A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.


Introduction
Fungal infection is one of the three main diseases of crops, other than bacteria and viruses that can result in reductions in agricultural output [1]. Green mold caused by the ascomycete fungi Prochloraz is a type of triazole fungicide that is widely used in Europe, Australia, Asia and South America for gardening and agriculture [25]. However, little is known about prochloraz resistance mechanisms of in P. digitatum. In this study, we report the identification and characterization of an ortholog of Aspergillus SrbA, SreA, in P. digitatum. By constructing an sreAdisrupted strain and a complemented strain, we analyzed the effects of SreA on full virulence, prochloraz (PRC) resistance and ergosterol biosynthetic genes. Our results provide further insight into the molecular mechanisms of fungicide resistance in P. digitatum.
Cloning and sequencing of sreA from P. digitatum Based on the DNA sequence of A. fumigatus SrbA (GenBank accession no.XM_744169), we identified an SREBP protein-encoding gene Pc20g05880 (GenBank accession no. XM_002563071) in P. chrysogenum. Given that the genome of P. digitatum and P. chrysogenum share high similarity [27], two pairs of specific primers sreA-a, sreA-b, sreA-c and sreA-d ( Table 1) were designed according to the relatively conserved sequences of A. fumigatus sreA and P. chrysogenum Pc20g05880 after sequence alignment with ClustalW. Two approximately 1000bp DNA fragments were amplified from genomic DNA of P. digitatum by PCR, and then cloned into pMD18-T vector (TaKaRa Biotech. Co., Dalian, China) for sequencing. Then an approximately 2000-bp DNA fragment of P. digitatum sreA was obtained after sequence-assembling. The 5' flanking DNA sequence of sreA was amplified by genome walking using the Genome Walking Kit (TaKaRa Biotech. Co., Dalian, China) with specific primers sreA-e, sreA-f and sreAg ( Table 1). The 3' flanking unknown DNA sequence of sreA was amplified using SMATer RACE 5'/3' Kit (TaKaRa Biotech. Co., Dalian, China) with specific primers sreA-h and sreA-i. The DNA sequence of sreA has been deposited in GenBank under accession number KJ939329.

DNA and protein analysis
The DNA sequence of sreA was analyzed using NCBI BLAST and BioEdit software. A protein analysis was performed using the NCBI BLAST and Interpro (http://www.ebi.ac.uk/interpro/) programs. TMHMM software (version 2.0) was used to predict the transmembrane domains of SreA.

Construction of an sreA disruption plasmid
The plasmid pTFCM containing the PtrpC promoter and TtrpC terminator from A. nidulans and carrying the hph gene which confers resistance to hygromycin B as selective marker was generously provided by Dr. Daohong Jiang (Huazhong Agricultural University, China). The sreA disruption plasmid was constructed by inserting the up-stream and down-stream flanking sequences of sreA into the pTFCM vector [28]. To this end, a 1-kb DNA fragment of the sreA 5' coding sequence was amplified from P. digitatum genomic DNA using primers sreA-1 and sreA-2 ( Table 1) and cloned into pMD18-T vector. After sequencing, the fragment containing sreA 5' coding sequence was digested by SpeI and XhoI and sub-cloned into pTFCM between SpeI and XhoI sites to generate the pTFCM-L plasmid. Next, another 1-kb fragment containing the sreA 3' coding sequence was amplified and cloned into the pMD18-T vector for sequencing using primers sreA-3 and sreA-4 ( Table 1). After digestion by restriction enzymes KpnI and SacI, the fragment containing sreA 3' coding sequence was inserted between the KpnI and SacI sites of pTFCM-L to generate the sreA disruption plasmid pTFCM-L-R (Fig. 1A).

Construction of sreA complementation plasmid
The sreA complementation plasmid was obtained by a PCR strategy (Fig. 1A). A DNA fragment containing the sreA open reading frame as well as its promoter and terminator (4385bp)  was amplified using genomic DNA of P. digitatum as a template and sreA-F1/sreA-R1 as primers ( Table 1). The PCR conditions were as follows: 3 min at 94°C followed by 30 cycles each of 30 s at 94°C, 1 min at 60°C, and 2 min at 72°C. A GeneAmp 9700 thermal cycler (Perkin-Elmer [PE] Applied Biosystems, Foster City, Calif., USA) was used with LA Taq polymerase (TaKaRa Biotech. Co., Dalian, China). The amplified fragment was digested by SpeI and cloned into plasmid pTFCM-neo to obtain the complementation plasmid pTFCM-neo-sreA. The pTFCM-neo plasmid was constructed by replacing the hph cassette between two XbaI sites in pTFCM with a neo cassette that confers resistance to the antibiotic G418.

Transformation of P. digitatum
Prior to the transformation, the recombinant plasmids pTFCM-L-R and pTFCM-neo-sreA were transformed into A. tumefaciens strain EHA105 by a heat-shock method [29]. Next, A. tumefaciens-mediated transformation was performed to obtain sreA disruption (ΔsreA) and complementation (COsreA) strains [28,30]. First, A. tumefaciens strains harboring the disruption plasmids were recovered on YEP plates at 28°C for two days. A single colony containing pTFCM-L-R plasmids was selected and cultured in MM medium before transfer into IM medium cointaining 200 μM acetosyringone; the cell density was adjusted to OD 600 = 0.15. After incubating at 28°C with shaking at 180 rpm for 6 h, equal volumes of A. tumefaciens culture and P. digitatum conidial suspensions (10 6 spores ml -1 ) were mixed and transferred to a lens paper on an IM agar plate for cultivation at 25°C for 3 days. Finally, the lens paper was transferred onto the PDA medium containing 50 μg/ml hygromycin B and 50 μg/ml cefoxitin to select for sreA disruption strains (ΔsreA). To obtain sreA complementation strains (COsreA), one selected ΔsreA strain was transformed with the complementation plasmid pTFCM-neo-sreA according to the protocol of ΔsreA strain construction, with the modification that G418 (200 μg/ml) was used to select transformants instead of hygromycin B. The ΔsreA and COsreA transformants were confirmed by PCR using primers sreA-F and sreA-R ( Table 1) and a Southern blot analysis.

Southern blot analysis
Genomic DNA was extracted from the HS-F6 wild-type, ΔsreA, and COsreA strains using Biospin Fungus DNA Extraction Kit (BioFlux, Tokyo, Japan). Approximately 30 μg genomic DNA was digested with HindIII at 37°C for 12 hours, electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane. A 1003-bp digoxigenin-labeled probe specific to the 5' region of sreA was generated using the DNA Probe Labeling Kit (TIANDZ, Beijing, China) by PCR with primers sreA-1 and sreA-2 ( Table 1) and hybridized with the genomic DNA blot. DIG Random Labeling and Detection Kit II (BOSTER, Wuhan, China) was used for color detection following the manufacturer's protocol.

Assays of vegetative growth and prochloraz EC 50
To compare the growth of HS-F6 wild-type, ΔsreA, and COsreA strains, a 0.8-mm mycelial plug was obtained from a PDA plate with 50 μl of the corresponding conidial suspension (1×10 6 spores ml -1 ) coated on the surface and then cultured on a new PDA plate. After four days' cultivation at 25°C, the diameters of different colonies were measured. EC 50 values of prochloraz (1-{N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]carbamoyl}; PRC) for the different strains were measured according to [31] with modifications. Briefly, 50 μl of a conidial conidia suspension (10 6 spores ml -1 ) of the HS-F6 wild-type and mutant strains was coated onto a PDA plate respectively and cultivated at 25°C for 24 h. Mycelial plugs (approximately 0.8 mm diameter) were then obtained from the plate using a punch and placed on the center of PDA plates containing different concentrations of prochloraz. After incubation at 25°C for 6 days, the diameters of the colonies were measured. Three replicates were used for each experiment. The average of the colony diameters in each independent test was used for EC 50 calculation by software SPSS 10.0.

Virulence assay
Mature citrus fruits (Citrus sinensis) were purchased from a fruit market in Hongshan district, Wuhan. The fruits were washed with distilled water and dried at room temperature before inoculation. Virulence assays for the HS-F6 and mutant strains were performed directly on citrus fruits. Firstly, a 2-mm deep hole was made on the pericarp using a 1-ml pipett tip. Then 3 μl of a conidial suspension (10 6 spores ml -1 ) of the HS-F6 wild-type or mutants was injected into the hole. After incubation at 25°C for three days, the diameters of the disease spots formed were measured and compared.

RNA extraction and quantitative real-time PCR (qRT-PCR)
qRT-PCR was used to analyze cyp51 gene expression. Before RNA extraction, 20 μl of a conidial suspension (10 6 spores ml -1 ) of P. digitatum HS-F6 and ΔsreA strains was cultured in PDB medium at 25°C for 72 h. In the prochloraz-treatment experiment, 7 μg/ml prochloraz (about the concentration of EC 50 ) was added to the PDB medium with shaking for an extra 6 h after cultured at 25°C for 48 h. The mycelia were then filtered and washed several times using double distilled water. Total RNA was extracted using RNAiso Plus (TaKaRa Biotech. Co., Dalian, China) according to the manufacturer's protocol. All RNA samples were treated with DNase I (TaKaRa Biotech. Co., Dalian, China). First-strand cDNA was prepared using All-in-one First strand cDNA Synthesis Kit (Genecopoeia, Guangzhou, China) following the manufacturer's protocol. qRT-PCR was performed using a BIO-RAD CFX96 q-PCR system with SYBR Green I fluorescent dye detection. The mRNA abundance was normalized using the housekeeping gene β-actin, and the relative expression levels were calculated using the 2 -ΔΔCt method [32]. The primers used to amplify cyp51A/B/C and β-actin genes in qRT-PCR are listed in Table 1.

Statistical analysis
All results presented with statistical significance were analyzed with an unpaired two-tailed Student's t test and two-way ANOVA. P<0.05 was considered significant.

Cloning and sequence analysis of sreA from PdHS-F6
A 3120-bp DNA fragment of sreA ORF was amplified from PdHS-F6 genomic DNA using primers sreA-F and sreA-R (Table 1), sequenced, and then analyzed by NCBI BLAST. The sreA ORF is predicted to encode a protein of 1040 amino acids that is a putative HLH transcription factor and an ortholog of A. fumigatus transcription factor SrbA. The nucleotide homology between sreA and srbA is 63%; protein homology is 61% (Fig. 1). The nucleotide homology between sreA and P. chrysogenum Pc20g05880 is 89%.
Two highly conserved domains in SreA were identified. A basic helix-loop-helix-leucine zipper (bHLH-zip) domain located in the N-terminus of the protein contains a unique tyrosine residue (at site 177) that distinguishes SREBPs from other bHLH transcriptional factors [33]; a domain DUF2014 of unknown function is located in the C-terminus of the protein. This domain is found at the C-terminus of a family of ER membrane bound transcription factors called sterol regulatory element binding proteins (SREBP). SreA is predicted to contain two transmembrane domains, suggesting that this protein is a membrane-bound transcriptional factor with a topology similar to vertebrate SREBPs.

Construction of sreA-disruption and -complementation strains
To analyze the function of SreA in P. digitatum, sreA disruption strains (ΔsreA) of HS-F6 (PdHS-F6m) were constructed ( Fig. 2A). Twenty putative mutants were selected on PDA medium supplemented with 50 μg/ml hygromycin B and screened by PCR using primers sreA-1 and sreA-4 ( Table 1). The 3.0-kb fragment of sreA in HS-F6 was replaced by a 4.0-kb fragment (the sreA gene disrupted with the hph gene) in the recombinant strains (ΔsreA) (as shown in Fig. 2B). Southern blot analysis further confirmed that a single copy was integrated. Only one strain was chosen and used for further study (Fig. 2C).  Table 1). C: Southern blot analysis of P. digitatum wild-type PdHS-F6 and ΔsreA, COsreA strains using a probe specific to the 5' region of Pdsre. 30μg genomic DNA was digested with HindIII and detected using a probe specific to the 5' region of sreA gene. Twenty complementation strains (COsreA) were obtained using G418 as selective marker. The insertion of sreA in the COsreA strains was confirmed by PCR using primers sreA-1 and sreA-4. The occurrence of a 3.0-kb band (sreA) and a 4.0-kb band (sreA disrupted with the hph) indicated the successful integration of the sreA gene into the genome of the ΔsreA strains (Fig. 2B). Southern blotting confirmed that the ΔsreA and COsreA strains were successfully constructed and that a single copy of sreA was integrated into the genome of the ΔsreA strain to generate the COsreA strain (Fig. 2C).
Deletion of sreA renders P. digitatum more susceptible to triazole drug prochloraz As stated above, PdHS-F6 is highly-resistant to triazole drug prochloraz and has an EC 50 value of 7.896 mg/l, which is more than 800 times higher than prochloraz-susceptible strains. As shown in Fig. 3, PdHS-F6 and ΔsreA strains showed similar growth on the PDA plate without prochloraz, the diameters of colonies were not significantly different. However, the diameters of colonies of ΔsreA strains on PDA plates supplemented with prochloraz (5 μg/ml, 10 μg/ml) were smaller than those of HS-F6. After being cultured on PDA plates supplemented with different concentration of prochloraz (0, 0.5, 1, 5, 10 μg/ml) for 6 days, the diameters of the colonies were measured (Fig. 3B), and the EC 50 value was calculated. The average EC 50 value of prochloraz for the ΔsreA strain was 3.2 mg/l, which was less than half of the value of HS-F6 (Fig. 3C). However, complementation of sreA (PdHS-F6c) restored the EC 50 value of the ΔsreA strain (average EC 50 value of prochloraz for the COsreA strain was 6.7 mg/l). These results demonstrated that the deletion of sreA renders P. digitatum more susceptible to triazole drug prochloraz, suggesting that sreA plays an important role in the resistance of P. digitatum to triazole drugs. Gene sreA is required for full virulence in P. digitatum To determine whether sreA plays a role in the virulence of P. digitatum toward citrus fruits, a virulence assay was performed directly on these fruits. The assay results demonstrated that the symptoms in the fruits incubated with conidial suspension of the ΔsreA strains developed more slowly than in the fruits incubated with the wild-type conidial suspension. The mean diameter of the macerated lesions of the fruits incubated with the ΔsreA conidial suspension was approximately 1.90 cm at 3 days post inoculation, whereas the mean macerated lesion diameter of the fruits incubated with the HS-F6 conidial suspension was about 3.4 cm (Fig. 4A, B). The virulence assay results revealed that the deletion of sreA rendered the ΔsreA strain less virulent compared with the wild-type HS-F6. A further experiment was performed to confirm this result. The virulence assay demonstrated that average diameter of the macerated lesions induced by COsreA was comparable to that of wild type HS-F6 (3.2 cm) (Fig. 4C, D). These results indicated that SreA is required for full virulence in P. digitatum.
Transcriptional abundance of the cyp51 genes is significantly decreased in the ΔsreA strain Because SrbA regulates the expression of erg11A gene in A. fumigatus [21], we hypothesized that its ortholog, SreA, plays a similar role in P. digitatum. To test this hypothesis, the mRNA abundance of the cyp51 genes in PdHS-F6 wild-type and the ΔsreA strain was analyzed using qRT-PCR (Fig. 5). The results showed that transcriptional abundance of the three cyp51 genes were all decreased in the ΔsreA strain, especially with regard to cyp51A. The normalized expression values of cyp51A, cyp51B, and cyp51C in the ΔsreA strain were 0.02, 0.29 and 0.47, respectively. Characterization of an SREBP in P. digitatum SreA is required for transcriptional response to prochloraz in P. digitatum To investigate the role of sreA in the transcriptional response of P. digitatum to prochloraz, a prochloraz induction experiment was performed with the ΔsreA and HS-F6 strains. Total RNA was isolated from the PdHS-F6 and ΔsreA strains with or without prochloraz treatment and used for qRT-PCR. In the case of wild-type HS-F6, the expression levels of the cyp51 genes were all increased after 6 h of prochloraz treatment (Fig. 6A); the normalized expression values in the cyp51A, cyp51B and cyp51C expression levels after prochloraz treatment were 5.3, 1.8, and 2.2, respectively. However, the induction of cyp51A and cyp51B by prochloraz was abolished in the ΔsreA strain. As shown in Fig. 6B, the normalized expression values for cyp51A  and cyp51B mRNA abundances were 0.56 and 0.65, respectively. Notably, the increasing foldchange in cyp51C mRNA abundance after prochloraz treatment was higher in the ΔsreA strain than in the wild-type strain HS-F6 (Fig. 6A, B), which might be due to regulation by other transcription factors that maintained cyp51C transcript level in response to prochloraz.

Discussion
A number of transcription factors associated with sterol metabolism and drug resistance have been well characterized in different clinical fungi. Upc2, a typical zinc cluster protein identified in S. cerevisiae and C. albicans, was the first documented transcription factor highly correlated with fungal drug resistance [34][35][36][37]. Deletion of CaUpc2 rendered C. albicans increased susceptibility to a range of common antifungal drugs used in clinical therapy, and this increased susceptibility is observed for drugs targeting the ergosterol biosynthesis pathway [16]. A previous report also provided direct evidence for Upc2 in transcriptional regulation of C. albicans erg genes involved in ergosterol biosynthesis, including erg7, erg11 and erg25 [38]. The erg11 overexpression in the Upc2 gain-of-function mutants led to strain resistance to azole fungicides [39]. SREBPs, functionally conserved in fungal kingdom, have been revealed to regulate sterol synthesis in fission yeast S. pombe as well as a number of fungal pathogens particularly grown under hypoxia conditions [20,40]. Sre1 undergoes sterol-dependent proteolytic activation and regulates genes required for maintaining cellular sterol homeostasis. Sre1 is also required for many other fungi to regulate sterol biosynthesis, and SREBP is required for mammalian cells to regulate cholesterol and fatty acid anabolism [41][42]. SrbA from the SREBP super family is associated with hypoxia, cell polarity, full virulence, and ergosterol homeostasis in A. fumigatus. It is notable that SrbA plays a critical role for triazole drug interactions in A. fumigates, which may have clinical importance [23][24]. In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by SREBPs. However, in yeasts such as S. cerevisiae and C. albicans, sterol synthesis is regulated by Upc2 instead [43]. Even though Upc2 functions similarly to SrbA, they are totally different from each other in terms of the protein structure: Upc2 is a zinc finger transcription factor with a typical Gal4-type zinc finger, while SREBPs contain a bHLH domain and a characteristic tyrosine residue.
In the present study, we characterized an SREBP transcriptional factor, SreA, in P. digitatum as an ortholog of SrbA that is associated with triazole susceptibility, full virulence, and regulation of cyp51 genes [24]. Our results demonstrated that SreA in P. digitatum may play a role similar to SrbA. The sreA deletion strains of P. digitatum (ΔsreA) were generated using A. tumefaciens-mediated genetic transformation based on the prochloraz-resistant P. digitatum strain PdHS-F6. Prochloraz is a triazole fungicide that is widely used in Europe, Australia, Asia and South America within gardening and agriculture [25]. However, with the extensive and excessive use of prochloraz, drug-resistant fungi have appeared. As reported in 2013, 78 strains of P. digitatum were isolated from citrus fruits collected in Hubei Province and 25 isolates were identified to be prochloraz-resistant, with a proportion as high as 32% [26]. For reducing the increasing economic loss due to fungicide resistance, understanding the molecular mechanisms of resistance is of practical significance.
Our results in P. digitatum demonstrated that the deletion of sreA increases susceptibility of PdHS-F6 to prochloraz. As shown in Fig. 3, the prochloraz EC 50 value of the ΔsreA strain was significantly lower than HS-F6. The growth assay on PDA plates revealed that the ΔsreA strain grew as well as PdHS-F6 without prochloraz. However, the growth rate of ΔsreA strain on PDA plates supplemented with prochloraz (5 μg/ml, 10 μg/ml) was much slower than that of the HS-F6 (Fig. 3A, B), revealing that drug response was altered in the ΔsreA strain. CYP51 proteins are the targets of triazole fungicides, and the susceptibility of fungi to triazole drugs is closely related with the mutations in or the expression level of cyp51 genes [44]. To confirm the role of SreA in the regulation of cyp51 gene expression, the transcriptional abundance of cyp51 genes in HS-F6 and ΔsreA strain were analyzed by qRT-PCR. The results revealed that the expression level of the three cyp51 genes was decreased in the ΔsreA strain, particularly cyp51A, which almost could not be detected (Fig. 6). Our study thus demonstrated that SreA is an important regulator of cyp51 genes: the deletion of sreA reduced the expression levels of cyp51 genes, which are the targets of trizole fungicide prochloraz, rendering the strain more susceptible to the drug. However, SreA is not the only transcription factor regulating the expression of cyp51 genes. In the ΔsreA strain, other regulators may control the expression of cyp51 genes in response to the drug and to some extent may compensate the loss of sreA. In support of this notion, the EC 50 value of the ΔsreA strain was not as low as the prochloraz-susceptible strain, and the ΔsreA strain could still grow on PDA plates supplemented with prochloraz at a relatively high concentration; nevertheless, the growth rate was much slower than that of HS-F6.
To determine whether sreA is associated with pathogenicity of P. digitatum, virulence assays were performed directly on citrus fruits using the HS-F6 wild-type and mutant strains. The results demonstrated that the ΔsreA strain was less virulent than the wild type. The symptoms in fruits incubated with a ΔsreA conidial suspension developed more slowly and the mean diameter of the macerated lesions was almost half of those produced by the HS-F6 wild-type strain (Fig. 4). As expected, the COsreA strain displayed symptoms similar to the wild type, indicating that the complementation of sreA restored the virulence of ΔsreA strain. Therefore, we can conclude that SreA is required for full virulence in P. digitatum.
Fungi have evolved sophisticated mechanisms to cope with environmental stresses, including antifungal drugs, and respond to triazole drugs by altering the expression levels of effector genes. DMI-resistant isolates were initially thought to exhibit fitness penalties that would preclude DMI resistance and becoming wide-spread in nature, as reported in the studies of several important fungal pathogens of different crops [45]. In the past few years, DMI-resistant strains have also been identified in human pathogenic fungi. For example, erg25 transcription is induced in response to itraconazole treatment in C. albicans [44]. Besides, the triazole-induced overexpression of cyp51 genes in fungi has been regarded as an important fungal self-protective mechanism towards a range of fungicides [24]. In this study, we compared the cyp51 mRNA expression in PdHS-F6 and ΔsreA strains before and after prochloraz treatments. In the HS-F6 wild-type strain, the expression level of cyp51 genes were all increased after prochloraz induction for 6 h (Fig. 6A). However, the increase in cyp51 expression levels, except cyp51C, was abolished in the ΔsreA strain. The further increased mRNA abundance of cyp51C in response to prochloraz-treatment in the ΔsreA strain might be attributed to the regulating by other transcription factors in response to the drug. However, the relatively decreased expression level of cyp51C in the ΔsreA strain demonstrated that sreA could regulate the expression of cyp51C to some extent. These results indicated that SreA is an important regulator of cyp51 genes. The deletion of sreA renders P. digitatum unable to cope with prochloraz properly, making it more susceptible to the antifungal drug.
With the widespread and constant use of triazloe antifungal drugs, resistance has been selected in many fungal species. Most of this resistance is due to the mutation and overexpression of target cyp51 genes or the mutation of their regulatory genes [5][6][18][19]. Besides, the overexpression of transporter-encoding genes also contributed to fungi resistance [46]. The documented alterations in transcription factors, particularly in their DNA binding sites, could affect drug resistance in the pathogenic yeast C. albicans, tumorigenesis in hosts, and for resistance of the malarial parasite Plasmodium vivax [18,[47][48][49][50][51]. Therefore, identifying new target genes and the designing of new drugs is of great significance to control the spread of resistant fungi. Previously, we reported the cloning, expression, and characterization of cyp51 from P. digitatum and Ustilago maydis [52][53][54][55]. The structural characteristics of the interaction between heterologous CYP51 and commercial azoles were also analyzed by binding assays. A series of new 2-azolyl-3, 4-dihydroquinazolines 6 was synthesized by the direct cyclization of imidazole or 1, 2, 4-triazole with carbodiimides 4, and the preliminary bioassay results demonstrated that these compounds exhibited well to significant fungicidal activity against P. digitatum [54]. The screening of new DMI fungicides based on optimized expression has been carried out for the first time in Ustilago maydis [55]. Recently, a cell-based high-throughput screen has identified small-molecule inhibitors of the Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The compounds were growth-inhibitory and could attenuate antifungal-induced sterol gene expression in vivo [56]. Thus, as a regulator of cyp51, SreA, with an essential function in P. digitatum fungicide resistance, represents a promising research direction to uncover a new fungus-specific antifungal drug targets.