The PHD transcription factor Cti6 is involved in the fungal colonization and aflatoxin B1 biological synthesis of Aspergillus flavus

Abstract

Aspergillus flavus and its main secondary metabolite AFB1 pose a serious threat to several important crops worldwide. Recently, it has been reported that some PHD family transcription factors are involved in the morphogenesis and AFB1 biological synthesis in A. flavus, but the role of Cti6, a PHD domain containing protein in A. flavus, is totally unknown. The study was designed to reveal the biological function of Cti6 in the fungus by deletion of cti6, and its two domains (PHD and Atrophin-1) through homologous recombination, respectively. The results showed that Cti6 might up-regulate the mycelium growth, conidiation, sclerotia formation and AFB1 biological synthesis of A. flavus by its PHD domain, while Atrophin-1 also improved the conidiation of the fungus. The qRT-PCR analysis showed that Cti6 increased the conidiation of the fungus through AbaA and BrlA mediated conidiation pathway, triggered the formation of sclerotia by orthodox sclerotia formation pathway, and improved the production of AFB1 by orthodox AFB1 synthesis pathway. Crops models analysis showed that A. flavus Cti6 plays vital role in colonization and the production of AFB1 on the host grains mainly via PHD domain. Bioinformatics analysis showed Cti6 is conservative in Aspergillus spp., and mCherry mediated subcellular localization showed that most Cti6 accumulated in the nuclei, which reflected that Cti6 performed its important biological function in the nuclei in Aspergillus spp.. The results of the current study elucidate the roles of PHD domain containing proteins in the mechanism of the infection of crops by A. flavus, and provided a novel target for effectively controlling the contamination of Aspergillus spp. to crops.

Keywords: AFB1; Aspergillus flavus; Colonization; Cti6; PHD domain.

Fig. 1

Bioinformatics analysis of Cti6. a The construction of phylogenetic relationship among 10 Cti6 homologs (from A. flavus, A. oryzae, A. terreus, A. fumigatus, A. nidulans, P. oryzae, S. cerevisiae, H. sapiens, M. musculus and A. thaliana) with MEGA5.0. b The domains of Cti6 from above 10 species were identified through NCBI, and the domains were further visualized by DOG2.0²

Fig. 2

Cti6 up-regulates the growth of fungal mycelium and conidiation in A. flavus. a The A. flavus strains were point-inoculated on PDA media for 5 d at 37 °C. b The colony diameters were measured and represented with column graph according to the result of (a) panel. c The number of conidia of each fungal strain on PDA medium. d The expression level of transcriptional factor abaA and brlA genes. The “*”, “**” and “***” represents significant difference levels: p < 0.01, p < 0.005 and p < 0.001, respectively. All experiments were carried out with three biological replicates, and repeated at least three times

Fig. 3

Cti6 is necessary in the formation of sclerotia in A. flavus. a The A. flavus strains were point-inoculated on CM media for 7 d at 37 °C. b The sclerotia number was counted, and the sclerotia formation ability of these fungal strains was compared. c The expression levels of transcriptional factor genes (nsdC, nsdD and sclR) were analyzed by qRT-PCR

Fig. 4

Cti6 is critical in AFB1 synthesis in A. flavus. a The production of AFB1 from above fungal strains was analyzed with TLC. b The relative quantity of the amount of AFB1 according to the result from above TLC analysis. c The expression levels of AFB1 synthesis regulator gene aflR and aflS were analyzed by qRT-PCR

Fig. 5

The role of Cti6 in the colonization of A. flavus on peanut and maize grains. a Colonization of these A. flavus strains on peanut and maize grains. b Cti6 and its PHD domain involved in the conidiation of A. flavus. c The AFB1 production capacity of these fungal strains was analyzed with TLC analysis. d Cti6 and its PHD domain positively regulated AFB1 production in A. flavus

Fig. 6

The subcelluar location of Cti6. a The construction strategy for mCherry and cti6 fusion expression A. flavus strain (mCherry-cti6). b The subcellular location of Cti6 was showed through co-expressed mCherry