The wild-type strain of F. pseudograminearum named 2035 was preserved by the Laboratory of Fungi Diseases in the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, PRC. The wild-type and mutant strains were activated and cultured on potato dextrose agar (PDA, 20% potato extract, 2% dextrose, and 1.5% agar) medium in this study.

The growth rates of different strains were expressed as colony radius per day on PDA medium at 25°C. For the conidiation assay, different strains were grown on carboxymethylcellulose sodium (CMC) medium for 4 days. A hemocytometer was used to determine the concentration of conidia (Chen et al., 2021). To assay stress responses, mutants and wild-type strains were grown on synthetic medium (STM) [0.05% yeast extract, 0.5% (NH₄)₂SO₄, salts (0.15% KH₂PO₄, 0.06% CaCl₂, and 0.06% MgSO₄), and trace amounts of metals (0.0005% FeSO₄ 7H₂O, 0.00016% MnSO₄ H₂O, 0.00037% CoCl₂, and 0.00014% ZnSO₄ 7H₂O)] containing NaCl (0.7 M), H₂O₂ (3 mM), Congo red (CR; 200 mg/L), or SDS (0.01%). Colony diameter was measured after incubation for 4 days. During gene deletion or complementarity, TB₃ (0.3% yeast extract, 0.3% casamino acids, 20% sucrose, and 1.5% agar) medium mended with hygromycin B (250 μg/ml, Calbiochem, La Jolla, CA) or geneticin (250 μg/ml, Sigma, St. Louis, MO) has been used to select resistant transformants.

The LaeB homology protein FpLaeB was identified via querying the F. pseudograminearum genomic sequence (GenBank accession NC_031951.1). The conserved domains of FpLaeB were predicted via the Conserved Domain Search Service (CD Search) in the National Center for Biotechnology Information (NCBI). The phylogenetic tree of FpLaeB and its homology proteins were constructed via the neighbour-joining method with the MEGA version 7.02 software package (Xia et al., 2021).

Open reading frame (ORF) was replaced by the hygromycin phosphotransferase gene to construct the deletion mutant of FpLaeB. Two primer pairs FpLaeB-1F/2R and FpLaeB-3F/4R were used to amplify the upstream and downstream flanking fragment of the FpLaeB gene. The hygromycin phosphotransferase (hph) gene was amplified via the primer pair HYG-F/R. The replacement fragment was constructed by joining the three fragments via double-joint polymerase chain reaction (PCR) (Yu et al., 2004). The FpLaeB replacement fragment was transformed into protoplasts of wild-type 2035 by the polyethylene glycol (PEG) approach (Liu and Friesen, 2012). Following screening by hygromycin, the transformants were screened and confirmed using PCR and Southern blot analyses, respectively (Tang et al., 2018). For complementation assays, XhoI-digested pFL2 and the FpLaeB fragments with promoters cotransformed into yeast strain XK1-25. FpLaeB–pFL2 plasmid was constructed by the yeast gap repair method (Zhang et al., 2017). Then, FpLaeB–pFL2 was transformed into the protoplasts of the FpLaeB deletion mutant by the PEG approach as well. After geneticin screening, the primer pair FpLaeB-5F/6R was used to confirm the complementation strain from geneticin-resistant transformants. Primers used for deletion, complementarity, and gene expression are listed in Supplementary Table S1.

For virulence on the wheat stem base, conidia were collected from CMC according to the method used in strains and culture conditions and then diluted to a concentration of 10⁵ conidia/ml. The inoculation procedure was as described by Li et al. (2009) with the following modifications: Seeds of susceptible cultivar Shixin 828 were germinated on wet filter paper saturated in Petri dishes. Germinated seeds were immersed in the spore suspension for 1 min. Then, treated seeds were sown in a pot with a diameter of 15 cm containing sterile soil mix. There were three replicates, with each pot containing 20 seedlings. The severity of Fusarium crown rot was assessed at 35 days post-inoculation (dpi) using a 0–5 scale (Li et al., 2009).

For virulence on wheat heads, conidia of different strains were diluted to a concentration of 10⁵ conidia/ml in 0.01% (vol/vol) Tween 20. A 20-μl aliquot of conidial suspension was injected into a floret of a wheat head of susceptible cultivar Shixin 828 at early anthesis. There were 30 replicates for each strain. The severity of head blight used a scale of 0–4 (Wang et al., 2015).

The severity of plants was determined using the disease index. The disease index (DI) was calculated as follows: DI = [∑ (number of diseased plants in this scale × value of this scale)/(total number of plants investigated × highest value of scale)] × 100.

Both wild-type and mutant strains were transferred to PDA and incubated at 25°C for 3 days. Mycelia samples of three biological repetitions were collected from the surface of the colony. Total RNA of the wild-type and FpLeaB deletion mutant were extracted using an RNA extraction kit. The process followed the manufacturer’s instructions (Qiagen, Hilden, Germany). Novogene Co., Ltd. (Tianjin, China) conducted the library preparation and sequencing procedure. Clean reads for each sample were mapped on the reference genome of F. pseudograminearum CS3096 (Gardiner et al., 2012) using the TopHat 2.0.8 software with default parameters. The mapped read counts were used to determine the number of reads per kilobase per million reads (RPKM). The HTSeq v0.9.1 software was used to identify the different expressions of genes between mutant and wild-type strains (Trapnell et al., 2010). The DESeq2 software was used to isolate the differentially expressed genes (DEGs) with false discovery rate adjusted p < 0.05 (Love et al., 2014). The RPKM value of the same gene was used to calculate the fold change (FC) in log2(FC) greater than 1.0 between mutant and wild type. Gene ontology (GO) annotation was implemented by the GOseq package software (Young et al., 2010). The clusterProfiler v3.8.1 software was used to analyse the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment with p < 0.05.

Quantitative real-time PCR (qRT-PCR) was used to determine the transcript levels of SM genes (Wang et al., 2020). Total RNA was isolated from the mycelium using the TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. First-strand complementary deoxyribo nucleic acid (cDNA) was derived from total RNA using the Fermentas 1st cDNA synthesis kit (Hanover, MD) according to the manufacturer’s instructions. All values were calculated and normalised using the 2^−ΔΔCT method and FpActin (FPSE_04141) gene, respectively (Livak and Schmittgen, 2001; Xia et al., 2021). Mean and standard deviation of data were collected from three biological replicates. Fisher’s least significant difference (LSD) in the Statistical Package for the Social Sciences (SPSS) was used for statistical analysis (p < 0.05). The primers are listed in Supplementary Table S1.

Three 6-mm-diameter agar plugs taken from the edge of the colony were inoculated into a 150-ml Erlenmeyer flask containing 30 ml of trichothecene biosynthesis induction (TBI) medium (Gardiner et al., 2009). After cultivating at 180 rpm in a shaker at 28°C for 3 days, the mycelium was collected for expression analysis of TRI5. The fermentation broth was filtered with a 0.22-μm aqueous filter at 14 dpi. DON was detected via ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) (Soleimany et al., 2012).

The FpLaeB protein (accession number XP_009259110.1) contains 738 amino acids (aa) and was identified to be 47.99% homologous to A. nidulans LaeB (AN4699). A polyadenylate binding protein domain (PABP-1234) and Sec24-related protein domain (PTZ00395) were predicted at 141–251 and 68–188 aa via CD Search in NCBI, respectively. The coding gene sequence of FpLaeB was interrupted by three introns at 289–378, 1,487–2,200, and 2,875–2,924 bp (Figure 1A). Phylogenetic analysis showed that FpLaeB is a fungal LaeB homologue with a very close genetic relationship to that of F. graminearum (Figure 1B).

In order to generate the FpLaeB deletion mutant (LDM), we used the hygromycin B phosphotransferase (hph) gene to replace the entire ORF of FpLaeB (Figure 2A). The transformants were confirmed by PCR amplification after preliminary screening by hygromycin. There is no PCR product when the primer pairs of ORF (FpLaeB-5F/6R) were used to amplify FpLaeB deletion mutants (Figure 2B). The genomic DNA of wild-type and mutant strains were further hybridised using the hph probe (Probe h). We found that only one 6.3-kb fragment band presented in the FpLaeB deletion mutant (Figure 2C). Hence, a homologous recombination event occurred in a single locus in the FpLaeB deletion mutant. The complementarity strain of FpLaeB deletion mutants showed an expected band (LDM-C, Figure 2D).

To evaluate whether FpLaeB was involved in the morphology formation of F. pseudograminearum, the deletion mutant of FpLaeB and wild-type strains were cultivated on PDA medium. The colonial morphology of FpLaeB deletion mutant strains was dramatically affected compared to wild type. There is scarcely any aerial hyphal growth in deletion mutants in contrast to the wild type. The colonies displayed a more compact appearance and a shorter peripheral edge in deletion mutants. Also, the hyphae are tenuous and curved under the microscope (Figure 3A). The hyphal growth rate of the FpLaeB deletion mutant was quantified to be 23% that of the wild type (Figure 3B). When the FpLaeB genes were reintroduced into the deletion mutant strain, the morphology formation of the complemented mutant was restored to that of the wild type (Figures 3A, B). Thus, the FpLaeB showed an important role on the growth phenotype of F. pseudograminearum.

To test whether FpLaeB affected conidiation, we assayed the conidia concentration of different strains in induced media. After 4 dpi, the number of conidia was 10⁷/ml in the wild-type strain. However, this amount was 10⁵/ml in the deletion mutant at the same time. When reintroducing the FpLaeB genes into deletion mutant strains, the phenomenon could be reversed. This result showed the regulated effect of FpLaeB on conidiation in F. pseudograminearum (Figure 3C).

To characterise whether FpLaeB affects sensibility to abiotic stress, the deletion mutants and wild-type strains were inoculated on STM mended with 3 mM H₂O₂ (oxidative stress), 0.7 M NaCl (Na⁺, osmotic pressure), 0.01% SDS (cell membrane damaging agent), or 200 mg/L CR (cell wall inhibitor, Figure 4A). We assayed the inhibition rates of FpLaeB deletion mutants, which were higher than those of wild type on STM mended with 0.01% SDS. The inhibition rates of FpLaeB deletion mutants were lower than those of the wild-type strain on STM mended with 200 mg/L CR. There are no significant differences in sensibility between deletion mutants and the wild-type strain when cultured on NaCl and H₂O₂ media (Figure 4B). The phenomenon was restored when the FpLaeB genes were reintroduced into the deletion mutant strain. These results indicated that FpLaeB is important to maintain cell wall and membrane integrity in F. pseudograminearum.

We assay pathogenicity tests with stem base and flowering wheat heads to characterise the role of FpLaeB in disease development. The wild-type strain developed typical crown rot and head blight symptoms at the stem base and heads at 21 dpi, respectively. Under the same condition, limited discolouration appeared only at the inoculation site of the FpLaeB deletion mutant (Figure 5A). The disease index of the FpLaeB deletion mutant was reduced by approximately 95% in the crown and head compared to that of wild type. To confirm these findings, we performed a complementation assay with the FpLaeB gene. The complementation led to a normal phenotype in both cases (Figure 5B, 5C). This result confirmed that FpLaeB is involved in virulence in F. pseudograminearum.

We analysed transcriptomes (RNA-seq) from wild type and the FpLaeB deletion mutant [raw sequence data for RNA-seq data are available in the NCBI Sequence Read Archive (SRA), accession number: PRJNA914495] to reveal the regulatory role of FpLaeB at a genome-wide scale. The absence of FpLaeB caused a significant change in expression levels in more than 3,200 genes in the F. pseudograminearum genome. The number of differentially downregulated genes was 1,748, while 1,456 genes showed an increase in their expression [all the expression analysis at p < 0.05, log2(FC) >1 or <−1] (Supplementary Table S2). Three classes, namely, “molecular function”, “cellular components”, and “biological process”, of the gene product were used to define the GO. In the class cellular components, the two most populated categories were “integral component of membrane” and “intrinsic component of membrane”. This result showed that the majority of both upregulated and downregulated DEGs were significantly associated with “membrane” (Figure 6A; Supplementary Table S3). We observed a broad range of transcripts encoding the biosynthesis of SMs that were enriched during Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figure 6B; Supplementary Table S4). There were 62 gene-encoded SMs with significantly downregulated expression while 53 genes were upregulated in FpLaeB (Figure 6C). To validate the expression profiles of these SMs, the expression levels of nine genes encoding SMs were used to verify the accuracy of transcriptomes. The results show that the expression levels (five downregulation and four upregulation) were basically the same between transcriptomes and qRT-PCR (Figure 6D).

Furthermore, we monitored the transcription level of the TRI5 gene and the production of DON in the FpLaeB deletion mutant and the wild-type strain in inducing medium. In comparison with the wild type, the expression levels of the TRI5 gene were downregulated 20 times in the FpLaeB deletion mutant (Figure 7A). After induced culture, the DON concentration of the culture solution in the deletion mutant strain was 60.34 µg/L. This value was significantly lower than that of the wild-type strain, which was 1,143.83 µg/L (Figures 7B, C). Therefore, FpLaeB appears to play an important role in regulating the TRI gene expression and DON production of F. pseudograminearum.