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FaSmi1 Is Essential for the Vegetative Development, Asexual Reproduction, DON Production and Virulence of Fusarium asiaticum

Smi1 is a protein required for cell cycle progression, morphogenesis, stress response and life span of Saccharomyces cerevisiae. FaSmi1 was identified as a Smi1 homolog in a wheat scab pathogenic fungus Fusarium asiaticum strain 2021. The deletion of FaSmi1 leads to defects in mycelial growth, asexual reproduction, and virulence. The FaSmi1 deletion mutant also exhibited increased sensitivity to osmotic stresses generated by NaCl and KCl, but increased tolerance to oxidative stresses and cell wall integrity inhibitors. All of these defects were restored by genetic complementation of the mutant with the whole parental FaSmi1 gene. Interestingly, the antioxidant system-associated genes exhibit a lower expression level and the mycotoxins’ DON content was decreased in the FaSmi1 deletion mutant compared with the parental strain 2021. These results indicate that FaSmi1 plays a critical role in the vegetative development, asexual reproduction, DON production and virulence of F. asiaticum.


Introduction
Fusarium head blight (FHB) caused by the Fusaruim complex species is a destructive plant disease and leads to large economic damage around the world. FHB quickly spreads in regions with high temperatures and humidity, causing 10-50% cereal yield loss, and the mycotoxins produced by Fusarium complex species in the infected wheat and other cereal crops pose a serious threat to human and animal health [1,2]. Despite the large economic impact of FHB, the main strategy for controlling FHB is the application of fungicides [3]. Only a few kinds of fungicides (including carbendazim azoxystrobin and triazoles) are available to reduce the FHB index, but they cannot control the accumulation of deoxynivalenol (DON) in cereals. Moreover, on account of the frequent and large application of fungicides to control FHB, the Fusarium complex species have developed resistance against various fungicides, leading to a weaker control effect on FHB and even failure [4,5]. Based on the current situation of FHB control, the exploitation of new types of inhibitors for the management of FHB is needed. Therefore, we explored key proteins required for vegetative development, DON production and virulence in Fusarium complex species, which might be considered a potential target protein for developing new fungicides to control FHB. Smi1, also known as Knr4, is an intrinsically disordered protein conserved in many fungi [6]. In Saccharomyces cerevisiae, Smi1 plays important roles in the Slt2 MAP kinase cell wall integrity pathway and the calcineurin phosphatase in the calcium-calcineurin pathway via physically interacting with the key components of two pathways [6,7]. Smi1 F. asiaticum strain 2021 was originally isolated from infected wheat ears in Zhejiang province of China. It was used as the wild-type strain to generate gene deletion mutants. Potato dextrose agar (PDA), complete medium (CM) and minimal medium (MM) were used for mycelia growth and stress sensitivity assays [19,20]. Mung bean liquid (MBL) liquid medium was used for conidiation assays [21]. Liquid trichothecene biosynthesis (LTB) liquid medium was used for DON production assays [22].

Sequence Analysis of FaSmi1 in F. asiaticum
FaSmi1 is a homologous protein of FgSmi1 (The accession number in Fusarium graminearum genome FGSG_06998.3) that was originally identified through BLASTP searches in the F. graminearum genome (available at http://www.broadinstitute.org/annotation/ genome/fusarium_group/Multi-Home.html, accessed on 16 March 2018), referring to the Smi1 from S. cerevisiae. Based on the sequence information of the FgSmi1 gene, full-length and cDNA of FaSmi1 was amplified from genomic DNA of wild-type strain 2021 for the sequence analysis.

Generation of FaSmi1 Deletion Mutants
To explore the roles of FaSmi1 in F. asiaticum, we generated FaSmi1 deletion mutants via the homology replacement method, as described previously [23]. Briefly, the gene replacement cassette was constructed, which carries the hygromycin resistance gene and herpes simplex virus thymidine kinase gene flanked by the 5 (upstream junction) and 3 (downstream junction) ends of the FaSmi1 gene. This cassette was constructed with double-joint PCR, as previously described [24]. The fragments 1.3 kb upstream and 1.2 kb downstream of FaSmi1 were amplified from the genomic DNA of strain 2021 using the primer pairs P1/P2 and P3/P4, respectively. The 3.5-kb fragment with trpC promoter of A. nidulans, hygromycin resistance gene and the thymidine kinase gene from the herpes simplex virus and the (HPH-tk) was amplified from pKHT plasmid using the primer pair HTF/HTR [23] (Table S1). After the abovementioned three fragments (up and downstream of FaSmi1 and HPH-tk) were purified by a gel purification kit, which was mixed with a molar ratio of 1:3:1 and used as a template to perform a double-joint PCR. After the fragment was amplified by PCR using 5 µL product of a double-joint PCR and primers P5/P6 (Table S1), the PCR product was purified and sequenced. Next, after the sequencing result was confirmed and the PCR product was transformed into protoplasts of F. asiaticum strain 2021 to generate FaSmi1 deletion mutant.

Complementation of FaSmi1 Deletion Mutants
The FaSmi1 deletion mutant (∆FaSmi1) was complemented with the full-length FaSmi1 gene to confirm that the phenotypic changes of the FaSmi1 deletion mutant were due to the disruption of the gene. The construct for the complementation of ∆FaSmi1 was generated by amplifying a fragment from the genomic DNA of strain 2021 with primer P1/P4 (Table S1). After being purified and sequenced, the PCR product was transformed into protoplasts of ∆FaSmi1.

Protoplast Preparation and Transformation of F. asiaticum
For preparation of protoplasts, conidia of the 2021 strain were harvested from sevenday-old cultures grown in MBL medium and inoculated into YEPD liquid medium (10 g peptone, 3 g yeast extract, 2 g glucose per 1 L ddH 2 O). Mycelia were isolated via gauze filtration after incubation at 175 rpm and 25 • C for 12 h. Then, mycelia were washed with 0.7 M NaCl buffer and incubated with lysing buffer (0.2 g lysing, 0.2 g helicase and 0.1 g driselase dissolved in 20 mL 0.7 M NaCl), and the protoplasts were isolated via gauze filtration after incubation at 75 rpm and 30 • C for 2 h. The protoplasts were washed twice using 0.7 M NaCl and STC (50 mM Tris pH 8.0, 0.8 M sorbitol, 0.05 M CaCl 2 ), respectively, then resuspended in STC-SPTC buffer [STC:SPTC = 4:1; SPTC (STC containing 40% PEG 6000)]. In the transformation, protoplasts (10 7 cells /mL), 3 mg target DNA and heparin sodium were added into 200 µL SPTC buffer and were mixed. After incubation for 30 min on ice, 1 mL SPTC was added and mixed, continuing incubation for 20 min at room temperature. Next, transformed protoplasts were added into 200 mL RM medium (274 g sucrose, 1 g yeast extract, 1 g casein hydrolyzate, 17 g agar, per 1 L ddH 2 O) at 43 • C. After sufficient mixing, RM medium containing protoplasts was poured into 9 cm diameter culture plates (15 mL per plate) and incubated at 25 • C for 12 to 16 h. Then, the plates were overlaid with 10 mL of SRM medium (342 g sucrose, 1 g yeast extract, 1 g casein hydrolyzate, 10 g agarose, per 1 L ddH 2 O) modified with 100 µg/mL hygromycin B. Transformants were obtained after 3-5-day incubation at 25 • C and were transferred onto PDA plates supplemented with 100 µg/mL hygromycin B (but complementation strains could not grow) and 0.2 µM floxuridine (but transformants could not grow). Complementation of ∆FaSmi1 with the wild-type FaSmi1 gene was performed as described above, except the selection agent was floxuridine.

Mycelial Growth, Conidiation and Stress Sensitivity Assay
The mycelial growth assay was conducted on PDA. Mycelial plugs of each strain taken from the margin of the colony were put onto PDA. The colony diameter of each strain was measured after incubation for 3 days at 25 • C in the dark. Each treatment had three replicates; the experiment was repeated three times.
We analyzed the sensitivity of the deletion mutant to osmotic stress, which was generated by NaCl and KCl, and cell wall integrity inhibitors (Congo red and caffeine), and cell membrane damage generated by SDS. The mycelial plugs of each strain were incubated on PDA plates supplemented with 1.2 M NaCl, 1.2 M KCl, 0.05% Congo red, 5 mM caffeine, and 0.05% SDS for 3 d at 25 • C in the dark. The relative inhibition growth rate was calculated by the following formula: [(A − B)/(A − 5)] × 100, where A and B are the colony diameter of control and treatment, respectively [25]. Each treatment had three replicates; the experiment was repeated three times.
In conidiation assay, eight mycelial plugs of each strain were put onto 250 mL broth containing 100 mL MBL medium incubated at 25 • C with shaking at 175 rpm for a 16-h photoperiod. After seven days, spores were counted with a hemocytometer [26]. Each treatment had three replicates; the experiment was repeated three times.

Quantitative RT-PCR (qRT-PCR)
The total RNA of each sample was isolated with a total RNA isolation Kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with the PrimeScript ® RT reagent kit (TaKaRa, Kusatsu, Japan). All qRT-PCR reactions were performed with a qRT-PCR assay Kit (Vazyme, Nanjing, China) and ABI 7500 real-time detection system (Applied Biosystems, Waltham, MA, USA). Primers used for qRT-PCR analysis are listed in Table S1. All data were normalized to actin gene expression, and relative changes in gene expression levels were analyzed with ABI 7500 SDS software (Applied Biosystems), which automatically set the baseline. Data from three biological replicates were used to calculate the means and standard deviations. The experiment was repeated three times.

Virulence Assay on Flowering Wheat Heads
After incubation in MBL medium for 7 days, conidia of each strain were collected by filtration through three layers of lens paper and subsequently resuspended in sterile distilled water and adjusted to a concentration of 10 6 conidia/mL. Wheat heads of the Zhenmai 22 cultivar were inoculated with 10 µL of a conidial suspension of each strain, as described by Gale [27]. After being moisturized for 2 days, the wheat plants were cultivated in a greenhouse. Each treatment had 20 wheat heads. After 15-day inoculation, the number of infected spikelets in each treatment was measured, and the data were assessed with the Fisher's LSD test. The experiment was repeated three times.

In Vitro DON Production Assay
For the in vitro total DON production analysis, conidia of each strain were inoculated into LTB liquid media (1 × 10 5 conidia/mL). The total DON production of each strain in LTB cultures was assayed with a competitive ELISA-based DON detection plate kit (Beacon Analytical Systems, Inc., Saco, ME, USA) after incubation at 28 • C for 7 days, as described [22]. The total DON production ability (DPA) of each strain was calculated by the formula: DPA = total DON production in LTB cultures (mg).

Identification of FaSmi1 in F. asiaticum
Using BLASTP, FaSmi1 was identified in the F. asiaticum genome, based on amino acid sequences of Smi1 in S. cerevisiae and F. graminearum genome data. FaSmi1 is highly homologous to its counterparts among various fungal species ( Figure S1). The sequencing data indicated that, in F. asiaticum, full-length FaSmi1 is 1678 bp, containing two introns, coding 571 amino acids, and the structural domain analysis showed that FaSmi1 has a SMI1/KNR4-conserved domain which is involved in the synthesis of β-1,3 glucan and cell wall integrity.

Deletion and Complementation of FaSmi1 in F. asiaticum
To investigate the functions of FaSmi1 in F. asiaticum, we generated the FaSmi1 deletion mutant (∆FaSmi1) by transforming the gene replacement construct containing an HPH-tk resistance cassette into the parental strain 2021 ( Figure S2A). The FaSmi1 deletion mutant was identified by PCR and southern blotting using special primer pairs and a probe, respectively ( Figure S2B,C). To ensure the phenotypes of deletion mutants were caused by deleting the corresponding gene, the FaSmi1 deletion mutant was complemented with the parental FaSmi1. The complementation strain (∆FaSmi1C) was confirmed by southern blotting ( Figure S2C).

Involvement of FaSmi1 in Hyphal Growth and Asexual Development of F. asiaticum
Compared to the parental 2021 strains, the mycelial growth rate of ∆FaSmi1 was decreased ( Figure 1A; Table 1) and the pink pigment production of ∆FaSmi1 was increased on PDA media ( Figure 1A). To explore whether the mycelial growth defects were associated with the medium, we incubated the 2021, ∆FaSmi1 and ∆FaSmiC strains on PDA, CM and MM media. Compared to 2021 and ∆FaSmi1C, the mycelial growth defects of ∆FaSmi1 were also observed on CM and MM medium ( Figure 1A; Table 1). Moreover, in the microscopic assay, the mycelia of ∆FaSmi1 were thicker and had thinner branches compared to 2021 and ∆FaSmiC ( Figure 1A). These results indicate that FaSmi1 plays a significant role in the mycelial growth and pigment generation of F. asiaticum.
probe, respectively (Figure S1B,C). To ensure the phenotypes of deletion mutants were caused by deleting the corresponding gene, the FaSmi1 deletion mutant was complemented with the parental FaSmi1. The complementation strain (ΔFaSmi1C) was confirmed by southern blotting ( Figure S1C).

Involvement of FaSmi1 in Hyphal Growth and Asexual Development of F. asiaticum
Compared to the parental 2021 strains, the mycelial growth rate of ΔFaSmi1 was decreased ( Figure 1A; Table 1) and the pink pigment production of ΔFaSmi1 was increased on PDA media ( Figure 1A). To explore whether the mycelial growth defects were associated with the medium, we incubated the 2021, ΔFaSmi1 and ΔFaSmiC strains on PDA, CM and MM media. Compared to 2021 and ΔFaSmi1C, the mycelial growth defects of ΔFaSmi1 were also observed on CM and MM medium ( Figure 1A; Table 1). Moreover, in the microscopic assay, the mycelia of ΔFaSmi1 were thicker and had thinner branches compared to 2021 and ΔFaSmiC ( Figure 1A). These results indicate that FaSmi1 plays a significant role in the mycelial growth and pigment generation of F. asiaticum.  In the conidiation assay, ∆FaSmi1 produced significantly fewer conidia compared to the 2021 and ∆FaSmi1C strains ( Figure 2A; Table 1). Moreover, microscopically, we found that the conidia length of ∆FaSmi1 was only about one third of the length of those of the 2021 and ∆FaSmi1C strains (Figure 2A). In a conidia germination assay, after incubation for 6 h, only 70% of the conidia of ∆FaSmi1 were germinated, while almost all conidia of the 2021 and ∆FaSmi1C strains were germinated under the same conditions ( Figure 2B). When the incubation time was extended to 10 h, all the conidia of ∆FaSmi1 were germinated, indicating that the deletion of FaSmi1 leads to defects in conidia germination. These results indicate that FaSmi1 plays a significant role in the conidial differentiation and germination in F. asiaticum. Mycelial linear growth rate was tested on PSA plate. The radial growth of each strain was measured after 3 days at 25 • C. Mean and standard deviations were calculated with results from three replicates. b Sporulation assay was conducted in MBB. c The length of brown lesions on diseased stems 7 days post inoculation. Ten coleoptiles were inoculated for each. Different uppercase letters are used to mark statistically significant differences between strains (p < 0.05). In the conidiation assay, ΔFaSmi1 produced significantly fewer conidia compared to the 2021 and ΔFaSmi1C strains (Figure 2A; Table 1). Moreover, microscopically, we found that the conidia length of ΔFaSmi1 was only about one third of the length of those of the 2021 and ΔFaSmi1C strains (Figure 2A). In a conidia germination assay, after incubation for 6 h, only 70% of the conidia of ΔFaSmi1 were germinated, while almost all conidia of the 2021 and ΔFaSmi1C strains were germinated under the same conditions ( Figure 2B). When the incubation time was extended to 10 h, all the conidia of ΔFaSmi1 were germinated, indicating that the deletion of FaSmi1 leads to defects in conidia germination. These results indicate that FaSmi1 plays a significant role in the conidial differentiation and germination in F. asiaticum.

Involvement of FaSmi1 in Cell Wall Integrity
The previous research showed that Smi1 is involved in the cell wall formation and osmotic stress sensitivity of S. cerevisiae. To explore the role of FaSmi1 in cell wall integrity, we analyzed the sensitivity of ∆FaSmi1 to the cell wall-damaging agents, Congo red (0.05%) and caffeine (5 mM). The results show that ∆FaSmi1 exhibits decreased sensitivity to Congo red and caffeine compared to strains 2021 and ∆FaSmi1C ( Figure 3A). Moreover, the number of protoplasts released from ∆FaSmi1 after mycelia incubation at 30 • C for 2 h and 4 h in lyase buffer was obviously lower than that from the 2021 and ∆FaSmi1C strains ( Figure 3B,C). These results suggest that FaSmi1 is required for the cell wall integrity of F. asiaticum.

Involvement of FaSmi1 in Cell Wall Integrity
The previous research showed that Smi1 is involved in the cell wall formation and osmotic stress sensitivity of S. cerevisiae. To explore the role of FaSmi1 in cell wall integrity, we analyzed the sensitivity of ΔFaSmi1 to the cell wall-damaging agents, Congo red (0.05 %) and caffeine (5 mM). The results show that ΔFaSmi1 exhibits decreased sensitivity to Congo red and caffeine compared to strains 2021 and ΔFaSmi1C ( Figure 3A). Moreover, the number of protoplasts released from ΔFaSmi1 after mycelia incubation at 30 °C for 2 h and 4 h in lyase buffer was obviously lower than that from the 2021 and ΔFaSmi1C strains ( Figure 3B,C). These results suggest that FaSmi1 is required for the cell wall integrity of F. asiaticum.

Involvement of FaSmi1 in Osmotic and Oxidative Stresses Sensitivity
To test the sensitivity to osmotic stress, each strain was incubated on CM medium supplemented with the osmotic stress generator 1.2 M NaCl or 1.2 M KCl. The results show that the relative growth inhibition of ∆FaSmi1 was higher compared to that of the 2021 and ∆FaSmi1C strains ( Figure 3D). To test the role of FaSmi1 in oxidative stress responses, the mycelia growth inhibition rate of each strain was analyzed on CM plates modified with oxidative stress generators, including H 2 O 2 and menadione. The results show that different concentrations of H 2 O 2 and menadione inhibited ∆FaSmi1 mycelial growth to a lower extent than that on 2021 and ∆FaSmi1C ( Figure 4A-D). Additionally, qRT-PCR analysis showed that, compared to 2021 and ∆FaSmi1C, the expression level of the four genes associated with antioxidant responses were significantly up-regulated in ∆FaSmi1, especially the superoxide dismutase gene, FaMnSOD1 ( Figure 4E). These results indicate that FaSmi1 is involved in the osmotic and oxidative stress response.

Involvement of FaSmi1 in Osmotic and Oxidative Stresses Sensitivity
To test the sensitivity to osmotic stress, each strain was incubated on CM medium supplemented with the osmotic stress generator 1.2 M NaCl or 1.2 M KCl. The results show that the relative growth inhibition of ΔFaSmi1 was higher compared to that of the 2021 and ΔFaSmi1C strains ( Figure 3D). To test the role of FaSmi1 in oxidative stress responses, the mycelia growth inhibition rate of each strain was analyzed on CM plates modified with oxidative stress generators, including H2O2 and menadione. The results show that different concentrations of H2O2 and menadione inhibited ΔFaSmi1 mycelial growth to a lower extent than that on 2021 and ΔFaSmi1C (Figure 4A-D). Additionally, qRT-PCR analysis showed that, compared to 2021 and ΔFaSmi1C, the expression level of the four genes associated with antioxidant responses were significantly up-regulated in ΔFaSmi1, especially the superoxide dismutase gene, FaMnSOD1 ( Figure 4E). These results indicate that FaSmi1 is involved in the osmotic and oxidative stress response.

Effect of FaSmi1 on the Virulence and DON Production of F. asiaticum
To explore the role of FaSmi1 in the virulence of F. asiaticum, we analyzed the infection ability of each strain on wheat spikes via inoculating conidial suspensions into flowering wheat heads [25]. After being inoculated for 15 days, the 2021 and ∆FaSmi1C had infected and caused typical blight symptoms on inoculated spikelets, the lesion area had expanded over 50% of the whole wheat heads, while, under the same conditions, the ∆FaSmi1 only infected spikelets at inoculation points, and the lesion cannot expand on the wheat heads ( Figure 5A; Table 1). In addition, after inoculation for 3 days with brick-shaped mycelial plugs, when the virulence of each strain was analyzed on corn stigmas, the ∆FaSmi1 failed to infect corn stigmas; however, the 2021 and ∆FaSmi1C had colonized and expanded on the corn stigmas ( Figure 5A). In the DON production assay, the DON production of the FaSmi1 deletion mutant was lower than that of 2021 ( Figure 5B). In addition, compared with the parental strain, the expression level of DON synthesis-associated genes FaTri4 and FaTri5 was significantly downregulated in ∆FaSmi1 ( Figure 5C). The results indicate that FaSmi1 is required for full virulence and DON synthesis in F. asiaticum.
are not significantly different at p = 0.05. (D) Colony morphology of each strain grown on PDA medium modified with different concentration menadione. (E) Relative expression levels of antioxidant system-related genes in 2021 and ΔFaSmi1. Line bars in each column denote standard errors of three repeated experiments. Values on the bars followed by the same letter are not significantly different at p = 0.05.

Effect of FaSmi1 on the Virulence and DON Production of F. asiaticum
To explore the role of FaSmi1 in the virulence of F. asiaticum, we analyzed the infection ability of each strain on wheat spikes via inoculating conidial suspensions into flowering wheat heads [25]. After being inoculated for 15 days, the 2021 and ΔFaSmi1C had infected and caused typical blight symptoms on inoculated spikelets, the lesion area had expanded over 50% of the whole wheat heads, while, under the same conditions, the ΔFaSmi1 only infected spikelets at inoculation points, and the lesion cannot expand on the wheat heads ( Figure 5A; Table 1). In addition, after inoculation for 3 days with brickshaped mycelial plugs, when the virulence of each strain was analyzed on corn stigmas, the ΔFaSmi1 failed to infect corn stigmas; however, the 2021 and ΔFaSmi1C had colonized and expanded on the corn stigmas ( Figure 5A). In the DON production assay, the DON production of the FaSmi1 deletion mutant was lower than that of 2021 ( Figure 5B). In addition, compared with the parental strain, the expression level of DON synthesis-associated genes FaTri4 and FaTri5 was significantly downregulated in ΔFaSmi1 ( Figure 5C). The results indicate that FaSmi1 is required for full virulence and DON synthesis in F. asiaticum.

Discussion
In budding yeast, Smi1 has been reported to be a transcriptional regulator of gene expression, affecting cell wall biosynthesis and maintenance [28,29]. However, the investigation of its function was not detailed. Smi1 has 533 genetic and 42 physical interaction partners, which are involved in various cellular processes, including cell wall biosynthesis and maintenance, cell cycle, metabolism and osmoregulation [6,7,10,30,31]. Besides maintaining cell wall integrity, Smil probably plays important roles in various cellular processes. The functions of Smi1 were mainly reported in S. cerevisiae. They were not explored in plant pathogenic fungi. In our study, mycelial growth was obviously decreased in the FaSmi1 deletion mutant, and spores of the FaSmi1 deletion mutant were malformed compared to the wild-type and complemented strain (Figure 2). The Smi1 deletion mutant of S. cerevisiae showed increased sensitivity to lysis, Congo red, caffeine and SDS [29]. In this study, we found that the sensitivity of ∆FaSmi1 to lysis, Congo red and caffeine was increased. The results indicate that the function of Smi1 on cell wall maintenance may be conserved among different fungi. In addition, we also found that the FaSmi1 deletion mutant exhibits increased tolerance to oxidative stress generated by H 2 O 2 and menadione (Figure 4), and the expression of the catalase genes FaCat6 and FaCcp1 and the superoxide dismutase gene FaMnSOD1 was obviously upregulated in ∆FaSmi1. Moreover, the previous study showed that Smi1 is involved in stress tolerance by affecting the expression level of associated genes [32]. Based on these results, we inferred that the deletion of FaSmi1 leads to upregulation of the oxidative balance-associated genes FaCat6, FaCcp1 and FaMnSOD1, which confers the tolerance ability of the FaSmi1 deletion mutant to oxidative stress.
The DON production is an essential factor in the virulence of F. asiaticum that was influenced by various factors; ROS is a key enhancement factor for DON production [33,34]. Moreover, the expression of trichothecene cluster genes is essential for DON production [35,36]; Smi1 regulates metabolic process by affecting transcription-associated genes [32]. In this study, compared with wild-type and complement strains, in the FaSmi1 deletion mutant, the trichothecene accumulation was obviously decreased ( Figure 5B). Moreover, the expression of the catalase genes FaCat6 and FaCcp1 and the superoxide dismutase gene FaMnSOD1 was upregulated ( Figure 4D); in addition, the expression of DON synthesis-associated genes Fa-Tri4 and FaTir5 in ∆FaSmi1 was downregulated ( Figure 5C). The results indicate that FaSmi1 involved in the regulation of DON may be associated to ROS balance. Taken together, we inferred that the deletion of FaSmi1 induces the expression level of FaCat6, FaCcp1 and FaMnSOD1, which was obviously upregulated, leading to a decreased ROS accumulation level. The lower level of ROS is responsible for the downregulated expression of FaTri4 and FaTir5, resulting in decreased DON production in F. asiaticum (Figures 4 and 5). The previous study showed that nutrient sources, light, pH and signal transduction pathways are important regulation factors for DON production [36]. In our study, the results show that the cell wall integrity pathway core protein FaSmi1 regulates DON production by affecting the expression gene in the ROS metabolism system. Our data indicate that the ROS level may be another significant regulation factor for DON production in fungi.
In the virulence assay, we found that only ∆FaSmi1 can infect the incubation site and loses the ability of extension in wheat ears, indicating the FaSmil is essential for the full virulence of F. asiaticum ( Figure 5A). Based on our results, we considered that many factors are involved in virulence defects of the FaSmi1 deletion mutant. First, the mycelial growth rate of ∆FaSmi1 was obviously decreased ( Figure 1A). Second, the FaSmi1 deletion mutant exhibited a significant defect on spore germination (Figure 2A). Third, the DON production of ∆FaSmi1 was obviously reduced ( Figure 5C). Therefore, we concluded that virulence defects in the FaSmi1 deletion mutant are closely associated with the defects on mycelial growth, spore germination and DON production. Taken together, our results indicate that FaSmi1 is essential for the vegetative development, asexual reproduction and virulence of F. asiaticum. Therefore, we thought that the FaSmi1 protein could be considered as a target for designing a new type of fungicide to control FHB caused by F. asiaticum.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8111189/s1, Figure S1: Protein sequence of the selected fungal FaSmi1 orthologs were aligned with the BioEdit 3. Figure S2: software The generation strategy and confirmation of FaSim1 deletion mutant and complement strains. Table S1: Primer used in this study.

Author Contributions:
All authors contributed to the study conception and design. Y.Z., C.C. and S.T. performed experimental design and wrote the manuscript draft. Y.Z., W.C., W.S. and D.S. conceived and performed all experiments and analyzed the results and corresponding data. All authors have read and agreed to the published version of the manuscript.