DOI: https://doi.org/10.21203/rs.3.rs-2083660/v1
In eukaryotes, Maf1 is an essential and specific negative regulator of RNA polymerase (Pol) III. Pol III, which synthesizes transfer RNAs (tRNAs), is suppressed by Maf1 under conditions of nutrient starvation or environmental stress. Here, we identified M. oryzae MoMaf1, a homolog of ScMaf1 in budding yeast. A heterogeneous complementation assay revealed that MoMaf1 fully restored growth defects in the ΔScmaf1 mutant under SDS stress. Disrupting MoMAF1 elevated the tRNA content and increased sensitivity to cell wall agents. Moreover, the ΔMomaf1 mutant exhibited reduced vegetative growth, conidiogenesis, and pathogenicity. Interestingly, we found that MoMaf1 undergoes nuclear–cytoplasmic shuffling, through which MoMaf1 accumulates in nuclei under nutrient deficiency or upon the interaction of M. oryzae with rice. Therefore, this study helps to elucidate the pathogenic molecular mechanism of M. oryzae.
In eukaryotic cells, the central growth regulator target of rapamycin (TOR) regulates the activity of RNA polymerase (Pol) III, which synthesizes transfer RNAs (tRNAs). RNA Pol III transcription activity varies with growth conditions. Dividing cells contain more tRNAs than resting cells, but the presence of too many tRNAs often has adverse effects in cells (Berns, 2008). For example, excess tRNAs in mammalian cells can increase the risk of carcinogenesis (Berns, 2008; Seton-Rogers, 2022). In cells, Maf1 is a conserved negative regulator of RNA Pol III transcription. Stressors such as rapamycin, nutrient starvation and DNA damage require Maf1 to repress Pol III transcription (Boguta and Graczyk, 2011).
In yeast, Maf1 is a phosphoprotein, and under favorable conditions, ScMaf1 is phosphorylated and mostly cytoplasmic (Karkusiewicz et al., 2011). However, under conditions of nutrient deprivation, ScMaf1 is dephosphorylated and moves the nucleus, where it binds Pol III and represses tRNA transcription. To activate tRNA transcription again, casein kinase II phosphorylates Maf1 to promote the dissociation of Maf1 from Pol III (Graczyk et al., 2011). Moreover, Maf1 phosphorylation by PKA regulates the suppression of Maf1 nuclear import. In addition, Sch9-mediated Maf1 phosphorylation is involved in the suppression of Pol III transcription (Moir et al., 2006).
The rice blast fungus Magnaporthe oryzae causes rice blast, a devastating global disease, and is also a widely adopted model organism for studying plant–pathogen interactions (Zhang et al., 2016). Studies have revealed that TOR signaling is important for regulating the virulence of M. oryzae and can engage in crosstalk with the cyclic adenosine monophosphate (cAMP), cell wall integrity (CWI) and autophagy pathways. We revealed that crosstalk between TOR and CWI signaling mediated by the protein phosphatase MoPpe1, a negative regulator of the TOR pathway, plays vital roles in vegetative growth, conidia formation and virulence (Qian et al., 2018). Although studies of TOR-dependent pathogenicity regulation have gradually been carried out, the underlying mechanisms by which TOR regulates tRNA synthesis during M. oryzae infection remain unclear.
Here, we identified a homolog of ScMaf1 in M. oryzae, which was named MoMaf1. We showed that MoMaf1 participates in tRNA transcription repression, vegetative growth, conidiogenesis and virulence in M. oryzae. Furthermore, the localization of MoMaf1 differs under different conditions, as MoMaf1 accumulates in nuclei under nutrient limitation and in the early infection phase but accumulates in the cytoplasm in the vegetative growth phase.
Fungal strains and culture conditions
All tested strains were cultured on CM at 28°C, and the wild-type Guy11 strain was used for transformation in this study (Qian et al., 2018; Qian et al., 2021). For vegetative growth, small blocks were cut and placed onto fresh medium, followed by incubation in the dark at 28°C for 7 days, and then the colony diameter was measured. Other media, including oatmeal agar medium and minimal medium, were prepared as described previously (Qian et al. 2018). For conidiation, mycelial blocks were inoculated on straw decoction and corn agar medium (SDC) at 28°C for 7 d in the dark, followed by 3 days of illumination under fluorescent light (Feng et al., 2021). Mycelia were harvested from liquid CM and used for DNA and RNA assays and protein extraction.
Complementation of the S. cerevisiae Δmaf1 mutant
The full-length cDNA of MoMAF1 was amplified using primers (Supporting Information Table S1), digested with XbaI and SacI, and then cloned into the yeast expression vector pYES2 (Invitrogen). After verification by sequencing and selection on SD medium lacking uracil, the MoMAF1-pYES2 vector was transformed into the yeast ΔMomaf1 mutant (BY4741, DYRE122C). Yeast strains were cultured on YPD medium and diluted to an OD600 of 0.1, after which 5 µl of 10-fold serial dilutions was grown on SD-Met-Leu-His-Ura (galactose) and SD-Met-Leu-His-Ura supplemented with 0.1% SDS plates at 30°C for 4 days before being photographed.
MoMAF1 gene deletion and complementation
The ΔMomaf1 mutants were generated by the one-step gene replacement strategy. Two 1.0-kb fragments flanking the targeted gene were PCR amplified with primer pairs (Table S1). The two 1.0-kb fragments were ligated to the two ends of the hygromycin resistance cassette (HPH 1.4 kb) by overlap PCR to form a 3.4-kb fragment. Then, the 3.4-kb fragment was transformed into Guy11 protoplasts by transformation (Yin et al., 2019). The putative mutants were screened by PCR after 7-10 days of incubation at 28°C and further confirmed by Southern blot analysis.
To generate a complementary pYF11-MoMAF1-GFP fusion construct, the gene sequence containing the MoMAF1 gene and 1.5-kb native promoter was amplified by PCR. Yeast strain XK1-25 was cotransformed with this sequence and the XhoI-digested pYF11 plasmid (containing a bleomycin resistance gene and GFP sequence) by the yeast gap repair approach. Then, the resulting yeast plasmid was expressed in E. coli. To generate the complementary strain, the pYF11-MoMAF1 construct containing the bleomycin resistance gene for the M. oryzae transformant screen was introduced into the ΔMomaf1 mutant.
Conidial germination and appressorium formation
Conidial germination and appressorium formation were measured on a hydrophobic surface. Conidial suspensions of 25 μl (5 × 104 spores/ml) were dropped onto a hydrophobic surface and placed in a humidified box at 28°C. The appressorium formation rate was counted at 24 h postinoculation (hpi) under a microscope, and more than 200 appressoria were counted for each strain.
Host penetration and pathogenicity assay
For the spraying assay, two-week-old rice seedlings (O. sativa cv. CO39) were sprayed with 5 ml of the conidial suspension and kept in a growth chamber at 28°C with high humidity in the dark for the first 24 h, followed by incubation under a 12-h light:12-h dark cycle (Liu et al. 2020, 2021). For the barley infection assay, 7-day-old barley leaves were inoculated with three droplets (25 μl) of the conidial suspension, and photographs were taken at 5 days after infection. Each experiment was repeated at least three times. To assess rice sheath penetration and invasive hyphal expansion, the conidial suspension (1×105 spores/ml) was inoculated into the sheaths. After incubation for 30 h at 28°C, the sheath cuticle cells were observed under a Zeiss Axio Observer A1 inverted microscope (Liu et al., 2019).
Western blot analysis of protein phosphorylation
The ΔMomaf1 mutant and wild-type strains were cultured in liquid CM for 2 days and then harvested, and 1 ml of protein lysis buffer (Qian et al. 2018) and 10 µl of protease inhibitor cocktail (Sangon, Shanghai, China) were added. After vortexing and homogenization, the lysate was centrifuged at 12 000 rpm for 10 min at 4°C. Then, 200 μl of the supernatant was mixed with an equal volume of 50 μl loading buffer and boiled for 5 min. The proteins were separated on SDS‒PAGE gels and transferred onto a polyvinylidene fluoride membrane with a Bio-Rad blotting apparatus. The intensity of the phosphorylated Mps1 signal was detected by the addition of an anti-phospho-p44/42 MAP kinase antibody (Cell Signaling Technology, Boston, MA, USA), with an anti-MAPK1 antibody (N-terminal anti-Mpk1) used as a control.
Chitin (N-acetylglucosamine, GlcNAc) content assay
The chitin (N-acetylglucosamine, GlcNAc) content was analyzed as described previously (Bulik et al., 2003; Song et al., 2010). First, mycelial samples were freeze-dried, and then 5 mg of the dried mycelia was resuspended in 1 mL of 6% KOH and heated at 80°C for 90 min. The samples were centrifuged (16 000 ×g, 10 min), and the pellets were washed with PBS over three cycles of centrifugation and resuspension (16 000 ×g, 10 min) before the final suspension in 0.5 mL of McIlvaine’s buffer (pH 6). An aliquot of 100 mL (13 units) of Streptomyces plicatus chitinase (Sigma, St. Louis, MO, USA) was added, and the mixture was incubated for 16 h at 37°C with gentle mixing; 100-mL samples were then combined with 100 mL of 0.27 M sodium borate (pH 9) and heated for 10 min at 100°C with the final addition of 1 mL of freshly diluted (1:10) Ehrlich’s reagent (10 g of p-dimethylaminobenzaldehyde in 1.25 mL of HCl and 8.75 mL of glacial acetic acid). After incubation at 37°C for 20 min, 1 mL of the sample was transferred to a 2.5-mL plastic cuvette (Greiner, Frickenhausen, Germany), and the absorbance at 585 nm was recorded. Standard curves were prepared with GlcNAc (Sigma). The experiment was repeated three times.
Observation of subcellular localized
To observe the subcellular localization of MoMaf1, we fused MoMaf1 with a GFP tag and a nuclear marker with a RFP tag. The green and red fluorescence signals in vegetative hyphae and IF were observed by dual fluorescence (Zeiss LSM710, 63× oil).
Identification and expression of MoMAF1
Examination of the M. oryzae genome database revealed that MGG_15675 and S. cerevisiae ScMaf1 exhibit high amino acid sequence homology, and we named the MGG_15675 sequence MoMaf1. We first expressed MoMAF1 in the ΔScmaf1 mutant using the yeast expression vector pYES2 and found that ΔScmaf1/MoMAF1 partially suppressed the defect in sensitivity to rapamycin in the ΔScmaf1 mutant, indicating that MoMaf1 is a functional paralog of ScMaf1 (Fig. 1a). In addition, transcription profile analysis of MoMAF1 at different developmental stages in M. oryzae showed that MoMAF1 was more highly expressed during the infection phase than during the mycelia stage, suggesting that MoMAF1 participates in the M. oryzae–rice interaction (Fig. 1b).
MoMAF1 is regulates tRNA levels
Maf1 is a negative regulator of Pol III that represses the synthesis of tRNA. In S. cerevisiae, deletion of MAF1 causes a substantial increase in tRNA levels (Pluta et al. 2001). To investigate the roles of MoMaf1 in M. oryzae, we generated ∆Momaf1 mutants and verified them by PCR amplification and southern blot hybridization (Additional file 1: Figure S1). We then further examined the function of MoMAF1 in tRNA synthesis. RNA extraction successfully yielded one small tRNA species and two large rRNA species (25S and 18S). The ∆Momaf1 mutant showed dramatically elevated tRNA levels compared with those of the wild-type and complemented strains (Fig. 2a, b). These results suggested that MoMAF1 regulates tRNA levels.
MoMaf1 is involved in vegetative growth and conidiation
Although MoMaf1 is a homolog of ScMaf1, the loss of MoMAF1 in M. oryzae led to a considerable defect in vegetable growth, but this defect was not observed in the ΔScmaf1 mutant (Fig. 3a, b). These results indicated that the functions of MoMaf1 and its ortholog in budding yeast differ (Pluta et al., 2001; Boguta, 2013). Additionally, the ΔMomaf1 mutant produced fewer conidia than the wild-type strain (Guy11) or the complemented strain (ΔMomaf1/MoMAF1) (Fig. 3c, d). Due to the function of Maf1 in inhibiting transcription, we examined the expression of six conidiation-related genes in the ΔMomaf1 mutant and Guy11 strain and found that the expression levels of MoCOM1, MoCON2, MoHOX2 and MoSTUA (Kim et al., 2009; Yang et al., 2010; Chen et al., 2018; Qian et al., 2021) were significantly lower in the ΔMomaf1 mutant, while MoCOS1 and MoCON7 (Zhou et al., 2009; Ruiz-Roldan et al., 2015) levels were not significantly different from those in the Guy11 strain (Additional file 1: Figure S2), indicating that MoMaf1 is involved in regulating conidiation-related genes.
MoMaf1 is required for penetration and infectious growth
To further examine the role of MoMaf1 in virulence, conidial suspensions of the Guy11 strain, the DMomaf1 mutant and the complemented strain were sprayed onto two-week-old rice seedlings (Oryza sativa cv. CO-39). After 7 days of inoculation, the mutant showed reduced virulence, with fewer and smaller lesions on the rice leaves in comparison to the numerous typical lesions caused by the wild-type strain (Guy11) and the complemented strain. A “lesion-type” scoring assay (Zhong et al., 2016) revealed that the numbers of all five types of lesions caused by the ΔMomaf1 mutant were significantly decreased (Fig. 4a, b). Similar results were obtained after the inoculation of conidial suspensions dropped on detached barley leaves, in which the ΔMomaf1 mutant caused more restricted lesions to form (Fig. 4c).
As the ΔMomaf1 mutant caused fewer and smaller lesions, we further investigated the role of MoMaf1 in penetration and infectious hyphal growth. Statistical analysis of the results showed that approximately 25% of the appressoria formed by the ΔMomaf1 mutant were unable to penetrate the rice cuticle (type 1), 40% of the penetration sites formed infectious hyphae (IH), but these IH were restricted to one cell with no branches or 1–2 branches (type 2 and type 3, respectively), and less than 15% of the IH extended to the neighboring cells (type 4). In contrast, there were only approximately 10% type 1 and over 70% type 3 and type 4 IH in the Guy11 strain and the complemented strain (Fig. 4d). These results indicated that MoMaf1 plays a critical role in penetration and infectious growth in rice blast fungus.
MoMaf1 regulates the generation of appressorium turgor pressure
Appressoria are critical structures for M. oryzae infection. The entire spore can be trafficked into the appressorium, where it undergoes maturation (Veneault-Fourrey et al., 2006). These coupled processes generate enormous hydrostatic turgor pressure in the appressorium, which has been measured at up to 8.0 MPa, to breach the rice leaf cuticle (Howard and Valent, 1996; deJong et al., 1997). As appressorium formation in the DMomaf1 mutant was no different from that in the wild type strain, we then examined whether the defect in turgor pressure generation resulted in a reduction in pathogenicity. An appressorium collapse assay was performed to test the appressorial turgor pressure using 1–4 M glycerol solutions (Tang et al., 2015). The appressoria of the ΔMomaf1 mutant exhibited higher collapse ratios than those of the wild-type and complemented strains (Additional file 1: Figure S3), suggesting that the reduced pathogenicity of the ΔMoMaf1 mutants may be related to the aberrant development of functional appressoria.
MoMaf1 is required for cell wall integrity (CWI)
We further investigated whether MoMaf1 is involved in modulating CWI. First, we assessed the effect of cell wall-degrading enzymes on mycelia in the ΔMomaf1 and Guy11 strains (Yin et al., 2016). Under the same conditions, the hyphae of the ΔMomaf1 mutant released more protoplasts than those of the wild-type strain or complemented strain (Fig. 5a, b). Then, we further quantified the chitin that accumulated in the cell wall and found that the ΔMomaf1 mutant had a higher chitin content than the wild-type strain (Guy11) (Fig. 5c). We also examined the expression levels of chitin synthase (CHS) genes (Kong et al., 2012) and found that the expression of six CHS genes was significantly reduced in the ΔMomaf1 mutant, but this was not the case for CHS2 (Fig. 5d). Additionally, the phosphorylation of MoMps1 was clearly decreased when compared with that of the wild-type Guy11 strain (Fig. 5e). In addition, the ΔMomaf1 mutants were more sensitive to the cell wall-perturbing agents CFW and Congo red (CR) (Additional file 1: Figure S4). Taken together, these results indicated that MoMaf1 is involved in maintaining CWI.
Subcellular localization of MoMaf1
To examine the subcellular localization of MoMaf1, we monitored the GFP-MoMaf1 fusion protein in the wild-type strain under different nutrient conditions. The GFP-MoMaf1 fluorescence signal was localized to the cytoplasm in nutrient-rich complete medium (CM), and GFP-MoMaf1 then translocated to the nucleus upon treatment with rapamycin to simulate nitrogen stress (Fig. 6). During infection, the GFP fluorescence signal in the cytosol was weaker than that in the nucleus, suggesting that MoMaf1 was transferred from the cytoplasm to the nucleus (Fig. 6). These results illustrated that the translocation of MoMaf1 from the cytosol to the nucleus is nutrient dependent and that tRNA transcription needs to be properly balanced to infect the host.
In this study, we characterized MoMaf1, a homolog of Maf1 in S. cerevisiae. We found that MoMaf1 is essential for the growth, conidiation and pathogenicity of M. oryzae. As observed in Candida albicans (Asghar et al., 2018), deletion of MoMAF1 led to a reduced growth rate and increased tRNA levels in M. oryzae. We also found that the ΔMomaf1 mutant was highly sensitive to different cell wall stressors (CFW and CR) and that the chitin content was lower than that of the wild-type Guy11 strain. Moreover, expression of the MoMaf1 gene partially repressed the sensitivity of the ΔScmaf1 mutant to rapamycin (Pluta et al., 2001; Asghar et al., 2018). These results indicated that the functions of the Maf1 protein are conserved.
Similar to that of other pathogenic fungi, the infection cycle of M. oryzae starts with conidia (Li et al., 2014). The ΔMomaf1 mutant produced fewer conidia than the wild-type strain, and we also found that the expression of four conidiation-related genes, MoCOM1, MoCON2, MoHOX2 and MoSTUA, was significantly reduced in the ΔMomaf1 mutant, which is consistent with the MoMaf1 protein expression data and indicated that MoMaf1 is involved in sporulation and conidial morphology by regulating the expression of these genes.
Importantly, we found that MoMaf1 undergoes cytoplasmic-to-nuclear translocation during infection or in response to nitrogen stress and that MoMaf1 affects tRNA synthesis and maturation. Similar interaction-dependent translocation has been reported before. Liu and colleagues revealed that MoYvh1 is translocated into the nucleus following oxidative stress to control the maturation of ribosomes, which promotes extracellular protein synthesis and secretion to scavenge rice reactive oxygen species (ROS) (Liu et al., 2018). Mature ribosomes carry out extracellular protein synthesis and secretion to scavenge ROS and modulate the rice defense response. Here, our findings revealed a novel link between tRNA synthesis and fungal virulence that is mediated by MoMaf1. We concluded that during early infection, M. oryzae may need tRNA synthesis to be properly inhibited for its pathogenicity. Taken together, our results have identified a pathogenic factor, MoMaf1, that plays an important role in growth, conidiation and pathogenicity in M. oryzae.
In this study, we reveal the function of Maf1 in M. oryzae. We found that MoMaf1 is involved in vegetative growth, conidiogenesis, and pathogenicity. We determined that MoMaf1 accumulates in the nucleus during the infectious phase and is located in the cytoplasm under normal growth conditions. Importantly, this study has revealed a novel link between tRNA synthesis and fungal virulence and facilitated elucidation of the pathogenesis of M. oryzae.
cell wall integrity
green fluorescent protein
mitogen-activated protein kinase
Magnaporthe oryzae
RNA polymerase III
red fluorescent protein
sodium dodecyl sulfate
transfer RNA
target of rapamycin
ACKNOWLEDGMENTS:
This research was supported by the Youth Program of the Natural Science Foundation of Jiangsu Province (BK20200543). We thank Prof. Zhengguang Zhang of Nanjing Agricultural University for providing the help of this study.
Author Contributions: BQ and CS designed the research, BQ and LG performed the research and analyzed the data, BQ wrote the paper, and CS and HJ revised the manuscript. All authors read and approved the final manuscript.
Funding: This research was supported by the Youth Program of the Natural Science Foundation of Jiangsu Province (BK20200543).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare that there are no conflicts of interest.