In this study, the wild-type strain Guy11 of rice blast fungus was used to investigate the functions of the MoNsr1. Phenotypic analysis of vegetative growth, chemical sensitivity, and asexual sporulation of rice blast fungus mutants was performed in a complete medium (CM) (Talbot et al., 1993). After the rice blast fungus was inoculated on CM plates and grown at 28 °C with 12-hour light-dark alternation for 9 days, conidia were obtained by washing the colonies with 10 ml sterile water and filtering them through 4 layers of sterile lens paper. After the rice blast fungus was inoculated into liquid CM medium and cultured in the dark at 28°C for 3 days, the culture was collected, and genomic DNA was recovered using EZNA^® Fungal DNA Mini Kit (Omega Bio-tek, Inc., Norcross, GA).

In order to investigate the subcellular localization of the MoNsr1 protein, an integrated expression plasmid for MoNsr1 and GFP was constructed using plasmid p1300BAR as the basic skeleton (Li et al., 2014). The whole MoNsr1 gene fragment (containing the 1.5kb promoter and MoNsr1 without a stop codon), as well as the GFP gene fragment, will be inserted between the EcoRI and XbaI restriction enzyme cleavage sites of plasmid p1300BAR. After the obtained plasmid pMoNsr1-GFP was sequenced and confirmed to be correct, the wild-type strain was introduced using the Agrobacterium tumefaciens-mediated transformation (AtMT) method described by (Rho et al., 2001). Green fluorescent transformants were obtained for subcellular localization investigation of MoNsr1 using CM plate screening containing 200 μg/ml glufosinate ammonium and fluorescence microscopy (ZEISS Imager. A2). The nuclear localization of rice blast fungus was performed using the DAPI staining method (Odenbach et al., 2009), and the subcellular localization of MoNsr1 was observed and recorded under a laser confocal microscope (ZEISS LSM780).

MoNsr1 gene-deletion strains were created according to the principle of homologous recombination. In short, the upstream and downstream fragments of the MoNsr1 gene were first amplified using a wild-type genomic DNA template (approximately 1.0 kb each) and cloned into the plasmid p1300-KO (Li et al., 2014), respectively, between the restriction enzyme cleavage sites EcoRI and SmaI, and between BamHI and XbaI. After obtaining the plasmid pKO-Nsr1 and confirming its correctness through sequencing, it was used for the AtMT transformation of wild-type strains. Gene deletion mutants were obtained through CM plate screening and PCR validation containing 200 μg/ml hygromycin B. The primers used for PCR validation are shown in Supplementary Table S1 . The MoNsr1 gene, to obtain a complementary mutant, was amplified using the wild-type genomic DNA as a template, including the promoter region (located 1.5 kb upstream of the start codon), and cloned between the EcoRI and XbaI restriction endonuclease sites of plasmid p1300BAR. The obtained complement plasmid was introduced into a MoNsr1 gene deletion mutant, and the complemented mutant was obtained through CM plate screening and PCR validation containing 200 μg/ml glufosinate-ammonium.

Referring to previous method (Liu et al., 2016a), pathogenicity testing was conducted on the susceptible rice (cv. CO-39) seedlings and 7-day-old barley (cv. ZJ-8) plants at 3-4 true leaf stage. The rice plants were inoculated with 0.25% gelatin suspension with a concentration of 1 × 10⁵ conidia/ml by spray. After inoculation, the rice plants were moved into a dark room with a humidity of 25 °C and above 95% for dark cultivation for 24 hours and then continued to grow alternately in light and dark for 16/8 hours at the same temperature until the wild-type inoculated plants showed typical symptoms. The disease severity of the leaves of inoculated rice plants was investigated and recorded to determine the pathogenicity of all tested strains. For the detached barley leaf inoculation assay, 20 µL of gradient diluted conidia suspension was spot inoculated on the barley leaves. The leaves were cultured in the dark at 25°C for 24 h and then illuminated until the wild-type inoculation treatment showed typical symptoms. The disease severity of each strain was compared and recorded. The experiment was carried out at least three times, with three replicates each time, to obtain similar results.

20 μL of 5 × 10⁴ conidia/ml suspension was inoculated onto a plastic coverslip (Thermo Fisher Scientific, Waltham, MA, USA) and cultured in the dark at 25°C. Conidia germination and appressorium differentiation were observed and recorded under a microscope for 2h, 4h, 8h, 12h and 24h after inoculation. The appressorium turgor pressure of rice blast fungus was determined according to the previous method (Dixon et al., 1999). The appressorium formed by induction on the hydrophobic surface for 24h was treated with 1-4 M glycerol for 10 minutes, and the collapse rate of mutant and wild-type appressorium after glycerol treatment was counted under a microscope. The test was repeated three times, and the number of conidia or appressorium observed for each strain was no less than 200. For the rice blast infection rate test, barley leaves grown for 7 days were selected and sprayed with 1 × 10^-5/mL rice blast conidia suspension. After 36 h or 48 h of inoculation, the barley leaves were decolorized with methanol, and the number of infected appressorium was counted under a light microscope. The experiment was repeated 3 times, and no less than 50 appressorium was observed for each strain.

The mycelium of all tested strains grown in liquid CM was used to extract the proteins. Western blotting was used to identify the amounts of phosphorylated Pmk1 and Mps1. To detect phosphor-Mps1 and phospho-Pmk1, a primary anti-phospho-p44/42 MAPK antibody (Cell Signaling Technology, USA) was employed. Two antibodies were applied to detect Mps1 and Pmk1, anti-ERK1/2 MAPK antibody (Cell Signaling Technology, USA) for Mps1 and anti-p44/42 MAPK antibody (Santa Cruz, USA) for Pmk1. A monoclonal anti-GAPDH antibody (HUABIO, China) was also used to detect the samples. Using the ImageJ program, the band intensity was calculated.

The relevant experimental data were processed with Excel 2016, and one-way ANOVA was analyzed using DPS (Data Processing System) v20.05. Significance levels were determined using Duncan’s new multiple-range test at p < 0.05.

The homologous genes of Saccharomyces cerevisiae ScNsr1 were searched in the EsdmblFungi (https://fungi.ensembl.org) database using the BLASTp program. The resultant MoNsr1 (MGG_01268) was obtained and comprised 486 amino acids. The MoNsr1-encoding gene comprises 1592 bp, with 2 introns and an exon. After amino acid sequence alignment, MoNsr1 showed 54% homology with ScNsr1 ( Figure 1A ), including 4 conserved domains. The N-terminus includes an acidic domain composed of serine and acidic residues. This structure may be related to the interaction between nucleolin and ribosomal proteins, histone H1, U3 snoRNP, etc (Erard et al., 1988; Ginisty et al., 1998; Sicard et al., 1998). The middle part contains two RRMs (RNA recognition motifs), which allow the protein to bind to RNA specifically, and the C-terminal GAR domain. The GAR domain is often found in nucleolar proteins, and the GAR domain in nucleolin interacts with ribosomal proteins (Bouvet et al., 1998).

In yeast, Nsr1 is localized in the nucleus and binds to the (TG1-3)_n telomeric DNA (Lee et al., 1991; Lin and Zakian, 1994). The MoNsr1-GFP fusion expression vector constructed in this study was transformed into the rice blast fungus strain Guy11 by AtMT, and 6 transformants with detectable green fluorescence were obtained. There was no significant difference in the obtained transformants growth and sporulation phenotypes compared with the wild-type. The hyphae and spores of the GFP-labeled transformants were stained with 0.8 μg/ml DAPI solution and observed under a fluorescence microscope. The blue nuclei labeled with DAPI fluorescence in the cells co-localized with the green fluorescence of the MoNsr1-GFP fusion expression protein ( Figure 1B ). The results showed that MoNsr1, like ScNsr1, is localized in the nucleus.

We performed gene knockout and complementation to investigate the function of the MoNsr1 gene. Inoculate the wild-type strain, gene deletion mutant ΔMoNsr1, and the complementary strain MoNsr1c onto CM medium with alternate light and dark cultivation at 28 °C for 12 hours for 6 days. The results showed that the average colony diameter of the ΔMoNsr1 mutant was significantly smaller than that of the wild-type (p<0.05) ( Figures 2A, C ). Rectangular mycelium blocks grown on CM medium for 4 days were cut and placed on a glass slide. The conidia induction experiment was carried out by alternating light and dark at 28 °C for 12 and 48 hours. Microscopic observation confirmed that compared with the wild-type and complemented strains, the ΔMoNsr1 mutant had fewer conidiophores and produced sparse conidia ( Figure 2B ). Conidia grown on CM medium for 6 days were collected for statistics. The results showed that the yield of conidia per unit area of the ΔMoNsr1 mutant was only 35% of that of the wild-type ( Figure 2D ). The above results indicate that the loss of the MoNsr1 gene is involved in the vegetative growth and asexual reproduction of rice blast fungus.

In order to clarify the role of the MoNsr1 in the pathogenesis of rice blast fungus, the wild-type strain Guy11, gene deletion mutant ΔMoNsr1, and compensatory strain MoNsr1c were used to inoculate barley leaves and 14-day-old susceptible rice seedlings (CO-39). The pathogenicity results of detached barley leaves showed that the pathogenicity of the ΔMoNsr1 mutant was lower than that of wild-type and complement treated with the same concentration of spore solution. Pathogenicity weakening became more pronounced with decreased spore concentration ( Figure 3A ). The results of inoculation of rice seedlings with 1 x 10⁵ spores/ml by spray showed that the mutant ΔMoNsr1 produced fewer disease spots, and the disease severity significantly decreased compared with that of the wild-type strain ( Figures 3B, C ).

In order to investigate the impact of MoNsr1 gene deletion on the pathogenicity of rice blast fungus, we analyzed the effects of gene deletion mutants on conidia germination, appressorium differentiation, and host epidermal penetration ability. The results of the conidial germination experiment showed that the conidia of the ΔMoNsr1 mutant could germinate and form an appressorium. Compared with the wild-type, the conidial germination rate and appressorium formation rate of the ΔMoNsr1 mutant were significantly reduced ( Figures 4A, B ). When the appressorium induced for 24 hours was treated with 1-4 M glycerol, the collapse rate of the appressorium in the ΔMoNsr1 mutant was significantly higher than that in the wild-type ( Figure 4C ). These results indicated that MoNsr1 is involved in conidial germination, appressorium differentiation, and maturation.

We analyzed the infection rate in barley leaves to determine whether the decrease in turgor pressure of the ΔMoNsr1 mutant caused the decrease in host pathogenicity. The results showed that after inoculating barley leaves with a suspension of 5 × 10⁴ spores/ml in vitro for 36 and 48 hours, the infection rates of the ΔMoNsr1 mutant on barley epidermal cells decreased by 31% and 26%, respectively, compared to that of the wild-type under a microscope ( Figure 4D ). The above indicates that the absence of the MoNsr1 leads to a decrease in mutant infection rate, resulting in a decrease in mutant pathogenicity. This result is consistent with the decrease in appressorial turgor pressure in the ΔMoNsr1 mutant.

We investigated the mechanism effects of the MoNsr1 on the cell wall integrity of the gene deletion mutant and the sensitivity of the ΔMoNsr1 mutant to cell wall disruptors. The results showed that the growth of the ΔMoNsr1 mutant was inhibited on CM plates containing 25 μg/mL Congo red (CR) and 12.5 μg/mL Calcolflour white (CFW), with inhibition rates of 24.6% and 12.1%, respectively, which were significantly higher than those of the wild-type ( Figures 5A, B ). The results indicate that gene deletion leads to defects in the integrity of the ΔMoNsr1 mutant cell wall. The cell wall integrity signaling pathway is a key mechanism for sensing and adapting to various environmental conditions, and the Mps1-mediated MAPK signaling pathway is its core component (Lu et al., 2023). The phosphorylation level of Mps1 in the ΔMoNsr1 mutant was analyzed, and the results showed that compared with the wild-type, the Mpsl phosphorylation level of the ΔMoNsr1 mutant was lower ( Figure 5C ). The above results indicate that MoNsr1 may respond to the cell integrity signaling pathway by regulating Mpsl phosphorylation. Surprisingly, the phosphorylation level of Pmkl in the ΔMoNsr1 mutant was also lower than that of the wild-type. Pmk1 is an important signaling pathway that regulates appressorium differentiation and infection in rice blast fungus (Zhang et al., 2011). Therefore, MoNsr1 may also participate in the differentiation of appressorium by regulating the phosphorylation of Pmk1.

In addition, we also found that the ΔMoNsr1 mutant is more sensitive to oxidants and cold stress. The relative growth inhibition rates of mutants on CM plates containing 2 mM methyl viologen (MV) or 7.35 mM H₂O₂ were 42% and 43%, respectively, significantly lower than that of the wild-type ( Figures 6A, B ). The relative growth inhibition rate of mutants cultured at 20 °C was 49%, significantly higher than that of the wild-type (40%) ( Figures 7A, B ). These findings indicate that MoNsr1 plays a positive role in resisting oxidative and low-temperature stress.