Peroxisome Proliferator FpPEX11 Is Involved in the Development and Pathogenicity in Fusarium pseudograminearum

Fusarium crown rot (FCR) of wheat, an important soil-borne disease, presents a worsening trend year by year, posing a significant threat to wheat production. Fusarium pseudograminearum cv. b was reported to be the dominant pathogen of FCR in China. Peroxisomes are single-membrane organelles in eukaryotes that are involved in many important biochemical metabolic processes, including fatty acid β-oxidation. PEX11 is important proteins in peroxisome proliferation, while less is known in the fungus F. pseudograminearum. The functions of FpPEX11a, FpPEX11b, and FpPEX11c in F. pseudograminearum were studied using reverse genetics, and the results showed that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction. After deleting FpPEX11a and FpPEX11b, cell wall integrity was impaired, cellular metabolism processes including active oxygen metabolism and fatty acid β-oxidation were significantly blocked, and the production ability of deoxynivalenol (DON) decreased. In addition, the deletion of genes of FpPEX11a and FpPEX11b revealed a strongly decreased expression level of peroxisome-proliferation-associated genes and DON-synthesis-related genes. However, deletion of FpPEX11c did not significantly affect these metabolic processes. Deletion of the three protein-coding genes resulted in reduced pathogenicity of F. pseudograminearum. In summary, FpPEX11a and FpPEX11b play crucial roles in the growth and development, asexual reproduction, pathogenicity, active oxygen accumulation, and fatty acid utilization in F. pseudograminearum.


Introduction
Fusarium crown rot (FCR) is a common wheat disease. In Australia, where it was first reported, the direct economic loss due to FCR was as high as AUD 56.3 million per year, with a potential loss of up to AUD 160 million. In the United States, FCR causes wheat yields to decrease by 10 million tons [1,2]. In recent years, the damage caused by FCR has gradually worsened in the Huang Huai wheat region of China; for example, the yield loss caused by FCR is up to 30-50% in many wheat-producing areas in the Henan Province. These data indicate that FCR is a serious threat to wheat grain security and should be controlled immediately. FCR is a typical soil-borne disease. It overwinters mainly in the form of mycelium in soil and diseased plant residues, which can be preserved for more than 2 years in general and longer in arid or semi-arid climates. It is mainly transmitted during cultivation, and its hosts mainly include weeds and various gramineous crops, such as wheat, barley, and corn. It generally does not infect dicotyledonous crops [3,4]. F. pseudograminearum shows heterothallism, and the sexual state of Gibberella coronicola is not easily inducible indoors and is difficult to find in the field [3]. Pathogens generally invade the roots and stem bases of the plants, and the specific infection site depends on the distribution of the pathogen source in the soil. Fusarium head blight could also be induced at the flowering stage in warm and humid climates [5]. The environment in the two to three members in most fungi, and up to five members in ascomycetes. For example, Magnaporthe oryzae contains three PEX11 family members, and deletion of the MoPEX11A gene has the greatest effect on its pathogenicity [31]. In S. cerevisiae, the C-terminal ends of PEX11 are similar to those of the homologous proteins, PEX25p (YPL112c) and PEX27 (YOR193w). PEX11 localizes to the peroxisomal membrane and may form homo-oligomers. These proteins form the S. cerevisiae PEX11 protein family, whose members are necessary for peroxisome biosynthesis, and play a role in the regulation of their size and number [32,33]. However, PEX25 is necessary for the de novo biosynthesis of peroxisomes, and PEX27 competes with PEX25 to inhibit peroxisomal proliferation [14]. Thus far, most reports are from studies on yeast and mammals, while the functions of PEX11 in F. pseudograminearum are largely unclear.
In this study, reverse genetics was used to investigate the functions of FpPEX11a, FpPEX11b, and FpPEX11c proteins. Our results indicate that FpPEX11a and FpPEX11b play important roles in the growth, asexual reproduction, active oxygen metabolism, and fatty acid utilization of F. pseudograminearum. However, the function of FpPEX11c could be confirmed by further study in the future. Most importantly, the absence of any FpPEX11 protein could reduce the pathogenicity of F. pseudograminearum.

Identification and Knockout of FpPEX11 in F. pseudograminearum
Local BLASTp sequence alignment was performed using three members of the PEX11 protein family of M. oryzae (MGG_08896, MGG_00648, and MGG_05271), and the PEX11 protein (NP_014494) from S. cerevisiae against the F. pseudograminearum genome database available in NCBI aided identification of the orthologous genes FPSE-09675, FPSE-09643, and FPSE-04578, which we named FpPEX11a, FpPEX11b, and FpPEX11c, respectively. Further analysis showed that this group of genes had the typical domain of the PEX11 protein in SMART, which was similar to the protein structure of homologous genes in S. cerevisiae, M. oryzae, and F. graminearum ( Figure 1). According to MEME, evolutionarily conserved motifs were found at the ends of FPSE-09675, FPSE-09643, and FPSE-04578 ( Figure S1A). Phylogenetic tree analysis was carried out using undetermined PEX11 proteins of Neurospora crassa, Ogataea angusta, Penicillium chrysogenum, S. cerevisiae, M. oryzae, Aspergillus oryzae, Aspergillus fumigatus, Homo sapiens, Candida albicans, and F. pseudograminearum ( Figure 1). When compared to yeast and other eukaryotes, FpPEX11 appeared to be closely related to F. graminearum and M. oryzae. Therefore, we designated FpPEX11a, FpPEX11b, and FpPEX11c as PEX11 protein family members.
To determine the function of FpPEX11 in F. pseudograminearum based on the principle of homologous recombination, several hygromycin-resistant transformants were obtained by PEG-mediated protoplast transformation ( Figure S1). Taking the FpPEX11a gene as an example, the transformant was first preliminarily determined using the detection primer (Table S1), and then the clean knockout mutant strain ∆FpPEX11a was identified by Southern blotting (Table S1). The complementary gene FpPEX11a was integrated into the plasmid pFL2 to obtain the complementary strain ∆FpPEX11a-C; ∆FpPEX11b, ∆FpPEX11c, ∆FpPEX11b-C, and ∆FpPEX11c-C were obtained in the same manner.

FpPEX11 Is Involved in Vegetative Growth and Asexual Reproduction
We compared the vegetative growth of the ∆FpPEX11a, ∆FpPEX11b, ∆FpPEX11c, WT, and complementation strains on PDA and CM media. When compared to WT, the colony diameter of ∆FpPEX11a on PDA medium was significantly lower, the color of the colony was darker, the aerial hyphae were sparser and more curved, and the edge of the ∆FpPEX11a aerial hyphae collapsed on CM medium, resulting in premature aging. There was no significant difference between ∆FpPEX11b colony diameter and that of WT, but the colony color was darker, the aerial hyphae were significantly reduced, the edge of ∆FpPEX11b aerial hyphae collapsed on CM, and the hyphae aged prematurely. No significant variation in vegetative growth of ∆FpPEX11c and WT was detected ( Figure 2). The results indicate that FpPEX11a and FpPEX11b regulate colony morphology and growth rate, which significantly affect the vegetative growth of F. pseudograminearum; however, FpPEX11c had little effect on vegetative growth. After 3 days of incubation, spore production of ∆FpPEX11a and ∆FpPEX11b growing in CMC media was reduced by 53.28% and 71.04%, respectively, when compared to that of the WT ( Table 1). The clustered conidiophore of ∆FpPEX11a and ∆FpPEX11b were less than those of ∆FpPEX11c. In ∆FpPEX11a and ∆FpPEX11b, abnormal spore morphology with constriction at the tip was noticed after 6 d incubation. There was no significant difference in spore production or morphology of ∆FpPEX11c compared to that of WT. When incubated in YEPD medium, the conidium germination of ∆FpPEX11a was reduced by 50% at 3 h post-incubation when compared to that of WT, and the germ tubes were shorter than those of WT. Moreover, ∆FpPEX11b and ∆FpPEX11c were not significantly different from the WT. From these data, it can be seen that the significant decrease in the number of spores of ∆FpPEX11a and ∆FpPEX11b was probably due to abnormal sporulation structure, and spore deformity further affected their germination efficiency; ∆FpPEX11c showed no significant change in asexual reproduction ability when compared to WT ( Figure 3). Summarily, FpPEX11a, FpPEX11b, and FpPEX11c played different roles in asexual reproduction of F. pseudograminearum. FpPEX11a was involved in the production, morphology, and germination of F. pseudograminearum spores to regulate asexual reproduction, while FpPEX11b was not involved in the germination of F. pseudograminearum spores and FpPEX11c had little effect on asexual reproduction.

FpPEX11 Regulates ROS Metabolism
To investigate whether FpPEX11 is involved in the response to oxidative stress, we analyzed the growth of ∆FpPEX11a and ∆FpPEX11b on CM medium containing 20 mM H 2 O 2 . Under these growth conditions, we observed obvious defects in the mutants when compared to WT, while ∆FpPEX11c was as resistant to oxidative stress as WT. The production of cellular ROS in each strain was qualitatively analyzed by NBT staining. As shown in Figure 4C, the edge color of the ∆FpPEX11a and ∆FpPEX11b colonies was darker than that of the WT, whereas the color change of ∆FpPEX11c was not significant. To verify this result, generation of ROS was visualized by using DHE. Compared to the wild type, the fluorescence of ∆FpPEX11b was the strongest, followed by ∆FpPEX11a and ∆FpPEX11c as the weakest, similar to the WT strain ( Figure S2). These results fall in line with NBT findings. This indicates that ROS accumulation and metabolism were inhibited in ∆FpPEX11a and ∆FpPEX11b, but not in ∆FpPEX11c. The above results indicate that the absence of FpPEX11a and FpPEX11b reduced the resistance of the strain to oxidative stress, affected the metabolism of ROS, and thereby caused ROS accumulation, ultimately damaging the detoxification function of peroxisomes. Deletion of FpPEX11c had little effect on the response of the strain to oxidative stress.

FpPEX11 Is Involved in Lipid Metabolism
Unlike short-chain fatty acids, which act as substrates for mitochondrial β-oxidation, the main substrate for peroxisome oxidation is medium-long-chain fatty acids [34]. The strain was cultured in MM containing C 14  The number of aerial hyphae of mutants was obviously less than that of the WT, and the longer the carbon chain length, the fewer aerial hyphae. When compared to the WT, the relative growth of ∆FpPEX11a and ∆FpPEX11b were significantly reduced, and only extremely sparse hyphae were observed in ∆FpPEX11b ( Figure 5A). No significant difference was noticed in the colony diameter extension of ∆FpPEX11b; ∆FpPEX11c was not significantly different from the WT strain ( Figure 5B). These results indicate that FpPEX11a and FpPEX11b can regulate the utilization of medium-, long-, and very-long-chain fatty acids, while FpPEX11c had little effect on the utilization of fatty acids.

FpPEX11 Is Involved in Responding to Cell Membrane and Cell Wall Stresses
Congo red (CR), a cell wall inhibitor, can hinder the normal assembly of cell walls to produce cell wall stress and inhibit fungal growth [35]. SDS destroys the stability of proteins and fungal cell walls. On CM medium supplemented with 0.01% SDS and 0.2% CR, the colony growth of the mutants was stunted, and the inhibition of ∆FpPEX11a and ∆FpPEX11b was significantly higher than that of the WT. The growth of aerial hyphae was abnormal, and the sensitivity to cell wall inhibitors increased; ∆FpPEX11c growth had no obvious defects when compared to WT and the complemented strain ∆FpPEX11c-C ( Figure 6). These results indicate that knockdown of FpPEX11a and FpPEX11b reduces the resistance of the cell membrane and cell wall to the external stress.

The ∆FpPEX11 Exhibits Abnormal Peroxisome Number
To investigate the effect of FpPEX11 deletion on peroxisomes, the peroxisomal membrane protein PMP70 was labeled with GFP. A green punctate distribution was displayed in the WT and all mutants, but fluorescence in ∆FpPEX11a and ∆FpPEX11b was significantly lower, which indicated that the deletion of FpPEX11a and FpPEX11b led to a decrease in the number of peroxisomes. The fluorescence intensity in ∆FpPEX11c increased, and its volume decreased slightly (Figure 7). This also indicates abnormal biochemical processes, such as active oxygen degradation and fatty acid metabolism, in ∆FpPEX11a and ∆FpPEX11b. In ∆FpPEX11c, the volume of peroxisomes decreased, but their number increased, leading to peroxisomes being supplemented with some functions.

FpPEX11 Regulates Expression of Peroxisome-Proliferator-Associated Gene
While further studying the effect of FpPEX11 on the pathogenicity and functional integrity of F. pseudograminearum at the expression level, we detected the effects of the deletion of FpPEX11a, FpPEX11b, or FpPEX11c on each other's expression levels, peroxisomal proliferation, and β-oxidation-related gene expression levels using quantitative PCR and analyzed their association with the phenotype of each strain.
Through analyzing the FpPEX11 gene expression difference between ∆FpPEX11 and WT, we found that the expression of PEX11b was downregulated and that of PEX11c was upregulated by 2.9 times in ∆FpPEX11a. The expression levels of PEX11a and PEX11c were both significantly upregulated in ∆FpPEX11b. The expression of both PEX11a and PEX11b was significantly downregulated in ∆FpPEX11c (Figure 8).
The number of peroxisomes increases through growth and division of pre-existing peroxisomes [36] or, when required, peroxisome biogenesis is initiated de novo from the ER [37]. PEX3 and PEX19 are key genes involved in the de novo biosynthesis of peroxisomes. PEX3 is responsible for the secretion of peroxisomal membrane proteins from the ER and acts as a docking protein for PEX19 [38,39]. As a membrane protein receptor, PEX19 recognizes and localizes to peroxisomes [40]. The expression of PEX3 and PEX19 was upregulated by 3-to 3.9-fold and 1.1-to 4.3-fold in each mutant when compared to the WT ( Figure 8C), but the number of peroxisomes in ∆FpPEX11a and ∆FpPEX11b was not enhanced (Figure 7). The other pathway for peroxisomal proliferation is the division and proliferation of existing peroxisomes, which occurs through polarized extension and membrane constriction and division. FIS1 is a division factor recruited during division [41], and the expression levels of FIS1 in the mutants were significantly lower than those in the WT ( Figure 8D). DNM1 is a dynamin-related protein involved in membrane constriction that promotes membrane division [42]. The expression of DNM1 was upregulated in each mutant when compared to that in the WT ( Figure 8D). The number of peroxisomes was significantly increased, and the volume was decreased in ∆FpPEX11c (Figure 7). Carnitine acetyltransferase plays an important role in fatty acid degradation. PTH2 is a peroxisome carnitine acetyltransferase that catalyzes the generation of acetyl-CoA from acetyl-CoA and facilitates the transport of acetyl-CoA between organelles. The expression of PTH2 was upregulated in each mutant compared to that in the wild type in each mutant ( Figure 8B). PIP2 is involved in fatty acid degradation. Induced by the fatty acid metabolism signal from the body, PIP2 binds to Adr1 to initiate expression and promote fatty acid degradation [43]. The expression of PIP2 in each mutant was upregulated when compared to that in the wild type ( Figure 8B).

FpPEX11 Is Essential for the Pathogenicity of F. pseudograminearum
To explore the effect of FpPEX11 knockdown on the pathogenicity of F. pseudograminearum in wheat, wheat coleoptiles were inoculated with spore suspensions of each strain. As shown in Figure 9, when compared to WT, the coleoptile infected length for ∆FpPEX11a and ∆FpPEX11b was reduced by 70% and 83.4%, respectively, while ∆FpPEX11c showed no significant difference when compared to the WT. Further inoculation at the blooming stage of wheat in the field was conducted, and spore suspensions of each strain were prepared in advance. After 10 days of investigation, the disease indices of ∆FpPEX11a, ∆FpPEX11b, and ∆FpPEX11c were significantly reduced when compared to those of the WT. These results indicate that FpPEX11a, FpPEX11b, and FpPEX11c are necessary for the pathogenicity of F. pseudograminearum (Figure 9). DON is an important factor in Fusarium pathogenicity. In recent years, F. pseudograminearum has been found to produce DON. We explored whether the altered pathogenicity change of ∆FpPEX11 is related to the abnormal DON synthesis. We tested DON yield, and the results are shown in Table 2. When compared to the WT, the yields of ∆FpPEX11a and ∆FpPEX11b are significantly reduced, and the yield of ∆FpPEX11c is even higher than that of the WT. The yield of DON in the culture medium was quantitatively determined using a beacon DON detection kit. The yields of ∆FpPEX11a and ∆FpPEX11b were 0. The yield of ∆FpPEX11c was 121.43 µg/mg, which was not significantly different from that of the WT (133.78 µg/mg), which is consistent with the trend determined by LC-MS/MS. To further verify this result, we analyzed the expression levels of TRI5, TRI6, and TRI10, which are related to DON synthesis. The results of fluorescence quantitative analysis showed that the expression levels of TRI5, TRI6, and TRI10 in ∆FpPEX11a and ∆FpPEX11b were all downregulated to different degrees, while the expression levels of TRI5 and TRI6 in ∆FpPEX11c were upregulated to different degrees. There was no significant difference between the expression levels of TRI10 and WT (Table 2). In summary, these results indicate that FpPEX11a and FpPEX11b are indispensable for the pathogenicity of F. pseudograminearum and affect the expression and biosynthesis of genes related to DON synthesis. The role of FpPEX11c in DON synthesis requires further investigation.

Discussion
In S. cerevisiae, the PEX11 protein family, consisting of PEX11, PEX25, and PEX27, has been shown to play a major role in peroxisome proliferation [44], and Huber demonstrated that PEX27 plays an inhibitory role in peroxisome proliferation in S. cerevisiae [14]. Deletion of PEX11 results in a smaller number of larger peroxisomes, whereas overexpression of PEX11 results in larger number of smaller peroxisomes. In M. oryzae, one of three PEX11 are required for peroxisomal proliferation [31]. To verify the presence or absence of peroxisome structures in the mutants, we determined the localization in the mycelia of a known peroxisomal membrane protein, PMP70, and the number of peroxisomes was found to be significantly reduced in ∆FpPEX11a and ∆FpPEX11b when compared with the WT (Figure 7). To compensate for the number of peroxisomes, the expression of the PEX3 and PEX19 genes associated with the de novo biosynthesis of peroxisomes was significantly upregulated, and the number and function of peroxisomes in ∆FpPEX11 did not recover well.
Downregulation of expression of the mitogen FIS1 involved in peroxisome proliferation and upregulation of DNM1 expression with PEX11 as the activator protein might be due to the feedback mechanism of the PEX11 protein ( Figure 8). These abnormalities in expression levels of PEX11-related genes also reflect changes in the number of peroxisomes in ∆FpPEX11. In ∆FpPEX11c, the number of peroxisomes increased, and the volume decreased. Expression analysis of FpPEX11 revealed a decreased number of peroxisomes when the expression of PEX11c was higher. It has been speculated that FpPEX11c and PEX27 [14] of yeast also play an inhibitory role in the peroxisome proliferation. FpPEX11a and FpPEX11b are also involved in the regulation of peroxisome proliferation. Therefore, the three PEX11 family members in F. pseudograminearum are involved in the regulation of peroxisome proliferation.
A variety of metabolic reactions occur in peroxisomes; these include fatty acid β-oxidation, the glyoxylic acid cycle, melanin biosynthesis, and glycerol accumulation, which are related to the growth and development of pathogens. Currently, it has been shown that the pathogenicity of many plant pathogens, including M. oryzae, Colletotrichum gloeosporioides, F. graminearum, Ustilaginoidea maydis, and Phaeosphaeria nodorum, has inevitable connection with peroxisomes [45]. In this study, the peroxisome metabolism of the PEX11 knockout was diminished, and the fatty acid β-oxidative metabolism of ∆FpPEX11a and ∆FpPEX11b was distinctly affected. In media containing medium-or long-chain fatty acids, hyphal growth was inhibited ( Figure 5), fatty acids could not be normally used, and supply of the metabolite acetyl-CoA was decreased. Fungal cell walls are mainly composed of chitin, β-1,3-glucan, β-1,6-glucan, and mannoproteins. Some studies have suggested that chitin and glucan in fungal cell walls are derived from acetyl-CoA [46]. Deletion of PEX5, PEX6, and PEX19 were all sensitive to Congo red and calcofluor-white in M. oryzae [47]. In this study, ∆FpPEX11a and ∆FpPEX11b were sensitive to cell wall inhibitor stress and showed impaired cell wall integrity (Figure 6), which reflects the impaired peroxisome function observed in ∆FpPEX11a and ∆FpPEX11b. The number of peroxisomes was significantly increased, and their volumes reduced in ∆FpPEX11c (Figure 7), hence, no significant change in the β-oxidation function of peroxisomes was observed. In S. cerevisiae, acetyl-CoA, a product of β-oxidation, can be transferred to the cytoplasm by carnitine acetyltransferase (CrAT2), or to the mitochondria by CrAT1, where it enters the tricarboxylic acid cycle [48]. The M. oryzae gene PTH2 (encoding CrAT1) plays a major role in acetyl-CoA metastasis. In the PTH2 deletion mutant, melanin was reduced, invasion was abnormal, and pathogenicity was lost [49]. In ∆FpPEX11a and ∆FpPEX11b, PTH2 satisfies the need of peroxisome enzyme metabolism by increasing its expression level (Figure 8). Being stimulated by fatty acid signals, PIP2 was upregulated. Fatty acid degradation was still severely impaired in ∆FpPEX11a and ∆FpPEX11b, especially the degradation of long-chain fatty acids. Therefore, FpPEX11a and FpPEX11b may play a significant role in regulating β-oxidation.
The early defense response of plants to pathogen infection involves the production of ROS to resist pathogen infection, and the ability of the pathogen to remove ROS is essential for successful invasion and colonization despite the host defense. The abundant enzymes in peroxisomes play an important role in regulating active oxygen balance. In yeast, peroxisome defects lead to ROS accumulation [50]. ROS are also produced during the interaction between Alternaria alternata and the host, and the destruction of the host ROS scavenging system leads to the reduction or loss of pathogenicity of A. alternata. [51]. In this study, it was found that the susceptibility of ∆FpPEX11a and ∆FpPEX11b to the oxidant H 2 O 2 was increased, the accumulation of ROS was increased in hyphae, and the removal of ROS was blocked (Figure 4). On the other hand, the abnormal response of F. pseudograminearum to ROS may also be associated with its reduced pathogenicity; ∆FpPEX11c showed normal ability to respond to oxidative stress ( Figure 4). Indoor coleoptile and field wheat inoculation showed that the pathogenicity of ∆FpPEX11a and ∆FpPEX11b significantly decreased (Figure 9), and the yield of DON decreased ( Table 2). Previous studies have shown that fungi can recognize the host environment and induce gene expression for DON biosynthesis during infection, leading to the production of DON. DON has also been detected in tissues inoculated with F. graminearum or F. pseudograminearum in the stalk portion and not inoculated at the apex [52]. Thus, the effects of the deletion of FpPEX11a and FpPEX11b on DON contamination in grains and stalks can be continued. In the laboratory conditions, ∆FpPEX11c had no significant effect on the pathogenicity when compared with the WT; however, in the field, the pathogenicity of ∆FpPEX11c was significantly decreased (Figure 9), which might be due to differences in the environmental factors of the host. The expression levels of TRI5, TRI6, and TRI10, which are related to DON biosynthesis, were significantly downregulated in ∆FpPEX11a and ∆FpPEX11b, while the expression of TRI5 was significantly upregulated in the ∆FpPEX11c, and the yield of DON was not significantly different from that in WT. Therefore, comprehensive knowledge about the function of FpPEX11c requires further study in the future.
In conclusion, our results indicate that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction of F. pseudograminearum, and act through regulation in the number and functions of peroxisomes, such as via fatty acid β-oxidation and active oxygen metabolism. After deletion of FpPEX11a and FpPEX11b, the pathogenicity and yield of DON in F. pseudograminearum was significantly weakened, and disease progress was hindered (Figure 9). After deletion of FpPEX11c, the number of peroxisomes increased, and their volume decreased (Figure 7). Therefore, the three members of the FpPEX11 family, FpPEX11a, FpPEX11b and FpPEX11c, perform their respective roles, and their mutual balance regulates the number of peroxisomes and metabolic activities. Furthermore, we characterized part of the function of the FpPEX11 family in F. pseudograminearum for the first time, which can aid in the understanding of mechanisms underlying pathogenicity of F. pseudograminearum and other filamentous phytopathogens.

Fungal Strain and Culture Medium
The F. pseudograminearum wild-type (WT) strain used in this study was the local isolate Fp3-3, which was reserved at Shandong Agricultural University. Solid potato dextrose agar (PDA; 200 g peeled potato, 20 g dextrose, 15 g agar, and 1000 mL water), complete medium (CM), yeast extract peptone dextrose (YEPD) medium (yeast extract, 10.0 g; peptone, 20.0 g; glucose, 20.0 g; and 1000 mL distilled water), and sporulation media consisting of carboxymethyl cellulose liquid media (CMC) preparations, as well as the culture conditions, were adopted from a previous report [53,54].

Strain Construction
At least 1 kb upstream and downstream fragments for each gene were amplified from genomic DNA, and the hygromycin-phosphotransferase (hph) gene was amplified from the pCB1003 plasmid using the primer pairs zH1-F/R and zH2-F/R and inserted into pCE-Zero to generate vectors pCE-aAH1, pCE-aH2B, pCE-bAH1, pCE-bH2B, pCE-cAH1, and pCE-cH2B. The fusion fragments were transformed into the WT strain to yield the transformants ∆FpPEX11a, ∆FpPEX11b, and ∆FpPEX11c by PEG-mediated protoplast transformation. Potential gene deletion mutants were further confirmed by Southern blot analysis [31].
For the complementation strains, genomic fragments containing full lengths of ORFs and 1.5 kb upstream of FpPEX11a, FpPEX11b, and FpPEX11c were amplified and inserted into pFL2-GRP to generate complementary vectors pFL2-GRP-aDW, pFL2-GRP-bDW, and pFL2-GRP-cDW, which were integrated into mutants. Candidate complementary strains were further determined by PCR.

Vegetative Growth Detection
In order to determine mycelial growth, each strain was cultured on PDA and CM at 25 • C for 3 days, and the colony diameter was measured by the perpendicular cross measurement method [55]. To determine cell wall sensitivity, each strain was cultured on CM containing 0.01% sodium dodecyl sulfate (SDS) and CM containing 0.2% Congo red for 3 days and then measured using the same method.
Utilization of carbon sources was measured in minimal medium (MM) supplemented with various carbon sources instead of sucrose at the following concentrations: 2.5 mM myristic acid (C14), 2.5 mM palmitic acid (C16), 2.5 mM oleic acid (C18), and 2.5 mM erucic acid (C22). Emulsifier NP40 was added to the MM. Colony diameters were measured after three days of incubation at 25 • C [56].
Three 5 mm bacterial cakes were cultured in CMC at a controlled environment of 25 • C and 150 rpm for 3 days to determine the conidia yield and observe the sporulation structure of each strain. Conidia were cultured in YEPD for 3 h, and the germination rate was calculated after microscopic observation.

ROS Detection
The strains were cultured in CM at 25 • C for 4 days to detect ROS by nitro tetrazolium blue chloride (NBT) staining. Each plate was stained with 15 mL of 0.2% (w/v) NBT solution and incubated in the dark at 25 • C for 45 min. The solution was drained, and the plates were washed with anhydrous ethanol. The plates were incubated for 30 min in the dark at 25 • C before imaging [16]. Generation of ROS was visualized by using DHE: mycelia harvested from YEPD cultures and were washed with phosphate-buffered saline (PBS). The samples were stained with DHE solution at 2.5 µg/mL in PBS and incubated in the dark for 5 min. The DHE stained cells were then examined under the same fluorescence filter set used for RFP.

Fluorescence Microscopy
Plasmids containing GFP-FpPMP70 were transformed into WT and mutants by PEG-mediated protoplast transformation to obtain fluorescently labeled strains. The fluorescence intensity of tagged proteins was observed using a Zeiss LSM880 confocal microscope (Gottingen, Niedersachsen, Germany). Image analysis was performed using ImageJ (http://rsb.info.nih.gov/nih-image/) (accessed on 20 February 2022) and Adobe Photoshop software. Peroxisome quantification was performed on 150 µm hyphae, and statistical data analysis was performed using Statistica 8.0.

Relative Quantitative Real-Time PCR
Total RNA was isolated from the hyphae of the WT, mutant, and complemented strains using TransZol Up (TransGen Biotech, Beijing, China). Relative quantification relates the PCR signal of the mutant transcript in a treatment group to that of another sample, such as the WT. For each sample, FP-β-tubulin was used as an internal control [57]. Quantitative results were calculated and analyzed using the 2 −∆∆Ct method [58]. The primers used are listed in the Supplementary Material.

Pathogenicity Test
The inoculum was prepared containing 1 × 10 7 conidia in 1 mL of sterile water, to which Triton 60 was added at a final concentration of 0.01% for better adherence of the inoculum to susceptible wheat Jimai 22 [59]. After inoculation, humidity was maintained with a sealing film for 48 h, and wheat heads were photographed and assayed [56] 10 days post-inoculation (dpi). Wheat coleoptiles were inoculated with inoculum and examined at 5 dpi and 25 • C [60].

Conflicts of Interest:
The authors declare no conflict of interest.