MoPtc5 plays synergistic roles with MoPtc1 and MoPtc2 in the vegetative growth, stress adaptation, and virulence of Magnaporthe oryzae

MoPtc5 plays synergistic roles with MoPtc1 and MoPtc2 in the vegetative growth, stress adaptation, and virulence of Magnaporthe oryzae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MoPtc5 plays synergistic roles with MoPtc1 and MoPtc2 in the vegetative growth, stress adaptation, and virulence of Magnaporthe oryzae Jules Biregeya, Frankine Jagero Otieno, Meilian Chen, Zifeng Yang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5080333/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Protein phosphatases are crucial enzymes that regulate key cellular processes such as cell cycle, gene transcription, and translation in eukaryotes. Seven PP2C protein phosphatases have been identified in Magnaporthe oryzae . However, their synergistic roles in the pathology and physiology of M. oryzae remain poorly investigated. By qRT-PCR analysis we found that PTC1 and PTC2 are significantly upregulated in the PTC5 deletion mutant. Double deletion of MoPTC5 / MoPTC1 and MoPTC5 / MoPTC2 genes significantly reduced hyphal growth, conidiophore formation, sporulation, and virulence in M. oryzae . In addition, the double knockout mutants were increasingly sensitive to different osmotic, oxidative, and cell wall stresses. Western blot analysis revealed that MoPtc5 plays a synergistic function with MoPtc1 and MoPtc2 in the regulation of MoMps1 and MoOsm1 phosphorylation levels. Lastly, appressorium formation and turgor generation were remarkably affected in the Δ Moptc5 Δ Moptc1 and Δ Moptc5 Δ Moptc2 double deletion mutants. These findings demonstrate the synergistic roles of PP2c protein phosphatase in the fungal development and pathogenesis of M. oryzae . Biological sciences/Microbiology/Fungi Biological sciences/Molecular biology Magnaporthe oryzae synergistic stress tolerance protein phosphatases pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Magnaporthe oryzae is a hemibiotrophic pathogen that threatens rice, wheat, and many other cereals production worldwide 1 , 2 . Early reports also demonstrated that rice blast fungus can infect some additional grass species, including Leersia , Pinicum , Setaria , and Lolium species. M. oryzae primarily causes a very devastating blast disease in rice, wheat, finger millet, and barley 3 that results in significant yield losses each year 4 . This fungus is accepted and recognized as a model organism for studying fungal and host interacting pathways 5 . Therefore, the availability of full genomic sequences of pathotypes and that of their hosts has made it more convenient to study fungal genomics 6 . M.oryzae infects the host via a structure known as appressoria. This structure accumulates turgor pressure, enabling the generation of mechanical force to penetrate plant cuticles using a penetration peg and release effectors into the plant tissues 7 - 9 . Several signaling pathways have been demonstrated to play a crucial role in appressorium formation in M. oryzae(ref). These pathways are regulated by protein kinases and protein phosphatases (ref). Rice blast poses a significant constraint to global cereals yield and extension services 10 . To manage and mitigate the severity of rice blast disease, integrated strategies have been implemented for durable crop resistance, including cultural practices, chemical control, and functional genomic analysis of host-pathogen interaction 11 - 13 . Protein phosphatases are very important components of cellular signaling pathways that regulate fungal development 14 . Type 2C protein phosphatases (PP2C) are subdivided within two classes such as, phosphoprotein and Mg 2+ -dependent protein phosphatases 15,16 . The functions of PP2C, includingPtc1, Ptc2, Ptc5, Ptc6 , and Ptc7, have been discussed extensively in Saccharomyces cerevisiae 17 . Previous research indicated that Ptc2 primarily involved in the Hog pathway 18 . Further studies also revealed that Ptc1 is involved in cell wall maintenance, conidiation, RNA splicing, and arrangement of subcellular organelles in S. cerevisiae 19-21 . Ptc2 and Ptc1 play critical roles in the dephosphorylating cyclin-dependent kinases, essential for the regulation of double-strand DNA breakdown by dephosphorylating Rad53 protein kinase 22,23 . In M. oryzae, mutants lacking both MoPTC1 and MoPTC2 exhibit defects in vegetative growth, virulence, protein phosphorylation, multistresses, and conidiogenesis 24,25 . Additionally, a study in Aspergillus flavus demonstratedthatdephosphorylation of PGK1 via Ptc1 and Ptc2 regulates aflatoxin level and autophagy assays. 26 . Early Publication has shown that Ptc5 protein is localized in the mitochondria of S. cerevisiae , C. albicans , and M. oryzae 30 . However, a single deletion of CaPTC5 in C. albicans did not affect tolerance towards various stress agents, including azoles 27 . In Arabidopsis, early reports showed that Ptc5 acts as a MAPK phosphatase, interacting precisely with MPK4, MPK6, and MPK3 in cells. Previous findings also indicated that in higher plants, Ptc5isinvolved in stomatal opening-closure, and regulating ABA biosynthesis genes 28 . Additionally, double knockout of PTC5 along with other type 2C protein phosphatase genes in S. cerevisiae revealed that PP2Cs are strongly important in responses to diverse abiotic stressors 29 . Recently, we revealed that PTC5 and PTC7 are important for stress maintenance, asexual reproduction and virulence in M. oryzae 30 . Type 2C protein phosphatases have been indicated to play overlapping and indispensable functions in filamentous fungi 29,31 . We applied molecular biology approach to explore the synergistic roles of MoPTC5 along with MoPTC1 or MoPTC2 in M. oryzae. Our study provided evidence that double mutation of MoPTC5 with MoPTC1 or MoPTC2 synergistically altered hyphal growth, fungal sporulation, cell wall integrity, regulation of MoMps1 and MoOsm1 phosphorylation , and weakened the virulence in M. o ryzae. Results Phylogenetic analyses and identification of MoPtc1, MoPtc2,and MoPtc5 The homologs of Ptc5, Ptc1, and Ptc2 in blast fungus were identified from the fungidb website ( http://fungidb.org/fungidb/ ) via BlastP search, based on the amino acid sequences from S. cerevisiae , and were termed MoPtc5, MoPtc1, and MoPtc2, respectively. Domain annotation of the proteins was generated using IBS software, while MEGAx software was used to build the phylogenetic tree with bootstrap analysis 36 . The analysis demonstrated that MoPtc5, MoPtc1, and MoPtc2 possess two conserved domains (PPCC2 domain and PP2C_SIG domain) (Fig. 1 A). Additionally, phylogenetic assay indicated that MoPtc1 and MoPtc2 are most closely related to their orthologs in Neurospora crassa , while MoPtc5 has the closest homology to N. crassa and Botrytis cinera (Fig. 1 B). ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 strongly affect hyphal growth and sporulation of M. oryzae We initially conducted qRT-PCR and observed significant up-regulation of MoPTC2 and MoPTC1 in the MoPTC5 mutant (Supplementary Figure S2), suggesting a redundancy of MoPTC5 , MoPTC1 , and MoPTC2 in M. oryzae . Subsequently, double genes knockout of MoPTC5 with either MoPTC1 or MoPTC2 was generated in the ∆ Moptc5 mutant, by replacing the ORF (Open reading flame) of MoPTC1 or MoPTC2 with neomycin resistance gene cassette. Culturing wild-type strain Guy11, mutant strains ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 , along with the complemented strain on CM revealed significantly reduced hyphae growth of the ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 double mutants compared to ∆ Moptc5 and Guy11 (Fig. 2 A,B). Conidiation was remarkably reduced in ∆ Moptc5 ∆ Moptc1 and remarkably reduced in ∆ Moptc5 ∆ Moptc2 in comparison to ∆ Moptc5 and wild-type cultured on solid rice bran media (RBM) (Fig. 2 D). Consistent with this result, ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 failed to produce conidiophores at diverse experimental time points 12, 24, and 36 hours (Fig. 2 C). Overall, the findings suggest the overlapping role of MoPTC5 with MoPTC1 or MoPTC2 in hyphal growth and asexual reproduction of blast fungus. MoPTC5 plays synergistic functions with MoPTC1 and MoPTC2 in cell wall maintenance and Mps1 phosphorylation level To further investigate the roles of MoPtc5 in conjunction with MoPtc1 or MoPtc2 in regulating cell wall maintenance of M. oryzae , we analyzed the growth inhibition rate of mutants grown on CM containing with different stress drugs including, DTT, SDS, CFW, and CR for ten days of incubation at 28 o C respectively. Results showed that the growth of ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 on CM supplemented with SDS, CR, CFW, and DTT was significantly inhibited. Especially, ∆ Moptc5 ∆ Moptc2 displayed a higher sensitivity response compared to other mutants when spotted on CM supplemented with SDS, CR, and CFW (Fig. 3 A,B). We then performed a western blotting to evaluate the activation of MoMps1 in the mutants. The results indicated an elevation of the phosphorylation rater of MoMps1 protein in ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 strains (Fig. 3 C). The findings demonstrate that MoPTC5 / MoPTC1 and MoPTC5 / MoPTC2 are synergistically involved in the integrity of cell wall and phosphorylation of MoMps1 in M.oryzae . Double knockout of MoPTC5 with MoPTC1 and MoPTC2 did not influence the cell wall thickness of rice blast fungus The cell wall serves as a robust protection safeguarding the plasma membrane and intracellular contents of plants and many microbes 37 . To investigate the potential involvement of MoPtc5 along with MoPtc1 or MoPtc2 in the cell wall thickness of blast fungus, we cultured strains on CM for three days. The fungal hyphae were then harvested, washed with ddH 2 O, dried with clean filter papers, and preserved in phosphate buffer. Transverse sections of hyphal cell walls were imaged using electron transmission microscopy (TEM). However, we observed no discernible difference in the thickness of cell walls among the wild-type strains, Δ Moptc5 , Δ Moptc5 Δ Moptc1 , and Δ Moptc5 Δ Moptc2 mutants (Fig. 4A,B). Chitin represents a vital constituent of fungal cell walls. Hence, we conducted further analysis on the transcription levels of chitin synthases in M. oryzae through qRT-PCR to gain a clearer understanding of how these proteins regulate cell wall stress. Our results showed a remarkably decrease expression of the chitin synthase-encoding genes in the mutants compared to wild-type (Fig. 4 C). ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 play important roles in osmotic, oxidative stress and MoOsm1 phosphorylation in M.oryzae To determine the functional redundancy of MoPTC5 , MoPTC1 , and MoPTC2 in M. oryzae under oxidative and hyperosmotic stresses, we cultured Guy11, Δ Moptc5 , Δ Moptc5 Δ Moptc1 , and Δ Moptc5 Δ Moptc2 on CM supplemented with osmotic and oxidative stressors drugs including, potassium chloride (1 M KCl), sodium chloride (1M NaCl), 5 mM H 2 O 2 , and 10 mM H 2 O 2 . Growth measurements were recorded after ten days post-incubation. Our findings demonstrated that both Δ Moptc5 Δ Moptc1 and Δ Moptc5 Δ Moptc2 exhibited higher sensitivity to hyperosmotic and oxidative stress induced by 1M NaCl, 1M KCl, 5mM H 2 O 2 or 10 mM H 2 O 2 in comparison to the Guy11 and Δ Moptc5 mutant (Fig. 5 A,B). A Previous study demonstrated that double deletion of ∆ Moptc5 ∆ Moptc7 reduced MoOsm1phosphplylation level. Consequently, we conducted western blotting assay to quantify the relative abundance of the osmoregulatory protein MoOsm1 in ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , ∆ Moptc5 ∆ Moptc2 , and Guy11. Our results revealed a low abundance of MoOsm1 in ∆ Moptc5 , and ∆ Moptc5 ∆ Moptc1 mutants but not in Δ Moptc5Moptc2 (Fig. 5 C). Altogether, we proposed that MoPtc5 , in conjunction with MoPtc1 and MoPtc2, significantly contributes to the hyperosmotic and oxidative tolerance of M.oryzae , as well as MoOsm1 phosphorylation. ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 mutants are remarkably delayed in appressoria formation The appressorium serves as the primary infectious structure facilitating the penetration of rice blast fungus. Therefore, to examine MoPTC5 ’s role alongside MoPTC1 or MoPTC2 in appressorium formation, conidial suspensions of ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , ∆ Moptc5 ∆ Moptc2 , and guy11 (wild type) were initially placed on hydrophobic coverslips. Appressoria formation was checked and viewed under a microscope at 4, 8 ,12, and 24 hours post-inoculation, respectively. Findings generated from this assay indicated that both double gene deletion mutants failed to develop appressorium at 4 h post-inoculation. However, the appressorium formation rate of ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 mutants gradually increases over time until comparable to the Guy11 (Fig. 6 ).Taken together, results demonstrated that the deletion of MoPTC5 , along with MoPTC1 or MoPTC2 , merely delayed the appressorium formation of M.oryzae . MoPTC5, MoPTC1,and MoPTC2 synergistically influence appressoria turgor generation and utilization of glycogen in M. oryzae To ascertain the synergistic roles of MoPtc5 in tandem with MoPtc1 and MoPtc2 during appressorium penetration in host epidermal cells, the appressorium turgor generation across all mutants and the wild type was examined, respectively. Conidia sourced from the aforementioned strains were inoculated onto hydrophobic coverslips, followed by exposure to varying concentrations of glycerol after 24 h. The percentage of collapsed appressoria were viewed and monitored under a microscope. Our findings demonstrated that a significant number of the appressorium produced by Δ Moptc5 Δ Moptc1 and Δ Moptc5 Δ Moptc2 were collapsed under diverse glycerol concentrations (Fig. 7 A,B). Moreover, appressorium formation of rice blast fungus necessitates the efficient transfer of nutrients, such as glycogen and lipid droplets, from conidia, acting as essential material sources for turgor generation post-maturation. Effective nutrient transport profoundly influences the virulence of blast fungus. Thus, to assess glycogen mobilization from conidia to appressoria, conidia were harvested and incubated at 28 o C for 2, 4, 8, 12, and 24 hours respectively, KI/I 2 was used to stain conidia before imaged under a microscope. The Findings unveiled a delaying of gylcogen mobilization from conidium to appressoria in the double mutants (Fig. 7 C,D). In summation, these findings underscore the pivotal contribution of MoPtc5 alongside MoPtc1 and MoPtc2 in appressorium turgor generation, primarily through the regulation of glycogen utilization. ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5∆Moptc2 strains weakened virulence to infect rice leaves Protein phosphorylation is an essential mechanism for infection-related morphogenesis 38 . Morever, the impact of M. oryzae protein phosphatase genes on virulence remains inadequately explored. To elucidate the combined effect of double mutations in MoPTC5 along with MoPTC1 and MoPTC2 on fungal virulence, we harvested spores from Guy11, ∆ Moptc 5, ∆ Moptc 5∆ Moptc 1, and ∆ Moptc 5∆ Moptc 2 and sprayed on the rice seedlings. A striking decrease in pathogenicity was examined in the double mutants in comparison to ∆ Moptc 5 and Guy11 (Fig. 8 A,B). Similarly, a significant reduction in virulence on barley leaves was evident using mycelial plugs (Fig. 8 C,D). Further analysis revealed a lower penetration rate of hyphae in cuticle cells of barley at different time points for the double gene deletion mutants (Fig. 8 E). Collectively, our findings demonstrated the collaborative influence of MoPTC5, MoPTC1, and MoPTC2 on the virulence of M. oryzae. Discussion The main purpose of this research was to reveal the synergistic functions of PP2C MoPtc5 along with MoPtc1 and MoPtc2 in the virulence and development of blast fungus , M.oryzae . To achieve this purpose, we initially deleted MoPTC5 and subsequently knocked out MoPTC1 and MoPTC2 in ∆ Moptc5 mutant, respectively. Our investigations indicated that all the mutant strains were viable on different growing mediums. Phylogenetic assay showed a close relationship among MoPtc5, MoPtc1, and MoPtc2 proteins and their homologs in other filamentous fungi, suggesting common ancestry. Spores formation is critical in filamentous fungi, with spores or conidia developing on vegetative hyphal structures known as conidiophores 39 . It was previously reported that ∆ Moptc5 single mutant demonstrated a slight decrease in fungal growth and sporulation 40 . Similarly, MoPtc1 and MoPtc2 are involved in M.oryzae growth and sporulation 24 , 25 . In this study, our findings revealed a very significant reduction in conidiation of ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 double mutants, suggesting a synergistic role of MoPTC5 , MoPTC1 , and MoPTC2 in sporulation regulation. This might be due to various environmental conditions including, aeration or low expression levels of conidiation-related genes which affect metabolic and physiological mechanisms that happen during sporulation 39 , 41 . The cell wall is important for cellular integrity and shielding against environmental signals 42 . In our research, we evaluated the impact of diverse cell wall stressors drugs, revealing higher sensitivity in the double mutant ∆ Moptc5 ∆ Moptc2 compared to ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and the wild-type strains. Previous research had indicated slight sensitivity of ∆ Moptc5 to cell wall stressors. 40 . Our findings demonstated that MoPTC5 and MoPTC2 play overlapping roles in stress maintenance, consistent with prior studies. 29 . Western blot analysis confirmed that ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 are crucial for the phosphorylation of Mps1, suggesting their involvement in MoMps1 phosphorylation activation in M. oryzae . Conclusively, MoPtc5, MoPtc1, and MoPtc 2 synergistically regulate cell wall maintenance and participate in signal transduction pathways essential for cell wall maintenance. Chitin was reported as an abundant component of fungal and insect cell walls, which protect the organisms against environmental stimuli 43 . In our study, we investigated and measured the cell wall sizes of ∆ Moptc5 , ∆ Moptc5 ∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 and the wild-type strains. The results unveiled combined effects of MoPTC5 , MoPTC1 , and MoPTC2 had no direct influence on the fungal cell wall thickness, which is inline with early result 40 . Morever, we observed downregulation of chitinase genes in the double mutants, suggesting that MoPTC5 in conjugation with MoPTC1 and MoPTC2 negatively regulates the expression of chitinase encoding genes, possibly through shared transcription factors for these orthologous genes 44 . Additionally, we investigated the synergistic functions of MoPTC5 with MoPTC1 and MoPTC2 in osmotic and oxidative responses. The double mutants ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 showed a higher inhibition rate on media containing 1M NaCl and 1M KCl 45 . Here, we speculated that MoPTC5 , MoPTC1 , and MoPTC2 might be required in the co-regulation of the cell sensitivity to multiple osmoregulatory agents and their pathways in the fungal development. Furthermore, the increased sensitivity of ∆ Moptc5∆Moptc2 to oxidative stress (10 mM H 2 O 2 ) suggests that these genes play important functions in the tolerance of oxidative and osmotic stresses 46 . To establish the genetic redundancy of these three protein phosphatases, we also checked the phosphorylation level of the osmoregulatory protein MoOsm1 in the single and double mutants. We found that MoOsm1 phosphorylation level significantly decreased in ∆ Moptc5 and ∆ Moptc5 ∆ Moptc1 , and here in, we speculated that MoPTC5 and MoPTC1 might negatively regulate MoOSM1 phosphorylation in M. oryzae. Generally, we concluded that the type 2C protein phosphatases are functionally redundant towards osmotic, oxidative stresses and are involved in the activation and regulation of osmolality glycerol pathways. Pathogenicity is crucial in fungi-host interaction. Some factors are important for the establishment of fungal virulence including, conidia attachment, germination on the surface of the host, and its colonization 47 . In this work, we assayed the pathogenicity levels of the various strains on susceptible rice cultivars and barley. Remarkably, we revealed a severe defect in virulence of the double mutants ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 . Our findings also indicated a high reduction of hyphal penetration and invasion of barley epidermal cells in double mutants. Previous publications showed that the single mutants ∆ Moptc5 , ∆ Moptc1 , and ∆ Moptc2 are required in the pathogenesis of the rice blast fungus , M.oryzae 24 , 48 , 49 . In this research, we reasoned that PP2C, type 2C protein phosphatases MoPTC5 , MoPTC1 , and MoPTC2 complementarily regulate fungal virulence. The double mutants ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 are impaired in appressorium formation at 4 h and 8 h after inoculation on hydrophobic coverslips, compared to the single mutant ∆ Moptc5 and the WT strains. This might be possibly caused by hyperactivation of Pmk1 pathway which has previously been revealed to cause abnormalities in appressorium morphogenesis and pathogenicity 50 . From these results, we suggested that MoPTC5 in collaboration with MoPTC1 and MoPTC2 synergistically regulates appressorium formation and maturation. Appressorium turgor pressure was also examined in various strains. We observed a higher percentage of collapsed appressoria and slowed glycogen mobilization and utilization from conidia to appressoria in ∆ Moptc5 ∆ Moptc1 , ∆ Moptc5 ∆ Moptc2 than in ∆ Moptc5 and wild-type. However, is still unclear about the pathways regulating the movement of conidium content to mature appressorium. Findings of incipient cytorrhysis are consistent with the defects of penetration and hyphae invasion in barley epidermal cells, which is also in consistent with early studies 51 , 52 . In summary, Rice blast disease is severe across the world, ultimately leading to a loss of 10% -30% for rice, wheat, and other cereals globally. This investigation carried out on PP2C, the type 2C protein phosphatases provided evidences of the synergy of MoPTC5 with MoPTC1 and MoPTC2 in the regulation of sporulation rate, hyphal growth, multi-stress adaptation, and virulence of M. oryzae . Conclusively, this research revealed new insights into the development and pathogenesis of M. oryzae . Materials and Methods Genes knockout and complementation assays The split marker assay was employed to knockout MoPTC5 in M. oryzae . Downstream and upstream fragments A and B were amplified respectively. These fragments were then fused with hygromycin flagments (HA and HB) by using SOE PCR. The ligated vectors were transformed into Guy11 (wild-type) protoplasts as presented in the early report 32 , 33 . The grown transformants were screened and confirmed by PCR. To get ∆ Moptc5 ∆ Moptc1 and ∆ Moptc5 ∆ Moptc2 double mutant constructs, flanking fragments A and B of MoPtc1 and MoPtc2 were amplified and ligated with the Neomycin resistant gene (NE and EO). These ligated constructs were then transformed into ∆ Moptc5 mutant protoplasts and positive transformants were selected on terrific broth 3 growing media (TB3) containing neomycin and colonies were verified by PCR. All primers used in this study are listed in supplementary Table S1 . Fungal culturing assay The M. oryzae Guy11 was employed in this research as a wild-type strain. Complete medium, starch yeast medium, and rice bran medium were used for culturing fungi. The hyphae morphology and growth assays were conducted as described in 34 . To discuss the influence of cell wall ,oxidative, and osmotic drugs on mutants in comparison to Guy11. The strains were spotted on CM media containing with 200 µg/mL Congo red, 200 µg/mL CFW, 0.01% SDS ,1M of sodium chrolide, 1 M of potassium chrolide, and 10 mM H 2 O 2 respectively. The strains were kept in incubator at 28℃, measurement of mycerial growth, and pictures were done after ten days post-inoculation. To ascetain the sporulation rate, mycelia plugs were grown on rice bran media at 28 ℃ and exposed to light. After ten days conidia were counted using light microscopy. Pathogenicity, host penetration, and analysis of appressoria turgor pressure For pathogenicity, Fungi were grown on solid rice bran media within ten days at 28 ℃, later the spores were collected, washed and the conidia suspension adjusted to 5x10 4 spores and sprayed onto the rice seedlings (CO-39), The seedlings were incubated at 25℃ and disease signs were monitored and examined after a week days. To investigate the host penetration potential of the mutants, 10 µL of conidial (5 × 10 4 spores/mL) from the mutants and guy11 were inoculated on barley leaves. The hyphae penetration rate were checked at 30, 48, and 60 hours post-inoculation. The Nikon microscope (Nikon, Nippon Kogaku kogyo kabushikigaisha optical industries Co., Ltd, Tokyo, Japan) were used to view hyphal growth in fungal epidermal cells. Incipient cytorrhysis (Appressoria collapsed due to loss of internal hydraulic turgor pressure) analyses were carried out to investigate generation of the appressorium turgor pressure in ∆ Moptc5 , ∆ Moptc 5∆ Moptc1 , ∆ Mopt5 ∆ Moptc2 , ∆ Moptc5_C , and wild-type strains. Conidia suspensions were dropped on hydrophobic coverslips (Fisher Scientific, Pittsburgh, PA 15275), and within 24 h conidia were mixed with glycerol solutions 1 M, 2 M, and 3 M respectively. Incipient cytorrhysis and glycogen mobilization were monitored under a microscope. RNA extraction ,quantitative real time PCR (qRT-PCR), and RT-PCR assays The mutants and wild type strains (Guy11, ∆ Moptc5 , ∆ Moptc 5∆ Moptc1 , and ∆ Moptc5 ∆ Moptc2 ) were inoculated in liquid CM, and shaken machine at 110 rpm, 28 o C for 3 days period. Mycelia were collected, washed with ddH 2 O, dried, and crushed into fine powder by using motar in the nitrogen solution. The extraction of total RNA from each strain was done following the protocol of an Eastep TM extraction kit (Promega, Beijing Biotech Co.Ltd, China). The quantitative real-time PCR (qRT-PCR) was conducted following Promega Super Real Premix kit instructions. The qRT-PCR was performed in an Eppendorf Realplex2 master cycler (Eppendorf China Ltd. Shanghai, China ), and analysis was done using (2 − ΔΔCT ) as previously reported by 35 ). Southern blotting assay For the Southern blotting, We firstly extracted fungal genomic from fungal strains using the cetytrimethylammonium bromide method (CTAB method). The digestion of the genomic DNA was done by using specific restriction enzymes PstI and ClaI respectively (New England Biolab Co., Ltd Beijing, China). Digested DNA product was separated by running a gel electrophorensis assay. The gel was later moved to a specific membrane (Merck) which is positively charged. Nucleic acid probes with sequences complementary to the region of targeted fragments were used for hybridization. During hybridization, the labeled probes were incubated overnight together with the DNA in a hybridizer machine at 42°C to promote the hybridization of the complementary sequences. Unhybridized probes were removed by washing the membrane with 2× SSC + SDS and with maleic acid + tween 20 solutions. Detection starter kit I (Merck KGa, Germany ,Darmstadt ) and images were captured using Tanon 5200 chemiluminescent imaging machine (Tanon Science &Technology Co.Ltd, China, Shangahai). Extration of proteins and western blotting assays The fungal mycelia plugs were cultured in the liquid CM media and shaken for 2–3 days at 28°C after that mycelia were collected and crashed powder by using motar and then 1–2 g of the mycelial powder was resuspended in 1 mL of protein lysis mixed with 10 µL of phenylmethyl sulphonyl fluoride (PMSF) and 10 µL of a proteinase inhibitor. The mixed solution was incubated in ice for a half hour and vortexed by inverting tubes within every 10 min, and afterward, the samples were centrifuged for 20 min at 4°C the the supernatants were sucked and added into 2 mL tubes, lastly sodium dodecyl sulfate buffer (SDS buffer) was added before storage at -21°C for future use. The western blot assay, SDS -PAGE (10% polyacrylamide) assay was used for the separation of proteins. The targeted proteins were detected using the primary antibodies (P-p38 MAPK, and P44/42MAPK) along with conjugated secondary antibodies (Goat antirabbit& mouse IgG-HRP) and an anti-beta actin mab was utilized as a control antibody. Finally, the protein phosphorylation signals were detected following the instructions of a western blotting Kit (Epizyme, SQ201, Shanghai, China), and photographs were taken by Imaging System (Tanon Science &Technology Co.Ltd,China Shanghai). Microscopic examination assays Conidiophores development, conidia formation, appressorium formation, invasive hyphae, conidia germination on the hydrophobic slides, glycogen mobilization in appressoria, and appressorial turgor generation were viewed under a Nikon TiE system (Nikon, Japan). Declarations Competing interests No conflict of interests were declared by authors. Author Contribution Z. W., W. T., Y. H., and J. B. designed the study plan, applied funding, and critically revised the manuscript; J. B., F. J. O., Z. Y., M. C., A. M. W. conducted the experiments and discussed about materials and methodology; J. B., R. Z analyzed all data and wrote the draft manuscript; J. B., A. F., N. O., Y. A., N. A. worked on editing of the final copy. All authors have approved the final manuscript copy. Acknowledgement Foundation of Natural Science of the Fujian Province (2022J01129) , Fujian provincial Science and Technology (2022NZ030014) and Exploratory Science, Technology Innovation Project of Fujian Academy of Agricultural Sciences (ZYTS202402) and Basic Research Special Project of Public Welfare Research Institutions in Fujian Province (2024R1022003) supported our study. Data Availability Data is provided within the manuscript or supplementary information files. References Talbot, N. J. On the trail of a cereal killer: exploring the biology of Magnaporthe grisea . Ann. Rev. Microbiol. 57 , 177–202. https://doi.org/10.1146/annurev.micro.57.030502.09095 (2003). Liu, X. et al. The putative protein phosphatase MoYvh1 functions upstream of MoPdeH to regulate the development and pathogenicity in Magnaporthe oryzae . M P M I . 29 , 496–507. https://doi.org/10.1094/MPMI-11-15-0259-R (2016). Yoshida, K. et al. 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Scie . 12 , 748120. https://doi.org/10.3389/fpls.2021.748120 (2021). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInfoFile.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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oxysporum, F. graminearum\u003c/em\u003e, \u003cem\u003eSaccharomyces\u003c/em\u003e \u003cem\u003ecerevisiae \u003c/em\u003e, \u003cem\u003eand Magnaporthe oryzae\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/524de5c4a6222f7c503a63b6.png"},{"id":68836795,"identity":"d57592c9-95ed-4783-9872-31d11a44f62e","added_by":"auto","created_at":"2024-11-12 14:29:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67887,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional redundancy of \u003cem\u003eMoPTC5\u003c/em\u003e with \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e in the development of \u003cem\u003eM. oryzae\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA,B\u003c/strong\u003e) Growth assay of \u003cem\u003e\u0026nbsp;\u003c/em\u003eGuy11\u003cem\u003e,\u003c/em\u003e ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e , and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e mutants. (\u003cstrong\u003eC\u003c/strong\u003e)Conidiophores producing ability of the mutants compared to Guy11. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eConidiation rate of mutant and Guy11 grown on rice bran media for ten days. The error bars displayed the standard mean from three biological repeats ,astriks indicated the significance differences , Data analysis was done by using one- way ANOVA with multiple comparison tests in the Graph prism software).Asteriks revealed the significances ( *P \u0026lt; 0.05 ; **P\u0026lt; 0 .01, and *** P\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/b5d3385ee12bfb2bab4fa27a.png"},{"id":68836796,"identity":"05a97fc4-f329-4e8a-ae0b-74b7d10d622b","added_by":"auto","created_at":"2024-11-12 14:29:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":209901,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003e∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e mutants towards diverse cell wall stressors. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eResponse of Guy11, ∆\u003cem\u003eMoptc5,\u003c/em\u003e ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1,\u003c/em\u003e ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2 \u003c/em\u003estrains cultured on the SDS, CR, CFW , and DTT.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;Analysis of\u003cstrong\u003e \u003c/strong\u003ethe inhibition rates of the individual strains due to the influence of the cell wall stress-inducing agents.(\u003cstrong\u003eC\u003c/strong\u003e) Western blotting analysis of Mps1 phosphorylation in ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e , and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2 \u003c/em\u003emutants in comparison to wild-type (Guy11) . The Error bars demonstrated the mean standard error from experimental replicates , Asteriks displayed the significance difference (** P\u0026lt; 0.01and *** P \u0026lt; 0.001 ; one-way ANOVA was used to analyze the data.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/6f1cde589bfa5e237c474292.png"},{"id":68836804,"identity":"1e9cbc83-befe-43b9-99fb-20e797c98367","added_by":"auto","created_at":"2024-11-12 14:29:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":200462,"visible":true,"origin":"","legend":"\u003cp\u003eDouble deletion of\u003cem\u003e MoPTC5\u003c/em\u003e with \u003cem\u003eMoPTC1 and MoPTC2 \u003c/em\u003edid not alter cell wall size or thickness. (\u003cstrong\u003eA\u003c/strong\u003e) Observation of cell wall thickness in hyphae harvested from Guy11, Δ\u003cem\u003eMoptc5\u003c/em\u003e, Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc\u003c/em\u003e mutants via transmission electron microscopy. (\u003cstrong\u003eB\u003c/strong\u003e) Bar Graphs presenting mycelial cell wall thickness for the WT strain and mutants. (\u003cstrong\u003eC\u003c/strong\u003e) Displaying expression levels of genes encoding for chitin synthase. The Actin gene was selected and used as a house keeping gene . Data are from three biological replicates. Error bars indicated standard deviations, and Asteriks demonstrated the significance differences.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/4a6c534fc8878829039b6be6.png"},{"id":68838364,"identity":"c65a25e8-c467-49b3-b3ae-fabb9ad9719f","added_by":"auto","created_at":"2024-11-12 14:45:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":309592,"visible":true,"origin":"","legend":"\u003cp\u003eTolerance\u003cstrong\u003e \u003c/strong\u003eof ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e \u003cem\u003eand \u003c/em\u003e∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e mutants to multiple hyperosmotic and oxidative stresses.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eMycerial growth of Guy11, ∆\u003cem\u003eMoptc5,\u003c/em\u003e ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1, \u003c/em\u003eand\u003cem\u003e \u003c/em\u003e∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2 \u003c/em\u003estrains on CM containng with oxidative and osmotic drugs.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eCalculation of\u003cstrong\u003e \u003c/strong\u003ethe inhibition rate of the mutants compared to Guy11 strains under the influence of osmotic and oxidative stresses. (\u003cstrong\u003eC\u003c/strong\u003e) Evaluation of MoOsm1 phosphorylation in Guy11, ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e , and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2 \u003c/em\u003emutants. Error bars represent the mean from replicates, one-way ANOVA was used for data analysis with multiple comparison tests in Graph Prism).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/4c66c75d30d34d51e890d364.png"},{"id":68837051,"identity":"ccde95ff-025c-4f0a-9c29-ebd9109209eb","added_by":"auto","created_at":"2024-11-12 14:37:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69038,"visible":true,"origin":"","legend":"\u003cp\u003eAppressorium formation rate of ∆\u003cem\u003eMoPtc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc\u003c/em\u003e1 and , ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e mutants, and wild-type strains on hydrophobic surface at 4 , 8 ,12 , and 24 hours post-inoculation. We used one- way \u0026nbsp;ANOVA to analyze the data with multiple comnparison tests in Graph prism 8).Asteriks indicated significance difference in comparison to wild type.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/ae014e7ce22ee06fafcfce3e.png"},{"id":68837050,"identity":"87fc964f-521f-4668-803a-e24780043be3","added_by":"auto","created_at":"2024-11-12 14:37:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":442094,"visible":true,"origin":"","legend":"\u003cp\u003eDouble gene deletion of \u003cem\u003eMoPTC5\u003c/em\u003ewith either \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e resulted in compromised turgor generation and\u003cstrong\u003e \u003c/strong\u003eglycogen mobilization during appressorium development in rice blast fungus. (\u003cstrong\u003eA,B\u003c/strong\u003e) Display appressoria images and percentages collapsed by treatment with glycerol . These appressoria were treated with different concentrations of glycerol solutions (1 M, 2 M, and 3 M), Collapse of the appressoria was observed and captured under a microscope.(\u003cstrong\u003eC,D\u003c/strong\u003e) illustrates the rate of glycogen mobilization in the indicated strains at 0, 4,8,12, and 24 hours time points during germination and appressorium formation. one-way ANOVA was used to analyze the data with multiple comparison tests in Graph Prism 8).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/fd8e93022301cadc5982e35e.png"},{"id":68836803,"identity":"90115e4e-d01c-43c1-8b6e-16f1ce6c8969","added_by":"auto","created_at":"2024-11-12 14:29:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":556470,"visible":true,"origin":"","legend":"\u003cp\u003eDouble deletion of MoPTC5 with either MoPTC1 or MoPTC2 underscores their redundant necessity for virulence in M. oryzae.(A,B) pathogenicity assay results and the corresponding lesion numbers observed on three-week-old rice seedlings (CO39 cultivar) by Guy11, ∆Moptc5, ∆Moptc5∆Moptc1, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e.(\u003cstrong\u003eC,D\u003c/strong\u003e) the infection assay conducted on barley leaves using mycelia plugs from Guy11, ∆\u003cem\u003eMoptc5\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e along with the quantification of lesion area. (\u003cstrong\u003eE) \u003c/strong\u003eThe development and \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003einvasion of hyphae in barley cells at different time points 12,24 , and 36 hours.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/169bb096ef6f5e0a84408428.png"},{"id":69218163,"identity":"198fe361-c56f-4cfa-a688-201636de8efd","added_by":"auto","created_at":"2024-11-18 06:47:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2910644,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/003f8ff2-94e4-4045-a8cf-ea12c4d9d023.pdf"},{"id":68836800,"identity":"9fb26297-bfef-46cf-a8bf-2540de17da9b","added_by":"auto","created_at":"2024-11-12 14:29:52","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":528417,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfoFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5080333/v1/e1e5e07a67720bccabc4c6c3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MoPtc5 plays synergistic roles with MoPtc1 and MoPtc2 in the vegetative growth, stress adaptation, and virulence of Magnaporthe oryzae","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eMagnaporthe oryzae\u003c/em\u003e is a \u0026nbsp;hemibiotrophic pathogen that threatens rice, wheat, and many other cereals production worldwide\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e. Early reports also demonstrated that rice blast fungus can infect some additional grass species, including\u003cem\u003e\u0026nbsp;Leersia\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Pinicum\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Setaria\u003c/em\u003e, and \u003cem\u003eLolium\u0026nbsp;\u003c/em\u003especies. \u003cem\u003eM. oryzae\u003c/em\u003e primarily causes a very devastating blast disease in rice, wheat, finger millet, and barley\u003csup\u003e3\u003c/sup\u003e that results in significant yield losses each year\u003csup\u003e4\u003c/sup\u003e. This fungus is accepted and recognized as a model organism for studying fungal and host interacting pathways\u003csup\u003e5\u003c/sup\u003e. Therefore, the availability of full genomic sequences of \u0026nbsp;pathotypes \u0026nbsp;and that of their hosts has made it more convenient to study fungal genomics\u0026nbsp;\u003csup\u003e6\u003c/sup\u003e. \u003cem\u003eM.oryzae\u003c/em\u003e infects the host via a structure known as appressoria. This structure accumulates turgor pressure, enabling the generation of mechanical force to penetrate plant cuticles using a \u0026nbsp; penetration peg and release effectors into the plant tissues\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e. Several signaling pathways have been demonstrated to play a crucial role in appressorium formation in M. oryzae(ref). These pathways are regulated by protein kinases and protein phosphatases (ref). \u0026nbsp;Rice blast poses a significant constraint to global cereals yield and extension services\u003csup\u003e10\u003c/sup\u003e. To manage and mitigate the severity of rice blast disease, integrated strategies have been implemented for durable crop resistance, including cultural practices, chemical control, and functional genomic analysis of host-pathogen interaction\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein phosphatases are very important components of cellular signaling pathways that regulate fungal development\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e.\u0026nbsp;Type 2C protein phosphatases (PP2C) are subdivided within two classes\u0026nbsp;such as, \u0026nbsp;phosphoprotein and\u0026nbsp;Mg\u003csup\u003e2+\u003c/sup\u003e-dependent protein phosphatases\u003csup\u003e15,16\u003c/sup\u003e. The functions of\u0026nbsp;PP2C, includingPtc1, Ptc2, Ptc5, Ptc6 , and Ptc7, have been discussed \u0026nbsp;extensively in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e \u003csup\u003e17\u003c/sup\u003e. Previous research indicated that Ptc2 primarily involved in the Hog pathway\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. Further studies also revealed that Ptc1 is involved in cell wall maintenance, conidiation, RNA splicing, and arrangement of subcellular organelles in \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003e\u003csup\u003e19-21\u003c/sup\u003e. Ptc2 and Ptc1 play critical roles in the dephosphorylating cyclin-dependent kinases, essential for the regulation of double-strand DNA breakdown by dephosphorylating Rad53 protein kinase\u0026nbsp;\u003csup\u003e22,23\u003c/sup\u003e. In \u003cem\u003eM. oryzae,\u003c/em\u003e mutants lacking both \u003cem\u003eMoPTC1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMoPTC2\u0026nbsp;\u003c/em\u003eexhibit defects in vegetative growth, virulence, protein phosphorylation, multistresses, and conidiogenesis\u0026nbsp;\u003csup\u003e24,25\u003c/sup\u003e. Additionally, a study in \u003cem\u003eAspergillus flavus\u003c/em\u003e demonstratedthatdephosphorylation of PGK1 via Ptc1 and Ptc2 \u0026nbsp;regulates \u0026nbsp;aflatoxin level and autophagy \u0026nbsp;assays.\u003csup\u003e26\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEarly Publication has shown that Ptc5 \u0026nbsp;protein is localized in the mitochondria of \u003cem\u003eS. cerevisiae\u003c/em\u003e,\u003cem\u003e\u0026nbsp;C. albicans\u0026nbsp;\u003c/em\u003e, and \u003cem\u003eM. oryzae\u0026nbsp;\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. However, a single deletion of \u003cem\u003eCaPTC5\u003c/em\u003e in \u003cem\u003eC. albicans\u003c/em\u003e did not affect tolerance towards various stress agents, including azoles\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e. In \u003cem\u003eArabidopsis,\u003c/em\u003e early reports showed that Ptc5 acts as a MAPK phosphatase, interacting precisely with MPK4, MPK6, and MPK3 in cells. Previous findings also indicated that in higher plants, Ptc5isinvolved in stomatal opening-closure, and regulating \u0026nbsp;ABA biosynthesis genes\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e. Additionally, double knockout of \u003cem\u003ePTC5\u003c/em\u003e along with other type 2C protein phosphatase genes in \u003cem\u003eS. cerevisiae\u003c/em\u003e revealed that PP2Cs are strongly important in responses to diverse abiotic stressors\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e. Recently, we revealed that \u003cem\u003ePTC5\u003c/em\u003e and \u003cem\u003ePTC7\u003c/em\u003e are important for stress maintenance, asexual reproduction and virulence in \u003cem\u003eM. oryzae\u0026nbsp;\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eType 2C protein phosphatases have been indicated to play overlapping and indispensable functions in filamentous fungi\u0026nbsp;\u003csup\u003e29,31\u003c/sup\u003e. We applied molecular biology approach to explore the synergistic roles of \u003cem\u003eMoPTC5\u003c/em\u003e along with \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e in \u003cem\u003eM. oryzae.\u003c/em\u003e Our study provided evidence that double mutation of\u0026nbsp;\u003cem\u003eMoPTC5\u0026nbsp;\u003c/em\u003ewith \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u0026nbsp;\u003c/em\u003esynergistically altered hyphal growth, fungal sporulation, cell wall integrity, regulation of MoMps1 and MoOsm1 phosphorylation , and weakened the virulence in \u003cem\u003eM. o\u003c/em\u003e\u003cem\u003eryzae.\u003c/em\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses and identification of MoPtc1, MoPtc2,and MoPtc5\u003c/h2\u003e \u003cp\u003eThe homologs of Ptc5, Ptc1, and Ptc2 in blast fungus were identified from the fungidb website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://fungidb.org/fungidb/\u003c/span\u003e\u003cspan address=\"http://fungidb.org/fungidb/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) via BlastP search, based on the amino acid sequences from \u003cem\u003eS. cerevisiae\u003c/em\u003e, and were termed MoPtc5, MoPtc1, and MoPtc2, respectively. Domain annotation of the proteins was generated using IBS software, while MEGAx software was used to build the phylogenetic tree with bootstrap analysis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The analysis demonstrated that MoPtc5, MoPtc1, and MoPtc2 possess two conserved domains (PPCC2 domain and PP2C_SIG domain) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, phylogenetic assay indicated that MoPtc1 and MoPtc2 are most closely related to their orthologs in \u003cem\u003eNeurospora crassa\u003c/em\u003e, while MoPtc5 has the closest homology to \u003cem\u003eN. crassa\u003c/em\u003e and \u003cem\u003eBotrytis cinera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc5\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc1\u003c/b\u003e \u003cb\u003eand ∆\u003c/b\u003e\u003cb\u003eMoptc5\u003c/b\u003e\u003cb\u003e∆\u003c/b\u003e\u003cb\u003eMoptc2\u003c/b\u003e \u003cb\u003estrongly affect hyphal growth and sporulation of\u003c/b\u003e \u003cb\u003eM. oryzae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe initially conducted qRT-PCR and observed significant up-regulation of \u003cem\u003eMoPTC2\u003c/em\u003e and \u003cem\u003eMoPTC1\u003c/em\u003e in the \u003cem\u003eMoPTC5\u003c/em\u003e mutant (Supplementary Figure S2), suggesting a redundancy of \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e in \u003cem\u003eM. oryzae\u003c/em\u003e. Subsequently, double genes knockout of \u003cem\u003eMoPTC5\u003c/em\u003e with either \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e was generated in the ∆\u003cem\u003eMoptc5\u003c/em\u003e mutant, by replacing the ORF (Open reading flame) of \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e with neomycin resistance gene cassette. Culturing wild-type strain Guy11, mutant strains ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e, along with the complemented strain on CM revealed significantly reduced hyphae growth of the ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e double mutants compared to ∆\u003cem\u003eMoptc5\u003c/em\u003e and Guy11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). Conidiation was remarkably reduced in ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and remarkably reduced in ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e in comparison to ∆\u003cem\u003eMoptc5\u003c/em\u003e and wild-type cultured on solid rice bran media (RBM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistent with this result, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e failed to produce conidiophores at diverse experimental time points 12, 24, and 36 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, the findings suggest the overlapping role of \u003cem\u003eMoPTC5\u003c/em\u003e with \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e in hyphal growth and asexual reproduction of blast fungus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMoPTC5 plays synergistic functions with MoPTC1 and MoPTC2 in cell wall maintenance and Mps1 phosphorylation level\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the roles of MoPtc5 in conjunction with MoPtc1 or MoPtc2 in regulating cell wall maintenance of \u003cem\u003eM. oryzae\u003c/em\u003e, we analyzed the growth inhibition rate of mutants grown on CM containing with different stress drugs including, DTT, SDS, CFW, and CR for ten days of incubation at 28\u003csup\u003eo\u003c/sup\u003eC respectively. Results showed that the growth of ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e on CM supplemented with SDS, CR, CFW, and DTT was significantly inhibited. Especially, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e displayed a higher sensitivity response compared to other mutants when spotted on CM supplemented with SDS, CR, and CFW (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B). We then performed a western blotting to evaluate the activation of MoMps1 in the mutants. The results indicated an elevation of the phosphorylation rater of MoMps1 protein in ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The findings demonstrate that \u003cem\u003eMoPTC5\u003c/em\u003e/\u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC5\u003c/em\u003e/\u003cem\u003eMoPTC2\u003c/em\u003e are synergistically involved in the integrity of cell wall and phosphorylation of MoMps1 in \u003cem\u003eM.oryzae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDouble knockout of MoPTC5 with MoPTC1 and MoPTC2 did not influence the cell wall thickness of rice blast fungus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cell wall serves as a robust protection safeguarding the plasma membrane and intracellular contents of plants and many microbes \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To investigate the potential involvement of MoPtc5 along with MoPtc1 or MoPtc2 in the cell wall thickness of blast fungus, we cultured strains on CM for three days. The fungal hyphae were then harvested, washed with ddH\u003csub\u003e2\u003c/sub\u003eO, dried with clean filter papers, and preserved in phosphate buffer. Transverse sections of hyphal cell walls were imaged using electron transmission microscopy (TEM). However, we observed no discernible difference in the thickness of cell walls among the wild-type strains, Δ\u003cem\u003eMoptc5\u003c/em\u003e, Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e, and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc2\u003c/em\u003e mutants (Fig. 4A,B). Chitin represents a vital constituent of fungal cell walls. Hence, we conducted further analysis on the transcription levels of chitin synthases in \u003cem\u003eM. oryzae\u003c/em\u003e through qRT-PCR to gain a clearer understanding of how these proteins regulate cell wall stress. Our results showed a remarkably decrease expression of the chitin synthase-encoding genes in the mutants compared to wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc5\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc1\u003c/b\u003e \u003cb\u003eand ∆\u003c/b\u003e\u003cb\u003eMoptc5\u003c/b\u003e\u003cb\u003e∆\u003c/b\u003e\u003cb\u003eMoptc2\u003c/b\u003e \u003cb\u003eplay important roles in osmotic, oxidative stress and MoOsm1 phosphorylation in\u003c/b\u003e \u003cb\u003eM.oryzae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the functional redundancy of \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e in \u003cem\u003eM. oryzae\u003c/em\u003e under oxidative and hyperosmotic stresses, we cultured Guy11, Δ\u003cem\u003eMoptc5\u003c/em\u003e, Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e, and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc2\u003c/em\u003e on CM supplemented with osmotic and oxidative stressors drugs including, potassium chloride (1 M KCl), sodium chloride (1M NaCl), 5 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Growth measurements were recorded after ten days post-incubation. Our findings demonstrated that both Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc2\u003c/em\u003e exhibited higher sensitivity to hyperosmotic and oxidative stress induced by 1M NaCl, 1M KCl, 5mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in comparison to the Guy11 and Δ\u003cem\u003eMoptc5\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B). A Previous study demonstrated that double deletion of ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc7\u003c/em\u003e reduced MoOsm1phosphplylation level. Consequently, we conducted western blotting assay to quantify the relative abundance of the osmoregulatory protein MoOsm1 in ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e, and Guy11. Our results revealed a low abundance of MoOsm1 in ∆\u003cem\u003eMoptc5\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e mutants but not in Δ\u003cem\u003eMoptc5Moptc2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Altogether, we proposed that \u003cem\u003eMoPtc5\u003c/em\u003e, in conjunction with MoPtc1 and MoPtc2, significantly contributes to the hyperosmotic and oxidative tolerance of \u003cem\u003eM.oryzae\u003c/em\u003e, as well as MoOsm1 phosphorylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc5\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc1 and\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e\u003cb\u003eMoptc5\u003c/b\u003e\u003cb\u003e∆\u003c/b\u003e\u003cb\u003eMoptc2\u003c/b\u003e \u003cb\u003emutants are remarkably delayed in appressoria formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe appressorium serves as the primary infectious structure facilitating the penetration of rice blast fungus. Therefore, to examine \u003cem\u003eMoPTC5\u003c/em\u003e\u0026rsquo;s role alongside \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e in appressorium formation, conidial suspensions of ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e, and guy11 (wild type) were initially placed on hydrophobic coverslips. Appressoria formation was checked and viewed under a microscope at 4, 8 ,12, and 24 hours post-inoculation, respectively. Findings generated from this assay indicated that both double gene deletion mutants failed to develop appressorium at 4 h post-inoculation. However, the appressorium formation rate of ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e mutants gradually increases over time until comparable to the Guy11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).Taken together, results demonstrated that the deletion of \u003cem\u003eMoPTC5\u003c/em\u003e, along with \u003cem\u003eMoPTC1\u003c/em\u003e or \u003cem\u003eMoPTC2\u003c/em\u003e, merely delayed the appressorium formation of \u003cem\u003eM.oryzae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMoPTC5, MoPTC1,and MoPTC2\u003c/b\u003e \u003cb\u003esynergistically influence appressoria turgor generation and utilization of glycogen in\u003c/b\u003e \u003cb\u003eM. oryzae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo ascertain the synergistic roles of MoPtc5 in tandem with MoPtc1 and MoPtc2 during appressorium penetration in host epidermal cells, the appressorium turgor generation across all mutants and the wild type was examined, respectively. Conidia sourced from the aforementioned strains were inoculated onto hydrophobic coverslips, followed by exposure to varying concentrations of glycerol after 24 h. The percentage of collapsed appressoria were viewed and monitored under a microscope. Our findings demonstrated that a significant number of the appressorium produced by Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc2\u003c/em\u003e were collapsed under diverse glycerol concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B). Moreover, appressorium formation of rice blast fungus necessitates the efficient transfer of nutrients, such as glycogen and lipid droplets, from conidia, acting as essential material sources for turgor generation post-maturation. Effective nutrient transport profoundly influences the virulence of blast fungus. Thus, to assess glycogen mobilization from conidia to appressoria, conidia were harvested and incubated at 28\u003csup\u003eo\u003c/sup\u003eC for 2, 4, 8, 12, and 24 hours respectively, KI/I\u003csub\u003e2\u003c/sub\u003e was used to stain conidia before imaged under a microscope. The Findings unveiled a delaying of gylcogen mobilization from conidium to appressoria in the double mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC,D). In summation, these findings underscore the pivotal contribution of MoPtc5 alongside MoPtc1 and MoPtc2 in appressorium turgor generation, primarily through the regulation of glycogen utilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc5\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e \u003cb\u003eMoptc1 and\u003c/b\u003e \u003cb\u003e∆\u003c/b\u003e\u003cb\u003eMoptc5∆Moptc2\u003c/b\u003e \u003cb\u003estrains weakened virulence to infect rice leaves\u003c/b\u003e\u003c/p\u003e \u003cp\u003eProtein phosphorylation is an essential mechanism for infection-related morphogenesis\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Morever, the impact of \u003cem\u003eM. oryzae\u003c/em\u003e protein phosphatase genes on virulence remains inadequately explored. To elucidate the combined effect of double mutations in \u003cem\u003eMoPTC5\u003c/em\u003e along with \u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e on fungal virulence, we harvested spores from Guy11, ∆\u003cem\u003eMoptc\u003c/em\u003e5, ∆\u003cem\u003eMoptc\u003c/em\u003e5∆\u003cem\u003eMoptc\u003c/em\u003e1, and ∆\u003cem\u003eMoptc\u003c/em\u003e5∆\u003cem\u003eMoptc\u003c/em\u003e2 and sprayed on the rice seedlings. A striking decrease in pathogenicity was examined in the double mutants in comparison to ∆\u003cem\u003eMoptc\u003c/em\u003e5 and Guy11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B). Similarly, a significant reduction in virulence on barley leaves was evident using mycelial plugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC,D). Further analysis revealed a lower penetration rate of hyphae in cuticle cells of barley at different time points for the double gene deletion mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Collectively, our findings demonstrated the collaborative influence of MoPTC5, MoPTC1, and MoPTC2 on the virulence of \u003cem\u003eM. oryzae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main purpose of this research was to reveal the synergistic functions of PP2C MoPtc5 along with MoPtc1 and MoPtc2 in the virulence and development of blast fungus ,\u003cem\u003eM.oryzae\u003c/em\u003e. To achieve this purpose, we initially deleted \u003cem\u003eMoPTC5\u003c/em\u003e and subsequently knocked out MoPTC1 and MoPTC2 in ∆\u003cem\u003eMoptc5\u003c/em\u003e mutant, respectively. Our investigations indicated that all the mutant strains were viable on different growing mediums. Phylogenetic assay showed a close relationship among MoPtc5, MoPtc1, and MoPtc2 proteins and their homologs in other filamentous fungi, suggesting common ancestry.\u003c/p\u003e \u003cp\u003eSpores formation is critical in filamentous fungi, with spores or conidia developing on vegetative hyphal structures known as conidiophores \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It was previously reported that ∆\u003cem\u003eMoptc5\u003c/em\u003e single mutant demonstrated a slight decrease in fungal growth and sporulation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Similarly, MoPtc1 and MoPtc2 are involved in \u003cem\u003eM.oryzae\u003c/em\u003e growth and sporulation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this study, our findings revealed a very significant reduction in conidiation of ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e double mutants, suggesting a synergistic role of \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e in sporulation regulation. This might be due to various environmental conditions including, aeration or low expression levels of conidiation-related genes which affect metabolic and physiological mechanisms that happen during sporulation \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cell wall is important for cellular integrity and shielding against environmental signals \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In our research, we evaluated the impact of diverse cell wall stressors drugs, revealing higher sensitivity in the double mutant ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e compared to ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and the wild-type strains. Previous research had indicated slight sensitivity of ∆\u003cem\u003eMoptc5\u003c/em\u003e to cell wall stressors. \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Our findings demonstated that \u003cem\u003eMoPTC5\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e play overlapping roles in stress maintenance, consistent with prior studies. \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Western blot analysis confirmed that ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e are crucial for the phosphorylation of Mps1, suggesting their involvement in MoMps1 phosphorylation activation in \u003cem\u003eM. oryzae\u003c/em\u003e. Conclusively, MoPtc5, MoPtc1, and MoPtc\u003cem\u003e2\u003c/em\u003e synergistically regulate cell wall maintenance and participate in signal transduction pathways essential for cell wall maintenance.\u003c/p\u003e \u003cp\u003eChitin was reported as an abundant component of fungal and insect cell walls, which protect the organisms against environmental stimuli \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In our study, we investigated and measured the cell wall sizes of ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e and the wild-type strains. The results unveiled combined effects of \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e had no direct influence on the fungal cell wall thickness, which is inline with early result \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Morever, we observed downregulation of chitinase genes in the double mutants, suggesting that \u003cem\u003eMoPTC5\u003c/em\u003e in conjugation with \u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e negatively regulates the expression of chitinase encoding genes, possibly through shared transcription factors for these orthologous genes \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, we investigated the synergistic functions of \u003cem\u003eMoPTC5\u003c/em\u003e with \u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e in osmotic and oxidative responses. The double mutants ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e showed a higher inhibition rate on media containing 1M NaCl and 1M KCl \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Here, we speculated that \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e might be required in the co-regulation of the cell sensitivity to multiple osmoregulatory agents and their pathways in the fungal development. Furthermore, the increased sensitivity of ∆\u003cem\u003eMoptc5∆Moptc2\u003c/em\u003e to oxidative stress (10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) suggests that these genes play important functions in the tolerance of oxidative and osmotic stresses \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To establish the genetic redundancy of these three protein phosphatases, we also checked the phosphorylation level of the osmoregulatory protein MoOsm1 in the single and double mutants. We found that MoOsm1 phosphorylation level significantly decreased in ∆\u003cem\u003eMoptc5\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, and here in, we speculated that \u003cem\u003eMoPTC5\u003c/em\u003e and \u003cem\u003eMoPTC1\u003c/em\u003e might negatively regulate MoOSM1 phosphorylation in \u003cem\u003eM. oryzae.\u003c/em\u003e Generally, we concluded that the type 2C protein phosphatases are functionally redundant towards osmotic, oxidative stresses and are involved in the activation and regulation of osmolality glycerol pathways.\u003c/p\u003e \u003cp\u003ePathogenicity is crucial in fungi-host interaction. Some factors are important for the establishment of fungal virulence including, conidia attachment, germination on the surface of the host, and its colonization \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In this work, we assayed the pathogenicity levels of the various strains on susceptible rice cultivars and barley. Remarkably, we revealed a severe defect in virulence of the double mutants ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e. Our findings also indicated a high reduction of hyphal penetration and invasion of barley epidermal cells in double mutants. Previous publications showed that the single mutants ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc2\u003c/em\u003e are required in the pathogenesis of the rice blast fungus ,\u003cem\u003eM.oryzae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In this research, we reasoned that PP2C, type 2C protein phosphatases \u003cem\u003eMoPTC5\u003c/em\u003e, \u003cem\u003eMoPTC1\u003c/em\u003e, and \u003cem\u003eMoPTC2\u003c/em\u003e complementarily regulate fungal virulence.\u003c/p\u003e \u003cp\u003eThe double mutants ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e are impaired in appressorium formation at 4 h and 8 h after inoculation on hydrophobic coverslips, compared to the single mutant ∆\u003cem\u003eMoptc5\u003c/em\u003e and the WT strains. This might be possibly caused by hyperactivation of Pmk1 pathway which has previously been revealed to cause abnormalities in appressorium morphogenesis and pathogenicity \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. From these results, we suggested that \u003cem\u003eMoPTC5\u003c/em\u003e in collaboration with \u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e synergistically regulates appressorium formation and maturation. Appressorium turgor pressure was also examined in various strains. We observed a higher percentage of collapsed appressoria and slowed glycogen mobilization and utilization from conidia to appressoria in ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e, ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e than in ∆\u003cem\u003eMoptc5\u003c/em\u003e and wild-type. However, is still unclear about the pathways regulating the movement of conidium content to mature appressorium. Findings of incipient cytorrhysis are consistent with the defects of penetration and hyphae invasion in barley epidermal cells, which is also in consistent with early studies \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In summary, Rice blast disease is severe across the world, ultimately leading to a loss of 10% -30% for rice, wheat, and other cereals globally. This investigation carried out on PP2C, the type 2C protein phosphatases provided evidences of the synergy of \u003cem\u003eMoPTC5\u003c/em\u003e with \u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC2\u003c/em\u003e in the regulation of sporulation rate, hyphal growth, multi-stress adaptation, and virulence of \u003cem\u003eM. oryzae\u003c/em\u003e. Conclusively, this research revealed new insights into the development and pathogenesis of \u003cem\u003eM. oryzae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenes knockout and complementation assays\u003c/h2\u003e \u003cp\u003eThe split marker assay was employed to knockout \u003cem\u003eMoPTC5\u003c/em\u003e in \u003cem\u003eM. oryzae\u003c/em\u003e. Downstream and upstream fragments A and B were amplified respectively. These fragments were then fused with hygromycin flagments (HA and HB) by using SOE PCR. The ligated vectors were transformed into Guy11 (wild-type) protoplasts as presented in the early report \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The grown transformants were screened and confirmed by PCR. To get ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc1\u003c/em\u003e and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e double mutant constructs, flanking fragments A and B of MoPtc1 and MoPtc2 were amplified and ligated with the Neomycin resistant gene (NE and EO). These ligated constructs were then transformed into ∆\u003cem\u003eMoptc5\u003c/em\u003e mutant protoplasts and positive transformants were selected on terrific broth 3 growing media (TB3) containing neomycin and colonies were verified by PCR. All primers used in this study are listed in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFungal culturing assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eM. oryzae\u003c/em\u003e Guy11 was employed in this research as a wild-type strain. Complete medium, starch yeast medium, and rice bran medium were used for culturing fungi. The hyphae morphology and growth assays were conducted as described in \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To discuss the influence of cell wall ,oxidative, and osmotic drugs on mutants in comparison to Guy11. The strains were spotted on CM media containing with 200 \u0026micro;g/mL Congo red, 200 \u0026micro;g/mL CFW, 0.01% SDS ,1M of sodium chrolide, 1 M of potassium chrolide, and 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e respectively. The strains were kept in incubator at 28℃, measurement of mycerial growth, and pictures were done after ten days post-inoculation. To ascetain the sporulation rate, mycelia plugs were grown on rice bran media at 28 ℃ and exposed to light. After ten days conidia were counted using light microscopy.\u003c/p\u003e\n\u003ch3\u003ePathogenicity, host penetration, and analysis of appressoria turgor pressure\u003c/h3\u003e\n\u003cp\u003eFor pathogenicity, Fungi were grown on solid rice bran media within ten days at 28 ℃, later the spores were collected, washed and the conidia suspension adjusted to 5x10\u003csup\u003e4\u003c/sup\u003e spores and sprayed onto the rice seedlings (CO-39), The seedlings were incubated at 25℃ and disease signs were monitored and examined after a week days. To investigate the host penetration potential of the mutants, 10 \u0026micro;L of conidial (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e spores/mL) from the mutants and guy11 were inoculated on barley leaves. The hyphae penetration rate were checked at 30, 48, and 60 hours post-inoculation. The Nikon microscope (Nikon, Nippon Kogaku kogyo kabushikigaisha optical industries Co., Ltd, Tokyo, Japan) were used to view hyphal growth in fungal epidermal cells. Incipient cytorrhysis (Appressoria collapsed due to loss of internal hydraulic turgor pressure) analyses were carried out to investigate generation of the appressorium turgor pressure in ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc\u003c/em\u003e5∆\u003cem\u003eMoptc1\u003c/em\u003e, ∆\u003cem\u003eMopt5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e, ∆\u003cem\u003eMoptc5_C\u003c/em\u003e, and wild-type strains. Conidia suspensions were dropped on hydrophobic coverslips (Fisher Scientific, Pittsburgh, PA 15275), and within 24 h conidia were mixed with glycerol solutions 1 M, 2 M, and 3 M respectively. Incipient cytorrhysis and glycogen mobilization were monitored under a microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction ,quantitative real time PCR (qRT-PCR), and RT-PCR assays\u003c/h2\u003e \u003cp\u003eThe mutants and wild type strains (Guy11, ∆\u003cem\u003eMoptc5\u003c/em\u003e, ∆\u003cem\u003eMoptc\u003c/em\u003e5∆\u003cem\u003eMoptc1\u003c/em\u003e, and ∆\u003cem\u003eMoptc5\u003c/em\u003e∆\u003cem\u003eMoptc2\u003c/em\u003e) were inoculated in liquid CM, and shaken machine at 110 rpm, 28\u003csup\u003eo\u003c/sup\u003eC for 3 days period. Mycelia were collected, washed with ddH\u003csub\u003e2\u003c/sub\u003eO, dried, and crushed into fine powder by using motar in the nitrogen solution. The extraction of total RNA from each strain was done following the protocol of an Eastep TM extraction kit (Promega, Beijing Biotech Co.Ltd, China). The quantitative real-time PCR (qRT-PCR) was conducted following Promega Super Real Premix kit instructions. The qRT-PCR was performed in an Eppendorf Realplex2 master cycler (Eppendorf China Ltd. Shanghai, China ), and analysis was done using (2 \u003csup\u003e\u0026minus; ΔΔCT\u003c/sup\u003e) as previously reported by \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSouthern blotting assay\u003c/h3\u003e\n\u003cp\u003eFor the Southern blotting, We firstly extracted fungal genomic from fungal strains using the cetytrimethylammonium bromide method (CTAB method). The digestion of the genomic DNA was done by using specific restriction enzymes PstI and ClaI respectively (New England Biolab Co., Ltd Beijing, China). Digested DNA product was separated by running a gel electrophorensis assay. The gel was later moved to a specific membrane (Merck) which is positively charged. Nucleic acid probes with sequences complementary to the region of targeted fragments were used for hybridization. During hybridization, the labeled probes were incubated overnight together with the DNA in a hybridizer machine at 42\u0026deg;C to promote the hybridization of the complementary sequences. Unhybridized probes were removed by washing the membrane with 2\u0026times; SSC\u0026thinsp;+\u0026thinsp;SDS and with maleic acid\u0026thinsp;+\u0026thinsp;tween 20 solutions. Detection starter kit I (Merck KGa, Germany ,Darmstadt ) and images were captured using Tanon 5200 chemiluminescent imaging machine (Tanon Science \u0026amp;Technology Co.Ltd, China, Shangahai).\u003c/p\u003e\n\u003ch3\u003eExtration of proteins and western blotting assays\u003c/h3\u003e\n\u003cp\u003eThe fungal mycelia plugs were cultured in the liquid CM media and shaken for 2\u0026ndash;3 days at 28\u0026deg;C after that mycelia were collected and crashed powder by using motar and then 1\u0026ndash;2 g of the mycelial powder was resuspended in 1 mL of protein lysis mixed with 10 \u0026micro;L of phenylmethyl sulphonyl fluoride (PMSF) and 10 \u0026micro;L of a proteinase inhibitor. The mixed solution was incubated in ice for a half hour and vortexed by inverting tubes within every 10 min, and afterward, the samples were centrifuged for 20 min at 4\u0026deg;C the the supernatants were sucked and added into 2 mL tubes, lastly sodium dodecyl sulfate buffer (SDS buffer) was added before storage at -21\u0026deg;C for future use. The western blot assay, SDS -PAGE (10% polyacrylamide) assay was used for the separation of proteins. The targeted proteins were detected using the primary antibodies (P-p38 MAPK, and P44/42MAPK) along with conjugated secondary antibodies (Goat antirabbit\u0026amp; mouse IgG-HRP) and an anti-beta actin mab was utilized as a control antibody. Finally, the protein phosphorylation signals were detected following the instructions of a western blotting Kit (Epizyme, SQ201, Shanghai, China), and photographs were taken by Imaging System (Tanon Science \u0026amp;Technology Co.Ltd,China Shanghai).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic examination assays\u003c/h2\u003e \u003cp\u003eConidiophores development, conidia formation, appressorium formation, invasive hyphae, conidia germination on the hydrophobic slides, glycogen mobilization in appressoria, and appressorial turgor generation were viewed under a Nikon TiE system (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eNo conflict of interests were declared by authors.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eZ. W., W. T., Y. H., and J. B. designed the study plan, applied funding, and critically revised the manuscript; J. B., F. J. O., Z. Y., M. C., A. M. W. conducted the experiments and discussed about materials and methodology; J. B., R. Z analyzed all data and wrote the draft manuscript; J. B., A. F., N. O., Y. A., N. A. worked on editing of the final copy. All authors have approved the final manuscript copy.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eFoundation of Natural Science of the Fujian Province (2022J01129) , Fujian provincial Science and Technology (2022NZ030014) and Exploratory Science, Technology Innovation Project of Fujian Academy of Agricultural Sciences (ZYTS202402) and Basic Research Special Project of Public Welfare Research Institutions in Fujian Province (2024R1022003) supported our study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTalbot, N. J. On the trail of a cereal killer: exploring the biology of \u003cem\u003eMagnaporthe grisea\u003c/em\u003e. \u003cem\u003eAnn. Rev. 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Scie\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 748120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.748120\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.748120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Magnaporthe oryzae, synergistic, stress tolerance, protein phosphatases, pathogenesis","lastPublishedDoi":"10.21203/rs.3.rs-5080333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5080333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein phosphatases are crucial enzymes that regulate key cellular processes such as cell cycle, gene transcription, and translation in eukaryotes. Seven PP2C protein phosphatases have been identified in \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e. However, their synergistic roles in the pathology and physiology of \u003cem\u003eM. oryzae\u003c/em\u003e remain poorly investigated. By qRT-PCR analysis we found that PTC1 and PTC2 are significantly upregulated in the PTC5 deletion mutant. Double deletion of \u003cem\u003eMoPTC5\u003c/em\u003e/\u003cem\u003eMoPTC1\u003c/em\u003e and \u003cem\u003eMoPTC5\u003c/em\u003e/\u003cem\u003eMoPTC2\u003c/em\u003e genes significantly reduced hyphal growth, conidiophore formation, sporulation, and virulence in \u003cem\u003eM. oryzae\u003c/em\u003e. In addition, the double knockout mutants were increasingly sensitive to different osmotic, oxidative, and cell wall stresses. Western blot analysis revealed that MoPtc5 plays a synergistic function with MoPtc1 and MoPtc2 in the regulation of MoMps1 and MoOsm1 phosphorylation levels. Lastly, appressorium formation and turgor generation were remarkably affected in the Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc1\u003c/em\u003e and Δ\u003cem\u003eMoptc5\u003c/em\u003eΔ\u003cem\u003eMoptc2\u003c/em\u003e double deletion mutants. These findings demonstrate the synergistic roles of PP2c protein phosphatase in the fungal development and pathogenesis of \u003cem\u003eM. oryzae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"MoPtc5 plays synergistic roles with MoPtc1 and MoPtc2 in the vegetative growth, stress adaptation, and virulence of Magnaporthe oryzae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-12 14:29:47","doi":"10.21203/rs.3.rs-5080333/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9755a77d-0d2e-4cca-9c66-fc63d9d92316","owner":[],"postedDate":"November 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39432907,"name":"Biological sciences/Microbiology/Fungi"},{"id":39432908,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-11-18T06:39:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-12 14:29:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5080333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5080333","identity":"rs-5080333","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}