repression Regulator Gene nmrA in Aspergillus flavus

Abstract

In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi.

Keywords: AreA; Aspergillus flavus; aflatoxins; nitrogen metabolism; nmrA.

FIGURE 1

Sequence alignment of nitrogen metabolite repressor NmrA proteins in Aspergillus clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, and A. terreus. DNAMAN 6.0.3.99 software was used for the alignment and presentation. Residues boxed in red mark the Rossmann fold motif.

FIGURE 2

Utilization of A. flavus ΔnmrA mutant on different nitrogen sources. (A) Radial growth of mycelia on YES, PDA, and GMM supplemented with 50 mM glutamine, ammonium, proline, alanine, sodium nitrate, and sodium nitrite, respectively, as a sole nitrogen source for 7 days at 28°C in the darkness. The photographs were taken on the 5th day. (B) Growth rate of WT, ΔnmrA and ΔnmrA::nmrA strains vegetated on YES, PDA, and GMM supplemented with different nitrogen sources on the 5th day at 28°C in the dark. Asterisk indicated statistical significance at P < 0.01.

FIGURE 3

Deletion of nmrA affected conidia production. Bars represent SE from three independent experiments with three replicates. (A) Deletion of nmrA resulted in conidiation augment on GMM supplemented with ammonium. Asterisk indicated statistical significance at P < 0.05. (B) Conidiophores were observed under a light microscope at 12 h after induction with illumination. Scale bar: 200 μm. (C) RT-qPCR analysis was performed in the indicated strains germinated on media as described in (A). The asterisks represented a significant difference level of P < 0.01.

FIGURE 4

Aflatoxin (AFB1) assessment of WT, ΔnmrA and ΔnmrA::nmrA strains grown on different culture media. (A) Aflatoxin production of WT, ΔnmrA and ΔnmrA::nmrA strains grown on media as described in Figure 2. (B) Quantification results of AFB1 of WT, ΔnmrA and ΔnmrA::nmrA strains grown on as (A) by Gene Tools analysis system software. SD means standard AFB1. The asterisks represented a significant difference level of P < 0.01.

FIGURE 5

Sclerotia production of ΔnmrA mutant. (A) One representative plate for the WT, ΔnmrA and ΔnmrA::nmrA strains grown on GMM supplemented with 50 mM glutamine and ammonium plus 2% sorbitol to induce sclerotia production at 37°C for 7 days in dark condition. (B) Quantity analysis of sclerotia among the strains listed. Asterisks indicated significant differences between each depletion strain relative to the WT and ΔnmrA::nmrA strains as determined by a Student T-test, with ^∗∗P < 0.01, ^∗∗∗P < 0.005.

FIGURE 6

Pathogenicity assay of ΔnmrA mutant. (A) Growth of A. flavus WT, ΔnmrA and ΔnmrA::nmrA strains on living peanut cotyledons after 5 days of inoculation. (B) Conidia production on peanut cotyledons after inoculation of 5 days. Asterisk indicates statistical significance at P < 0.05. (C) TLC measurements of aflatoxin (AFB1) extracted from seed in (A). SD means standard AFB1.

FIGURE 7

Sensitivity of the ΔnmrA strain to multiple stress reagents. (A) Sensitivity of WT, ΔnmrA and ΔnmrA::nmrA strains to 200 ng/mL rapamycin (specific inhibitor of TOR, Target of Rapamycin), 0.02% MMS (methyl methanesulfonate), 1 M NaCl and 1.2 M sorbitol added in YES agar. (B) Inhibition of mycelial growth in the ΔnmrA strain to multiple stress reagents. Asterisk indicates statistical significance at P < 0.01.

FIGURE 8

Effect of nitrogen source and ΔnmrA on transcript levels of nitrogen regulatory and catabolic genes. (A) Interaction network analyzed by SMART website. (B) RT-qPCR analysis was performed in the indicated strains germinated 30 h on GMM supplemented with 50 mM ammonium or NaNO2 as the sole nitrogen source. Transcript levels of nitrogen regulatory genes areA, areB, nmrA, meaB, sreA, and nirA as well as nitrogen catabolic genes niaD and niiA in ΔnmrA strain (light-green columns) were expressed compared to transcript levels of the WT (pink columns), and ΔnmrA::nmrA strains (yellow columns). The A. flavus actin gene was used as an internal control to normalize the expression data. Bars represent SE from three independent experiments with four replicates each. Asterisks indicated significant differences between each depletion strain relative to the WT and ΔnmrA::nmrA strains as determined by a Student T-test, with ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.005.