Identification and phenotypic characterization of MoSRP1

To identify the Srp1 orthologue in M. oryzae, we first performed BLASTp search against the protein database of the 70–15 and P131 strains using the S. pombe Srp1 as a query [54, 56]. The search showed that Srp1 had the highest hit with 52% identity and 55% coverage to MGG_04482 in 70–15 and P131_08116 in P131 that represented a single protein named MoSrp1. To functionally characterize MoSRP1 in M. oryzae, two deletion mutants of the gene, srp1ko1 and srp1ko2, were generated in P131 background by the homologous recombination approach (S1 Fig). The phenotypic assay showed that vegetative mycelial growth of srp1ko1 and srp1ko2 was significantly reduced compared to that of P131 (Fig 1A and 1B). Re-introduction of the MoSRP1-eGFP allele into srp1ko1 generated more than 30 transformants, and all of them, including cSRP1, restored the wild-type mycelial growth (Fig 1A and 1B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. MoSrp1 is important for vegetative growth, conidia morphology, and conidiation.

(A) Colonies formed by the wild-type (WT) strain P131, the ΔMosrp1 mutant srp1ko1, and its complementation transformant cSRP1 on OTA plates cultured for 5 d. (B) Statistics on colony diameters of strains P131, srp1ko1, and cSRP1 at 5 dpi. The mean and standard deviations were calculated based on three independent experiments each with three plates. Asterisk marks significant difference of the mutant from P131 and cSRP1 using t-test (p < 0.05). (C) Conidia per petri dish produced by strains P131, srp1ko1, and cSRP1. The mean and standard deviations were calculated based on three independent experiments each with three plates. Asterisk marks a significant difference of srp1ko1 from WT and cSRP1 using t-test (p < 0.05). (D) Conidia of strains P131 and srp1ko1 co-stained with CFW and DAPI. At the right of the images were percentages of conidia with 1-, 2-, and 3-cell formed by strains P131 and srp1ko1. At least 300 conidia were calculated for each strain. Bar, 10 μm. (E) Relative expression level of seven cell cycle-related genes in vegetative hyphae of strains P131 and srp1ko1. The gene expression level in P131 was arbitrarily set to 1. The actin gene was used as the internal control. The data were obtained from three replicates. Asterisk marks a significant difference of srp1ko1 from P131 using t-test (p < 0.05).

https://doi.org/10.1371/journal.ppat.1011036.g001

We further measured the conidium formation of the three strains. As shown in Fig 1C, the MoSRP1 deletion mutant srp1ko1 produced 1.2×10⁷ conidia per OTA plate, which was about 60% of the wild-type P131 and the complemented cSRP1 strains. Notably, 52% of the conidia produced by srp1ko1 had less than three cells (Fig 1D). RT-qPCR analysis showed that conidium morphology-related genes were significantly down-regulated in srp1ko1, including ACR1, MoCDC15, CDC42, COM1, CON7, RAC1, and RHO3 (Fig 1E). These results indicated that MoSRP1 is not only important for conidiation but also involved in conidium morphogenesis.

MoSRP1 is required for full virulence by regulating invasive hyphal growth

We also evaluated the effect of MoSRP1 on pathogenicity by spray-inoculation. As shown in Fig 2A, srp1ko1 produced only tiny lesions on the leaves of seedlings of rice and barley. In contrast, P131 and cSRP1 formed typical susceptible lesions. Statistics analysis indicated that these strains were not significantly different in the number of total disease lesions formed on both leaves (Fig 2B), suggesting that MoSRP1 is important for fungal proliferation in plant tissues but not for pre-penetration developments. We thereby performed further inoculation of abraded rice leaves with mycelial plugs, which showed that both P131 and cSRP1 formed larger typical disease lesions beyond the wounded sites, while srp1ko1 produced smaller lesions around the abraded sites (Fig 2C and 2D). These results indicated that MoSRP1 is indispensable for full virulence.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. MoSrp1 is essential for full virulence.

(A) Representative disease lesions on leaves of susceptible barley cultivar (left panel) and rice cultivar (right panel) sprayed with conidia of P131, srp1ko1, and cSRP1. (B) Statistics on disease lesions formed by P131, srp1ko1, and cSRP1 on susceptible rice cultivar ‘LTH’. Lesions with gray centers and larger than 2-mm in diameter were counted as expanded lesions. Means and standard deviations were calculated from the lesions formed on the middle 5-cm of the fourth leaves. Three independent experiments were conducted with at least 10 leaves in each replicate. (C) The top leaves of rice seedlings at the four-leaf stage were abraded and inoculated with mycelium plugs of P131, srp1ko1, and cSRP1. Representative disease symptoms were photographed at 5 dpi. (D) Statistics on length of individual disease lesions formed by P131, srp1ko1, and cSRP1 on abraded rice leaves as described in (C). Means and standard deviations were calculated from three independent experiments, in each of which at least 10 leaves were inoculated for each of the strains. (E) Appressorial penetration and invasive hyphal growth of P131, srp1ko1, and cSRP1 in barley epidermis at 24 and 36 hpi. Asterisk marks appressorium. Bar, 10 μm. (F) Statistics on percentages of appressoria with distinct types of invasive hyphae (IH), primary IH, IH with 1–2 branches, and IH with more than 2 branches at 24 and 36 hpi. Means and standard deviations were calculated from three independent experiments each with at least 50 appressoria scored.

https://doi.org/10.1371/journal.ppat.1011036.g002

To investigate how MoSRP1 affects plant infection, we compared the infection process of srp1ko1 with that of P131 and cSRP1 on barley leaf epidermis. These three strains were indistinguishable in conidial germination, appressorium formation and penetration (S2 Fig). However, compared with P131 and cSRP1, srp1ko1 was severely impaired in developing invasive hyphae (IH) in host cells (Fig 2E). At 24 hpi, srp1ko1 remained in primary IH at 60% of infection sites, whereas P131 and cSRP1 developed the secondary IH at 70% of infection sites, at more than half of which, IH formed two branches (Fig 2F). At 36 hpi, srp1ko1 developed the secondary IH with two branches at 60% infection sites but was confined to the first infected epidermal cells, while the P131 and cSRP1 IH had more than three branches and occupied the initial penetrated plant cells at almost every infection site, at some infection sites, IH expended into the neighboring plant cells (Fig 2E and 2F). These observations indicated that MoSRP1 is important for invasive hyphal growth of M. oryzae.

MoSrp1 interacts with some components of the exon junction complex in the nucleus

PSORTII analysis indicated that MoSrp1 has two potential nuclear localization signals at its middle region, suggesting that MoSrp1 is likely a nuclear protein. To verify the localization, we performed fluorescence microscopy with cSRP1. The GFP signals were concentrated in the nuclei of vegetative hyphae, conidia, appressoria, and infection hyphae of cSRP1 (Fig 3A). As a control, the GFP signals were even distributed in the cytoplasm of the transformants expressing eGFP driven by the promoter of MoSRP1 (S3 Fig). So, MoSrp1 is a protein functioning in the nuclei.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. MoSrp1 is a nuclear protein and interacts with three RNA-binding proteins of the putative EJC complex.

(A) MoSrp1-GFP fusion vector under the native promoter was introduced into srp1ko1 and a transformant restored to the WT phenotypes was subsequently used to observe the GFP signals with a Nikon 90i fluorescence microscope. Photos show the images of GFP signals localized in nuclei of vegetative hyphae (HY), conidia (CO), appressorium (AP), and invasive hyphae at 24 hpi (IH24h). Bar, 10 μm. (B) Three MoSrp1-interacting proteins identified by immunoprecipitation and yeast library screen assays. (C) The yeast two-hybrid assays show interactions between MoSrp1 and MoRnps1, MoGrp1, or MoThoc1, and between MoRnps1 and MoGrp1 or MoThoc1. The interaction between AD-T and BD-Lam is used as a negative control, and the interaction between AD-T and BD-53 as a positive control. (D) Co-immunoprecipitation assays between MoSrp1-GFP and MoRnps1-HA or MoThoc1-HA, and between MoGrp1-GFP and MoSrp1-HA. The total and eluted proteins were immunoblotted and detected with an anti-GFP or an anti-HA antibody, and the expected sizes of hybridized bands were indicated on the right. (E) Co-localization of MoSrp1-RFP with MoRnps1-GFP, MoGrp1-GFP, and MoThoc1-GFP in conidia. Bar, 10 μm.

https://doi.org/10.1371/journal.ppat.1011036.g003

To determine the molecular function of MoSrp1, we first identified MoSrp1-interacting proteins through IP-MS (co-immunoprecipitation coupled with mass spectrometry) and the Y2H library screening. Five putative nuclear proteins were identified among the immunoprecipitated proteins (S1 Table; Fig 3B), including MGG_02580 (named as MoRnps1), which is likely an RNA-binding protein with RRM and serine-rich domains similar to the human Rnps1, a component of the exon junction complex (EJC) [57–59]. From the Y2H library screening, eight nuclear-localized MoSrp1-interacting proteins were identified (S1 Table), and MGG_04399 and MGG_07511 were selected for further analysis (Fig 3B). MGG_04399 encodes an RNA-binding protein similar to THOC1, which is involved in EJC [60] and was named MoThoc1. MGG_07511 encodes a glycine-rich RNA-binding protein MoGrp1 that was recently identified as a novel splicing factor regulating virulence and growth in M. oryzae [61]. We further conducted the Y2H and Co-IP assays to validate the interactions of MoSrp1 with MoRnps1, MoThoc1, and MoGrp1. For the Y2H assays, the yeast transformants expressing each of the three combinatorial proteins grew on the selection medium, indicating that MoSrp1 physically interacts with MoRnps1, MoThoc1, and MoGrp1 (Fig 3C). Moreover, MoRnps1 physically interacted with MoThoc1 and MoGrp1 (Fig 3C). For the Co-IP assay, transformants expressing MoSrp1-GFP with either MoRnps1-HA or MoThoc1-HA were generated. As expected, in the total protein extracts and the anti-GFP affinity proteins, the 53-kD MoSrp1-GFP was detected with the 47-kD MoRnps1-HA or the 36-kD MoThoc1-HA (Fig 3D). Similarly, MoSrp1-HA was co-IPed with MoGrp1-GFP in vivo (Fig 3D). As a control, MoRnps1-HA or MoThoc1-HA did not interact with GFP, and MoGrp1-GFP could not interact with a non-related protein MoMas1 (S4 Fig).

We also generated transformants expressing MoSrp1-RFP fusion with MoRnps1-, MoThoc1-, or MoGrp1-GFP fusion. In conidia of the transformants, the GFP signals were clearly overlapped with the RFP signals in the nuclei, confirming that MoSrp1 is co-localized in the nucleus with MoRnps1, MoThoc1, and MoGrp1 (Fig 3E).

MoSrp1 is an important dual-function factor enhancing or suppressing splicing

Since the above-mentioned data suggested that MoSrp1 is likely a splicing factor, we investigated how MoSrp1 affects pre-mRNA splicing, first by comparing the mycelial transcriptomes of srp1ko1 and P131 and by analyzing defects in mRNA splicing resulting from the loss of MoSRP1. The comparative analyses revealed that the two strains expressed a total of 8,812 genes with ≥1 FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) in the CM cultured mycelia, including 2,048 genes that were differentially expressed between the two strains (591 genes had Log2≥0.5, and 1457 genes had Log2≤-0.5; p <0.05) (S2 Table), suggesting that MoSrp1 is important for the expression of these genes. With the RNA-seq data, we further performed analysis on different splicing events between the two strains and identified 1,301 aberrant splicing events those had occurred in transcripts of 905 genes in srp1ko1 (PSI (Percent spliced in) >0.05) (S3 Table). These aberrant splicing events comprised 37 exon skipping events (ES), 88 5’-alternative splicing site events (A5SS), 110 3’-alternative splicing site events (A3SS), and 1,066 intron retention events (IR) in transcripts of 33, 78, 101 and 806 genes, respectively. To validate the bioinformatics results, we performed RT-PCR analysis on the splicing of six introns. As shown in Fig 4A, three less efficiently spliced introns revealed by the bioinformatics analyses were more retained in the transcripts of srp1ko1 than in that of P131, including one intron that was not spliced in srp1ko1. In contrast, the other three introns were more efficiently spliced in srp1ko1 than in P131 (Fig 4B). These results suggested that MoSrp1 could enhance or suppress intron splicing as a dual-function splicing factor.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. MoSrp1 regulates intron splicing efficiency.

RT-PCR analyses show (A) enhanced splicing efficiencyand (B) suppressed splicing efficiency of three introns in the wild-type P131, the ΔMosrp1 mutant srp1ko1, and its complementation transformant cSRP1^K78R/E80Q expressing the mutated sumoylation site (srp1ko1/Srp1^K78R/E80Q-GFP) allele. The corresponding genomic DNA (gDNA) fragment amplified was used as a control with the un-spliced intron. The ratio between the spliced and the total introns was shown at the bottom of each line.

https://doi.org/10.1371/journal.ppat.1011036.g004

MoSrp1 binds to the 5’-GUAG-3’ consensus in RNAs

To understand how MoSrp1 is involved in the regulation of alternative splicing, we identified RNA sequence bound by MoSrp1 with the RIP-seq technique by using two strains Srp1-flag-3 and Srp1-flag-7 that expressed the MoSrp1-3Flag fusion in srp1ko1. Two strains expressing 3Flag were used as a control. With the two duplicated RIP-seq assays, a short sequence, 5’-GUAG-3’ was reproducibly identified as the top consensus in the MoSrp1-bound mRNA fragments compared to the control (Fig 5A). To confirm the binding of GUAG motif by MoSrp1, RNA-EMSA assay was performed with MoSrp1-pHAT2 fusion protein expressed in E. coli (S5 Fig) and biotin-tagged RNA oligomers containing the GUAG motif. An obvious hybridization band was detected in lanes containing both MoSrp1 and the biotin-tagged RNA fragment, and was strengthened with the increased amount of MoSrp1 (Fig 5B). Moreover, the addition of unlabeled RNA oligomers with GUAG motif into the reaction mixture reduced the hybridization, more competitors were added, and fewer hybridization signals were detected. Thus, MoSrp1 was verified to directly bind to the 5’-GUAG-3’ motif in an RNA transcript.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. MoSrp1 binds to the GUAG consensus in pre-mRNAs.

(A) Bit image of the top MoSrp1-binding motif by analyzing the RIP-Seq data. (B) RNA-EMSA assay showing the binding of MoSrp1 to the GUAG consensus. Lane 1–4: 2 nM biotin-tagged RNA fragment of 5’-GUAG-3’ with increased amount of MoSrp1 (0.3, 0.6, 0.9, and 1.2 μg); Lane 5–8: 2 nM biotin-tagged RNA fragment and 0.5 μg MoSrp1 with increased amount of unlabeled RNA oligomer (0, 8, 20, and 40 nM); Lane 9: 2 nM biotin-tagged 5’-GUAG-3’ RNA fragment without unlabeled RNA oligomer and MoSrp1. MoSrp1 was expressed and purified from E. coli, and the arrow indicates the binding band. (C) Different types (Type A to N) of aberrantly spliced transcripts with or without the GUAG consensus in the introns and their neighboring exons. A total of 1,066 introns retained in the ΔMosrp1 mutant srp1ko1 were used for analysis. (D) The number of intron retention events with PSI > 0.03 in srp1ko1 in comparison with WT P131. The number of introns with up- and down-regulated PSI was shown in red and yellow, respectively. (E) Distributions of the GUAG consensus within intron (maximum length of the MoSrp1-regulated introns is 330 nt) and its neighboring 5’- and 3’-exon with 100 nt.

https://doi.org/10.1371/journal.ppat.1011036.g005

To correlate the GUAG motif with alternative splicing, we analyzed the distribution of the GUAG motif in 905 genes that had aberrant alternative splicing events in srp1ko1. The analysis revealed that for most of the aberrant splicing events, there existed one or more GUAG motifs in a proximate position (S3 Table). Among the 1,066 introns with aberrant splicing in srp1ko1, 267 introns contained the GUAG motif, many of which also have the motifs in the proximate upstream and/or downstream exons (in 232 genes, Types A-D shown in Fig 5C and 5D and S3 Table); Surprisingly, 526 introns themselves lack the motif but in replace have a motif in their proximate upstream and/or downstream the exons (in 455 genes, Types E-G shown in Fig 5C and 5D and S3 Table), suggesting that the GUAG motif in exons may affect splicing of their proximate introns. In total, 793 of 1,066 introns had the motif in introns and/or neighboring exons (Fig 5D). Similarly, most of the exon skipping events and aberrant 5’ or 3’ splicing site events were also closely related to the existence of the GUAG motif (S3 Table). Furthermore, we analyzed the location of the GUAG motif in the introns and/or in the exons showing aberrant splicing. The frequency of the GUAG motif around the -80~30 bp of branch point sequence (BPS) is obviously higher than others, and nearly 42% of the GUAG motifs are distributed within the 100 bp upstream and 40 bp downstream of BPS in introns (Fig 5E), revealing that the GUAG motif was enriched around the branching point sites.

MoSrp1 modulates alternative splicing at different developmental stages

When co-analyzing the RNA-seq and RIP-seq data, we found that the MoSrp1-bound GUAG consensus was distributed in the transcripts of most genes (7,799 out of 8,812 genes) expressed in mycelia of M. oryzae, but only a small portion (905 genes) of the expressed genes displayed aberrant alternative splicing in srp1ko1. So, we questioned whether the rest genes with the GUAG consensus may have alternative splicing at other developmental stages affected by MoSrp1. To check this idea, we selected several reported infection-related genes that were not significantly different between the two strains in the alternative splicing of mycelial cells, including MgFOW1 (MGG_07201), MoKDCDH (MGG_05814), MoVELA (MGG_08556), MoLYS20 (MGG_01092), and MoRHO3 (MGG_10323) [62–66] for RT-PCR analysis (Figs 6 and S6). At the conidiophore stage, the introns from MgFOW1 and MoVELA were less efficiently spliced in srp1ko1. At the conidia stage, the introns from MgFOW1, MoKDCDH, MoVELA, and MoRHO3 were less efficiently spliced in srp1ko1, whereas MoLYS20 was obviously more spliced in srp1ko1. At the infection hyphae stage, the introns of MgFOW1, MoKDCDH, MoVELA, and MoLYS20 were less efficiently spliced in srp1ko1; in contrast, MoRHO3 was less efficiently spliced in P131. Taken together, the RT-PCR analyses indicated that MoSrp1 is a global splicing factor that modulates alternative splicing at growth, development, and infection stages.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. MoSrp1 regulates alternative intron splicing at different developmental stages.

RT-PCR analyses show splicing efficiency of the introns of five reported genes, MgFOW1, MoKDCDH, MoVELA, MoLYS20, and MoRHO3, in strains P131 and srp1ko1 at different developmental stages, including mycelium (MY), conidiophores (CP), conidium (CO), and invasive hyphae (IH). The corresponding genomic DNA (gDNA) fragment amplified was used as a control with an un-spliced intron. The ratio between the spliced and the total introns was shown at the bottom of each line.

https://doi.org/10.1371/journal.ppat.1011036.g006

MoSrp1 enhances or suppresses intron splicing of genes important for colony growth, conidiation, and virulence

To verify how MoSrp1 functions as a dual-function splicing factor to facilitate its biological roles, we checked whether any of the previously characterized genes had aberrant intron splicing in srp1ko1. Among the 905 genes that had aberrant intron splicing in srp1ko1, 68 genes have been reported to be essential or important for colony growth, conidiation or/and virulence (S4 Table), including MoATF1 (MGG_08212) and PPG1 (MGG_01690) [67]. MoATF1 encodes a basic leucine zipper (bZIP) transcription factor and is important for growth, conidiation and virulence in M. oryzae [68]. In MoATF1 pre-mRNA, the first intron with 224-bp contains a GUAG motif around 10-nt downstream of 5’SS (Fig 7A). When a pair of primers across this intron was used for RT-PCR, a 400-bp band was amplified in P131 while a bigger band with 624-bp was produced in addition to the 400-bp band in srp1ko1 (Fig 7B), showing that the intron was retained in part of MoATF1 mRNAs in srp1ko1. Moreover, a synthesized biotin-labeled RNA fragment originated from this intron with the GUAG motif exhibiting strong binding by MoSrp1 (Fig 7C). To further determine whether the GUAG motif mediates the first intron splicing of MoATF1, we mutated the GUAG to CAAC in MoATF1 and transformed this mutation allele into the MoATF1 knockout mutant. This mutation led to the retention of the first intron in half the transcripts (Fig 7D). In contrast, the intron in P131 and a complementation transformant with the wild-type MoATF1 allele was fully spliced from the pre-mRNAs (Fig 7D). Moreover, MoATF1 with the mutation was dysfunctional, unable to rescue colony growth, conidiation, and plant infection although the mutant intron was removed from half of the transcripts (Fig 7E–7H). Taken together, the first intron of MoATF1 was a direct target of MoSrp1 and its splicing efficiency was enhanced by MoSrp1, which was essential for MoATF1 transcripts to be functional.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. MoSrp1 enhances the intron splicing efficiency of a virulence gene MoATF1.

(A) Position of the 5’-GUAG-3’ motif in the MoATF1 pre-mRNA. (B) RT-PCR analysis showing splicing efficiency of the first intron of MoATF1 in the ΔMosrp1 mutant srp1ko1 as compared with the wild-type P131. The ratio between the spliced and the total introns was shown at the bottom of each line. (C) RNA-EMSA assay showing the binding strength between MoSrp1 and the 5’-GUAG-3’ consensus in the first intron of MoATF1. (D) RT-PCR analysis showing splicing efficiency of MoATF1 or MoATF1^CAAC in strains wild-type (WT) Guy11, ΔMoatf1, MoATF1/ΔMoatf1, and MoATF1^CAAC/ΔMoatf1 (mutation of 5’-GUAG-3’ to 5’-CAAC-3’ in MoATF1 transcript). Guy11 was used as the wild-type strain to generate the ΔMoatf1 mutant. The ratio between the spliced and the total introns was shown at the bottom of each line. (E) Colony formed by strains WT, ΔMoatf1, MoATF1/ΔMoatf1, and MoATF1^CAAC/ΔMoatf1 on OTA plates at 5 dpi. (F) Statistics on colony diameters of strains WT, ΔMoatf1, MoATF1/ΔMoatf1, and MoATF1^CAAC/ΔMoatf1. (G) Conidia per petri dish produced by strains WT, ΔMoatf1, MoATF1/ΔMoatf1, and MoATF1^CAAC/ΔMoatf1 on OTA plates. The means and standard deviations in (F and G) were calculated based on three independent experiments each with three plates measured. Asterisk marks a significant difference between the Guy11 and mutant strains using t-test (p < 0.05). (H) Representative disease lesions on leaves of susceptible barley sprayed with conidia of strains WT, ΔMoatf1, MoATF1/ΔMoatf1, and MoATF1^CAAC/ΔMoatf1.

https://doi.org/10.1371/journal.ppat.1011036.g007

To exemplify the intron splicing negatively regulated by MoSrp1, we selected the second intron in the MGG_07843 for analysis, which is an uncharacterized gene of M. oryzae encoding a putative MFS superfamily transporter protein with a GUAG motif at its end and was named MoMTP1 (Fig 8A). As shown in Fig 8B, about 22% of the MoMTP1 transcript retained the intron in P131, whereas only about 7% of the intron was retained in srp1ko1. Moreover, a synthesized biotin-labeled RNA fragment originated from the second intron of MoMTP1 with the GUAG motif was bound by MoSrp1 (Fig 8C). To further explore whether the GUAG motif is responsible for the second intron splicing of MoMTP1, we generated two deletion mutants of MoMTP1, mtp1ko1 and mtp1ko2 (S1 Fig), and phenotypic analysis showed that the ΔMomtp1 mutant exhibited obvious defects on colony growth, conidiation, and virulence (Fig 8E–8H). Because the GUAG motif was overlapped with the 3’ splice site (3’SS) of the second intron, we generated four types of complementation transformants in the ΔMomtp1 mutant mtp1ko1 background: one with the wild-type MoMTP1, and the other three with different types of mutation (CUAG, CAAG, and CAAC) in the second intron of MoMTP1. These three types of mutations resulted in varying degrees of retention (ranging from 33% to 52%) of the second intron in the MoMTP1 transcripts, whereas the intron in P131 and a complementation transformant with the wild-type MoMTP1 allele had about 20% retention rate (Fig 8D). Moreover, the complementation transformants expressing the three types of mutation alleles exhibited similar defects on colony growth, conidiation, and virulence as the ΔMomtp1 mutant (Fig 8E–8H). Taken together, the second intron of MoMTP1 was a direct target of MoSrp1 and its splicing efficiency was suppressed by MoSrp1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. MoSrp1 suppresses the intron splicing efficiency of a novel virulence gene MoMTP1.

(A) Position of the 5’-GUAG-3’ motif in the MoMTP1 pre-mRNA. (B) RT-PCR analysis showing splicing efficiency of the second intron of MoMTP1 transcript in the ΔMosrp1 mutant srp1ko1 as compared with the wild-type P131. The ratio between the spliced and the total introns was shown at the bottom of each line. (C) RNA-EMSA assay showing binding of MoSrp1 to the 5’-GUAG-3’ consensus in the MoMTP1 intron. (D) RT-PCR analysis on splicing efficiency of the second intron of the MoMTP1 transcript in strains P131, ΔMomtp1, and MoMTP1/ΔMomtp1, the MoMTP1^CUAG transcript in strain MoMTP1^CUAG/ΔMomtp1, the MoMTP1^CAAG transcript in strain MoMTP1^CAAG/ΔMomtp1, and the MoMTP1^CAAC transcript in strain MoMTP1^CAAC/ΔMomtp1. The ratio between the spliced and the total introns was shown at the bottom of each line. (E) The colony formed by strains P131, ΔMomtp1, MoMTP1/ΔMomtp1, MoMTP1^CUAG/ΔMomtp1, MoMTP1^CAAG/ΔMomtp1 and MoMTP1^CAAC/ΔMomtp1 strains on OTA plates at 5 dpi. (F) Statistics on colony diameters of the strains described in (E). (G) Conidia per petri dish produced by the strains described in (E) on OTA plates. The means and standard deviations in (F and G) were calculated based on three independent experiments each with three plates measured. Asterisk marks significant differences between P131 and mutant strains using t-test (p < 0.05). (H) Representative disease lesions on leaves of susceptible barley sprayed with conidia of the strains described in (E).

https://doi.org/10.1371/journal.ppat.1011036.g008

The RRM domain is essential to the full functions of MoSrp1 but the RD/E region is important for regulating virulence and responses to stresses

To dissect the roles of the RRM at 7–80 aa and RD/E-rich domain at 96–172 aa, we divided MoSrp1 into two regions, MoSrp1^1-90 with the RRM and MoSrp1^91-206 with the RD/E-rich domain, and tested which region mediates the interaction with MoRnps1, MoThoc1, and MoGrp1 by yeast two-hybrid assay. The MoSrp1^1-90 but not the MoSrp1^91-206 interacted with these three proteins (Fig 10A). Then, two constructs, MoSrp1^△1–90 and MoSrp1^△91–206 that lacked the RRM and the RD/E-rich domain, respectively, were individually introduced into srp1ko1, and 20 transformants were randomly picked for each construct. Phenotypic assays showed that the MoSrp1^△1–90 (cSRP1^91-206) transformants exhibited similar mycelial growth as srp1ko1 (Figs 10B–10G; S8) while the MoSrp1^△91–206 (cSRP1^1-90) transformants almost restored the capacities on colony growth and conidiation on OTA plates (Fig 10B–10D). However, the cSRP1^1-90 transformants were similar to srp1ko1 in virulence (Fig 10E), and were more sensitive than WT P131 and cSRP1 strains to chemical agents, including 1 M sorbitol, 100 μg/ml CFW, 200 μg/ml CR, MM, MM-N, and MM-C (Figs 10F–10G and S8). Therefore, the RRM domain is essential for the full functions of MoSrp1, and the RD/E-rich region is important for MoSrp1 to regulate virulence and response to stresses, albeit not important for mycelial growth and conidiation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 10. The RRM domain is essential to all the MoSrp1 functions but the SR region is important for stress responses.

(A) The yeast two-hybrid assay showing that the interactions between N-terminus (1–90 aa) or the C-terminus (91–206 aa) of MoSrp1 and MoRnps1, MoGrp1, and MoThoc1. The interaction between AD-T and BD-Lam is used as the negative control, and the interaction between AD-T and BD-53 is used as the positive control. (B) The colonies formed on OTA plates at 5 dpi by the strains P131, srp1ko1, cSRP1 and srp1ko1 transformants expressing the N-terminal fragments cSRP1^1-90 (1–90 aa), cSRP1^1-130 (1–130 aa), the C-terminus fragment cSRP1^91-206 (91–206 aa), mutated MoSrp1^S117A (M117), mutated MoSrp1^S119A (M119), and mutated MoSrp1^S117AS119A (M117/119) of MoSrp1. (C) Statistics on colony diameters of the strains described in (B). (D) Conidia per petri dish produced on OTA plates by the strains described in (B). (E) Representative disease lesions on the leaves of susceptible barley sprayed with conidia of the strains described in (B), and photographed at 5 dpi. (F) Statistical analysis of colony growth reduction rates of the strains in (B) cultured on CM plates supplemented with different stress agents (1 M sorbitol, 100 μg/ml CFW, or 200 μg/ml CR) at 5 dpi. (G) Statistical analysis of colony growth reduction rates of the strains described in (B) under MM, MM-N, and MM-C conditions. The means and standard deviations in (C, D, F and G) were calculated based on three independent experiments with measuring three plates for each replicate. Asterisk marks significant difference between P131 and mutant strains using t-test (p < 0.05).

https://doi.org/10.1371/journal.ppat.1011036.g010

Based on the observations that the MoSrp1^1-90 could not fully rescue defects of srp1ko1, we speculated that its longer sequence might fulfill its full functions and searched the conservation regions between MoSrp1 and its orthologues. The results showed that the 1–130 aa of MoSrp1 is conserved among its orthologues from Pezizomycontina fungi. So, the truncated MoSrp1^1-130 was introduced into srp1ko1, and the resulting transformants, including cSRP1^1-130, exhibited the wild-type phenotypes on colony growth, conidiation, and virulence (Fig 10B–10E). These findings suggested that the MoSrp1^1-130 is enough to achieve the roles in normal growth, development and plant infection. However, the cSRP1^1-130 transformants only partially restored tolerance to several stresses (Figs 10F–10G and S8), further indicating that the RD/E-rich region is important for regulating responses to stresses.

Previous proteomics studies showed that MoSrp1 is phosphorylated at S117 and S119 within the RD/E-rich region [71,72]. To determine whether phosphorylation at these sites affects the functions of MoSrp1, we generated constructs of MoSRP1 with the single and double mutations of serine to alanine and introduced them into srp1ko1. Transformants with two single-site mutation alleles, M117 and M119, and the double site mutation allele, M117/M119, exhibited the wild-type phenotypes on colony growth, conidiation, and plant infection (Fig 10B–10E). However, similar to the cSRP1^1-130 transformants, the single and double mutation alleles were obviously reduced in colony growth under stresses, including CM with 1 M sorbitol, 100 μg/ml CFW, 200 μg/ml CR, MM-N, and MM-C (Figs 10F–10G and S8). Thereby, phosphorylation of MoSrp1 within the RD/E-rich region is important for regulating stress tolerances.

MoSrp1 orthologues in Pezizomycotina fungi are functionally similar but divergent from those in other eukaryotes

BLASTp searches suggested that plants and animals had three MoSrp1 orthologues, while fungi contained only one. With these MoSrp1 orthologues, we performed a phylogenetic analysis, which showed that MoSrp1 orthologues were widely distributed among eukaryotes but with high divergences (Fig 11A). Multiple sequence alignment of MoSrp1 orthologues from fungi showed that all the orthologues were similar in the RRM domain, but only those from Pezizomycotina fungi were conserved on the RD/E-rich domain (Fig 11B). Notably, MoSrp1 had the highest similarity with the orthologues in F. graminearum. Interestingly, the SUMOylation motif that was essential to MoSrp1 was conserved only in the orthologues from Pezizomycotina fungi but missed in the orthologues of fungi outside Pezizomycotina, suggesting that Srp1 orthologues may be functionally diversified even among subphylum of Ascomycota. To test this speculation, we individually introduced MoSrp1 orthologues from different species, including F. graminearum FgSrp1, S. pombe SpSrp1 and SpSrp2, and A. thaliana AtRBP1 and AtSC35 into srp1ko1. As expected, only FgSrp1 completely rescued defects of srp1ko1 on colony growth, conidiation, and plant infection (Fig 11C–11F), confirming that MoSrp1 orthologues are functionally conserved only in Pezizomycotina fungi.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 11. MoSrp1 homologues in Pezizomycotina fungi are functionally similar but divergent from those in other eukaryotes.

(A) Phylogenetic analysis of MoSrp1 and its orthologues from different fungi (red dot): Fusarium graminearum (FgSrp1), Sclerotinia sclerotiorum (SsSrp1), Botrytis cinerea (BcSrp1), Alternaria alternata (AaSrp1), Aspergillus nidulans (AnSrp1), Neurospora crassa (NcSrp1), Puccinia striformis (PsSrp1), Ustilago maydis (UmSrp1), Schizosaccharomyces pombe (Srp1), Pneumocystis carinii (PcSrp1), and Candida albicans (Slr1), plants (yellow dot): Arabidopsis thaliana (AtSc35, AtRS41, and AtRBP1) and Oryza sativa (OsRS2Z32, OsRS2Z33, and OsRS2Z36), animals (blue dot): Drosophila melanogaster (DmSC35, DmSRp55, and DmB52) and Mus musculus (MmSRSF6, MmSRSF10, and MmSRSF12), and human Homo sapiens (HsSRSF6, HsSRSF10, and HsSRSF12). (B) Sequence alignment of MoSrp1 and its counterparts from other fungi including Pezizomycotina (P) FgSrp1, SsSrp1, BcSrp1, AaSrp1, AnSrp1, and NcSrp1, Taphrinamycotina (T) Srp1 and PcSrp1, Saccharomycotina (S) Slr1 and ClSrp1, and Basidiomycota (B) PsSrp1 and UmSrp1. The conserved amino acids among different fungi were highlighted. The RRM domain and RD/E-rich domain of MoSrp1 were underlined. An asterisk marks the SUMOylation site. (C) Colonies formed on OTA plates at 6 dpi by the strains P131, srp1ko1, cSRP1 and srp1ko1 transformants expressing AtRBP1 (AtRBP1/srp1ko1), AtSC35 (AtSC35/srp1ko1), SpSrp2 (SpSrp2/srp1ko1), SpSrp1 (SpSrp1/srp1ko1), FgSrp1 (FgSrp1/srp1ko1), the MoSrp1-SpSrp1 chimeric structures: SpSrp1^1-130 (the N terminal 130 aa residues of SpSrp1), MoSrp1^1-60-SpSrp1^61-130 (the N terminal 60 aa residues of MoSrp1 fused with the 61–130 aa residues of SpSrp1), SpSrp1^1-60-MoSrp1^61-130 (the N terminal 60 aa residues of SpSrp1 fused with the 61–130 aa residues of MoSrp1), and SpSrp1^80K/81I (SpSrp1 mutated with the sumoylation site). (D) Statistics on colony growth of the strains described in (C). (E) Statistics on conidiation of the strains described in (C). The means and standard deviations in (D-E) were calculated based on three independent experiments by measuring three plates for each replicate. Asterisk marks significant difference between P131 and mutant strains using t-test (p < 0.05). (F) Representative disease lesions on the leaves of susceptible barley sprayed with conidia of the strains described in (C).

https://doi.org/10.1371/journal.ppat.1011036.g011

To further delimit which regions of MoSrp1 result in the function diversification, we generated mosaic constructs of MoSrp1 with SpSrp1, and individually introduced them into srp1ko1 (S9 Fig). The SpSrp1^1-130, MoSrp1^1-60-SpSrp1^61-130, and SpSrp1^1-60-MoSrp1^61-130 alleles failed to rescue defects of srp1ko1 on colony growth, conidiation, and plant infection (Fig 11C–11F). In addition, SpSrp1^80K81I, a mutated construct generated to possess the same sumoylation site at the 80th aa as MoSrp1 also failed to rescue srp1ko1 (Fig 11C–11F). Together, these results suggested that multiple sites in the RRM domain of Srp1 proteins may be involved in functional diversification.

Fungal culture conditions and nucleic acid manipulations

All fungal strains (S5 Table) were cultured and maintained on oatmeal tomato agar (OTA) plates [88]. Fungal protoplast preparation and transformation, colony growth, and conidiation were performed as described [89]. To test stress responses, colony diameter was measured at five days post-inoculation (dpi) onto minimal medium (MM), MM-N, MM-C plates, and complete medium (CM) plates supplemented with 200 μg/ml Congo Red (CR), 100 μg/ml Calcofluor White (CFW), and 1 M Sorbitol. Fungal genomic DNA was extracted from vegetative mycelia with the cetyltrimethylammonium bromide (CTAB) protocol [90]. Plasmid isolation, nucleic acid extraction, DNA gel blots, enzymatic manipulation, and sequencing were performed as described [91]. Probes for DNA gel blot were labeled with a Random Primer Labeling Kit (Takara). PCR primers (S6 Table) were synthesized by Sangon. Phylogenetic and molecular evolutionary analyses were conducted using MEGA [92]. Domains were predicted by InterproScan [93].

Plant infection and microscopy assays

The conidial suspension was adjusted to 2×10⁴ conidia/mL in a 0.025% Tween 20 and used to spray-inoculate seedlings of four-week-old susceptible rice cultivar ‘LTH’ and eight-day-old barley cultivar ‘E9’. The infected plants were incubated in a moist chamber and maintained [94]. Lesion formation was examined at 5 to 7 dpi. An infection assay with mycelial plugs on abraded leaves was performed as described [94]. 1×10⁵ conidia/mL conidia were inoculated onto cover glass slides to observe conidial morphology and inoculated onto barley leaves to observe infection process [95]. Inoculated samples were incubated in a moist chamber at 25°C, and invasive hyphal growth was examined at 24 and 36 hpi with a Nikon 90i microscope [61]. Staining on septa and nuclei of fungal conidia and hyphae was performed as described [95].

Gene knockout, gene complementation, and subcellular localization assays

The upstream and downstream fragments of MoSRP1 were amplified with P1/P2 and P3/P4, and cloned into pKOV21 [67], and the resulting vector was transformed into P131 protoplasts. Putative ΔMosrp1 mutants were screened by PCR with the paired primers P5/hu and P6/hd, and then confirmed by DNA gel blot hybridization with the probe amplified by P3/P4. A split marker approach was used for generating the gene deletion mutants of MoMTP1 [52]. Putative ΔMomtp1 mutants were screened and confirmed by PCR with the paired primers LB-CK/hu, RB-CK/hd, and inf/inr. For gene complementation of the ΔMosrp1 mutant, a fragment containing MoSRP1 and its 1.5-kb promoter region was amplified with Srp1DWF/Srp1DWR and cloned into pGTN [89]. The same strategy was used to generate the GFP fusion constructs of MoSrp1 mutation alleles. For gene complementation of the ΔMoatf1 or ΔMomtp1 mutant, a fragment containing MoATF1 or MoMTP1 and its 1.5-kb promoter region was amplified with ATF1HB-F/ATF1HB-R or MTP1HB-F/MTP1HB-R and cloned into pKN [89]. The same strategy was used to generate the MoAtf1^CAAC and MoMtp1^CAAC mutation alleles. For heterologous complementation, the cDNA fragments containing FgSRP1, SpSRP1, SpSRP2, AtRBP1, and AtSC35 were individually cloned into pKNRG. The same strategy was used to generate the SpSrp1^1-130, MoSrp1^1-60+SpSrp1^61-130, SpSrp1^1-60+MoSrp1^1-130, and SpSrp1^80K/81I alleles. The resulting constructs were transformed into a ΔMosrp1 mutant. The MoTHOC1-eGFP, MoGRP1-eGFP and MoRNPS1-eGFP constructs and MoSrp1-RFP were co-transformed into P131. All neomycin-resistant transformants were confirmed by PCR analysis and screened for fluorescence expression under a Nikon 90i microscope.

Yeast two-hybrid (Y2H) assays

For Y2H library screening, the pGBKT7-MoSrp1 as bait was transformed into S. cerevisiae Y2H Gold strain with the MATCHMAKER Gal4 Two-Hybrid System (Clontech). After checking protein expression and no self-activation, correct yeast strain was used for mating with the yeast library constructed from the cDNA of M. oryzae vegetative mycelia. The positive colonies can grow under the SD medium lacking Histidine (SD-L/W/H), and PCR products of the positive colonies were selected for sequencing. Different bait and prey constructs were generated by cloning the full-length cDNA or truncated alleles of MoSRP1, MoTHOC1, MoGRP1, and MoRNPS1 into pGBKT7 and pGADT7, respectively. The paired resulting bait and prey constructs were co-transformed into the Gold strain, and the Trp⁺Leu⁺ transformants were selected and were tested their growth on SD/Trp^-/Leu^-/His^- medium. Yeast strains harboring the pairs of pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T were used as positive and negative controls, respectively.

Coimmunoprecipitation (Co-IP) assays

For identifying MoSrp1 co-immunoprecipitated proteins, the MoSRP1-3Flag constructs were introduced into strain srp1ko1. Total proteins were extracted from the resulting transformants and incubated with anti-Flag M2 beads (Sigma), and then proteins bound to the beads were eluted for LC-MS/MS analysis. For protein-protein interaction, the MoSRP1-eGFP and MoRNPS1-HA constructs were co-introduced into strain P131, the total proteins were extracted from the resulting transformant and then incubated with GFP-Trap beads (Chromotek). Proteins bound to GFP-Trap beads were eluted according to the protocol, and western blots of the total proteins and the elution from GFP-Trap beads were performed with an anti-GFP antibody and an anti-HA antibody, respectively (Sigma). The same approach was used to assay the interaction between MoSrp1-eGFP and MoThoc1-HA, MoGrp1-eGFP and MoSrp1-HA, MoGrp1-eGFP and MoThoc1-HA, and MoGrp1-eGFP and MoRnps1-HA.

RT-PCR and quantitative RT-PCR (RT-qPCR)

Total RNAs were extracted from vegetative hyphae grown in liquid CM, conidiophores and conidia formed on OTA plate, and invasive hyphae in barley leaves, pretreated with DNase I and then reverse transcribed with a PrimeScript RT-PCR Kit (Takara). RT-PCR was performed to evaluate the intron splicing efficiency of individual transcripts between P131 and the ΔMosrp1 mutant. RT-qPCR was performed on an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions. The actin gene (MGG_03982) was used as a control. Relative abundance of gene expression level was calculated using the 2^-ΔΔCt method as described previously [96].

RNA-seq and RNA-immunoprecipitation and sequencing (RIP-seq) assays

RNA extract, high-throughput cDNA libraries construction, and comparative transcriptomic analysis were performed as described [61,95] with mycelia grown in liquid CM for two days. The transcriptomes were firstly compared with the annotated genes in the reference genome of 70–15, and then differences in exon skipping, alternative 3’SS selection, alternative 5’SS selection, and intron retention were examined between P131 and the ΔMosrp1 mutant. Alternative splicing events were detected and quantified by using SplAdder [97]. For each event in each gene, percent-spliced-in (PSI) values were computed based on the quantified splicing graphs. The PSI values between strains P131 and srp1ko1 were compared. The events, whose PSI maximum value (> 0.03) of WT was higher than the minimum value of mutant, or otherwise, were filtered.

RNA-immunoprecipitation and cDNA library construction were performed as described [98] with the abovementioned CM-cultured mycelia. Two independent strains expressing the MoSrp1-3Flag fusion protein (Srp1-flag-3 and Srp1-flag-7) were compared with two control strains expressing the empty vector (RP27-flag-1 and RP27-flag-2), respectively. Two grams of mycelia from each sample were harvested for protein extraction, and anti-Flag beads were used to immunoprecipitate the MoSrp1-3Flag protein. The resulting complexes (beads, antibody, MoSrp1-3Flag, and RNAs) were then collected by using a magnet. Further, total RNAs from the complexes were isolated by using the phenol-chloroform-isoamyl alcohol extraction method. cDNA was then synthesized with reverse transcriptase (SuperScript IV Reverse transcriptase) following the manufacturer’s protocol. A “no reverse transcription” control (no SuperScript IV added) was prepared for each sample. The resulting cDNA was used immediately for library preparation of high-throughput sequencing. For high-throughput sequencing, the cDNA libraries were applied to the Illumina Hiseq2000 system for 150 nt paired-end sequencing by ABLife company. The RIP-seq data analysis was performed as described [99]. Two replicates were performed for both the RNA-seq and RIP-seq assays.

RNA electrophoretic mobility shift (RNA-EMSA) assays

The pHAT2-MoSrp1 construct with the N-terminal 6His was generated and expressed in Escherichia coli BL21(DE3). The induced E. coli cells were harvested and lysed by sonication. Supernatants from the cell lysates were applied to a Ni-Chelating Sepharose Fast Flow column and after washing with lysis buffer, His-tagged proteins were eluted with elution buffer. The eluted proteins were then purified by gel filtration chromatography on a Superdex 75 10/300GL column (GE Healthcare). The MoSrp1-GFP and MoSrp1^K78R/E80Q-GFP protein were extracted from P131 and incubated with GFP-Trap beads (Chromotek). Proteins bound to GFP-Trap beads were eluted according to the manual provided by the manufacturer. The protein-RNA binding assays were performed with the Light Shit Chemiluminescent RNA EMSA Kit according to the manual provided by manufacturer (Thermo Pierce).