The wild-type strain PH-1 (NRRL 31084) was chosen as parental strain for generating all deletion mutants (Cuomo et al., 2007). All strains were cultured on complete medium (CM), potato dextrose agar (PDA), minimal medium (MM), and yeast extract peptone dextrose (YEPD) medium at 25°C to assay growth rate.

The gene deletion mutants were constructed by double-joint PCR method, the primer pairs which were used to amplify the upstream and downstream sequence of the targeted gene are shown in Supplementary Table 1. The resulting products were then transformed into the protoplasts of wild-type PH-1 as described previously (Tang et al., 2020). To complement the ΔFgspe3, the Fgspe3-GFP cassette (GFP, green fluorescent protein) was constructed, a Fgspe3 fragment which included the promoter and gene fragment (without stop codon) was amplified from wild-type genomic DNA with specific primers listed in Supplementary Table 1. The resulting complementation fragments and Xho1 digested pYF11 vector were co-transformed into yeast XK1-25. The recombined FgSPE3-GFP fusion vector was transformed into Escherichia coli for amplification and then transformed into the ΔFgspe3 mutant, and the generating transformant was named as the ΔFgspe3-C. By means of the same methods, the FgSPE1-GFP, FgSPE2-GFP, and FgSPE4-GFP recombinant plasmids were transformed into the mutants, respectively, and generated the ΔFgspe1-C, ΔFgspe2-C, and ΔFgspe4-C complementation strains.

For conidia production, fresh mycelial plugs of each tested strain were inoculated in liquid carboxymethyl cellulose (CMC) medium (0.1% CMC, 2 mM MgSO₄, 4 mM NH₄NO₃, 10 mM KH₂PO₄, and 1 L water). The 50-mL flasks were cultured in a shaker (180 rpm) for 4 days at 25°C. The number of conidia for each strain in the medium was counted using a hemacytometer (Sigma-Aldrich, United States). The conidial morphology was observed by Calcofluor White (CFW) staining and photographed with a confocal microscope (Gottingen, Niedersachsen, Germany). Perithecium production was inoculated on carrot agar medium (CA) with a 12 h/12 h light/dark cycle at 25°C.

To determine the growth rate under different stress conditions for deletion mutants, fresh mycelia plugs (5 mm in diameter) of each strain were inoculated on PDA medium containing various stress compounds described in the figure legends. Exogenous NO₃^–, NH₄⁺, Put, Spd, and Spm (Sigma-Aldrich, United States) at 5 mM were separately added into minimum medium minus nitrogen (MM-N) medium to evaluate the growth of the ΔFgspe mutants. MM-N medium which did not include any nitrogen source served as a control.

Conidia were collected from a CMC medium and adjusted to 10⁵ conidia/mL with sterile water. A 10-μL conidial suspension was inoculated into a spikelet of a 6-week-old flowering wheat head. For each strain, 15 replicates were injected into spikelets for analysis. The infected spikelet was characterized 15 days after inoculation. To further analyze the pathogenicity of the ΔFgspe mutants in detail, a fresh mycelia plug of wild-type and mutants was placed on fresh corn silks. Then in 5 days after incubation at 25°C, the extent of discoloration was recorded. For wheat leaf infection assays, the 4-week-old fresh leaves were inoculated with mycelial plugs at 25°C. Pictures were taken 6 days after inoculation. For wheat coleoptile infection assays, 3-day-old seedlings of wheat were used. Conidial suspension was injected and recorded symptoms 7 days after inoculation. For each strain, there were 15 replicates in each experiment.

For detection of DON production, ΔFgspe3 strain was grown in liquid trichothecene biosynthesis induction (TBI) medium at 28°C for 1 week in the dark. The supernatant was harvested by three layers of filter paper and then quantified by a liquid chromatography tandem–mass spectrometry (LC-MS/MS) system (Tang et al., 2018).

The wild-type and ΔFgspe3 were cultured into TBI medium for 2 days at 28°C. The mycelia of each strain were harvested, and then total RNA was isolated using the TaKaRa RNAiso Extraction Kit, and then used RevertAid H Minus First Strand cDNA Synthesis Kit to synthesize cDNA (Fermentas Life Sciences, Burlington, ON, Canada). The transcriptional levels of TRI1, TRI5, and TRI6 of PH-1 were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using the method previously described (Liu et al., 2019). ACTIN (FGSG_07335) gene was served as a control. The primers were listed in Supplementary Table 1.

The conidia of deletion mutant were stained by 0.1 mg/mL CFW for 30 s and observed with a fluorescent microscope (Gottingen, Niedersachsen, Germany). Hyphal branching patterns were examined with a Nikon microscope.

For deep transcriptome sequencing, 2-day-old mycelia of wild-type and ΔFgspe3 strain were harvested from liquid YEPD shaken at 180 rpm, washed three times, and ground in liquid nitrogen. For each strain, there are three biological replicates independently. Total RNA was isolated, and cDNA libraries were constructed by NEBNext^® Ultra^TM Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, United States) using the manufacturer’s instructions. The cDNA libraries were sequenced by an Illumina Hiseq Novaseq platform with paired-end reads (150 bp) by Novogene Corporation (Beijing, China). Reads were mapped to the genome of F. graminearum PH-1 strain. DESeq R package was used for differential expression gene analysis by Novogene Corporation (Beijing, China). Genes with log₂ Fold change > 1 or < −1 and P-value < 0.05 were divided into differentially expressed. Raw data of the RNA-seq experiments generated in this article were deposited in the National Center for Biotechnology Information under the accession number PRJNA759842¹.

Graphs were plotted by GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, United States). The statistical experiments were evaluated using SPSS Statistics 21.0 software. Statistical analysis was performed one-way ANOVA and Fisher’s LSD test (P < 0.05).

Combined with the other fungi, plants, and animals, we firstly inferred the pathway of polyamine biosynthesis in F. graminearum (Supplementary Figure 1). Further, using the S. cerevisiae polyamine biosynthesis (Spe) protein sequence to query F. graminearum genome, the heterologous genes involved in polyamine biosynthesis which included FGSG_05903 (designated as Fgspe1), FGSG_09035 (designated as Fgspe2), FGSG_13725 (designated as Fgspe3), and FGSG_09525 (designated as Fgspe4) were revealed in this fungus. To further confirm the Spe protein evolution relationships within the organism, phylogenetic analysis of the Spe protein sequence was performed and indicated that the Spes protein was highly conserved in fungi. Interestingly, most Spe homologs were notably clustered into one distinct subclade (Figure 1A). Meanwhile, the InterPro database and tools for protein domain analysis indicated that FgSpes protein harbored conserved domains that are unique to Spe proteins (Figure 1B). In general, The domain characteristic and the phylogenetic tree indicated Spe homologs proteins were conserved and might have similar biological functions in other fungi.

Polyamines play an essential role to maintain normal growth and development in a living organism (Igarashi and Kashiwagi, 2019). To further study the function of polyamine biosynthesis gene in F. graminearum, we constructed deletion mutants, ΔFgspe1, ΔFgspe2, ΔFgspe3, and ΔFgspe4, using homologous recombination strategy by replacing open reading frame (ORF) of FgSPE gene with hygromycin B phosphotransferase (hph) gene cassette. Transformants in each mutant showed consistent phenotypes on the same conditions. To verify that the phenotypic defects in mutants were due to gene deletion, we constructed the complemented vector for each mutant and generated the ΔFgspe1-C, ΔFgspe2-C, ΔFgspe3-C, and ΔFgspe4-C complemented strains. As shown in Figures 2A,B, the loss of Fgspe1 resulted in significantly reduced growth in CM medium and MM medium that were polyamine-free mediums. However, the ΔFgspe1 exhibited similar mycelial growth rates compared with the wild-type strain in PDA and YEPD medium that may be included by trace amounts of polyamines. The ΔFgspe2 and ΔFgspe3 mutants showed obviously hyphal growth defects on PDA, CM, MM, and YEPD medium. Moreover, ΔFgspe2 and ΔFgspe3 also showed a reduction in aerial hyphae. However, ΔFgspe4 showed no obvious phenotypic defects. It might indicate that Fgspe4 encoding spermine synthase was not important for the growth of F. graminearum (Figures 2A,B). Subsequently, microscopic observation exhibited that the hyphal morphology of ΔFgspe2 and ΔFgspe3 mutants were highly branched and misshapen (Figure 2C). The dry weight biomass of ΔFgspe2 and ΔFgspe3 were significantly reduced compared to wild-type in YEPD medium for 2 days (Figure 2D). As shown in Supplementary Figure 1, Fgspe2 and Fgspe3 are essential for Spd synthesis. Therefore, our result indicates that the endogenous Spd is vital for the hyphal growth of F. graminearum.

As shown in Figure 3, wild-type PH-1 showed as seriously growth defective in the MM-N medium in which was not included any nitrogen source. To further confirm the function of the polyamine in F. graminearum therefore, we firstly testified the growth rate of wild-type PH-1 in MM-N medium by exogenous supplemented polyamine, NO₃^– and NH₄⁺ served as the control. In Figure 3, the phenotypic defects of wild-type PH-1 in MM-N medium showed better recovery by exogenous supplementation of Spd in comparison with Put and Spm, indicating that Spd exhibits a more important role for the growth of F. graminearum. The ΔFgspe1, ΔFgspe2, and ΔFgspe3 mutants showed the seriously defective growth in CM, MM, and MM-N mediums that were free of polyamines (Figures 2A, 3). Put, Spd, and Spm are the downstream metabolites of ornithine decarboxylase (Vanrell et al., 2013). To explore the defective phenotypes of ΔFgspe mutants and whether they could be recovered by the addition of exogenous polyamines, the ΔFgspe mutants were grown on MM-N medium supplemented with Put, Spd, and Spm at 5 mM concentration, respectively. NO₃^– and NH₄⁺ served as the control. The results showed that NO₃^– and NH₄⁺ were not conducive for growth restoration of ΔFgspe1, ΔFgspe2, and ΔFgspe3 mutants. When Put was added to MM-N medium, defects in hyphal growth of ΔFgspe1 mutant were restored (Figure 3). When exogenous Spd was added to an MM-N medium, the vegetative growth of the ΔFgspe2 and ΔFgspe3 could be fully rescued. In general, our data further indicates that the Spd plays a vital role in hyphal growth in this fungus.

Sexual and asexual reproduction play a vital role in the infection cycle of F. graminearum. Subsequently, the conidial and perithecia production of ΔFgspe mutants was observed. Firstly, to rule out the effect of growth defects on asexual and sexual reproduction, the growth of ΔFgspe mutants was determined. As shown in Figures 4A,B, the ΔFgspe mutants and complementary strains showed a consistent phenotype in conidiation (CMC) and sexual reproduction (CA) in this medium in comparison with the PDA medium (Figure 2A). Hence, we further observed the conidial and perithecia production of ΔFgspe mutants in CMC and CA medium. Compared to wild-type, our result indicated that the ΔFgspe2 and ΔFgspe3 exhibited an obvious reduction in conidiation after incubation for 4 days in CMC medium (Figure 4C). To further observe conidial morphology, the conidia of all mutants were stained by CFW dye. The conidial morphology was recorded with a fluorescence microscope. The conidia size of ΔFgspe2 and ΔFgspe3 was shorter in comparison with wild-type (Figure 4D). Moreover, the ΔFgspe2 and ΔFgspe3 showed dramatic reduction in perithecia formation on the CA agar medium in comparison with wild-type (Figure 4E). Thus, our results indicate that endogenous Spd is critical for the asexual and sexual reproduction of F. graminearum.

A previous study conducted by Yamaguchi et al. (2007) has indicated that polyamine is important to the response to various environmental stresses. Therefore, we detected the sensitivities of the ΔFgspe mutants to different environmental stress agents, including sodium dodecyl sulfate (SDS), Congo Red, H₂O₂, and temperature. The sensitivity assays indicated that the ΔFgspe2 and ΔFgspe3 showed significantly increased sensitivity to various stresses compared to the wild-type. Moreover, the ΔFgspe2 and ΔFgspe3 mutants could not grow at the high temperature and oxidative stress generated by 30°C and H₂O₂, respectively. ΔFgspe1 shows weakly increased sensitivity to environmental stress in Figure 5A. However, ΔFgspe4 showed a similar susceptibility in the wild-type toward the above stress (Figures 5A,B). The complementation strains exhibited the same sensitivities as wild-type under various stress conditions. Aurofusarin of Fusarium fungi played an important role in responding to environment adaptation (Xu et al., 2019). Therefore, we observed the aurofusarin production of ΔFgspe mutants and found that ΔFgspe2 and ΔFgspe3 mutants showed increased yellow pigments on PDA medium (Supplementary Figure 2). In general, our results indicate that Spd shows a vital role in the response to abiotic and biotic stresses.

To further investigate the function of polyamine in this pathogenic progress, the virulence of the ΔFgspe mutants was evaluated in flowering wheat heads, the leaves of wheat seedlings, corn silk, and wheat coleoptiles. Notably, the wild-type, ΔFgspe1, ΔFgspe4, and complemented strains could develop on the injected spikelets and quickly spread to the neighboring wheat head, causing discoloration symptoms 2 weeks after inoculation. However, the ΔFgspe2 and ΔFgspe3 mutants were not capable of penetrating the spikelet (Figure 6A). Additionally, the ΔFgspe2 and ΔFgspe3 demonstrated attenuated virulence on seedling leaves, corn silk, and wheat coleoptiles (Figures 6B–D). To demonstrate that Spd is the main cause of the reduction in virulence of ΔFgspe3, furthermore, we testified whether exogenous Spd supplement assay could rescue the virulence defects of ΔFgspe2 and ΔFgspe3. Our result showed that the addition of exogenous Spd can indeed rescue the virulence defects of the ΔFgspe2 and ΔFgspe3 mutants in planta (Figure 7). These results demonstrate that Spd is important for virulence in F. graminearum.

As DON is proposed as a vital virulence factor and assists the fungus spread during F. graminearum infection process (Seong et al., 2009), we guessed that the reduced pathogenicity of ΔFgspe2 and ΔFgspe3 was also partly due to a reduction in DON production. Therefore, the transcriptional levels of TRI genes which are essential for DON synthesis in the ΔFgspe2 and ΔFgspe3 were detected by RT-qPCR assay after incubation for 2 days in liquid TBI medium. These results indicated that the expression levels of TRI6, TRI5, and TRI1 genes were significantly down-regulated in the ΔFgspe2 and ΔFgspe3 compared with wild-type (Figure 8A). Furthermore, the DON synthesis of ΔFgspe2 and ΔFgspe3 mutants showed a dramatic reduction after incubation for 7 days in a TBI medium (Figure 8B). These results suggest that ΔFgspe2 and ΔFgspe3 are essential for DON biosynthesis in F. graminearum.

To further explore the function of Spd, we performed RNA-seq to wild-type and ΔFgspe3 strain in YEPD medium. For each strain, we plotted three biological replicates. For most genes, the replicates produced similar FPKM values, and all points fall near a line with a slope of 1 (Supplementary Figure 3). Transcriptome analysis revealed that 6,957 genes were differentially expressed in the ΔFgspe3 compared with the wild-type, including 3,331 up-regulated and 3,626 down-regulated genes (Figure 9A). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the function of the differentially expressed genes (DEGs). GO enrichment analysis identified that genes that are significantly expressed in the ΔFgspe3 mutant were mainly involved in biological processes such as organonitrogen compound biosynthetic, peptide metabolic and translation; cellular components such as ribosome, intracellular ribonucleoprotein complex, cytoplasm, and mitochondrion; and molecular functions such as structural constituents of ribosomes, and RNA and ATP binding (Figure 9B). Furthermore, KEGG enrichment analysis showed a list of the top 20 regulated pathways in ΔFgspe3, including ribosome, DNA replication, oxidative phosphorylation, RNA polymerase, and the citrate cycle (TCA cycle) (Figure 9C). Then, we tested transcriptional expression patterns of nine representative DEGs by qRT-PCR, and the trends in gene expression profiles from qRT-PCR were positively correlated with the transcriptomic data, indicating that expression levels based on RNA-seq data were believable (Figure 9D). Taken together, these results indicate that Fgspe3 might modulate the essential cellular process involved in ribosomes, DNA replication, and carbon metabolism in F. graminearum.