Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus

The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in Aspergillus flavus, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in A. flavus. Deletion of ssu72 resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of A. flavus. Furthermore, the Δssu72 mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in A. flavus, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.


Introduction
Aspergillus flavus is a saprophytic plant pathogenic fungus that infects a range of seed crops (maize, peanuts, cottonseed and tree nuts) before and after harvest [1,2]. In addition, A. flavus is also a human opportunistic pathogen, causing invasive aspergillosis in mammals and humans [3,4]. Aflatoxin (AF), which is mainly synthesised by A. flavus, is one of the most toxic and carcinogenic secondary metabolites in nature, posing a huge threat to economic development, food safety and human health [5,6]. Long-term intake of low doses of AFs may result in a number of health problems, such as growth impairment, lung and liver cancer or even death in many mammals and humans [7,8]. Therefore, it is essential to explain and clarify the regulatory mechanism of this fungus in pathogenicity and aflatoxin biosynthesis.
Previous studies have shown that aflatoxin biosynthesis and pathogenicity of A. flavus are regulated by multiple factors, such as temperature [9], water activity [10] and post-translational modifications (PTMs) including phosphorylation [11], acetylation [12], succinylation [13], methylation [14] and SUMOylation [15]. Reversible phosphorylation catalyzed by kinases and phosphatases, is one of the most common PTMs, and has been shown to regulate various biological processes [16,17]. In eukaryotes, reversible phosphorylation mainly occurs on three amino acids (serine, threonine and tyrosine) [18,19]. Phosphatase-mediated dephosphorylation plays a critical role in signal transduction in eukaryotic cells [20]. Based on sequence homology, structure and catalytic specificity, phosphatases can be classified into two major superfamilies: serine/threonine (S/T) phosphatases and tyrosine phosphatases (PTPs) [21]. The PTPs have demonstrated to be involved in the regulation of various cellular processes in eukaryotes, including cell cycle, signal transduction, transcriptional activation, development and secondary metabolism [22][23][24].
In eukaryotes, transcription of all coding genes is mainly regulated by RNA polymerase II (RNAP II) complex, which consist of 12 different subunits [25]. The carboxy-terminal domain (CTD) is one of the largest RNAP II subunits and composed of the evolutionarily conserved reiterate heptapeptide sequence (YSPTSPS), and reversible phosphorylation of this sequence is involved in distinct stages of transcription [26,27]. Ssu72 is identified as a highly conserved CTD phosphatase and negatively regulate the phosphorylation of specific Ser (5) and Ser(7) [28]. In Saccharomyces cerevisiae, Ssu72 is well studied due to its role in transcription initiation, mRNA processing, transcription termination and sister-chromatid cohesion [29,30]. In fission yeast, phosphatase Ssu72 is known to be essential for growth [31]. In addition, Ssu72 is found to be involved in RNA 3 processing and the regulation of phosphate homeostasis in Schizosaccharomyces pombe [32,33]. In plant pathogenic fungus Fusarium graminearum, it has been demonstrated that the ortholog of phosphatase Ssu72 is involved in asexual development and virulence [34]. Despite the roles of phosphatase Ssu72 in yeast have been well studied, the function of Ssu72 in Aspergillus species is still poorly understood.
In this study, we identified a putative phosphatase Ssu72 of A. flavus, and then systematically characterized the function of Ssu72 in A. flavus. Our results reveal that the putative phosphatase Ssu72 plays critical roles in the regulation of fungal development, aflatoxin biosynthesis, stresses response and pathogenicity in A. flavus. This will provide a new insight that Ssu72 may be used as a potential target for preventing the hazard of A. flavus.

Identification of Putative Phosphatase Ssu72 in A. flavus
To identify the ortholog of Ssu72 phosphatase in A. flavus, the Ssu72 protein sequence of model organism S. cerevisiae was used to search with a basic local alignment search tool (BLAST) in the A. flavus genome database. A putative Ssu72 phosphatase protein in A. flavus was identified, predicting to encode 290 amino acids and present 45% homology with S. cerevisiae Ssu72. Then, a similar approach was performed to obtain Ssu72 homologous protein sequences in some fungi (A. nidulans, A. fumigatus, F. graminearum, Magnapoethe oryzae, Neurospora crassa, Candida albicans). Our sequence alignment results showed that the A. flavus Ssu72 protein displayed 99% similarity to A. oryzae, 95% similarity to A. nidulans, and 93% similarity to A. fumigatus. The phylogenetic analysis indicated that the Ssu72 protein is evolutionarily conserved in Aspergillus species ( Figure 1A), and the A. flavus Ssu72 protein exhibited a high similarity with the ortholog of Ssu72 in Aspergillus species. Domain analysis revealed that the Ssu72 homologous proteins all contained a Ssu72-like phosphatase domain ( Figure 1B), and this domain was mainly concentrated on the C-terminal of Ssu72 protein, suggesting that Ssu72-like phosphatase domain is highly conserved in fungi. These results indicate that the putative Ssu72 phosphatase protein is highly conserved in evolution and may have similar function in fungi.

Construction of the ssu72 Mutant Strains
To investigate the potential function of Ssu72 phosphatase gene in A. flavus, the ssu72 deletion (Δssu72) and the ssu72 complementation (Δssu72-Com) strains were constructed by a homologous recombination approach (Figure 2A). The specific primers and sequence lengths are displayed in Figure 2A. For knockout strain construction, three fragments (1392 bp ssu72 5′UTR, 1220 bp 3′UTR and 1890 bp pyrG) were amplified with the specific primers and then fused together. PyrG from A. fumigatus was used to replace the ssu72 gene, and open reading frame (ORF) of ssu72 gene was reinserted into the genome of the knockout strain to construct complementary strains. Then, the deletion and complementation transformants were verified by polymerase chain reaction (PCR) analysis ( Figure 2B), and the result showed that ORF fragment could be detected in WT (wild type) and Δssu72-Com strains, but not in Δssu72 strain. While AP (5′UTR+pyrG) and BP (5′UTR+pyrG) fragments ( Figure 2A) were amplified from ssu72 deletion and complementation mutants but not from WT strain. Furthermore, expression levels of ssu72 gene in WT, Δssu72 and Δssu72-Com strains

Construction of the ssu72 Mutant Strains
To investigate the potential function of Ssu72 phosphatase gene in A. flavus, the ssu72 deletion (∆ssu72) and the ssu72 complementation (∆ssu72-Com) strains were constructed by a homologous recombination approach (Figure 2A). The specific primers and sequence lengths are displayed in Figure 2A. For knockout strain construction, three fragments (1392 bp ssu72 5 UTR, 1220 bp 3 UTR and 1890 bp pyrG) were amplified with the specific primers and then fused together. PyrG from A. fumigatus was used to replace the ssu72 gene, and open reading frame (ORF) of ssu72 gene was reinserted into the genome of the knockout strain to construct complementary strains. Then, the deletion and complementation transformants were verified by polymerase chain reaction (PCR) analysis ( Figure 2B), and the result showed that ORF fragment could be detected in WT (wild type) and ∆ssu72-Com strains, but not in ∆ssu72 strain. While AP (5 UTR+pyrG) and BP (5 UTR+pyrG) fragments ( Figure 2A) were amplified from ssu72 deletion and complementation mutants but not from WT strain. Furthermore, expression levels of ssu72 gene in WT, ∆ssu72 and ∆ssu72-Com strains were analyzed by reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR), and the results indicated that the transcripts of ssu72 were not detected in ∆ssu72 strain, whereas ssu72 gene was expressed in the WT and ∆ssu72-Com strains ( Figure 2C,D). All these results showed that both ssu72 knockout mutant (∆ssu72) and ssu72 complementation strain (∆ssu72-Com) were successfully constructed. were analyzed by reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR), and the results indicated that the transcripts of ssu72 were not detected in Δssu72 strain, whereas ssu72 gene was expressed in the WT and Δssu72-Com strains ( Figure 2C,D). All these results showed that both ssu72 knockout mutant (Δssu72) and ssu72 complementation strain (Δssu72-Com) were successfully constructed.

Ssu72 is Involved in Vegetative Growth in A. flavus
To investigate the roles of ssu72 in vegetative growth of A. flavus, the WT, Δssu72 and Δssu72-Com strains were inoculated in yeast extract-sucrose (YES), potato dextrose agar (PDA) and yeast glucose trace-elements (YGT) media and cultured for 5 days. As shown in Figure 3A,B, our result revealed that the Δssu72 mutant displayed significant growth reduction on YES, PDA and YGT media when compared with WT and complementation strains (Δssu72-Com). Moreover, microscopic examination showed that abnormal and shorter aerial hyphal were observed in Δssu72 mutant, while the abnormal hyphal defect of Δssu72 mutant was restored in the Δssu72-Com strain ( Figure 3C). These observations suggest that phosphatase Ssu72 plays important roles in vegetative growth of A. flavus.

Ssu72 Is Involved in Vegetative Growth in A. flavus
To investigate the roles of ssu72 in vegetative growth of A. flavus, the WT, ∆ssu72 and ∆ssu72-Com strains were inoculated in yeast extract-sucrose (YES), potato dextrose agar (PDA) and yeast glucose trace-elements (YGT) media and cultured for 5 days. As shown in Figure 3A,B, our result revealed that the ∆ssu72 mutant displayed significant growth reduction on YES, PDA and YGT media when compared with WT and complementation strains (∆ssu72-Com). Moreover, microscopic examination showed that abnormal and shorter aerial hyphal were observed in ∆ssu72 mutant, while the abnormal hyphal defect of ∆ssu72 mutant was restored in the ∆ssu72-Com strain ( Figure 3C). These observations suggest that phosphatase Ssu72 plays important roles in vegetative growth of A. flavus.

Ssu72 is Critical for Conidiation in A. flavus
Conidia is mainly produced by aerial hyphae and plays critical roles in asexual development and infection processes of A. flavus [35]. To assess the effect of ssu72 in conidiation, the WT, Δssu72 and Δssu72-Com strains were cultured on PDA medium for 5 days, and we found that the amount of conidia was obviously reduced in the Δssu72 mutant in comparison with WT and Δssu72-Com strains ( Figure 4A). About one tenth of conidia was observed in the knockout strain, when compared with the WT and complementation strains ( Figure 4B). Furthermore, microscopic examination revealed that abnormal head of conidiophores was observed in Δssu72 mutant ( Figure  4C). To further explore the role of ssu72 in conidiation, the expression levels of two key transcript factors (brlA and abaA) were analyzed by qRT-PCR. The consequence showed that the expression levels of brlA and abaA were both significantly down-regulated in Δssu72 mutant when compared to WT and Δssu72-Com strains ( Figure 2D). These results suggest that phosphatase Ssu72 is involved in the conidia production of A. flavus.

Ssu72 Is Critical for Conidiation in A. flavus
Conidia is mainly produced by aerial hyphae and plays critical roles in asexual development and infection processes of A. flavus [35]. To assess the effect of ssu72 in conidiation, the WT, ∆ssu72 and ∆ssu72-Com strains were cultured on PDA medium for 5 days, and we found that the amount of conidia was obviously reduced in the ∆ssu72 mutant in comparison with WT and ∆ssu72-Com strains ( Figure 4A). About one tenth of conidia was observed in the knockout strain, when compared with the WT and complementation strains ( Figure 4B). Furthermore, microscopic examination revealed that abnormal head of conidiophores was observed in ∆ssu72 mutant ( Figure 4C). To further explore the role of ssu72 in conidiation, the expression levels of two key transcript factors (brlA and abaA) were analyzed by qRT-PCR. The consequence showed that the expression levels of brlA and abaA were both significantly down-regulated in ∆ssu72 mutant when compared to WT and ∆ssu72-Com strains ( Figure 2D). These results suggest that phosphatase Ssu72 is involved in the conidia production of A. flavus.

Ssu72 is Required for Sclerotia Formation in A. flavus
In A. flavus, a sexual structure-sclerotia is formed to survive under harsh environment [5]. To investigate the bio-function of ssu72 in sclerotia formation, the WT, Δssu72 and Δssu72-Com strains were cultured on the Wickerham (WKM) medium in the dark for 7 days. As shown in Figure 5A,B, lots of sclerotia were discovered in WT and Δssu72-Com strains. However, no sclerotia was observed in Δssu72 strain. To further confirm these findings, qRT-PCR analysis was performed to analyze the transcript levels of two sclerotia formation key genes (nsdC and nsdD). The result indicated that the transcript levels of nsdC and nsdD were both dramatically reduced in the Δssu72 mutants when compared to WT and Δssu72-Com strains ( Figure 5C). All the above findings suggest that phosphatase Ssu72 is required for sclerotia formation of A. flavus.

Ssu72 Is Required for Sclerotia Formation in A. flavus
In A. flavus, a sexual structure-sclerotia is formed to survive under harsh environment [5]. To investigate the bio-function of ssu72 in sclerotia formation, the WT, ∆ssu72 and ∆ssu72-Com strains were cultured on the Wickerham (WKM) medium in the dark for 7 days. As shown in Figure 5A,B, lots of sclerotia were discovered in WT and ∆ssu72-Com strains. However, no sclerotia was observed in ∆ssu72 strain. To further confirm these findings, qRT-PCR analysis was performed to analyze the transcript levels of two sclerotia formation key genes (nsdC and nsdD). The result indicated that the transcript levels of nsdC and nsdD were both dramatically reduced in the ∆ssu72 mutants when compared to WT and ∆ssu72-Com strains ( Figure 5C). All the above findings suggest that phosphatase Ssu72 is required for sclerotia formation of A. flavus.

Ssu72 Plays a Positive Role in Regulation of Aflatoxin Biosynthesis
Aflatoxin (AF) is one of the most toxic secondary metabolites of A. flavus, and poses a huge threat to human health [36,37]. Thus, it is necessary to determine whether phosphatase Ssu72 is involved in aflatoxin biosynthesis. Then, the strains were cultured in YES liquid medium for 6 days, and thin-layer chromatography (TLC) analysis showed that deletion of ssu72 gene resulted in a significant decrease in aflatoxin B1 (AFB1) production when compared to WT and Δssu72-Com strains ( Figure 6A,B). Subsequently, qRT-PCR was performed to analyze the expression levels of AFB1 biosynthesis cluster genes. We found that the transcript levels of some key genes (regulatory genes aflR and aflS, structural genes aflP, aflQ, aflO and aflK) in Δssu72 mutant were significantly lower than those in WT and Δssu72-Com strains ( Figure 6C). Overall, these results indicate that phosphatase Ssu72 positively regulates AFB1 biosynthesis in A. flavus.

Ssu72 Plays a Positive Role in Regulation of Aflatoxin Biosynthesis
Aflatoxin (AF) is one of the most toxic secondary metabolites of A. flavus, and poses a huge threat to human health [36,37]. Thus, it is necessary to determine whether phosphatase Ssu72 is involved in aflatoxin biosynthesis. Then, the strains were cultured in YES liquid medium for 6 days, and thin-layer chromatography (TLC) analysis showed that deletion of ssu72 gene resulted in a significant decrease in aflatoxin B1 (AFB1) production when compared to WT and ∆ssu72-Com strains ( Figure 6A,B). Subsequently, qRT-PCR was performed to analyze the expression levels of AFB1 biosynthesis cluster genes. We found that the transcript levels of some key genes (regulatory genes aflR and aflS, structural genes aflP, aflQ, aflO and aflK) in ∆ssu72 mutant were significantly lower than those in WT and ∆ssu72-Com strains ( Figure 6C). Overall, these results indicate that phosphatase Ssu72 positively regulates AFB1 biosynthesis in A. flavus.

Ssu72 Contributes to Pathogenicity in Crop Seeds in A. flavus
Since A. flavus infects crops and causes huge economic losses, the effect of phosphatase Ssu72 on the pathogenicity to crop seeds was characterized in this study. After cultivation for 5 days, a lot of conidia on surface of peanut and maize seeds were found in the WT and Δssu72-Com strain ( Figure 7A). However, no obvious colonization was observed on peanuts and maize seeds after inoculated with Δssu72 mutant (Figure 7A), indicating that deletion of ssu72 resulted in a significant decrease in pathogenicity to peanut and maize seeds. Conidia were then harvested from the infected seeds, and Δssu72 mutant produced less amount of conidia on infected seeds than WT and Δssu72-Com strain ( Figure 7B). Subsequently, TLC assay was used to detect aflatoxin production in the infected seeds, and the result revealed that no AFB1 was observed in peanut and maize seeds infected with Δssu72 strain (Figure 7C,D). All these data suggest that phosphatase Ssu72 contributes to its pathogenicity to crop seeds in A. flavus.

Ssu72 Contributes to Pathogenicity in Crop Seeds in A. flavus
Since A. flavus infects crops and causes huge economic losses, the effect of phosphatase Ssu72 on the pathogenicity to crop seeds was characterized in this study. After cultivation for 5 days, a lot of conidia on surface of peanut and maize seeds were found in the WT and ∆ssu72-Com strain ( Figure 7A). However, no obvious colonization was observed on peanuts and maize seeds after inoculated with ∆ssu72 mutant (Figure 7A), indicating that deletion of ssu72 resulted in a significant decrease in pathogenicity to peanut and maize seeds. Conidia were then harvested from the infected seeds, and ∆ssu72 mutant produced less amount of conidia on infected seeds than WT and ∆ssu72-Com strain ( Figure 7B). Subsequently, TLC assay was used to detect aflatoxin production in the infected seeds, and the result revealed that no AFB1 was observed in peanut and maize seeds infected with ∆ssu72 strain (Figure 7C,D). All these data suggest that phosphatase Ssu72 contributes to its pathogenicity to crop seeds in A. flavus.

Ssu72 Response to Multiple Stresses in A. flavus
In A. flavus, phosphatases have been proven to be involved in multiple stresses response [38,39]. To explore the role of putative phosphatase Ssu72 in response to osmotic and oxidative stresses, the strains (WT, Δssu72 and Δssu72-Com) were cultured on PDA medium supplemented with different stress reagents. As observed in Figure 8A,B, the relative inhibition of growth rate in Δssu72 induced by 1 M NaCl was obviously higher than that of WT and Δssu72-Com strains, suggesting that Δssu72 mutant displayed increased sensitivity to osmotic stresses. Additionally, the growth of Δssu72 mutant was completely inhibited after added with higher osmotic stresses (5 mM H2O2) ( Figure 8A,B). All these results demonstrate that Ssu72 participates in osmotic and oxidative stresses in A. flavus.

Ssu72 Response to Multiple Stresses in A. flavus
In A. flavus, phosphatases have been proven to be involved in multiple stresses response [38,39]. To explore the role of putative phosphatase Ssu72 in response to osmotic and oxidative stresses, the strains (WT, ∆ssu72 and ∆ssu72-Com) were cultured on PDA medium supplemented with different stress reagents. As observed in Figure 8A,B, the relative inhibition of growth rate in ∆ssu72 induced by 1 M NaCl was obviously higher than that of WT and ∆ssu72-Com strains, suggesting that ∆ssu72 mutant displayed increased sensitivity to osmotic stresses. Additionally, the growth of ∆ssu72 mutant was completely inhibited after added with higher osmotic stresses (5 mM H 2 O 2 ) ( Figure 8A,B). All these results demonstrate that Ssu72 participates in osmotic and oxidative stresses in A. flavus.

Discussion
As one of the largest RNAP II subunits, carboxy-terminal domain (CTD) is known to be involved in the regulation of transcriptional initiation/termination and pre-mRNA processing in eukaryotes [25,40]. Ssu72 is identified as a conserved CTD-related phosphatase and well characterized in yeast [33]. However, the biological functions of Ssu72 in other fungi, especially in Aspergillus species, are still unclear. In this study, a putative phosphatase Ssu72 of A. flavus was identified. The C-terminus of the ssu72 protein in fungi all contained a conserved Ssu72-like

Discussion
As one of the largest RNAP II subunits, carboxy-terminal domain (CTD) is known to be involved in the regulation of transcriptional initiation/termination and pre-mRNA processing in eukaryotes [25,40]. Ssu72 is identified as a conserved CTD-related phosphatase and well characterized in yeast [33]. However, the biological functions of Ssu72 in other fungi, especially in Aspergillus species, are still unclear. In this study, a putative phosphatase Ssu72 of A. flavus was identified. The C-terminus of the ssu72 protein in fungi all contained a conserved Ssu72-like phosphatase domain, indicating that the ssu72 protein is evolutionarily conserved, and may have similar functions in fungi. In S. cerevisiae, the ssu72 protein has been shown to play important roles in regulation of multiple processes, so we speculate that Ssu72 may be involved in various processes in A. flavus. Next ssu72 gene deletion and complementation strains were constructed to characterize its function in A. flavus. Our findings reveal that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity of A. flavus.
Our previous studies have concluded that tyrosine phosphatases contribute to vegetative growth and conidiation in A. flavus [38]. In this study, our results demonstrated that knockout of putative phosphatase gene ssu72 resulted in a severe defect in vegetative growth ( Figure 3A), which is consistent with the results in budding yeast [31]. Our microscopic observation on hyphae tips showed that shorter aerial hyphal was found in ∆ssu72 strain, which is in a good agreement with the above conclusion ( Figure 3C). In addition, the amount of conidia was significantly reduced in ∆ssu72 strain ( Figure 4A), which is consistent with the defect that was observed in ssu72 homolog mutants in F. graminearum and N. crassa [34,41]. This finding is well supported by the down-regulation of key transcription factors brlA and abaA [42]. Sclerotia is a sexual structure to survive in unfavorable environment in A. flavus [43]. However, no sclerotia was discovered in ∆ssu72 strain when compared to WT and complementation strains ( Figure 5A), suggesting that phosphatase Ssu72 is essential for sclerotia formation. Furthermore, transcript levels of nsdC and nsdD genes, which have been known to be critical for sclerotia formation [44], was also significantly decreased in ∆ssu72 strain in this study. It is possible that deletion of ssu72 decrease the expression levels of nsdC and nsdD genes, and then regulate the sclerotia production. Therefore, phosphatase Ssu72 plays important roles in the regulation of vegetative growth, conidiation and sclerotia formation in A. flavus.
As one of the most toxic secondary metabolites in nature, aflatoxin contamination poses a serious threat to food safety, human and animal health [45]. Recent studies have revealed that kinases and phosphatases play vital roles in regulation of aflatoxin production in A. flavus [39,46,47]. Here, we observed that aflatoxin production was dramatically reduced in ∆ssu72 mutant, and this finding is in accordance with the down-regulation of aflatoxin biosynthesis regulatory and structural genes ( Figure 6). A similar result was also found that deletion of ssu72 homolog gene led to a lower production of mycotoxin-DON in plant pathogenic fungi F. graminearum [34]. A 70-kb DNA cluster is associated with aflatoxin biosynthesis, and regulatory and structural genes play important roles in this cluster [48]. As a conserved CTD phosphatase in fungi, Ssu72 has been proven to be involved in multiple cellular processes, including mRNA processing, transcription initiation and termination [29,32]. So we speculated that phosphatase Ssu72 may affect the transcription process of some key aflatoxin biosynthesis related genes, and then regulate the production of aflatoxin. These findings suggest that phosphatase Ssu72 positively regulates AFB1 biosynthesis in A. flavus.
Tyrosine phosphatases are well conserved and play critical roles in fungal pathogenicity in filamentous fungi [24,39]. In F. graminearum, homologues of Ssu72 have been demonstrated to be essential for plant infection [34]. However, the role of phosphatase Ssu72 in seeds infection is still unknown in A. flavus. Here, seeds infection assays showed that deletion of ssu72 resulted in a severe defect in pathogenicity on maize and peanuts ( Figure 7A), which is consistent with the previous finding in F. graminearum [34]. In addition, fewer conidia and aflatoxins were observed in the infected seeds of ∆ssu72 mutant ( Figure 7B,C). In A. flavus, pathogenicity is related to various factors, such as vegetative growth, conidiation, mycotoxins and environment stresses [43]. Therefore, we believed that the severe defects in development and aflatoxin production are the main reasons for the decreased pathogenicity to seeds. Taken together, these data suggest that phosphatase Ssu72 displays a vital role in pathogenicity to seeds of A. flavus.
Mitogen-activated protein kinase (MAPK) related phosphatases have been identified to be involved in multiple stresses response by regulating the phosphorylation level of MAP kinases (Hog1, Slt2 and Fus3) in A. flavus [39]. In this study, we found that the putative phosphatase Ssu72 is critical to respond to osmotic stress ( Figure 8A). Similarly, the orthologue of Ssu72 has also been known to response to osmotic stress in N. crassa, and a higher phosphorylation level of Hog1 kinase was observed in ssu72 deletion mutant [41]. Additionally, ∆ssu72 mutants exhibited more sensitive to oxidative stress than WT and ∆ssu72-Com strains ( Figure 8B). Reactive oxygen species (ROS) have been characterized to be highly related to oxidative stress [43]. In plant pathogenic fungi, elimination of ROS is critical to seeds infection [34]. Due to lower pathogenicity found in ∆ssu72 strain, we speculate that ROS scavenging ability of ∆ssu72 strain may be reduced. And this hypothesis is consistent with the result that the ∆ssu72 mutant displayed more sensitive to oxidative stress in A. flavus.
In conclusion, a putative phosphatase Ssu72 was identified in A. flavus, and our results indicate that Ssu72 is involved in the regulation of development, aflatoxin biosynthesis and pathogenicity. This is the first report on the biofunction of phosphatase Ssu72 in Aspergillus species. We believe that our discoveries could improve the understanding of Ssu72 in filamentous fungi, and may provide a novel insight for developing new control strategies to this fungus.

Strains and Culture Conditions
Strains of A. flavus used and constructed were listed in Table 1. All strains were cultured on YES, PDA and YGT media for vegetative growth and conidiation analysis, and on WKM medium for sclerotia formation analysis [49]. YES liquid medium was used for aflatoxin production as described before [50].

Mutant Strains Construction
The homologous recombination approach was used to generate the ssu72 knockout mutant (∆ssu72) [4]. Three fragments (1392 bp ssu72 5 UTR, 1220 bp 3 UTR and 1890 bp pyrG) were amplified with specific primers and fused together. Then, the fusion PCR products were transformed into A. flavus CA14 protoplasts as described earlier [51]. As the pyrG gene is inserted into the knockout strain, the knockout strain could grow in a medium without urea. This feature could be used to screen the knockout strain. Afterwards, the ssu72 complementation strain (∆ssu72-Com) was constructed using a previously described method [50]. Briefly, an overlap PCR product which contains ssu72 coding region was introduced into the ∆ssu72 protoplasts. Finally, all the selected transformants were identified by PCR, and further confirmed by RT-PCR and qRT-PCR assays. At least two or three positive transformants were used for further phenotypic analysis. Primers are listed in Table 2. Table 2. Primers used in this study.

Phenotpic Assays
To explore the roles of ssu72 in growth and conidiation in A. flavus, 10 4 spores were spotted on YES, PDA and YGT media, and cultured at 37 • C for 5 days. Colony diameter was measured after 5 days of cultivation. Subsequently, conidia were collected from PDA medium and quantified using a hemocytometer as previously described [52]. For sclerotia production assay, all strains were incubated on WKM medium at 37 • C for 7 days. Then, 75% ethanol was used to wash away the mycelia and conidia, and the sclerotia were harvested and counted by a light microscope as described earlier [53].

Aflatoxin Production Assays
To determine aflatoxin production, 10 4 conidia of strains were inoculated on YES liquid medium and cultured at 29 • C for 6 days in the dark, then aflatoxins were extracted by chloroform according to a previously described approach [38]. TLC was performed to analyze aflatoxin in a solvent system (chloroform:acetone = 9:1) and examined under 365 nm UV light [13].

Pathogenicity Assays
Pathogenicity analysis on peanuts and maize seeds were conducted as described earlier [46]. Briefly, the sterilized crop seeds were inoculated with 10 6 spores of each strain at 29 • C for 5 days. The infected seeds were collected and transferred to 50 mL centrifuge tubes with 15 mL sterile water. Then, the conidia amount and aflatoxin production of the infected seeds were analyzed as the methods mentioned before.

Stress Assay
To investigate the role of Ssu72 in various stresses response, the WT, ∆ssu72 and ∆ssu72-Com strains were inoculated onto PDA medium supplemented with 1 M NaCl or 5 mM H 2 O 2 at 37 • C for 4 days. The inhibition of growth rates was calculated as described before [38].

RNA Extraction and Quantitative Real-Time PCR Analysis
RNA extraction and cDNA synthesis were conducted according to the published references in our lab [39]. Mycelia were collected from PDA and WKM media cultured for 48 h, then TRIzol reagent (Biomarker Technologies, Beijing, China) was used for total RNA isolation. cDNA was synthesized with First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). Subsequently, cDNA was used as a template for qRT-PCR analysis. All the qRT-PCR primers used in this study are listed in Table 3. The relative transcript levels of related genes were calculated with the 2 -∆∆Ct method [54], and β-actin was used as internal control. Table 3. Primers used for qRT-PCR analysis.