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/lp/springer-journals/a-lipid-droplet-associated-protein-nem1-regulates-appressorium-0XQpCb0kqV
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- Springer Journals
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- Copyright © The Author(s) 2023
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- 2662-1738
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- 10.1007/s42994-023-00098-5
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Abstract
aBIOTECH https://doi.org/10.1007/s42994-023-00098-5 aBIOTECH RESEARCH ARTICLE A lipid droplet-associated protein Nem1 regulates appressorium function for infection of Magnaporthe oryzae 1 1 2 3 1 Deng Chen , Xuan Cai , Junjie Xing , Shen Chen , Juan Zhao , 1 1 1 1 1 Zhiguang Qu , Guotian Li , Hao Liu , Lu Zheng , Junbin Huang , 1& Xiao-Lin Chen State Key Laboratory of Agricultural Microbiology and Provincial Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China Received: 23 November 2022 / Accepted: 1 February 2023 Abstract Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection. However, the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown. Here, in Magnaporthe oryzae, we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization. Nem1 was highly expressed in conidia, but lowly expressed in appressoria, and its encoded protein was localized to lipid droplets. Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol (DAG) or triacylglycerol (TAG). NEM1 was required for asexual development especially conidia production. The Dnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth. Remarkably, Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process. The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1. Together, our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus. Keywords Lipid biogenesis, TOR signaling, cAMP-PKA signaling, Conidium formation, Appressorium, Magnaporthe oryzae INTRODUCTION eukaryotic cells, are important organelles responsible for storing and providing lipid to meet required energy Most eukaryotic cell organelles are enclosed by a (Graef 2018; Henne et al. 2019). LDs are often consid- membrane consisted of proteins and a lipid bilayer ered originally form the endoplasmic reticulum (ER) (Pillai et al. 2017). The major components of the lipid membrane and are evolutionarily conserved. The main bilayer are phospholipids which play a crucial role in forms of lipids stored in LDs are neutral lipids con- membrane biogenesis and lipid metabolism (Eastmond taining triacylglycerols (TAGs) and/or sterol esters et al. 2010; Nakamura et al. 2009; Pillai et al. 2018). (SEs). Phosphatidate (PA) is the key precursor of lipid Cytoplasmic lipid droplets (LDs), existing in almost all metabolism (Henne et al. 2019; Karanasios et al. 2013). On the one hand, dephosphorylation of PA produces diacylglycerol (DAG) which can be acylated to TAG to be & Correspondence: chenxiaolin@mail.hzau.edu.cn (X.-L. Chen) The Author(s) 2023 aBIOTECH stored in rich nutrients. On the other hand, PA can be from adhesion of conidia on the host plants. In proper converted to cytidine diphosphate diacylglycerol (CDP- environment, conidia geminate to form a specialized DAG), which forms membrane phospholipids including structure known as appressorium. The appressorium phosphatidylserine (PS), phosphatidylethanolamine accumulates enormous turgor pressure to form a pen- (PE), and phosphatidylcholine (PC) to promote cell etration peg to rupture plant cuticles. Inside the host growth and proliferation (Barbosa et al. 2015; Dubots cell, the primary invasive hypha will differentiate to et al. 2014; Karanasios et al. 2013). Because phospho- bulbous biotrophic invasive hypha for expansion and lipids and TAG share the same precursor, it is necessary blast lesions formed with numerous conidia at the late for cells to coordinate the two pathways in which many necrotrophic stage (Fernandez et al. 2018; Martin- important proteins are involved. Urdiroz et al. 2016; Wilson et al. 2009). Lipins, large proteins mainly found in the cytosol, are The appressorium formation and maturation is a key 2? Mg -dependent phosphatidate phosphatases, which process required for successful infection. Several sig- catalyzes the conversion from PA to DAG by promoting naling pathways have been reported to play vital roles dephosphorylation of PA (Santos-Rosa et al. 2005). In in the appressorium formation, including cAMP-PKA, mammals, three LIPIN genes, LIPIN1-3, were identified Pmk1-MAPK, and TOR signaling pathways (Wilson et al. to have different but overlapping expression patterns. 2009). During the appressorium formation and matu- Disrupting lipins genes led to lipid metabolic disorders, ration, a series of cellular processes, such as utilization rhabdomyolysis, peripheral neuropathy, and inflamma- of lipids, are activated (Chen et al. 2017; Eseola et al. tion (Chen et al. 2015; Santos-Rosa et al. 2005). In 2021; Kubo 2013; Yan et al. 2016). However, the Caenorhabditis elegans, low lipin expression affected the underlined mechanism linked lipid biogenesis in coni- dynamics of the peripheral ER and nuclear envelope dium and the utilization in appressorium remain to be (Jung et al. 2020). In Saccharomyces cerevisiae, a single revealed. In this study, a M. oryzae lipid droplet protein lipin orthologue named PAH1 was found. Loss of PAH1 Nem1 was identified and found to be involved in lipid in yeast led to slow growth, abnormal expansion of droplets formation during conidia formation and lipid nuclear/ER membrane, and disordered lipid metabo- utilization during appressoria formation. Importantly, lism (Fang et al. 2014; Hsu et al. 2021; Kwiatek et al. we found that Nem1 was by both the TOR signaling 2020). Activated PAH1 required to be dephosphorylated pathway and the cAMP-PKA signaling pathway. Further, by the nuclear/ER membrane-associated protein phos- a phosphorylation site of Nem1 at Ser303 found to be phatase complex consisting of Nem1 (catalytic subunit) regulated by cAMP-PKA signaling pathway and neces- and Spo7 (regulatory subunit) (Liu et al. 2019). The two sary for full function of Nem1. Our results revealed a subunit proteins both possess two transmembrane- novel regulatory mechanism of lipid droplets during the spanning domains. Nem1 binds to Spo7 through its appressorium formation of M. oryzae. conserved C-terminal domain, and this association is responsible for the formation of the complex in the membrane bilayer (Siniossoglou et al. 1998). Nem1 is a RESULTS member of the haloacid dehalogenase superfamily, and its phosphatase activity depends on the DXDX(T/V) Identification of NEM1 catalytic motif within its HAD-like domain. Spo7, which binds to the catalytic domain of Nem1, is essential for In S. cerevisiae, a protein named Nem1 (nuclear envel- the activity of the phosphatase complex (Dubots et al. ope morphology protein 1) is found to be required for 2014; Kim et al. 2007). Nem1-Spo7-mediated dephos- Pah1 dephosphorylation or activation and is essential phorylation of Pah1 activates the catalytic efficiency of for lipid metabolism and TAG synthesis (Bahmanyar Pah1, but meanwhile primes it for proteasome-depen- 2015; Karanasios et al. 2013; Liu et al. 2019; Zhang et al. dent degradation. Therefore, dephosphorylated Pah1 is 2018). Through a BLAST search, we identified a both active and unstable, which is likely to be a con- homologous protein of Nem1 in M. oryzae, MGG_06001. straint step preventing excess PA into the synthesis of M. oryzae Nem1 contains 536 amino acids with a long TAG (Bahmanyar 2015; Karanasios et al. 2013; Zhang CPDc (catalytic domain of ctd-like phosphatases) et al. 2018). The function of the phosphatase Nem1- domain near its C-terminus (Fig. 1A). Phylogenetic Spo7 axis is important, but the underlined upstream analysis shows that Nem1 is highly conserved among regulatory mechanism is largely unknown. fungi especially the plant pathogenic fungi, such as Magnaporthe oryzae, the causal agent of rice blast, Fusarium oxysporum, Gaeumannomyces tritici, and Ver- leads to severe yield loss of rice every year (Dean et al. ticillium dahliae (Fig. 1B). 2012; Ebbole 2007). Infection of this ascomycete starts The Author(s) 2023 aBIOTECH Fig. 1 Protein property of Nem1. A Protein domain analysis of Nem1. CPDc, catalytic domain of ctd-like phosphatases. B Evolutional analysis of Nem1 among fungi. Nem1 of M. oryzae was labelled in red. C Relative gene expression of NEM1 at different developmental stages. HY, hypha; CO, conidium; GT, germ tube; AP, appressorium at 12 hpi; IH24H, invasive hypha at 24 hpi; IH48H, invasive hypha at 48 hpi. D Nem1 is localized to the lipid droplets in the hypha and conidium. The transformant expressing Nem1-GFP was stained with Nile red and observed under a confocal microscope. Scale bar: 10 lm. HY, hypha; CO, conidium; AP, appressorium. E Line-scan graph analysis showed that Nem1-GFP was co-localized with Nile red-stained lipid droplets To investigate function of Nem1 in M. oryzae,we conidia and germ tube (immature appressorium) stages deleted NEM1 in the wild-type strain P131 using a split- (Fig. 1C), suggesting that NEM1 may play roles in marker approach (Fig. S1A). Two independent deletion conidia formation and appressoria formation. mutants, NEM1KO1 and NEM1KO2, were gained and confirmed by a PCR-mediated verification (Fig. S1B). We NEM1 is localized in lipid droplets also obtained the complementary strains by introducing the vector pKN-NEM1, which contains the coding region To further study the molecular function of NEM1, sub- of NEM1 driven by its native promoter, to NEM1KO1 cellular localization observation was conducted in M. (Fig. S2A). All transformants have been verified by PCR oryzae. Since Nem1 is predicted to be involved in lipid amplification of the NEM1 gene, and their phenotypes synthesis, we hypothesized that M. oryzae Nem1 is were all similar to P131 (Fig. S2B-E). One of them, localized to the lipid droplet, an organelle responsible cNEM1, was chosen for further analysis. for lipid storage. The Nem1 protein driven by its native To investigate the possible biological function in M. promoter fused with the green fluorescent protein oryzae, the expression profile of NEM1 was detected at (GFP) was expressed in P131. Transformants expressing different developmental and infection stages, including Nem1-GFP were selected to observe fluorescent signals. vegetative hyphae (HY), conidia (CO), germ tubes (GT), The lipophilic dye Nile red was used as a control to stain appressoria (AP), the early (IH18H) and late (IH48H) lipid droplets in cells of the transformant strain. Strong stage of invasive hyphae. NEM1 was highly expressed at punctate Nem1-GFP signal was well co-localized with The Author(s) 2023 aBIOTECH Nile red-stained lipid droplets in vegetative hyphae, indicated by spot areas (Fig. S3). Lipidomics analysis conidia, and appressoria (Fig. 1D). The two fluorescence demonstrated that the content of PA, PG, and PI were signals were well overlapped with the line-scan graph all significantly reduced in Dnem1 compared with that analysis (Fig. 1E). Therefore, we concluded that Nem1 is in P131. Meanwhile, the level of PC, LPC (lysophos- a lipid droplet localized protein. phatidyl choline), and LPE (lysophosphatidyl ethanola- mine) in Dnem1 was obviously higher than that in P131. Nem1 is required for lipid accumulation More importantly, the relative contents of DAG (diacyl- in mycelium and conidium glycerol) and TAG (triacylglycerol) were both signifi- cantly reduced in Dnem1 (Fig. 2B). These results Because Nem1 is localized to lipid droplets, we then indicate that Nem1 affects accumulation and composi- investigated whether Nem1 is required for lipid bio- tion of lipids in M. oryzae. genesis. BODIPY 493/503, a lipophilic fluorescent probe, was used to label lipid droplets in mycelium and Nem1 is important for asexual development of M. conidium. A great number of lipid droplets were oryzae observed in mycelium of P131, while far fewer lipid droplets were detected in the Dnem1. In the conidium, Because Nem1 is required for lipid biogenesis of the - numerous lipid droplets were observed in P131, far mycelium and conidium, we then tested if it affects more than that of Dnem1 (Fig. 2A). These results asexual development of M. oryzae. When grown on demonstrated that lipid content was significantly oatmeal agar (OTA) plates for 5 days, the colony diam- reduced in Dnem1 compared with P131. Further, thin- eter of NEM1KO1 or NEM1KO2 was around 2.5 cm, far layer chromatography (TLC) assay was conducted to smaller than that of P131 or cNEM1 (* 4.0 cm) detect lipid contents between P131 and Dnem1. Lipid (Fig. 3A, B). Moreover, the cell wall and septa of the composition including PA (phosphatidic acid), PC hyphal tips were stained with CFW to determine the (phosphatidylcholine), PE (phosphatidylethanolamine), hyphal cell length. The average length of P131 or PS (phosphatidylserine), PI (phosphatidylinositol), PG cNEM1 hyphae was significantly longer than those of (phosphatidyl glycerol), CL (cardiolipin), DGDG (di- NEM1KO1 or NEM1KO2 (Fig. 3C, D). Cell numbers of galactosyl diglyceride), and SQDG (sulphoquinovosyl conidium were also determine using CFW to stain the diglyceride) was segregated, and several compositions conidial septa. Around 80% of P131 or cNEM1 conidia (PA, PC, PS, PE, PG, and PI) showed difference in content had three cells, whereas the percentage of NEM1KO1 or Fig. 2 Nem1 is important for lipid accumulation and composition. A Lipid droplets were stained by BODIPY 493/503 in hyphae (HY) and conidia (CO) of WT and Dnem1. White arrows indicate lipid droplets strained by BODIPY 493/503. Scale bars: 10 lm. B Relative content of lipid composition in WT and Dnem1 by lipidomics analysis. Asterisks indicate statistically significant differences (P \ 0.05). PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidyl glycerol; PS, phosphatidylserine; PI, phosphatidylinositol; MGDG, monogalactosyl diglyceride; DGDG, digalactosyl diglyceride; LPC, lysophosphatidyl choline; LPE, lysophos- phatidyl ethanolamine; DAG, diacylglycerol; TAG, triacylglycerol The Author(s) 2023 aBIOTECH Fig. 3 Nem1 is necessary for asexual development of M. oryzae. A Colonies of P131, NEM1KO1, NEM1KO2, and cNEM1 grew on OTA plate for 5 days. B Statistical analysis of the colony diameter of the indicated strains. Asterisks indicate statistically significant differences (P \ 0.01). C Hyphal tip of P131, NEM1KO1, NEM1KO2, and cNEM1 were stained by CFW. White arrows show septa of hyphae. Scale bar: 20 lm. D Statistical analysis of near apical hyphal tip cell length of the indicated strains. Asterisks indicate statistically significant differences (P \ 0.01). E Statistical analysis of percentages of conidia with three cells, two cells, and one cell. Asterisks indicate statistically significant differences (P \ 0.01). Scale bar: 10 lm. F Conidiophores of P131, NEM1KO1, NEM1KO2, and cNEM1 were observed under the microscope. Scale bar: 20 lm. G Statistical analysis of conidiation of the indicated strains. P131, the wild-type strain; NEM1KO1 and NEM1KO2, deletion mutants of NEM1; cNEM1, the NEM1 complementary strain. Asterisks indicate statistically significant differences (P \ 0.01) NEM1KO2 was only 20%. The ratios of conidia with two hardly produce on leaves inoculated with the Dnem1 cells and one cell were much higher than that of the mutants (Fig. 4A, B). Further, inoculations on scratched wild-type or complementary strains (Fig. 3E). Further, rice leaves were also performed by mycelial blocks. conidia production was detected among these strains. Compared with the large-sized lesions caused by P131 Numerous conidia were formed on conidiophores of or cNEM1, almost no expanded lesions were found on P131 or cNEM1, but sparse conidia were observed on rice leaves inoculated with NEM1KO1 or NEM1KO2 conidiophores of the deletion mutants (Fig. 3F). Con- (Fig. 4C, D). To further determine which infectious stage sistent with this, the conidiation of the Dnem1 mutants was affected in the mutants, the infection process was (approximately 2.5 9 10 /mL per 6-cm-diame- observed on barley epidermis inoculated with different ter plate) was only about 2% of P131 or cNEM1 (ap- strains. At 24 h post-inoculation (hpi), the majority of proximately 1.7 9 10 /mL per 6-cm-diameter plates) P131 or cNEM1 appressoria had formed invasive (Fig. 3G). Taken together, our results demonstrated that hyphae (IH). However, the Dnem1 mutants still main- Nem1 is important for vegetative growth, conidial tained at the appressoria stage. Later at 30 hpi, the morphology, and conidia production. percentage of IH caused by P131 or cNEM1 reached more than 90%, while it was no more than 40% caused Nem1 is required for virulence of M. oryzae by the Dnem1 mutants. At 36 hpi, there were still around 80% appressoria that were unable to form To investigate the role of Nem1 in pathogenicity, coni- branched IH, while the majority of P131 or cNEM1 had dial suspension of P131, NEM1KO1, NEM1KO2, and formed IH with more than one branch (Fig. 4E, F). In cNEM1 were spray-inoculated on barley leaves and rice conclusion, Nem1 is required for virulence and invasive seedlings. Numerous lesions were observed on leaves growth of M. oryzae. infected with P131 or cNEM1, whereas lesions could The Author(s) 2023 aBIOTECH Fig. 4 Nem1 is required for full virulence of M. oryzae. A Barley leaves were inoculated by spraying with conidial suspension of P131, NEM1KO1, NEM1KO2, and cNEM1. B Rice seedlings were inoculated by spraying with conidial suspension of the indicated strains. C Scratched rice leaves were inoculated by conidial suspension of the indicated strains. D Statistical analysis of length of lesion produced from wounded sites in (C). Asterisks indicate statistically significant differences (P \ 0.01). E Invasive growth of P131, NEM1KO1, NEM1KO2, and cNEM1 at different time points were observed under a microscope. White arrows indicate appressoria. Scale bar: 10 lm. F Statistical analysis of percentages of AP and branched IH in (E). AP, appressorium; IH, invasive hypha Nem1 is required for the appressorium lipid droplets in the wild-type strain were quickly uti- formation and appressorial lipid droplets lized and degraded during the process of appressoria utilization formation, and gradually disappeared at 15 hpi. While in the Dnem1 mutant, lipid droplets were still observable As lipid turnover is necessary for appressoria formation at 15 hpi (Fig. 5C). We also noticed that at 10 hpi, lipid of M. oryzae, we then tested whether appressoria for- droplets in WT appressoria were much smaller than mation was affected in the Dnem1 mutants. Conidial that in the Dnem1 mutant (Fig. 5D), further indicating suspension of different strains was dropped on the that the lipid droplets degradation process was affected hydrophobic surface. At 24 hpi, the appressoria forma- in the mutant. These results demonstrate that Nem1 is tion percentages of P131 and cNEM1 were nearly 90%, required for lipid droplets utilization during the while it was less than 20% of the Dnem1 mutants. On appressorium maturation, which may affect appresso- onion epidermis, the tendency of AP formation was in rial turgor accumulation and penetration. consistence with that on the hydrophobic surface (Fig. 5A, B). These results showed that Nem1 is essen- Nem1 is important for cell wall integrity tial for appressoria formation. and stress response We also intended to detect whether appressorial turgor of the Dnem1 mutant was affected, however, it To determine if Nem1 plays roles in stress response, cell was hard for us to perform the cytorrhysis assay. wall integrity perturbing agents (Calcofluor white However, we were able to detect if utilization of lipid [CFW], Congo red [CR], and sodium dodecyl sulfate droplets was affected. On the hydrophobic surface, the [SDS]), and osmotic pressure agents (0.7 M NaCl and The Author(s) 2023 aBIOTECH Fig. 5 Nem1 is required for appressoria formation. A Observation of appressoria formation of P131, NEM1KO1, NEM1KO2, and cNEM1 on the cover glass. White arrows indicate appressoria. B Statistical analysis of the AP formation rate in (A). Asterisks indicate statistically significant differences (P \ 0.01). C Lipid turnover during appressoria formation of P131 and Dnem1 was stained with Nile red at different time points. Scale bar: 10 lm. D Lipid droplets were stained by BODIPY 493/503 in the appressorium of WT and Dnem1 at 10 hpi. White arrows indicate the strained lipid droplets. Scale bar: 10 lm 1.0 M Sorbitol) were separately added to the complete (Fig. S2G). These results suggest that Nem1 plays an medium (CM) plates for testing. The Dnem1 mutant was important role in stress response and scavenging host more sensitive to 0.1 mg/mL CFW or 0.2 mg/mL CR, ROS. but not 0.005% SDS. The Dnem1 mutant was slightly sensitive to 0.7 M NaCl, while it was more resistant to Nem1 regulates autophagy process 1.0 M Sorbitol. This result shows Nem1 is involved in cell wall integrity and osmotic pressure response Since Nem1 is required for appressorium maturation, (Fig. S4A, B). we wondered if Nem1 regulates autophagy in M. oryzae. Since the NEM1 deletion mutants are defective in A GFP-Atg8 fusion construct was respectively trans- invasive growth, we speculated during infection, the formed into WT and Dnem1. Resulting transformants mutants encountered inhibition from host reactive confirmed by Western blotting were cultured in mini- oxygen species (ROS). As expected, compared with WT, mum medium without nitrogen (MM-N). When cultured the Dnem1 mutant was more sensitive to different in MM-N for 6 h, compared with that in wild-type cells, concentrations of H O (10 mM, 15 mM, or 20 mM), less GFP-Atg8 was localized to the vacuole in Dnem1 2 2 suggesting Nem1 plays a role in ROS response (Fig. S4C, cells (Fig. 6A, B). This result indicated the autophagy D). Cellular ROS produced from host plants infected by process was affected in Dnem1. Total protein levels of P131, Dnem1, and cNEM1 at 30 hpi was detected with GFP-Atg8 in WT or Dnem1 cultured in MM-N for 2 h and 3,3’-diaminobenzidine (DAB). Compared with P131 or 6 h were also quantified to determine autophagy level cNEM1, ROS accumulation was more often observed in by immunoblot. The extent of autophagy was estimated barley epidermal cells infected with Dnem1 (Fig. S2E, F). by the intensity ratio of free GFP to the intact GFP-Atg8 Therefore, limited invasive growth of Dnem1 might be and free GFP together (GFP/[GFP ? GFP-Atg8]). When caused by defect in scavenging host ROS. To confirm the WT mycelium was treated with MM-N, the ratio this, diphenyleneiodonium (DPI) was used to inhibit the remarkably increases from 0.7 (0 h) to 0.79 (2 h) and host NADPH oxidase activity, which is necessary for host 0.94 (6 h). By contrast, in Dnem1, the value (GFP/ ROS generation (Liu et al. 2021). Compared with the [GFP ? GFP-Atg8]) slightly increased from 0.8 (0 h) to DMSO treatment control, 0.5 lM DPI treatment resulted 0.85 (6 h) (Fig. 6C). This result means autophagy is in a partial recovery of the invasive growth of Dnem1 The Author(s) 2023 aBIOTECH Fig. 6 Nem1 regulates autophagy in M. oryzae. A Observation of GFP-Atg8 localization in WT and Dnem1 cultured in CM for 48 h followed by MM-N for 0 h and 6 h. White arrows indicate vacuoles. Scale bar: 20 lm. B Statistical analysis of percentage of cells with vacuole localized Atg8. Asterisks indicate statistically significant differences (P \ 0.01). C Western blotting analysis of GFP-Atg8 and free GFP in the transformant WT/ GFP-Atg8 and Dnem1/ GFP-Atg8 cultured in MM-N for 2 h and 6 h. The numerical value showing gray value ratio of free GFP to the sum of free GFP and fused GFP-Atg8. GAPDH was loaded as a control Fig. 7 Nem1 is regulated by the TOR signaling pathway. A P131, Dnem1, and cNEM1 were cultured on CM plates added with 6.25 nM, 12.5 nM, 25 nM, 100 nM, or 500 nM rapamycin for 5 days. B Statistical analysis of growth inhibition rate of the indicated concentration of rapamycin. Asterisks indicate statistically significant differences (P \ 0.05). C Statistical analysis of relative DAG and TAG content of P131 and Dnem1 with or without rapamycin induction. Asterisks indicate statistically significant differences (P \ 0.01). ns, no significance. D Cell extracts of transformants expressed Nem1-HA in WT induced with rapamycin for 30 min and 60 min were subjected to Phos-tag SDS-PAGE analysis. The normal SDS-PAGE was used as a control blocked in the Dnem1 mutant, indicating the autophagy complex 1 (TORC1). Gradient concentrations of rapa- process of M. oryzae is regulated by Nem1. mycin from 6.25 nM to 500 nM were added in the CM plates to detect colony diameter of different strains. As Nem1 is involved in the TOR signaling pathway expected, compared to that of P131 and cNEM1, the Dnem1 was more sensitive to rapamycin in every con- Because the TOR signaling is important for the centration (Fig. 7A, B). Since Nem1 is a protein related appressorium formation which is strongly affected by to lipid metabolism, we hypothesized that DAG and TAG Nem1, we then tested whether the Dnem1 mutant was contents may be affected by rapamycin. To test this sensitive to rapamycin, which can inactivate the TOR hypothesis, total lipid in the mycelium of P131 and The Author(s) 2023 aBIOTECH Dnem1 treated with 25 nM rapamycin was extracted for phosphatase-sensitive and slow-moving band was not lipidomics analysis. The result of lipidomics showed observed in extracts of untreated Dcpka mutant cells that the TAG content of P131 treated by rapamycin was (Fig. 8A), suggesting that CPKA regulates phosphoryla- almost two-fold of the untreated one. By contrast, no tion of Nem1. significant difference of TAG content of Dnem1 was To further confirm that if appressoria formation of detected between rapamycin treated and untreated Dnem1 was regulated by cAMP-PKA signaling pathway, conditions. Relative DAG content of the untreated P131 exogenous 10 mM cAMP or 2.5 mM IBMX were added or Dnem1 was considerable to that treated by rapamy- to observe whether Dnem1 recovered in appressoria cin (Fig. 7C). This result indicated that Nem1-mediated formation. At 12 hpi and 24 hpi on hydrophobic surface, TAG synthesis is affected by the TOR signaling pathway. the appressoria formation rate of Dnem1 was around In budding yeast, Pah1 phosphatidate phosphatase 10%, equivalent to that of Dnem1 treated with cAMP or activity is regulated by TORC1. In parallel, Pah1 activa- IBMX. While the percentage of the wild-type strain was tion requires the Nem1-Spo7 phosphatase complex above 80% (Fig. 8B, C). Similar phenomenon was which in turn is often phosphorylated in the cell observed on the onion epidermis (Fig. 8D, E). This (Dubots et al. 2014;Suetal. 2018). We wondered if result indicates addition of cAMP or IBMX is unable to phosphorylation of Nem1 was affected by TORC1 in M. restore the appressoria formation defect of the Dnem1 oryzae. To verify this assumption, the Nem1-HA fusion mutant, consistent with above founding that Nem1 is construct was transformed into WT. The resulting strain downstream of the cAMP-PKA signaling pathway. WT/Nem1-HA was cultured in CM treated with 20 nM rapamycin for 30 min or 60 min. Total proteins were Nem1 is phosphorylated by CPKA at Ser303 extracted and subjected to be analyzed on gels con- taining the Phos-tag, which retards the mobility of In budding yeast, Nem1 was proved to be phosphory- phosphoproteins. As shown in Fig. 7D, Western blotting lated at both Ser140 and Ser210. By homologous using anti-HA as an antibody showed the mobility shift sequence comparison, Ser303 was speculated to be a of the WT/Nem1-HA. The shift of Nem1-HA treated with candidate phosphorylated site of Nem1 in M. oryzae.To rapamycin for 30 min or 60 min was slower than that of confirm this phosphorylation site, the serine was the untreated one, suggesting a phosphorylation of mutated to alanine (S303A). We expressed the S303A Nem1 (Fig. 7D). Together, phosphorylation of Nem1 is Nem1 -HA in Dnem1 and obtained the Dnem1/ S303A regulated by the TOR signaling pathway in M. oryzae. Nem1 -HA transformants. We then examined the S303A phosphorylation of Nem1 -HA using the Phos-tag S303A Nem1 is regulated by cAMP-PKA signaling method. The mobility shift of Nem1 -HA was similar pathway as Nem1-HA expressed in Dcpka, but faster than Nem1- HA expressed in WT (Fig. 9A). This result showed that Considering that Nem1 is required for appressorium Nem1 was phosphorylated at Ser303. formation in M. oryzae, it is possible that Nem1 func- To clarify the biological role of the Nem1 phospho- tions downstream of cAMP-PKA signaling pathway, such rylation site (Ser303) in M. oryzae, phenotypes of S303A as phosphorylated by PKA. To test this possibility, we Dnem1/NEM1 -HA were determined. The colony of S303A first deleted CPKA, which encodes the catalytic subunit Dnem1/NEM1 -HA was significantly smaller than of PKA, using the split-marker approach (Fig. S1A). One that of WT and the complementary strain Dnem1/ mutant termed Dcpka was obtained after PCR verifica- NEM1, although obviously greater than that of the tion for further study (Fig. S1C). The Nem1-HA con- Dnem1 mutant (Fig. 9B, C). Conidiation of Dnem1/ S303A struct was respectively transformed into the wild-type NEM1 -HA was also obviously recovered to the strain and the Dcpka mutant. The resulting transfor- wild-type level (Fig. 9D). Moreover, the virulence of S303A mants WT/Nem1-HA and Dcpka/Nem1-HA were ana- Dnem1/NEM1 -HA was evidently recovered com- lyzed on gels containing the Phos-tag. Cell extracts of paring with that of the Dnem1 mutant (Fig. 9E, F). On WT/Nem1-HA and Dcpka/Nem1-HA were treated either hydrophobic surface, the appressorium formation rate S303A with the phosphatase or the phosphatase inhibitor, fol- of Dnem1/NEM1 -HA was around 67%, significantly lowed by examination of mobility shifts by higher than that of the Dnem1 mutant (Fig. 9G, H). immunoblotting with the anti-HA antibody. The reduced Altogether, these results indicate that Ser303 of Nem1 mobility form of Nem1-HA was present in untreated plays key roles for development and virulence of the wild-type cells but not in the phosphatase-treated ones. rice blast fungus. Nem1-HA of wild-type cells treated with phosphatase inhibitor also exhibited slow mobility. The similar The Author(s) 2023 aBIOTECH Fig. 8 Nem1 is regulated by the cAMP-PKA signaling pathway. A Cell extracts of transformants expressed Nem1-HA in WT and Dcpka were subjected to Phos-tag SDS-PAGE. The normal SDS-PAGE was used as a control. B Observation of appressoria formation of Dnem1 with cAMP or IBMX treatment. Bars, 20 lm. C Statistical analysis of appressoria formation rate in (B). Asterisks indicate statistically significant differences (P \ 0.01). AP, appressoria. D Observation of appressoria formation of Dnem1 with cAMP or IBMX treatment on the onion epidermis. Scale bar: 20 lm. E Statistical analysis of appressoria formation rate in (D). Asterisks indicate statistically significant differences (P \ 0.01) DISCUSSION As shown in the TLC assay, deletion of NEM1 altered the lipid profile. Nine different kinds of lipid were iso- Lipid droplet storages in the fungal conidia have been lated and exhibited various sizes, though the overall found to play a key role during appressorium-mediated distribution of the lipid is similar. Lipidomics analysis infection of the plant pathogenic fungi, but the regula- showed relative content of 11 lipids, among which, PA, tory mechanism of lipid biogenesis and utilization is not PG, PI, DAG, and TAG were reduced in Dnem1 compared clear. In this study, our results showed a crucial role of with that in WT. Because PA tends to synthesize mem- Nem1 in conidial lipid droplets biogenesis and appres- brane phospholipids in a nutrient-rich condition such as sorial lipid droplets’ utilization. These two processes are in the CM medium, it is explainable for the reduction of well coordinated by phosphorylation of Nem1, which is PA although loss of NEM1 impedes transition from PA to regulated by TOR and cAMP-PKA signaling pathways. DAG. In a recent study (Zhao et al. 2022), deletion of Our study provides novel insight into the regulatory Pah1 led to obviously increased PA content. This may be mechanism of lipid droplet, as well as appressorium- caused by blocked transfer of PA to DAG and TAG for mediated infection of M. oryzae. lipid storage. The medium used was the nutrient star- vation minimum medium, which facilitated PA to The Author(s) 2023 aBIOTECH Fig. 9 Phosphorylation at Ser303 of Nem1 is important for asexual development and virulence. A Cell extracts of the transformant WT/ S303A Nem1-HA, Dcpka/Nem1-HA, and Dnem1/NEM1 -HA were subjected to Phos-tag SDS-PAGE. The normal SDS-PAGE was used as a S303A control. B WT, Dnem1, Dnem1/NEM1, and Dnem1/NEM1 -HA were cultured on OTA plates for 5 days. C Statistical analysis of the colony size in (B). Asterisks indicate statistically significant differences (P \ 0.01). D Statistical analysis of conidiation of the indicated strains. Asterisks indicate statistically significant differences (P \ 0.01). E Virulence test of WT, Dnem1, Dnem1/NEM1, and Dnem1/ S303A NEM1 -HA on barley leaves. F Statistical analysis of lesion area in (E). Asterisks indicate statistically significant differences S303A (P \ 0.01). G Appressoria formation of WT, Dnem1, Dnem1/NEM1, and Dnem1/NEM1 -HA on the cover glass at 12 hpi. H Statistical analysis of AP formation rates in (G). Asterisks indicate statistically significant differences (P \ 0.01) synthesize TAG for storage. Notably, both contents of delayed autophagy of Dnem1 in this study might resul- TAG and DAG were reduced in the NEM1 deletion ted from altered lipid composition and defected vacuole mutant, which is consistent with the result of lipid fusion. These can be explained by the result that lipid droplets staining assay. droplets in the Dnem1 mutant was delayed in utilization In yeast, Pah1 localizes to the nuclear membrane on (Fig. 5D). either side of NVJ (nuclear vacuole junction) and is Nutrient starvation and inactivation of rapamycin associated with lipid droplets (Graef 2018; Henne et al. complex 1 (TORC1) are considered two ways to pro- 2019). Phospholipids stored in lipid droplets can be mote autophagy. TORC1 is the core component of the subjected to autophagy by entering the vacuole in case TOR signaling pathway regulating processes including of excess nutrient. Additionally, phospholipids consti- lipid, protein, and nucleotide metabolism (Rahman et al. tute cellular membrane system including the vacuole. 2018a). TORC1 kinase controls nutrient availability, one The yeast Pah1 mutant was defective in vacuole fusion, way which affects lipid synthesis or storage by directly which is necessary for autophagy. Moreover, disruption phosphorylating the Nem1/Spo7–Pah1 axis (Dubots of the M. oryzae PAH1 led to delayed autophagy. Since et al. 2014). In budding yeast, nutrition starvation and Nem1 was localized to lipid droplets and dephospho- inactivation of TORC1 protein kinase promote macro- rylated PAH1 to be functional, thus, we considered that autophagy, a crucial process in cellular activities The Author(s) 2023 aBIOTECH through degrading cytoplasmic components and orga- pathway during the appressorium formation stage. nelles in lysosomes/vacuoles (Rahman et al. 2018b; Yin Phosphorylated Nem1 may be functional to facilitate the et al. 2020). In our study, autophagy of the Dnem1 lipid droplets degradation through the autophagy pro- mutant was blocked upon nitrogen starvation. This cess or enzymatic degradation. Upon nutrient starvation finding is consistent with that in S. cerevisiae.In F. or TORC1 inactivation, phosphorylation of Nem1 can be graminearum, rapamycin treatment led to significant inhibited by responding to the TOR signaling pathway, increase of LD biogenesis (Liu et al. 2019). In our study, leading to prevention of the appressorium formation we also proved that rapamycin treatment increases TAG process. A similar regulatory mechanism can be also content rather than DAG. This probably because TAG is found in perilipin LDP1, another lipid droplet-associ- the main form of lipid stored in LDs and DAG is the ated protein in M. oryzae (Cai et al. 2020). Therefore, intermediate of TAG. This effect was eliminated in the Nem1-mediated regulatory mechanism may be helpful loss of Nem1, indicating Nem1-mediated lipid synthesis for developing fungicide in future. is regulated by the TOR signaling. Disruption of NEM1 remarkably affected appresso- rium formation. In M. oryzae, several well-studied sig- MATERIALS AND METHODS naling pathways have been addressed to regulate this important biological process. Among them, the cAMP- Strains and culture conditions PKA signaling pathway regulates the early stage of appressorium formation. In yeast, Nem1 was reported The strain P131 was used as the M. oryzae wild-type to be phosphorylated by PKA (Su et al. 2018). In this (Xue et al. 2012). All strains listed in Table S1 were study, we deleted CPKA, the catalytic subunit of PKA, cultured on OTA plates at 28 C in the incubator. Colony and verified CPKA also phosphorylated Nem1. Exoge- diameters on the OTA plates were measured at 5 days nous addition of cAMP or IBMX was unable to rescue post-inoculation (dpi). Conidia from 7-day-old colonies appressorium formation, suggesting Nem1 was down- cultured on OTA plates were harvested for testing. stream of the cAMP-PKA signaling pathway. Lipid uti- Strains cultured in liquid complete medium (CM) at lization was required for appressorium formation (Cai 28 C were used for DNA and RNA extraction. et al. 2020; Chen et al. 2022). Here, we provided a novel To test fungal response to environmental stresses, regulatory mechanism of the cAMP-PKA signaling mycelial blocks were inoculated onto the CM plates pathway regulated lipid utilization and appressorium supplemented with 0.2 mg/mL Congo red (Sigma- formation. Aldrich, St. Louis, MO, USA), 0.1 mg/mL CFW (Sigma- Phos-tag SDS-PAGE analysis shows Cpka phosphory- Aldrich, St. Louis, MO, USA), 0.005% SDS, 0.7 M NaCl, lates NEM1 at Ser303. The site mutant S303A was 1.0 M sorbitol, 5–20 mM H O , 6.25–500 nM rapamycin 2 2 generated to study the biological function of this phos- (53,123–88-9, Merck, Darmstadt, Germany), respec- phorylation site. Pleiotropic defects including vegetative tively. The colony diameters were measured at 5 dpi. growth, conidiation, appressorium formation, and viru- lence of the site mutant were all partially restored. This Phylogenetic analysis probably results from more than one residue phos- phorylated by CPKA since the Ser303 serine was found Nem1 protein sequences in various species including M., through homologous sequence alignment with S. cere- Colletotrichum truncatum, V. dahlia, F. oxysporum, G. visiae, in which Ser140 and Ser210 were both phos- tritici, Neurospora crassa, Aspergillus melleus, phorylated to be functional. A more Schizosaccharomyces pombe, S. cerevisiae, and Candida suitable phosphorylation site confirmation method albicans were downloaded from the NCBI database. The should be used, such as mass spectrometry analysis. neighbor-joining phylogenetic tree was constructed In conclusion, this study has demonstrated that using MEGA 7.0 in the p-distance model with 1000 Nem1, a lipid metabolism-related gene, is important for bootstrap replicates. asexual development, appressorium formation, and pathogenicity. We proposed a model to understand the Quantitative real-time PCR analysis Nem1-mediated regulatory mechanism in M. oryzae (Fig. S5). In this model, Nem1 is a de-phosphorylated To evaluate the expression profile of NEM1, conidia pattern in the conidium (or in the hypha), which is produced from OTA plates, appressoria (3 hpi and 12 important for lipid droplets biogenesis and accumula- hpi) harvested from Teflon films (0.2 mm thickness), tion under the nutrient-rich condition. While Nem1 can and invasive hyphae (18, 24, and 48 hpi) collected from be phosphorylated at Ser303 by cAMP-PKA signaling inoculated epidermis of barley leaves were harvested to The Author(s) 2023 aBIOTECH extract total RNA for preparing cDNA templates. The were counted as the number of appressoria produced in qRT-PCR was performed using an SYBR Green PCR every 100 conidia. By evaluating effect of cAMP and Master Mix kit (Takara, Dalian, China) on an ABI 7500 IBMX on appressorium formation, 10 mM cAMP and real-time PCR system (Applied Biosystems, Foster City, 2.5 mM IBMX were separately added to the conidial CA, USA). The M. oryzae GAPDH was used as the refer- suspension drops followed by inoculated on the cover ence gene. glass or onion epidermis. Gene deletion and complementation Staining assays The split-PCR strategy was used for gene disruption as The conidia of different strains were collected from OTA previously described (Goswami 2012; Shi et al. 2022). plates and stained with 10 lg/mL CFW solution (Sigma- For deleting NEM1 or CPKA, hygromycin (HYG) was used Aldrich, St. Louis, MO, USA). The stained conidia were as a bridge gene to generate recombinant fragments. observed and photographed under a fluorescence PEG-mediated genetic transformation was adopted, and microscope (Ni90 microscope, Nikon, Tokyo, Japan). The transformants were screened by 250 lg/mL hygro- proportion of conidia with different number of septa mycin B (Roche Diagnostics, Indianapolis, IN, USA). Left was counted. The hyphae grew on the coverslip, which boarder (LB) and right boarder (RB) of NEM1 or CPKA was previously inserted to the colony edge on the OTA from transformants were confirmed by PCR using the plate. Hyphae tips on the coverslip were stained with LBCK/ HPT-up and RBCK/HPT-down primer pairs CFW solution for 5 min. The samples were washed (Table S3). Gene deletion was further verified by PCR of twice with PBS buffer before observed and pho- an internal fragment of NEM1 or CPKA. For obtaining tographed under the fluorescence microscope (Ni90). complementary strains, a vector containing 1.5 kb The ROS staining assay was performed as previously native promoter region, the NEM1 or CPKA gene coding described (Chen et al. 2014). Barley leaves infected by region and the adjacent 0.5 kb downstream region were the indicated strains at 30 hpi were stained with 1 mg/ separately transferred into the Dnem1 and Dcpka mL DAB (Sigma-Aldrich) solution (pH 3.8) for 12 h mutant. The CM plates supplemented with 400 lg/mL under darkness, followed by de-stained with ethanol/ G418 were used to select the complementary transfor- acetic acid solution (v/v, 94:4) for 24 h on the shaker mants followed by PCR and phenotype verification. with de-stained buffer changing. 0.5 lM diphenyleneiodonium (DPI) was added onto the coni- Observation of conidiophores and conidia dial droplets to eliminate ROS. The whole leaf was observed and photographed under the fluorescence Mycelia of P131, NEM1 deletion mutants and the com- microscope (Ni90). plementary strain grown on OTA media for 5 days were For lipid droplet staining assay, vegetative hyphae scraped to a sterilized tube added with 1 mL of steril- and conidia were collected in a 2 mL centrifuge tube ized ddH O in the tube. The mycelia were broken with a and incubated with 2 lM BODIPY 493/503 (Sigma- vibrator for 30 s and transferred to a new thicker OTA Aldrich) solution in PBS in the dark by shaking gently plate, and evenly smeared on the surface of the medium. for 5 min at the room temperature. Samples were then The mixture was dried and cultured at 28 C for 36 h transferred to the cover slide and washed with a quick till new hyphae grew on the surface. The hyphae were rinse using PBS before observation under a confocal stirred and washed with sterile water, and then dried. microscope (TCS SP8; Leica). The maximum wavelength The sterilized blade was used to cut the long piece of of excitation/emission is 493/503 nm. Appressoria mycelium at the edge of the colony, put it on the slide were stained on the hydrophobic slide with 2 lM with the front facing up and keep moisture, followed by BODIPY 493/503 adding on the conidial suspension incubated it in the 28 C incubator under light, and took drop cultured for 8 h. photos under the microscope after 18 h. Virulence test assays Appressorium formation Rice seedlings (Oryza sativa cv. LTH) grown for four Conidial suspension drops (2 9 10 spores/mL) were weeks and the barley (cv. E9) grown for one week were inoculated on the microscope cover glass (12540A, used for virulence tests. For spraying inoculation, coni- ThermoFisher, Pittsburgh, PA, USA) or onion epidermis dial concentration was adjusted to 3 9 10 conidia/mL and incubated at 28 C under darkness for 12 h and in 0.025% Tween 20 to spray barley and 1.5 9 10 to 24 h before observation. Appressorium formation rates spray rice leaves. The leaves were incubated under a full The Author(s) 2023 aBIOTECH humidity condition at 28 C with 12 h of darkness and After loading total lipid extracted from the samples, 12 h of light successively in a day. The inoculated leaves TLC plates were incubated at 110 C for 90 min and were observed and photographed 4 days later. For samples were spotted at one corner of the plates. infection process observation, conidia suspension Chloroform/ethanol/ammonium hydroxide (65:25:2, (2 9 10 ) drops were inoculated onto barley leaves. v/v/v) and chloroform/ethanol/acetic acid/water The leaf epidermis was collected and observed at 24, 30, (85:15:10:3, v/v/v/v) were used for the first and sec- and 36 hpi under the fluorescence microscope (Ni90). ond dimensional separation, respectively. After drying, the plates were exposed to iodine vapor for 90 s in the Subcellular localization observation tank. The lipid contents were measured as described using an Agilent HPLC system coupled with a triple The gene coding region of NEM1 linked with the native quadrupole/ion trap 4000 QTrap mass spectrometer promoter was amplified and ligated into the N-terminus (Applied Biosystems) (Nakamura et al. 2014; Zhao et al. of the GFP gene in the vector pGTN (Table S1; Table S2). 2022). The subsequent vector pGTN-NEM1 was transformed into Dnem1. Transformants at different developmental Phos-tag analysis and infection stages were used to observe subcellular localization under the confocal laser scanning micro- Phos-tag assays were performed as previously descri- scope (TCS SP8; Leica). bed (Li et al. 2017). The Nem1-HA fusion construct was transferred into the wild-type strain and the Dnem1 Autophagy analysis mutant. The positive transformants were cultured in liquid CM for 48 h. For protein isolation, 200 mg of Autophagy analysis was conducted as previously mycelia was ground into powder in liquid nitrogen and described (Chen et al. 2022; Ren et al. 2022; Yin et al. re-suspended in 1 mL of extraction buffer (10 mM Tris– 2020). The construct of MoAtg8 fused with GFP at its N- HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40) terminus was transformed into the WT and Dnem1 to which 1 mM PMSF, 10 lL of protease inhibitor mutant. Transformants were screened by Western cocktail (Sigma), and 10 lL of phosphatase inhibitor blotting, followed by cultured in CM for 48 h. Mycelia cocktail 3 (Sigma) had to be freshly added. For the were rinsed with water and transferred to the MM with preparation of the phosphatase-treated cell lysates, the nitrogen starvation (MM-N) for 2 h and 6 h to induce phosphatase inhibitor cocktail was omitted for 2.5 nonselective autophagy. The fluorescence signals were U/mL alkaline phosphatase (P6774; Sigma), and the observed under a confocal microscope (TCS SP8; Leica). sample was incubated for 1 h with the addition of 1 mM Proteins were extracted and analyzed by Western blot- MgCl (37C). The samples were further resolved on 8% ting with the anti-GFP antibody. The amount of free GFP SDS–polyacrylamide gels prepared with 50 lM acry- and GFP-Atg8 was quantified by densitometric analysis lamide-pendant Phos-tag ligand and 100 lM MnCl with the ImageJ software. according to the instructions provided by the Phos-tag consortium. Gels were electrophoresed at 60 V/gel for Lipid analysis 5 h. Prior to transfer, gels were first equilibrated in transfer buffer containing 5 mM EDTA for 20 min three Lipid was extracted as previously described (Bligh et al. times and then in transfer buffer without EDTA for 2? 1959; Khoomrung et al. 2013). Mycelia were harvested 10 min by shaking. Protein transfer from the Mn - TM from liquid CM cultured at 28 C with shaking phos-tag acrylamide gel to the PVDF membrane was (160 rpm) for 48 h and freeze-dried. The samples were performed overnight at 80 V at 4 C, and then the further incubated in hot isopropanol (75 C) containing membrane was analyzed by Western blotting using anti- 0.05% (v/v) butylated hydroxytoluene (Sigma-Aldrich) HA antibodies. for 15 min. Total lipid was extracted using chloroform/ methanol (2:1, v/v) added with 0.01% butylated Statistical analysis hydroxytoluene. This step was repeated five times with shaking. Then 1.0 M KCl and ddH O were added to the All values represent the mean of at least three biological sample for centrifugation, and the upper phase was replicates. Error bars indicate the standard deviation. discarded. The solvent was evaporated under a nitrogen Statistical comparisons were performed using one-way gas stream and re-dissolved in chloroform (5 mg/mL) analysis of variance (ANOVA) (P \ 0.05) in SPSS 19.0 before detecting by TLC and lipidomics analysis. software (IBM, New York, USA). The Author(s) 2023 aBIOTECH Supplementary InformationThe online version contains Chen XL, Shen M, Yang J, Xing Y, Chen D, Li Z, Zhao W, Zhang Y supplementary material available at https://doi.org/10.1007/ (2017) Peroxisomal fission is induced during appressorium s42994-023-00098-5. formation and is required for full virulence of the rice blast fungus. Mol Plant Pathol 18:222–237. https://doi.org/10. 1111/mpp.12395 Acknowledgements We thank Prof. Li Qiang from Huazhong Agricultural University for lipidomics analysis. This work was Chen D, Hu H, He W, Zhang S, Tang M, Xiang S, Liu C, Cai X, Hendy supported by the National Natural Science Foundation of China A, Kamran M, Liu H, Zheng L, Huang J, Chen XL, Xing J (2022) (Grants 32072365 and 32272476) and the Open Research Fund of Endocytic protein Pal1 regulates appressorium formation the State Key Laboratory of Crop Gene Exploration and Utilization and is required for full virulence of Magnaporthe oryzae. Mol in Southwest China (SKL-KF202216). Plant Pathol 23:133–147. https://doi.org/10.1111/mpp. Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro Data availability The data that support the findings of this study A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster are available from the corresponding author upon reasonable GD (2012) The Top 10 fungal pathogens in molecular plant request. pathology. Mol Plant Pathol 13:414–430. https://doi.org/10. 1111/j.1364-3703.2011.00783.x Declarations Dubots E, Cottier S, Pe´li-Gulli MP, Jaquenoud M, Bontron S, Schneiter R, De Virgilio C (2014) TORC1 regulates Pah1 Conflict of interest The authors declare no conflict of interest phosphatidate phosphatase activity via the Nem1/Spo7 exists. protein phosphatase complex. PLoS ONE. https://doi.org/ 10.1371/journal.pone.0104194 Open Access This article is licensed under a Creative Commons Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas Attribution 4.0 International License, which permits use, sharing, AR (2010) Phosphatidic acid phosphohydrolase 1 and 2 adaptation, distribution and reproduction in any medium or for- regulate phospholipid synthesis at the endoplasmic reticulum mat, as long as you give appropriate credit to the original in Arabidopsis. Plant Cell 22:2796–2811. https://doi.org/10. author(s) and the source, provide a link to the Creative Commons 1105/tpc.109.071423 licence, and indicate if changes were made. The images or other Ebbole DJ (2007) Magnaporthe as a model for understanding third party material in this article are included in the article’s host-pathogen interactions. Annu Rev Phytopathol Creative Commons licence, unless indicated otherwise in a credit 45:437–456. https://doi.org/10.1146/annurev.phyto.45. line to the material. If material is not included in the article’s 062806.094346 Creative Commons licence and your intended use is not permitted Eseola AB, Ryder LS, Ose´s-Ruiz M, Findlay K, Yan X, Cruz-Mireles by statutory regulation or exceeds the permitted use, you will N, Molinari C, Gardun˜ o-Rosales M, Talbot NJ (2021) Investi- need to obtain permission directly from the copyright holder. To gating the cell and developmental biology of plant infection view a copy of this licence, visit http://creativecommons.org/ by the rice blast fungus Magnaporthe oryzae. Fungal Genet licenses/by/4.0/. Biol. https://doi.org/10.1016/j.fgb.2021.103562 Fang Z, Wang S, Du X, Shi P, Huang Z (2014) Phosphatidate phosphatase-1 is functionally conserved in lipid synthesis and storage from human to yeast. Acta Biol Hung References 65:481–492. https://doi.org/10.1556/ABiol.65.2014.4.11 Fernandez J, Orth K (2018) Rise of a cereal killer: the biology of Magnaporthe oryzae biotrophic growth. Trends Microbiol Bahmanyar S (2015) Spatial regulation of phospholipid synthesis 26:582–597. https://doi.org/10.1016/j.tim.2017.12.007 within the nuclear envelope domain of the endoplasmic Goswami RS (2012) Targeted gene replacement in fungi using a reticulum. Nucleus 6:102–106. https://doi.org/10.1080/ split-marker approach. Methods Mol Biol 835:255–269. 19491034.2015.1010942 https://doi.org/10.1007/978-1-61779-501-5_16 Barbosa AD, Sembongi H, Su WM, Abreu S, Reggiori F, Carman GM, Graef M (2018) Lipid droplet-mediated lipid and protein home- Siniossoglou S (2015) Lipid partitioning at the nuclear ostasis in budding yeast. FEBS Lett 592:1291–1303. https:// envelope controls membrane biogenesis. Mol Biol Cell doi.org/10.1002/1873-3468.12996 26:3641–3657. https://doi.org/10.1091/mbc.E15-03-0173 Henne WM, Reese ML, Goodman JM (2019) The assembly of lipid Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction droplets and their roles in challenged cells. EMBO. https:// and purification. Can J Biochem Physiol 37:911–917. https:// doi.org/10.15252/embj.2019101816 doi.org/10.1139/o59-099 Hsu WH, Huang YH, Chen PR, Hsieh LS (2021) NLIP and HAD-like Cai X, Yan J, Liu C, Xing J, Ren Z, Hendy A, Zheng L, Huang J, Chen domains of pah1 and lipin 1 phosphatidate phosphatases are XL (2020) Perilipin LDP1 coordinates lipid droplets forma- essential for their catalytic activities. Molecules 26:5470. tion and utilization for appressorium-mediated infection in https://doi.org/10.3390/molecules26185470 Magnaporthe oryzae. Environ Microbiol 22:2843–2857. Jung Y, Kwon S, Ham S, Lee D, Park HH, Yamaoka Y, Jeong DE, Artan https://doi.org/10.1111/1462-2920.15019 M, Altintas O, Park S, Hwang W, Lee Y, Son HG, An SWA, Kim Chen XL, Shi T, Yang J, Shi W, Gao X, Chen D, Xu X, Xu JR, Talbot NJ, EJE, Seo M, Lee SV (2020) Caenorhabditis elegans Lipin 1 Peng YL (2014) N-glycosylation of effector proteins by an a- moderates the lifespan-shortening effects of dietary glucose 1,3-mannosyltransferase is required for the rice blast fungus by maintaining x-6 polyunsaturated fatty acids. Aging Cell. to evade host innate immunity. Plant Cell 26:1360–1376. https://doi.org/10.1111/acel.13150 https://doi.org/10.1105/tpc.114.123588 Karanasios E, Barbosa AD, Sembongi H, Mari M, Han GS, Reggiori F, Chen Y, Rui BB, Tang LY, Hu CM (2015) Lipin family proteins–key Carman GM, Siniossoglou S (2013) Regulation of lipid droplet regulators in lipid metabolism. Ann Nutr Metab 66:10–18. and membrane biogenesis by the acidic tail of the phosphati- https://doi.org/10.1159/000368661 date phosphatase Pah1p. Mol Biol Cell 24:2124–2133. https://doi.org/10.1091/mbc.E13-01-0021 The Author(s) 2023 aBIOTECH Khoomrung S, Chumnanpuen P, Jansa-Ard S, Sta˚hlman M, modification regulates autophagy and is required for infec- Nookaew I, Bore´n J, Nielsen J (2013) Rapid quantification tion of the rice blast fungus. New Phytol 235:247–262. of yeast lipid using microwave-assisted total lipid extraction https://doi.org/10.1111/nph.18117 and HPLC-CAD. Anal Chem 85:4912–4919. https://doi.org/ Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S 10.1021/ac3032405 (2005) The yeast lipin Smp2 couples phospholipid biosyn- Kim Y, Gentry MS, Harris TE, Wiley SE, Lawrence JC Jr, Dixon JE thesis to nuclear membrane growth. EMBO J 24:1931–1941. (2007) A conserved phosphatase cascade that regulates https://doi.org/10.1038/sj.emboj.7600672 nuclear membrane biogenesis. Proc Natl Acad Sci U S A Shi W, Yang J, Chen D, Yin C, Zhang H, Xu X, Pan X, Wang R, Fei L, Li 104:6596–6601. https://doi.org/10.1073/pnas.0702099104 M, Qi L, Bhadauria V, Liu J, Peng YL (2022) The rice blast Kubo Y (2013) Function of peroxisomes in plant-pathogen fungus SR protein 1 regulates alternative splicing with interactions. Subcell Biochem 69:329–345. https://doi.org/ unique mechanisms. PLoS Pathog. https://doi.org/10.1371/ 10.1007/978-94-007-6889-5_18 journal.ppat.1011036 Kwiatek JM, Carman GM (2020) Yeast phosphatidic acid phos- Siniossoglou S, Santos-Rosa H, Rappsilber J, Mann M, Hurt E phatase Pah1 hops and scoots along the membrane phos- (1998) A novel complex of membrane proteins required for pholipid bilayer. J Lipid Res 61:1232–1243. https://doi.org/ formation of a spherical nucleus. EMBO J 17:6449–6464. 10.1194/jlr.RA120000937 https://doi.org/10.1093/emboj/17.22.6449 Li L, Chen X, Zhang S, Yang J, Chen D, Liu M, Zhang H, Zheng X, Su WM, Han GS, Dey P, Carman GM (2018) Protein kinase A Wang P, Peng Y, Zhang Z (2017) MoCAP proteins regulated by phosphorylates the Nem1-Spo7 protein phosphatase complex MoArk1-mediated phosphorylation coordinate endocytosis that regulates the phosphorylation state of the phosphatidate and actin dynamics to govern development and virulence of phosphatase Pah1 in yeast. J Biol Chem 293:15801–15814. Magnaporthe oryzae. PLoS Genet. https://doi.org/10.1371/ https://doi.org/10.1074/jbc.RA118.005348 journal.pgen.1006814 Wilson RA, Talbot NJ (2009) Under pressure: investigating the Liu N, Yun Y, Yin Y, Hahn M, Ma Z, Chen Y (2019) Lipid droplet biology of plant infection by Magnaporthe oryzae. Nat Rev biogenesis regulated by the FgNem1/Spo7-FgPah1 phos- Microbiol 7:185–195. https://doi.org/10.1038/nrmicro2032 phatase cascade plays critical roles in fungal development Xu X, Okamoto K (2018) The Nem1-Spo7 protein phosphatase and virulence in Fusarium graminearum. New Phytol complex is required for efficient mitophagy in yeast. Biochem 223:412–429. https://doi.org/10.1111/nph.15748 Biophys Res Commun 496:51–57. https://doi.org/10.1016/j. Martin-Urdiroz M, Oses-Ruiz M, Ryder LS, Talbot NJ (2016) bbrc.2017.12.163 Investigating the biology of plant infection by the rice blast Xue M, Yang J, Li Z, Hu S, Yao N, Dean RA, Zhao W, Shen M, Zhang H, fungus Magnaporthe oryzae. Fungal Genet Biol 90:61–68. Li C, Liu L, Cao L, Xu X, Xing Y, Hsiang T, Zhang Z, Xu JR, Peng https://doi.org/10.1016/j.fgb.2015.12.009 YL (2012) Comparative analysis of the genomes of two field Nakamura Y, Koizumi R, Shui G, Shimojima M, Wenk MR, Ito T, isolates of the rice blast fungus Magnaporthe oryzae. PLoS Ohta H (2009) Arabidopsis lipins mediate eukaryotic pathway Genet. https://doi.org/10.1371/journal.pgen.1002869 of lipid metabolism and cope critically with phosphate Yan X, Talbot NJ (2016) Investigating the cell biology of plant starvation. Proc Natl Acad Sci U S A 106:20978–20983. infection by the rice blast fungus Magnaporthe oryzae. Curr https://doi.org/10.1073/pnas.0907173106 Opin Microbiol 34:147–153. https://doi.org/10.1016/j.mib. Nakamura Y, Teo NZ, Shui G, Chua CH, Cheong WF, Parameswaran 2016.10.001 S, Koizumi R, Ohta H, Wenk MR, Ito T (2014) Transcriptomic Yin Z, Feng W, Chen C, Xu J, Li Y, Yang L, Wang J, Liu X, Wang W, Gao and lipidomic profiles of glycerolipids during Arabidopsis C, Zhang H, Zheng X, Wang P, Zhang Z (2020) Shedding light flower development. New Phytol 203:310–322. https://doi. on autophagy coordinating with cell wall integrity signaling org/10.1111/nph.12774 to govern pathogenicity of Magnaporthe oryzae. Autophagy. Pillai AN, Shukla S, Rahaman A (2017) An evolutionarily https://doi.org/10.1080/15548627.2019.1644075 conserved phosphatidate phosphatase maintains lipid droplet Zhang Z, He G, Han GS, Zhang J, Catanzaro N, Diaz A, Wu Z, Carman number and endoplasmic reticulum morphology but not GM, Xie L, Wang X (2018) Host Pah1p phosphatidate nuclear morphology. Biol Open 6:1629–1643. https://doi. phosphatase limits viral replication by regulating phospho- org/10.1242/bio.028233 lipid synthesis. PLoS Pathog. https://doi.org/10.1371/jour Rahman MA, Mostofa MG, Ushimaru T (2018a) The Nem1/Spo7- nal.ppat.1006988 Pah1/lipin axis is required for autophagy induction after Zhao J, Sun P, Sun Q, Li R, Qin Z, Sha G, Zhou Y, Bi R, Zhang H, Zheng TORC1 inactivation. FEBS J 285:1840–1860. https://doi.org/ L, Chen XL, Yang L, Li Q, Li G (2022) The MoPah1 10.1111/febs.14448 phosphatidate phosphatase is involved in lipid metabolism, Rahman MA, Terasawa M, Mostofa MG, Ushimaru T (2018b) The development, and pathogenesis in Magnaporthe oryzae. Mol TORC1-Nem1/Spo7-Pah1/lipin axis regulates microau- Plant Pathol 23:720–732. https://doi.org/10.1111/mpp. tophagy induction in budding yeast. Biochem Biophys Res 13193 Commun 504:505–512. https://doi.org/10.1016/j.bbrc.2018. 09.011 Ren Z, Tang B, Xing J, Liu C, Cai X, Hendy A, Kamran M, Liu H, Zheng L, Huang J, Chen XL (2022) MTA1-mediated RNA m6 A The Author(s) 2023
Journal
aBIOTECH – Springer Journals
Published: Feb 18, 2023
Keywords: Lipid biogenesis; TOR signaling; cAMP-PKA signaling; Conidium formation; Appressorium; Magnaporthe oryzae