The PHD Transcription Factor Rum1 Regulates Morphogenesis and Aflatoxin Biosynthesis in Aspergillus flavus

Abstract

Aspergillus flavus produces mycotoxins especially aflatoxin B₁ and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. flavus through the construction of rum1 deletion mutants and rum1 complementation strains with the method of homologous recombination. It was found, in the study, that Rum1 negatively regulates conidiation through abaA and brlA, positively regulates sclerotia formation through nsdC, nsdD, and sclR, triggers aflatoxin biological synthesis, and enhances the activity of amylase. Our findings suggested that Rum1 plays a major role in the growth of mycelia, conidia, and sclerotia production along with aflatoxin biosynthesis in A. flavus.

Keywords: Aspergillus flavus; Rum1; aflatoxin; conidiation; sclerotia.

Figure 1

Characterization of the PHD transcription factor Rum1 in A. flavus. (A) Phylogenetic relationship of 12 Rum1 homologs from different species was analyzed with MEGA5.0. (B) The domain structure of Rum1 homologs was identified by SMART and were visualized using software IBS 1.0.

Figure 2

Strategy and confirmation of the mutant strains. (A) The scheme of rum1 deletion and complementation strategy (H: HindIII, probe: the probe used in southern blot analysis). (B) Gene knockout and complemented strains were verified by PCR analysis. (The rum1 ORF was confirmed by primers rum1-p9 and rum1-p10, AP fragment was confirmed by primers rum1-p1 and P801, and fragment BP was confirmed by primers P1020 and rum1-p4). (C) q-PCR verification of rum1 gene deletion and complementation strains. The actin gene was used as an inner reference. (D) Southern blot analysis for ∆rum1 mutant. Genomic DNA from above strains was digested with HindIII and hybridized with a 1.4 kb probe of the downstream region fragment of rum1 (3′-UTR) (The probe fragment was amplified with primers rum1-p3 and rum1-p4), (E) q-PCR analysis of the expression level of rum1 gene in ∆rum1 WT and ∆rum1-C strains. *** represents significant difference (p < 0.001).

Figure 2

Strategy and confirmation of the mutant strains. (A) The scheme of rum1 deletion and complementation strategy (H: HindIII, probe: the probe used in southern blot analysis). (B) Gene knockout and complemented strains were verified by PCR analysis. (The rum1 ORF was confirmed by primers rum1-p9 and rum1-p10, AP fragment was confirmed by primers rum1-p1 and P801, and fragment BP was confirmed by primers P1020 and rum1-p4). (C) q-PCR verification of rum1 gene deletion and complementation strains. The actin gene was used as an inner reference. (D) Southern blot analysis for ∆rum1 mutant. Genomic DNA from above strains was digested with HindIII and hybridized with a 1.4 kb probe of the downstream region fragment of rum1 (3′-UTR) (The probe fragment was amplified with primers rum1-p3 and rum1-p4), (E) q-PCR analysis of the expression level of rum1 gene in ∆rum1 WT and ∆rum1-C strains. *** represents significant difference (p < 0.001).

Figure 3

The roles of Rum1 in mycelium growth and conidiation in A. flavus. (A) The colonies of A. flavus strains grew on PDA medium at 29 °C. (B) The histogram of colony diameter calculated according to the result of A. (C) The conidiophores of A. flavus strains were observed under a microscope (20×). (D) The conidia of each plate were suspended with 5 mL of spore eluate. The conidia number of A. flavus strains in 1 ml spore eluate was calculated using hemocytometer. (E) The q-PCR analysis of brlA and abaA and the transcriptional factor genes for conidiation in the related A. flavus strains. ** and *** denote statistical significant levels of p < 0.01 and p < 0.001.

Figure 4

The role of Rum1 in sclerotia formation. (A) WT, ∆rum1, and ∆rum1-C strains were inoculated on WKM medium for 7 days and then the plates were sprayed with 75% ethanol to make sclerotia visible. (B) The histogram showing the amount of sclerotia according to (A), (C) Transcriptional expression levels of nsdC, nsdD, and sclR, which are the genes of positive regulators in sclerotia formation. *** represents significant difference (p < 0.001).

Figure 5

The role of Rum1 in aflatoxin biosynthesis. (A) AFB₁ production of WT, ∆rum1, and ∆rum1-C strains was detected by Thin-Layer Chromatography (TLC). All strains were cultivated in liquid YES medium for 6 days at 29 °C in the dark (5 μL of each sample was allotted for the TLC analysis. Three lanes of the same strain represent biological repeats, respectively). (B) The quantity of AFB₁ produced by related A. flavus strains was quantitated according to (A), (C) Transcriptional levels of aflatoxin biosynthesis related gene aflR, aflS, aflC, and aflO in the strains mentioned above. *** represents a significant difference (p < 0.001).

Figure 6

The role of Rum1 in A. flavus colonization. (A) Photographs presented the maize and peanut kernels colonized by WT, ∆rum1, and ∆rum1-C strains. (B) Conidia production of all the strains on seeds (The conidia of each plate were suspended with 5 mL of spore eluate and the conidia number of A. flavus strains in 1 mL spore eluate was calculated with hemocytometer). (C) The production of AFB₁ from colonized kernels was detected by TLC (5 μL from each sample was loaded for the TLC analysis. Three lanes of the same strain represent three biological repeats, respectively). (D) Relative AFB₁ production in (C) was quantitated. (E) Amylase activity was analyzed with iodine solution and 1% starch (Red arrows point to the degradation zones). (F) The relative degradation rate of starch of WT, ∆rum1, and ∆rum1-C strains, according to the results of (E). *** represents significant difference (p < 0.001).