MoNap1, a Nucleosome Assemble Protein 1, Regulates Growth, Development, and Pathogenicity in Magnaporthe oryzae

Nap1 is an evolutionarily conserved protein from yeast to human and is involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle regulation. In this paper, we identified nucleosome assemble protein MoNap1 in Magnaporthe oryzae and investigated its function in pathogenicity. Deletion of MoNAP1 resulted in reduced growth and conidiation, decreased appressorium formation rate, and impaired virulence. MoNap1 affects appressorium turgor and utilization of glycogen and lipid droplets. In addition, MoNap1 is involved in the regulation of cell wall, oxidation, and hyperosmotic stress. The subcellular localization experiments showed that MoNap1 is located in the cytoplasm. MoNap1 interacts with MoNbp2, MoClb3, and MoClb1 in M. oryzae. Moreover, deletion of MoNBP2 and MoCLB3 has no effects on vegetative growth, conidiation, and pathogenicity. Transcriptome analysis reveals that MoNAP1 is involved in regulating pathogenicity, the melanin biosynthetic process. Taken together, our results showed that MoNap1 plays a crucial role in growth, conidiation, and pathogenicity of M. oryzae.


Introduction
Nucleosome assembly protein 1 (Nap1) is an integral component in the establishment, maintenance, and dynamics of eukaryotic chromatin [1]. Nap1 family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes [2]. Nap1 was originally identified in HeLa cells as a histone chaperone [3]. Further, homologs of Nap1 have been identified in multiple organisms, such as Saccharomyces cerevisiae, Drosophila melanogaster, tobacco, and rice [4][5][6]. In S. cerevisiae, deletion of NAP1 leads to alterations in the gene expression of about 10% of the genome [7], but no significant phenotype was observed in NAP1 knockout yeast strains [8]. On the contrary, a NAP1 knockout in D. melanogaster resulted in either embryonic lethality or poorly viable adults [9]. In plants, different plant NAP1-like proteins display distinct functions in vivo, which was supported by their distinct intracellular localization, e.g., both NtNAP1_L3 and NtNAP1_L4 were localized exclusively in the cytoplasm, whereas OsNAP1_L1 was localized in both the cytoplasm and the nucleus [4].
Previous studies indicated that Nap1 is a chromatin-assembly factor and histone chaperone protein [3,10]. Chromatin assembly involves the initial deposition of a tetramer of histones H3 and H4 onto DNA, followed by the addition of H2A-H2B heterodimers to form the histone octamer [11,12]. In this process, Kap114p is required for nuclear import of H2A and H2B, and Nap1p can bind directly Kap114p [13]. In addition, Nap1 has also been implicated in cell-cycle regulation [14], transcription regulation [15], incorporation In S. cerevisiae, Nap1 has been reported to be a nucleocytoplasmic shuttling protein [8]. To determine the subcellular localization of MoNap1 in M. oryzae, the fusion cassette MoNap1-GFP under control of its native promoter was constructed and then co-expressed the MoH2B-RFP in the wild-type (WT) strain. We found MoNap1 was mostly located in cytoplasm in conidium, appressorium, vegetative mycelia, and infection mycelia ( Figure  2A,B). This result suggested that MoNap1 was localized in cytoplasm during each growth and development stage of M. oryzae. In S. cerevisiae, Nap1 has been reported to be a nucleocytoplasmic shuttling protein [8]. To determine the subcellular localization of MoNap1 in M. oryzae, the fusion cassette MoNap1-GFP under control of its native promoter was constructed and then co-expressed the MoH 2 B-RFP in the wild-type (WT) strain. We found MoNap1 was mostly located in cytoplasm in conidium, appressorium, vegetative mycelia, and infection mycelia (Figure 2A,B). This result suggested that MoNap1 was localized in cytoplasm during each growth and development stage of M. oryzae. i 2023, 9,50

Targeted Gene Deletion and Complementation of MoNAP1
To determine the roles of MoNAP1 in growth development and pathogeni oryzae, we generated the targeted gene deletion mutant of MoNAP1 in the wild-ty Guy11 background using a homologous recombination strategy ( Figure S1A). F tive transformants of the MoNAP1 deletion were identified by polymerase chain (PCR) using primer pairs (Table S1) ( Figure S1B). These transformants were fur firmed by Southern blot assay and reverse transcription polymerase chain reac PCR) assay ( Figures S1C,D). Corresponding results obtained from these assays that MoNAP1 was successfully replaced with a hygromycin-resistance cassette generate MoNAP1 deletion ΔMonap1 (#1, #3, #4, and #7). In addition, we also per

Targeted Gene Deletion and Complementation of MoNAP1
To determine the roles of MoNAP1 in growth development and pathogenicity of M. oryzae, we generated the targeted gene deletion mutant of MoNAP1 in the wild-type strain Guy11 background using a homologous recombination strategy ( Figure S1A). Four putative transformants of the MoNAP1 deletion were identified by polymerase chain reaction (PCR) using primer pairs (Table S1 and Figure S1B). These transformants were further confirmed by Southern blot assay and reverse transcription polymerase chain reaction (RT-PCR) assay ( Figure S1C,D). Corresponding results obtained from these assays showed that MoNAP1 was successfully replaced with a hygromycin-resistance cassette (HPH) to generate MoNAP1 deletion ∆Monap1 (#1, #3, #4, and #7). In addition, we also performed a complementation experiment to generate the complementation strains ∆Monap1/MoNAP1 by introducing the native promoter-driven MoNAP1 coding region into ∆Monap1 mutant (#1). The complementation strains ∆Monap1/MoNAP1 was verified by RT-PCR ( Figure S1D) and recovered to WT in basic phenotypic. The ∆Monap1 mutant (#1) and ∆Monap1/MoNAP1 were used for assessment in some phenotypes.

MoNAP1 Is Involved in Vegetative Growth, Melanin Biosynthesis, and Conidia Production
To analyze the roles of MoNAP1 in vegetative growth, we observed the colony growth and mycelial morphology of the ∆Monap1 mutant. Compared with WT and the ∆Monap1/MoNAP1, we found a significant growth reduction and fluffier aerial mycelia in the ∆Monap1 mutant ( Figure 3A-C). Meanwhile, we noticed that the ∆Monap1 mutant colony showed less pigment and whiter hyphae compared with WT strain and ∆Monap1/MoNAP1 after culturing in CM medium for seven days ( Figure 3A). This result prompted us to speculate that MoNAP1 could affect the expression of pigmentation biosynthesis-related genes. As expected, qRT-PCR analysis revealed that ALB1 and BUF1 were down-regulated in the mutant ∆Monap1 in contrast to the WT and ∆Monap1/MoNap1 strains ( Figure 3D).

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ΔMonap1/MoNAP1, we found a significant growth reduction and fluffier aerial mycelia in the ΔMonap1 mutant ( Figure 3A-C). Meanwhile, we noticed that the ΔMonap1 mutan colony showed less pigment and whiter hyphae compared with WT strain and ΔMonap1/MoNAP1 after culturing in CM medium for seven days ( Figure 3A). This resul prompted us to speculate that MoNAP1 could affect the expression of pigmentation bio synthesis-related genes. As expected, qRT-PCR analysis revealed that ALB1 and BUF1 were down-regulated in the mutant ΔMonap1 in contrast to the WT and ΔMonap1/MoNap1 strains ( Figure 3D).  To determine whether MoNAP1 plays role in asexual development in M. oryzae, the WT strain Guy11, ∆Monap1, and ∆Monap1/MoNAP1 were cultured on RDC medium for 8 days, and then, the number of conidia was counted. We found that conidiation of ∆Monap1 mutant was drastically reduced compared with the WT strain Guy11 and the complementation strain ∆Monap1/MoNAP1 ( Figure 3F). To validate this result, we further revealed that MoNAP1 deletion affected the development of conidiophore. We noticed that the number of conidia per conidiophore produced was significantly reduced in the ∆Monap1 mutant ( Figure 3E). These results strongly indicated that the loss of MoNAP1 causes slower vegetative growth, thicker aerial hyphae, decreased melanin formation, and reduced conidiation.

MoNAP1 Is Required for M. oryzae Pathogenicity
To evaluate the role of MoNAP1 in the pathogenicity of M. oryzae, we conducted pathogenicity assays on different plant hosts. The WT Guy11 and the complementation strain ∆Monap1/MoNAP1 caused severe lesions, while the ∆Monap1 mutant produced smaller lesions after inoculated on isolated intact and wounded barley leaves with mycelial agar plugs for 5 days ( Figure 4A). After inoculated on detached intact and wounded barley leaves with 1 × 10 5 conidia ml −1 conidial suspensions of Guy11, ∆Monap1, and ∆Monap1/MoNAP1 for 5 days, the ∆Monap1 mutant also resulted in smaller disease lesions compared to Guy11 and ∆Monap1/MoNAP1 ( Figure 4B). In addition, conidia suspensions (1 × 10 5 conidia ml −1 ) of tested strains were sprayed onto 14-day-old rice seedlings (Co39), and at 5 days post incubation (dpi), only a few very small lesions were observed on rice leaves infected with the ∆Monap1 mutant. Instead, more necrotic lesions were produced by Guy11 and ∆Monap1/MoNAP1 ( Figure 4C). These results suggested that MoNAP1 is essential for M. oryzae pathogenicity.
To explore the reasons for pathogenicity attenuation in the ∆Monap1 mutant, appressoriummediated penetration assay was performed using conidia of all tested strains on detached barley leaves, and expansion ability of the infected hyphae was observed in barley epidermis cell. We found that approximately 70% IH displayed type III, 20% showed type II, and 10% showed type I at 24 hpi in WT and complemented strains ∆Monap1/MoNAP1 ( Figure 4D,E). However, in the ∆Monap1 mutant, less 20% IH showed type III, 40% showed type II, and 20% showed type I. These results suggested that MoNAP1 is involved in infectious growth ( Figure 4D,E). Taken together, these results suggested that MoNAP1 is required for pathogenicity, and reduced invasive hyphae extension ability of ∆Monap1 mutant strain may be the reason for the significant reduction in pathogenicity of M. oryzae.

∆Monap1 Results in Accumulation of Reactive Oxygen Species (ROS) Produced by Host Cells
As mentioned in the above results, most infected hyphae of ∆Monap1 had more difficulty in colonizing adjacent cells ( Figure 4D,E). Therefore, we hypothesized that the ability to scavenge host reactive oxygen species was prevented in ∆Monap1. A 3,3diaminobenzidine (DAB) staining assay of the penetrated plant cells was performed. As shown in Figure 4F,G, approximately 70% of host cells penetrated by ∆Monap1 mutant possessed ROS accumulation with reddish-brown color, while only about 30% of host cells infected with WT strain were stained by DAB, which suggested that the ability of ∆Monap1 to eliminate host-produced ROS was decreased. Our result provided a demonstration that MoNAP1 is involved in suppressing host oxidative burst. ΔMonap1/MoNAP1 for 5 days, the ΔMonap1 mutant also resulted in smaller disease lesions compared to Guy11 and ΔMonap1/MoNAP1 ( Figure 4B). In addition, conidia suspensions (1 × 10 5 conidia ml −1 ) of tested strains were sprayed onto 14-day-old rice seedlings (Co39), and at 5 days post incubation (dpi), only a few very small lesions were observed on rice leaves infected with the ΔMonap1 mutant. Instead, more necrotic lesions were produced by Guy11 and ΔMonap1/MoNAP1 ( Figure 4C). These results suggested that MoNAP1 is essential for M. oryzae pathogenicity.  (G) Statistical analysis for proportion of the infected cells stained by DAB. For each strain, at least 100 invaded cells were observed, and the numbers of stained cells were counted. Error bars represent standard deviation, and asterisks above the columns indicate significant differences between WT and ∆Monap1 estimated by Student's t-test (** p < 0.01).

MoNAP1 Is Required for Appressorium Formation and Utilization of Glycogen and Lipid Droplets
The appressorium-mediated penetration is key for M. oryzae pathogenicity. To determine whether the MoNAP1 was involved in the appressorium formation, we performed appressorium assay on hydrophobic surfaces. The appressorium formation rates of the wild-type strain Guy11, the ∆Monap1 mutant, and the complementation strain ∆Monap1/MoNAP1 were observed on the hydrophobic surfaces after 4, 6, 8, 12, and 24 hpi. We found that the appressorium formation rate was significantly delayed in the ∆monap1 mutant after 4, 6, 8, and 12 hpi compared with Guy11 and the complementation strain ∆Monap1/MoNAP1, while this delay became indistinguishable after 24 h ( Figure 5A,B). For each strain, 100 conidia and appresso were observed, and the numbers of stained conidia and appressoria were counted. Error bars r resent standard deviation, and asterisks above the columns indicate significant differences betwe WT and ΔMonap1 estimated by Student's t-test (** p < 0.01).
The formation of appressorium is accompanied by degradation of glycogen and lip droplets. Considering that the appressorium formation rate of ΔMonap1 was decreas we supposed that the translocation and degradation of nutriments were also delay Hence, we observed the cellular distribution of glycogen and lipid droplets at 0, 2, 8, and 24 h, while glycogen and lipid droplets were stained with I2/KI and Nile red soluti respectively. In WT, the glycogen was rapidly consumed after 16 h and disappeared at h, while in ΔMonap1 mutant, the glycogen was utilized more slowly and was still notic at 24 h ( Figure 5C-E). Similarly, at 16 hpi, more than 80% of the ΔMonap1 conidia co tained lipid droplets; however, only about 40% of Guy11 conidia contained lipid drople Even at 24 hpi, lipid droplets could be observed in approximately 70% of the ΔMona For each strain, 100 conidia and appressoria were observed, and the numbers of stained conidia and appressoria were counted. Error bars represent standard deviation, and asterisks above the columns indicate significant differences between WT and ∆Monap1 estimated by Student's t-test (** p < 0.01).
The formation of appressorium is accompanied by degradation of glycogen and lipid droplets. Considering that the appressorium formation rate of ∆Monap1 was decreased, we supposed that the translocation and degradation of nutriments were also delayed. Hence, we observed the cellular distribution of glycogen and lipid droplets at 0, 2, 8, 16, and 24 h, while glycogen and lipid droplets were stained with I 2 /KI and Nile red solution, respectively. In WT, the glycogen was rapidly consumed after 16 h and disappeared at 24 h, while in ∆Monap1 mutant, the glycogen was utilized more slowly and was still noticed at 24 h ( Figure 5C-E). Similarly, at 16 hpi, more than 80% of the ∆Monap1 conidia contained lipid droplets; however, only about 40% of Guy11 conidia contained lipid droplets. Even at 24 hpi, lipid droplets could be observed in approximately 70% of the ∆Monap1 appressoria, whereas only 15% of the Guy11 appressoria contained lipid droplets ( Figure 5F,G). These results indicated that the deletion of MoNAP1 affects appressorium formation and metabolism of nutrients, which is important for sufficient turgor pressure for host penetration.

MoNAP1 Is Involved in Responses to Multiple Stressors
In eukaryotes, a normal osmotic environment is essential for fungal growth and development. To determine the role of MoNAP1 in hyperosmotic stress response, we tested the sensitivities of ∆Monap1 mutant to different osmotic stress agents. The tested strains were cultured on CM plates containing 1 M sorbitol, 0.7 M NaCl, and 0.6 M KCl for 7 days. As shown in Figure 6A,B, ∆Monap1 was more sensitive to all above-mentioned osmotic stresses compared with WT and ∆Monap1/MoNAP1, which indicates that MoNAP1 plays an important role in responding to different osmotic stresses. In addition, the production of reactive oxygen species (ROS) is one of the most important defense responses in plants. According to the Figure 4F,G, we speculated that MoNAP1 affects the sensitivity in responses to oxidative stresses. To confirm this, the relative inhibition rates of WT, ∆Monap1, and ∆Monap1/MoNAP1 on CM with 5 mM and 10 mM H 2 O 2 were examined. As expected, ∆Monap1 was more sensitive to H 2 O 2 and showed a more significant inhibition rate ( Figure 6C,D). The cell wall integrity signaling pathway is important for blast fungus to resist external environmental stress. To test the sensitivity of ∆Monap1 in response to cell wall integrity stress, WT, ∆Monap1, and ∆Monap1/MoNAP1 were inoculated on CM plates supplemented with 200 µg/mL calcofluor white (CFW), 600 µg/mL Congo red (CR), and 0.004% sodium dodecyl sulfate (SDS). Figure 6E,F shows that ∆Monap1 was more sensitive than the wild type and ∆Monap1/MoNAP1 to CFW and CR but more resistant to SDS, indicating that the MoNAP1 is involved in regulating the cell wall integrity pathway. Taken together, our results obtained from these assays suggested that the deletion of MoNAP1 results in increased sensitivity to different stresses.

MoNap1 Interacts with Cell-Cycle-Related Proteins in M. oryzae
The Nap1 protein interacts with Nbp2 [25], Clb3 [8], Clb1 [8], Cdc28, Gin4 [21], Ats1 [32], and Cyc1 [8] Figure S3 indicate that MoNap1 interacts with MoNbp2, MoClb1, and MoClb3, respectively, in vivo. To further explore the connection between MoNap1 and its partner proteins, ∆Monbp2 and ∆Moclb3 mutant strains were generated ( Figure S4). The deletion strain of MoCLB1 was unsuccessful even when more than 300 knock-out transformants were examined, which suggests that MoCLB1 is essential for the survival of M. oryzae. The pathogenicity of these mutant strains was analyzed, and results showed that the deletion of MoNBP2 or MoCLB3 has no effect on the pathogenicity of M. oryzae ( Figure S4). and 0.004% sodium dodecyl sulfate (SDS). Figure 6E,F shows that ΔMonap1 was sensitive than the wild type and ΔMonap1/MoNAP1 to CFW and CR but more resista SDS, indicating that the MoNAP1 is involved in regulating the cell wall integrity path Taken together, our results obtained from these assays suggested that the deletio MoNAP1 results in increased sensitivity to different stresses.  explore the connection between MoNap1 and its partner proteins, ΔMonbp2 and ΔMoclb3 mutant strains were generated ( Figure S4). The deletion strain of MoCLB1 was unsuccessful even when more than 300 knock-out transformants were examined, which suggests that MoCLB1 is essential for the survival of M. oryzae. The pathogenicity of these mutant strains was analyzed, and results showed that the deletion of MoNBP2 or MoCLB3 has no effect on the pathogenicity of M. oryzae ( Figure S4). Previous studies reported that deletion of NAP1 results in the unnormal function of B-type cyclin Clb2 and causes prolonged mitotic delays in S. cerevisiae [8,14]. To investigate if NAP1 affects cell cycle progression in M. oryzae, we transferred H1-RFP plasmid into WT and ΔMonap1 mutant and then observed the localization of H1 during the vegetative hyphae stage. As shown in Figure 8A,B, there was only one nucleus in most WT cells but two or more cell nucleuses in more than 30% of the ΔMonap1 cells. Our result showed that loss of MoNAP1 significantly causes a disordered cell cycle in M. oryzae.  Previous studies reported that deletion of NAP1 results in the unnormal function of B-type cyclin Clb2 and causes prolonged mitotic delays in S. cerevisiae [8,14]. To investigate if NAP1 affects cell cycle progression in M. oryzae, we transferred H1-RFP plasmid into WT and ∆Monap1 mutant and then observed the localization of H1 during the vegetative hyphae stage. As shown in Figure 8A,B, there was only one nucleus in most WT cells but two or more cell nucleuses in more than 30% of the ∆Monap1 cells. Our result showed that loss of MoNAP1 significantly causes a disordered cell cycle in M. oryzae. explore the connection between MoNap1 and its partner proteins, ΔMonbp2 and ΔMoclb3 mutant strains were generated ( Figure S4). The deletion strain of MoCLB1 was unsuccessful even when more than 300 knock-out transformants were examined, which suggests that MoCLB1 is essential for the survival of M. oryzae. The pathogenicity of these mutant strains was analyzed, and results showed that the deletion of MoNBP2 or MoCLB3 has no effect on the pathogenicity of M. oryzae ( Figure S4). Previous studies reported that deletion of NAP1 results in the unnormal function of B-type cyclin Clb2 and causes prolonged mitotic delays in S. cerevisiae [8,14]. To investigate if NAP1 affects cell cycle progression in M. oryzae, we transferred H1-RFP plasmid into WT and ΔMonap1 mutant and then observed the localization of H1 during the vegetative hyphae stage. As shown in Figure 8A,B, there was only one nucleus in most WT cells but two or more cell nucleuses in more than 30% of the ΔMonap1 cells. Our result showed that loss of MoNAP1 significantly causes a disordered cell cycle in M. oryzae.  (B) Statistical analysis for proportion of the abnormal quantity of nucleus in a single cell. For each strain, at least 100 vegetative hypha cells were observed and counted. Error bars represent standard deviation, and asterisks above the columns indicate significant differences between WT and ∆Monap1 estimated by Student's t-test (** p < 0.01).

Transcriptional Analysis for Identification of Differentially Expressed Genes (DEGs) in a Comparison of the ∆Monap1 and Guy11
To identify the potential target genes regulated by MoNAP1, we performed transcriptome analysis between Guy11 and ∆Monap1 mycelia. A total of 1783 DEGs were identified between Guy11 and the ∆Monap1 mutant using the standard criterion of |log 2 FoldChange|values > 1 and Padj < 0.05, which include 1121 down-regulated and 662 up-regulated genes ( Figure 9A,B). Gene ontology (GO) enrichment analysis of the genes up-regulated and down-regulated showed that the biological processes of transmembrane transport, carbohydrate metabolic process, pathogenesis, secondary metabolite biosynthetic process, and melanin biosynthetic process were enriched. For cellular components, integral component of membrane, extracellular region, plasma membrane, integral component of plasma membrane, mitotic spindle, and spindle were enriched ( Figures 9C,D and S5). We also selected two pathogenesisrelated genes to perform qRT-PCR to further identify the authenticity of RNA-seq results, and the results in Figure S6 suggest that the expression level of MoZFP6 and MoDIT2 were severely decreased compared to WT (Table S2). These results obtained from GO enrichment analysis further confirmed that MoNAP1 is involved in utilization of glycogen, in the cell cycle, and in response to different stresses. ponent of plasma membrane, mitotic spindle, and spindle were enriched ( Figures 9C,D  and S5). We also selected two pathogenesis-related genes to perform qRT-PCR to further identify the authenticity of RNA-seq results, and the results in Figure S6 suggest that the expression level of MoZFP6 and MoDIT2 were severely decreased compared to WT (Table  S2). These results obtained from GO enrichment analysis further confirmed that MoNAP1 is involved in utilization of glycogen, in the cell cycle, and in response to different stresses.  In addition, a total of 1783 genes were mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the top 30 significantly enriched (Padj < 0.05) pathways are shown in Figure S7, which include amino acid metabolism (beta-alanine metabolism, tryptophan metabolism, phenylalanine metabolism, glutathione metabolism, and tyrosine metabolism), lipid metabolism (fatty acid degradation), betalain biosynthesis, and metabolism of xenobiotics by cytochrome P450. The KEGG pathway enrichment analysis indicated that MoNAP1 is important for amino acid metabolism and lipid metabolism in M. oryzae.

Discussion
Nucleosome assembly protein 1 (Nap1) plays important roles in diverse biological processes [2]. However, the biological functions of MoNAP1 in filamentous fungus have rarely been reported. In this study, we identified the nucleosome assembly protein MoNap1 in M. oryzae. We found MoNap1 is essential for vegetative growth, asexual reproduction, appressorium formation, and pathogenicity in M. oryzae. Moreover, we identified and verified three proteins that interact with MoNap1. Additionally, transcriptome data suggested important roles of MoNAP1 in pathogenicity and pigmentation biosynthesis-related pathways.
In the yeast S. cerevisiae, deletion of NAP1 causes no phenotypes or very limited phenotypes [8,22]. Our results showed that deletion of MoNAP1 reduced the growth rate of colony and spore quantity in M. oryzae. The colonies of the mutant appear whiter than the WT and complemented strains. The expression levels of MoALB1 and MoBUF1 that regulate the formation of melanin were tested. The result indicated that MoNAP1 is involved in the pathway related to melanin formation. Further experiments showed that the deletion of MoNAP1 caused a significant reduction in pathogenicity on different plants and restricted the extension of invasive hyphae in plant cells.
Considering that infection sites of the ∆Monap1 mutant are much fewer than in Guy11 and ∆Monap1/MoNAP1, we speculated that the appressorium formation rate of ∆Monap1 was significantly reduced compared to Guy11 and ∆Monap1/MoNAP1. As expected, the appressorium formation rate of ∆Monap1 was approximately 84% at 8 h, which confirmed our hypothesis. In addition, rapid degradation of glycogen and lipid droplets occurred during conidia germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. According to the above results, we confirmed the slower degradation of glycogen lipid droplets. As shown in our results, the degradation of nutriments was delayed in ∆Monap1 mutant compared to WT and complemented strains, which confirmed our hypothesis.
Stress-signaling pathways are evolutionarily conserved and play an important role in the maintenance of homeostasis. These pathways are also critical for adaptation to new cellular environments [33]. The cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis [34]. Here, we explored the roles of MoNAP1 in response to cell-wall-integrity stress. As result, the ∆Monap1 mutant is more sensitive than WT and complemented strains in CFW and CR but less in SDS, which suggests MoNAP1 is involved in cell wall integrity. The ability of cells to adapt to hyperosmotic stress involves early responses in which ions move across cell membranes and late responses characterized by increased synthesis of either membrane transporters or enzymes involved in their synthesis. The goal of these responses is to return the cell to its normal size and maintain cellular homeostasis [35]. To test the sensitivity of ∆Monap1 to hyperosmotic stress, ∆Monap1, Guy11, and ∆Monap1/MoNAP1 were treated with hyperosmotic stress factors. Our results showed that ∆Monap1 is more sensitive to these factors, which indicated the importance of the adaptation of MoNAP1 to hypertonic stresses in M. oryzae. During plant infection, both pathogenic and beneficial fungi experience a host-derived oxidative burst of reactive oxygen species (ROS) [36]. This burst is part of the plant's innate immune response and must be subdued in order to avoid triggering more robust plant defenses. Besides overcoming plant-derived ROS, fungi must also overcome ROS produced as a byproduct of aerobic respiration [37]. Given that, we detected the sensitivity of ∆Monap1 to H 2 O 2 , and our data showed that the ∆Monap1 mutant was more sensitive to H 2 O 2 . From these findings, we concluded that MoNAP1 plays an indispensable role in regulating oxidative stress.
Nap1 is generally localized in the cytoplasm except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development [6]. In the yeast Saccharomyces cerevisiae, yNap1 is also a nucleocytoplasmic shuttling protein [38]. Here, we verified that MoNap1 was located in cytoplasm but not nucleus in both conidia, appressorium, vegetative mycelia, and infection mycelia. In yeast, deletion of the nuclear export sequence (NES) makes yNap1 locate to the cell nucleus [38]. However, when NES was deleted in MoNAP1 of M. oryzae, the localization of MoNap1 appears to be impervious [39]. The reason for this contradiction remains to be investigated.
Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid assay [25]. Nbp2 regulates mitotic cell growth at high temperature and the cell-wall-integrity pathway [25]. Furthermore, the interaction between B-type cyclins and Nap1 proteins is highly conserved during evolution [8]. In this study, we identified that MoNap1 also interacts with MoNbp2, MoClb1, and MoClb3 in M. oryzae. Deletion of MoNbp2 and MoClb1 did not affect the pathogenicity in M. oryzae. In addition, our failure to obtain an MoCLB3 deletion mutant implies that MoCLB3 is likely to be an essential gene. Furthermore, transcriptome data suggested that deletion of MoNAP1 significantly reduced the expression level of many pathogenesis-related and melanization-related genes.
In conclusion, our study provides new proof that MoNAP1 is required for growth, asexual reproduction, and pathogenicity, and MoNAP1 regulates the appressorium formation rate, melanization, and cell cycle. Meanwhile, we identified three MoNap1-interacting proteins, which may be of benefit in further exploring the function of MoNAP1 in M. oryzae.

Strains and Culture Conditions
The M. oryzae WT strain Guy11 and all strains obtained in this study were cultured on complete medium (CM) at 28 • C. To extract DNA, RNA, and protein, mycelia of all tested strain were inoculated in liquid CM medium for 48 h. For growth assay, equal-sized plugs of each strain tested were cultivated on CM agar at 28 • C for 7 days, and diameter of colony was measured. For conidiation, conidia from colonies cultured on RDC medium at 28 • C for 5 days in dark followed by 3 days of continuous illumination under fluorescent light were harvested for experiment. For appressorium formation assay, a suspension of conidia of all tested strains was cultured on hydrophobic surface to induce formation of appressorium at 28 • C in dark. The rate of appressorium formation was counted at 4 h, 6 h, 8 h, 12 h, and 24 h, respectively, and the images were obtained with inverted fluorescence microscopy (Nikon, Tokyo, Japan).
For sensitivity assays, strains were inoculated on CM plates supplemented with different stress agents (1 M sorbitol, 0.7 M NaCl, 0.6 M KCL, 200 µg/mL CFW, 600 µg/mL Congo red, 0.004% SDS, and 10 mM H 2 O 2 ) at 28 • C in dark. The colony diameters were measured after 7 days post inoculation (dpi). All treatments were performed in three independent biological experiments with three replicates. The significance analysis was performed using t-test formula in Excel software.

Plasmid Constructs and Genetic Transformation
To generate ∆Monap1 deletion mutants, a high-throughput target-gene deletion system was used to construct gene deletion vector [40]. The 1.2 kb upstream fragment (UF) and 1.2 kb downstream fragment (DF) of the targeted ORF were, respectively, amplified from the M. oryzae genome using primers pairs listed in Table S1. The hygromycin-resistance cassette (HPH) sequence was amplified from the plasmid pFGL821 using pairs listed in Table S1. All of these fragments (UF, DF, and HPH) were cloned into vector pKO1B with the Xba I/Hind III-linearized by one-step cloning kit (Vazyme Biotech Co. C113, Nanjing, China). After PCR and sequencing verification, the correct plasmid was transferred into M. oryzae WT strain Guy11 through Agrobacterium tumefaciens-mediated transformation (ATMT). The transformants were screened using CM medium containing 250 µg/mL hygromycin B and further verified by PCR and RT-PCR using pairs listed in Table S1. The positive transformants were confirmed by Southern blot analysis. Four independent mutants with similar phenotype were obtained, and one of all mutants was used for further analysis.
For complementation of the mutants ∆Monap1 and subcellular localization of MoNap1, the complement fragments containing the native promoter and ORF without stop codon were amplified by PCR using pairs listed in Table S1 and inserted into pYF11 as previously described [41]. The construct was transformed into deletion mutant and WT through protoplast transformation, respectively. The transformants were screened using CM medium containing 60 µg/mL bleomycin and further verified by RT-PCR using pairs listed in Table S1. To generate a nucleus marker construct MoH2B-mCherry, fragments including the MoH2B without stop codon and its native promoter were amplified from Guy11 genomic DNA by PCR using pairs listed in Table S1, and the mCherry fragment was amplified using pairs listed in Table S1. All of these fragments (MoH2B and mCherry) were cloned into vector pKNT with the Xho I/BamH I-linearized by Clonexpress Multis One-Step Cloning Kit (Vazyme Biotech Co. C113, China). After PCR and sequencing verification, the correct plasmid MoH2B-mCherry was transferred into was transformed into strain-expressed MoNAP1-GFP in background using PEG-mediated method. The transformants were screened using CM medium containing 200 mg/mL G418 or confirmed by the presence of mCherry signals.

Pathogenicity and Infection Penetration Assays
To determine the virulence of all tested strains, we performed pathogenicity and infection assays on 7-day-old barley (Golden Promise) and 14-day-old rice seedlings (CO39). First, mycelial blocks or spore suspensions of all tested strains were inoculated onto intact and injured barley leaves and kept in the dark incubator for 24 h at 28 • C with 90% humidity, followed by 12 h light:12 h dark photoperiod for 5 days. Then, the disease spots were observed and imaged. In addition, spore suspension (5 × 10 4 conidia/mL) collected from all tested strains were sprayed onto 14-day-old rice seedlings (CO39). The disease spots were observed and imaged after 5 days. Furthermore, for infection penetration, 10 µL spore suspension per drop was inoculated onto the back of the barley and then cultured at 28 • C for 24 h under dark conditions. After that, the barley epidermis was observed by using inverted fluorescence microscope (Nikon, Japan). Each assay was repeated three times.

Nucleic Acid Manipulation, Sothern Blot Analysis, and RT-PCR and Real-Time qRT-PCR Analysis
Genomic DNA of all tested strains were extracted from the mycelia following the SDS-CTAB method. The resulting genomic DNA was used for PCR or Sothern blot analysis. Sothern blot assay was performed as in the previous study [41]. The flank sequence of target gene was used as the specific probe, which was labelled with the DIG High Prime. Hybridization and detection were performed according to manufacturer's instructions (Roche Applied Science, Penzberg, Germany). Total RNA was isolated from fresh mycelia using RNeasy Mini Kit (Qiagen, 74104, Hilden, Germany). Then, 5 mg of total RNA of each sample returned was reverse-transcribed to cDNA using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China). RT-PCR was performed to confirm the deletion and the complementation of targeted gene with gene-specific primers (Table S1). qRT-PCR was performed with 2×RealStar Green Fast Mixture (Genstar, San Francisco, CA, USA) using CFX Connect Real-Time PCR Detection System. The ACTIN gene (MGG_03982) was used as an endogenous reference. The experiment was repeated three times with three replicates each time. All primers used in these assays are listed in Table S1.

Glycogen and Lipid Droplet Straining
Glycogen and lipid droplet staining was observed at 0 h, 2h, 8 h, 16 h, and 24 h using KI/I 2 solution and Nile red solution, respectively. A laser scanning confocal microscope (Nikon, Tokyo, Japan) was used to image red fluorescence.

Subcellular Localization
The transformant co-expressed MoH2B-mCherry and MoNap1-GFP was used to observed subcellular localization at different stages using a Nikon TIE system (Nikon, Japan). The assay was repeated three times.

Transcriptome Analysis
The wild-type strain Guy11 and ∆Monap1 mutant were incubated in CM liquid medium at 28 • C for 48 h. Three replicates were performed for each strain. The mycelium pellets were collected and snap-frozen with liquid nitrogen. Total RNA from the wild-type strain Guy11 and ∆Monap1 mutant was extracted using Qiagen RNAeasy Mini kit (Qiagen, 74104) according to the manufacture's protocol. The integrity of RNA was evaluated using the RNA nano 6000 assay kit (Agilent Technologies, Santa Clara, CA, USA) of Agilent 2100 Bioanalyzer. The RNA with poly-A was enriched by TIANSeq mRNA Capture Kit (TIAN-GEN). Then, using the captured RNA as the starting sample, TIANSeq Fast RNA Library Kit (Illumina, San Diego, CA, USA) was used to construct the transcriptome sequencing libraries, and sequencing was performed on an Illumina Novaseq platform by (Beijing Tangtang Tianxia Biotechnology Co., Ltd., Beijing, China).
Differentially expressed genes were analyzed by the DESeq2 package, and expression with log2|FC| > 1 with <0.05 padj values was defined as DEGs [42]. Ontology enrichment analysis was performed using the topGO R package, and p-adjust values < 0.05 were considered significantly enriched by DEGs [43]. KEGG enrichment analysis was performed using clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways [44].