A. flavus strains used in the study were listed in Table 1. The primers used in the study were showed in Table 2. YPD (1% yeast extract, 2% peptone, 2% glucose, and 1.5% agar) and YES (2% yeast extract, 150 g/L sucrose, 1 g/L MgSO₄·7H₂O) were prepared for the cultivation of A. flavus strains in the study. Supplements (Uracil, and uridine) for auxotrophic marker (pyrG-) were added as required (Yang et al., 2016; Li et al., 2017). Strains were stored in 30% glycerol at −80°C.

The homologs of PbsB from (A. flavus, A. oryzae, A. clavatus, A. fumigatus, A. niger, A. terreus, A. nidulans, N. crassa, and S. cerevisiae) were downloaded from NCBI (http://www.ncbi.nlm.nih.gov), and further aligned with Clustal X. Protein domains of above 9 species were analyzed with InterPro (http://www.ebi.ac.uk/interpro/scan.html) and edited with IBS 1.0. Phylogenetic tree of PbsB homologs from these 9 species was constructed with MEGA5.1 by an algorithm of 1,000 times Neighbering comparison.

The pbsB deletion mutants were constructed according to Han et al. (2016) with minor modification. The deletion cassette was constructed by fusion PCR. In details, 5′- untranslated regions (5′-UTR) and 3′-UTR were amplified from A. flavus genomic DNA with two pairs of primers (pbsB-AF and pbsB-AR, pbsB-BF and pbsB-BR in Table 2). The intermediate A. fumigatus pyrG was amplified from A. fumigatus genomic DNA by primers pyrG-PF and pyrG-PR (Table 2) as transformation selecting marker. 5′-UTR and 3′-UTR of pbsB and pyrG were fused together by fusion PCR with nesting primers OverlapF and OverlapR (Table 2). Polyethylene glycol-mediated transformation of CA14Δku70ΔpyrG protoplasts was carried out as described by Cary et al. (2005). The pyrG prototroph strain (ΔpbsB: Δku70, ΔpbsB::pyrG showed in Table 1), in which the entire pbsB coding region was replaced by pyrG, was further tested with PCR (primers listed in Table 2), and the flanking regions of confirmed ΔpbsB strain was further sequenced by BioSune (Shanghai, China). The pbsB overexpression strains were prepared by homologous recombination (Nie et al., 2016). After four DNA fragments were amplified with four pairs of primers: pbsB-AF and pbsB-AR for 5′HR, pyrG-PF and pyrG-PR for pyrG, gpdA-F and gpdA-R for gpdA(p), and OE-F and OE-R for 3′HR, they were overlapped with nesting primers: OE-overlap-F and OE-overlap-R (Table 2). And pbsB overexpression strains (OE) were prepared by Polyethylene glycol-mediated transformation of CA14Δku70ΔpyrG protoplasts with the overlap PCR production as mentioned above. The OE strains were further confirmed with primer gpdA-F and OE-R for 2,000 bp OE-ORF fragment, and with primer OE-overlap-F and OE-R for 4,730 bp OR-ORF fragment as shown in Table 2. Finally, the expression level of pbsB in A. flavus strains mentioned above was further assayed with qRT-PCR analysis.

The qRT-PCR was conducted according to the protocol provided by Zhang et al. (2016). Three micrograms of total RNA were treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) to remove possible G-DNA contamination. One microgram G-DNA free RNA was reverse transcribed by using the Revert Aid First-strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was carried out by using the SYBR Green Premix kit (Takara, Dalian, China) with Mx3000p thermocycler (Agilent Technologies). The expression levels of target gene were evaluated with the 2′ ΔΔCt method.

The effect of PbsB in AFB1 production was performed after 5 d cultivation according to the method by Dhingra et al. (2013). 25 mL of liquid YES medium was inoculated with 10⁴ conidia/mL, and the cultures were shaking at 180 r/min at 28°C. The supernatant samples were analyzed by thin-layer chromatography (TLC). A volume of 3 μL supernatant was spotted onto a TLC plate (Si250F, J.T. Baker), and the plate was developed in acetone-chloroform (2:8, vol/vol), and dried at 80°C for 10 min, then the aflatoxin B1 was examined under UV light.

Peanut and maize kernels were prepared to discover the role of PbsB in plant infection following the method described by Yang et al. (2016) with minor modification. Each peanut and maize kernel was sterilized with 0.05% sodium hypochlorite. Viable cotyledon was dried and placed on sterile Petri dish plate. The cultures were incubated at 28°C in dark for 7 d. For AFB1 analysis, a Petri dish plate of kernels was soaked in double distill water for 10 h. After chloroform (half of the water volume) was added, the mixture was shacked at 180 r/min for 1 h, and kept still for another 2 h. The chloroform layer was collected and dried by evaporation. The sediment was re-suspended with 1 mL chloroform, and re-dissolved in 30 μL chloroform after dried by evaporation. Finally, 5 μL sample was analysis by TLC.

The data in the study was presented as the means ± standard deviation (SD). The presence of statistical differences was determined by one-way ANOVA, and statistical significance was recognized when P < 0.05.

PbsB protein in A. flavus and its orthologs in other 8 species were aligned by Clustal X, and the result showed that PbsB in A. flavus and A. oryzae had the highest similarity (83.27%), and the lowest homology was found between A. flavus and S. cerevisiae (29.70%) in Figure 1A. The protein domain in PbsB was further analyzed with IBS 1.0, and a catalytic domain (Serine/Threonine protein kinases) was found in all 9 species (Figure 1B), which meant that the catalytic domain was very conservative. Phylogenetic tree among these 9 species was further constructed with MEGA5.1 as shown in Figure 1C. The genetic relationship of PbsB from A. flavus and A. oryzae was found to be the closest, compared with the relationship of the protein from A. flavus and any other species, and the protein sequences of PbsB from Aspergillus spp. were classified into one cluster compared with the species from other genera (N. crassa and S. cerevisiae).

The A. flavus pbsB deletion strains (ΔpbsB) and over expression strains (OE) were constructed by transforming the protoplasts of WT (pyrG-) with the fusion PCR productions, respectively (Figures 2A,C), and the resulting transformants were confirmed by PCR analysis (Figures 2B,D). The result of PCR showed that the ORF of pbsB has been deleted from pbsB deletion mutants, and the amplification of AP and BP from ΔpbsB meant that ORF of pbsB has been replaced by pyrG from A. fumigatus (Figure 2B). The franking regions between pyrG and 5′-UTR and 3′-UTR of pbsB of the PCR confirmed ΔpbsB mutant were further sequenced and aligned with DNAMAN (Version: 6.0.40), the results (100% identity, Figure 2E) showed pbsB had been deleted as the scheme showed in Figure 2A. The results in Figure 2D showed that 2,000 bp DNA fragment (OE-ORF) only could be amplified from OE strain, and the OR-ORF fragment from OE strain was 4,730 bp, but it was only 1,960 bp for WT strain (Figures 2C,D). By qRT-PCR analysis, it was found that not pbsB activity could be detected in ΔpbsB, and the expression level of pbsB in OE was dramatically improved compared with WT (Figure 2F). These results showed that both ΔpbsB and OE strains of A. flavus were successfully constructed. In order to evaluate the role of PbsB played in the growth of mycelium, the conidia (10⁴ spores/mL) from A. flavus strains (WT, ΔpbsB and OE) were point inoculated onto YES agar in dark at 37°C for 5 d. The results showed that the colony diameter of ΔpbsB mutant was obvious smaller than that of WT from 1 to 5th d, and opposite situation was found in OE strain compared to WT strain (Figure 2G). The diameter of each mycelium colony was measured from 1 to 5 day, and the results of the histogram in Figure 2H revealed that the PbsB significantly improved the growth of A. flavus.

Aspergillus spp. always produces a massive number of conidia which are easily dispersed in the air in breeze. Conidiation is an important means to spread contamination for A. flavus, so the capacity of asexual reproduction—conidiation is a critical index to amplify the detriment of A. flavus. To explore the role of PbsB in conidiation, A. flavus strains (WT, ΔpbsB, and OE) were point inoculated onto YES at 37°C in dark, and phenotype was observed after 4 d cultivation. ΔpbsB mutant showed the decreased conidial production compared to WT strain (Figures 3A,B). Meanwhile, more conidiophores were observed in WT strain when compared to ΔpbsB mutant, and the conidiophores from ΔpbsB mutant were obvious dysplasia (Figure 3C). When pbsB overexpressed, significantly (P < 0.005) more conidia and conidiophores was observed in OE strain compared with WT strain (Figure 3). The result showed that PbsB improved the production of conidia, and the absence of PbsB significantly (P < 0.05) repressed the asexual reproduction of A. flavus.

Sclerotia are readily produced by single strain of A. flavus, and sclerotia formation is commonly considered to be survival structure of A. flavus against unfavorable conditions. To reveal the bio-function of PbsB in the formation of sclerotia, A. flavus strains (WT, ΔpbsB, and OE) were point inoculated onto YPD at 37°C in dark for 7 d. After spraying with 75% ethanol, and the number of sclerotia on each plate was counted, respectively. The result showed that lack of PbsB significantly (P < 0.01) decreased the production of sclerotia, and the overexpression of pbsB obviously (P < 0.01) increased sclerotia production compared with WT (Figures 4A,B). The expression level of sclerotia regulator (nsdC) was also analyzed by qRT-PCR, and the results showed that nsdC was significantly (P < 0.01) down-regulated at 72 h when PbsB was absent, and was dramatically (P < 0.005) up-regulated when PbsB was overexpressed (Figure 4C). All these results indicate that PbsB is important in sclerotia formation in A. flavus.

The role of PbsB in AFB1 production was analyzed by cultivating the WT, ΔpbsB and OE strains in the YES liquid medium, and the samples were collected at 5th day. The result of TLC analysis showed that AFB1 production was significantly (P < 0.005) decreased when pbsB was deleted, and the mycotoxin was obviously (P < 0.005) elevated when pbsB overexpressed in OE strain compared with WT strain (Figures 5A,B). The expression levels of aflatoxin bio-synthesis gene aflC, aflD, aflK, and aflQ, and regulatory gene aflR at 48 h were further analyzed by qRT-PCR, and the results showed that the expression levels of the aflatoxin bio-synthesis and regulatory genes were all significantly down-regulated in ΔpbsB mutant and up-regulated in OE strain compared to WT strain (Figure 5C). These results reflected that PbsB positively regulated the aflatoxin bio-synthesis through aflatoxin bio-synthesis and regulatory genes (aflR, aflC, aflD, aflK, and aflQ).

PbsB was reported to play an important role in the resistance of C. albicans to hyperosmotic conditions (Arana et al., 2005). To assess the role of PbsB under hyperosmotic stress in A. flavus, WT, ΔpbsB and OE strains were inoculated onto YES agar with 1.2 mol/L sorbitol for 4 d. It was found that the inhibition rate of ΔpbsB mutants dramatically (P < 0.01) greater than that of WT and OE strains (Figure 6). The results revealed that pbsB deletion mutant was more susceptible to hyperosmotic stress, and PbsB was one of the critical factors in A. flavus to fight against hyperosmotic conditions.

Peanut and maize kernels were inoculated with 10⁴ spores/mL of WT, ΔpbsB and OE strains in Petri dish plate for 7 d, respectively. It was shown in Figures 7A,B,E,F that dramatically (P < 0.005) reduced conidiation was observed in ΔpbsB mutant, and significant (P < 0.05) increased sporulation yield was found in OE strain compared to that in WT strain. TLC analysis showed that AFB1 bio-synthesis capacity of A. flavus on peanuts was significantly (P < 0.005) repressed when PbsB was absent from ΔpbsB mutant, and was obviously (P < 0.005) improved when PbsB was over-produced in OE strain (Figures 7C,G). To maize kernel, similar results were observed (Figures 7D,H). All these results of the study indicated that PbsB played an essential role in crops infection.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.