NsdC and NsdD affect Aspergillus flavus morphogenesis and aflatoxin production

Abstract

The transcription factors NsdC and NsdD are required for sexual development in Aspergillus nidulans. We now show these proteins also play a role in asexual development in the agriculturally important aflatoxin (AF)-producing fungus Aspergillus flavus. We found that both NsdC and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Conidiophores in nsdC and nsdD deletion mutants had shortened stipes and altered conidial heads compared to those of wild-type A. flavus. Our results suggest that NsdC and NsdD regulate transcription of genes required for early processes in conidiophore development preceding conidium formation. As the cultures aged, the ΔnsdC and ΔnsdD mutants produced a dark pigment that was not observed in the wild type. Gene expression data showed that although AflR is expressed at normal levels, a number of aflatoxin biosynthesis genes are expressed at reduced levels in both nsd mutants. Expression of aflD, aflM, and aflP was greatly reduced in nsdC mutants, and neither aflatoxin nor the proteins for these genes could be detected. Our results support previous studies showing that there is a strong association between conidiophore and sclerotium development and aflatoxin production in A. flavus.

Fig 1

Colony growth, pigmentation, and sclerotium production in A. flavus CA14 ΔnsdC and ΔnsdD mutants and ΔnsdC and ΔnsdD complementation strains. CA14, ΔnsdC 17 and ΔnsdD 3 mutants, and genetically complemented ΔnsdC C5 and ΔnsdD C4 strains were grown on YGT-U medium for 5 days with fluorescent light illumination. (A) View from top of colony. Conidial pigmentation was altered in the Δnsd mutants. Decreased growth of the ΔnsdD 3 strain was observed. (B) View of underside of colony. Pigmentation was readily observed on the undersides of the ΔnsdC 17 and ΔnsdD 3 mutant colonies. (C) YGT-U plates demonstrating sclerotium production were grown for 14 days in the dark. The plates were sprayed with 70% ethanol to allow visualization of sclerotia. Sclerotia were absent in the ΔnsdC and ΔnsdD mutants and were produced in the wild-type CA14 and ΔnsdC and ΔnsdD complementation strains. S, sclerotia.

Fig 2

Light microscopy of Δnsd mutants reveals altered conidiophore morphology. (A) Column 1, examination of conidiophore stipe length. Fungal mycelia were harvested from PDA-U plates and placed in mounting medium solution on a microscope slide. Magnification, ×400. Arrows denote the conidiophore stipe. Note that the CA14 image does not demonstrate the total stipe (see column 2). Column 2, examination of conidial head structure. Images were captured from cultures growing on surfaces of PDA-U agar plates. Magnification, ×60. Column 3, examination of conidiophore production under submerged culture in PDA-U broth. Magnification, ×200. Phialides (P) and conidia (C) on the degenerate conidiophores are indicated. (B) Analysis of stipe length was done by microscopy on the wild-type CA14, the ΔnsdC 17 and ΔnsdD 3 mutants, and the ΔnsdC C5 and ΔnsdD C4 complementation strains. Values are the means and standard deviations of 20 stipe length measurements for each Δnsd mutant, each complementation isolate, and the wild-type CA14 isolate. Bars with different letters have values that are significantly different (P < 0.001).

Fig 3

Deletion of nsdC and nsdD results in reduced conidiation. The wild-type CA14, three ΔnsdC (16, 17, and 21) and ΔnsdD (2, 3, and 7) mutants, and two ΔnsdC (C2 and C5) and ΔnsdD (C4 and C5) complementation isolates were grown on YGT-U agar for 5 days at 30°C with illumination. Values are the means and standard deviations from the two samplings each of conidia from the three separate transformants of the deletion mutants and two transformants of the complementation isolates. Bars with different letters have values that are significantly different (P < 0.003).

Fig 4

qPCR of the conidiation-specific transcription factors brlA and abaA. Gene expression levels at each time point were normalized (ΔΔCT analysis) to A. flavus 18S rRNA gene expression levels utilizing the gene expression analysis software package for the Bio-Rad iQ5.

Fig 5

Aflatoxin production and expression of aflatoxin biosynthetic genes in Δnsd mutants and Δnsd complementation strains. (A) Extracts of the wild-type CA14, ΔnsdC 17 and ΔnsdD 3 mutants, and ΔnsdC (C2 and C5) and ΔnsdD (C4 and C5) genetically complemented strains grown for 3 days on PDB-U. Extracts (5 μl) were spotted onto 250-μm silica gel TLC plates, and metabolites were separated in toluene-ethyl acetate-formic acid (5:4:1, vol/vol/vol). Aflatoxin standards were also spotted on the plate. The plates were visualized under 310-nm UV light. (B) qPCR of aflatoxin gene transcripts from 24-, 48-, and 72-h cultures. Gene expression levels at each time point were normalized (ΔΔCT analysis) to A. flavus 18S rRNA gene expression levels utilizing the gene expression analysis software package for the Bio-Rad iQ5. Bars: white, CA14; gray, ΔnsdD 3 mutant; black, ΔnsdC 17 mutant. (C) Western blot analysis of AF enzymes (AflM, AflD, and AflP) present in total protein extracts of 48-h cultures of strains grown in PDB-U shake cultures.