Publisher Correction: The glycogen synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal development and pathogenicity in Magnaporthe oryzae

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Phytopathogenic fungi pose a constant threat to the global food security 1 . Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease, is the most devastating plant pathogen on rice and causes up to 30% yield loss of rice every year 2 . Beside rice, it also causes yield losses in other cereals such as wheat, barley and finger millet [3][4][5] . The infection cycle of the fungus starts when the three-celled conidia land on the leaf surface. Upon landing on cuticle, the spores form a polarized germ tube. Recognition of environmental cues such as surface hydrophobicity and toughness induces swelling at the tip of germ tube, which then differentiates into the specialized infection cell called an appressorium. The appressorium generates enormous turgor pressure by accumulating glycerol with the help of melanised cell wall. This pressure is converted into mechanical force to penetrate the plant surface, followed by further invasive hyphal expansion to colonize plant tissues 6 .
Perception of cues on leaf surface is the key to initiation of cellular responses mediating appressorium development. Coordination of these morphogenetic events is governed by conserved MAP kinase pathways 7 . In M. oryzae, there are three MAPK pathways namely Pmk1, Hog1 and Mps1. Pmk1 is a central regulator of appressorium development and the pmk1 mutant is unable to form appressoria and cause disease 8 . Hog1 is dispensable for fungal virulence but is necessary for adaptation to osmotic stress 9 . In contrast to the essential role of Pmk1 in appressorium formation, Mps1 is required for appressorium penetration of plant surface via modulating the fungal cell wall integrity 10 . Beside its role in pathogenesis, Mps1 is active in conidiation and cellular stress responses in M. oryzae 10 . Identification of downstream effectors of the Mps1 pathway is critical for understanding the signaling network required for fungal pathogenesis. To date two effectors of the Mps1 pathway have been characterized in M. oryzae. Mig1 is a MADS-box transcription factor, which has been shown to interact directly with Mps1 in the yeast two-hybrid assay. The mig1 mutants in M. oryzae are able to form appressoria that penetrate the plant surface and elaborate into primary infectious hyphae but fail to expand to the neighboring cells, causing loss of pathogenicity 11 . The M. oryzae Swi6 is an APSES family transcription factor that also physically interacts with Mps1 in both in vivo and in vitro assays. The Δswi6 mutant forms deformed and non-melanized appressoria that are unable to penetrate plant surface due to the impaired cell wall integrity 12 .
In addition to targeting transcriptional factors in the nucleus, the Mps1 homolog in Saccharomyces cerevisiae Mpk1 has been shown to function in the cytoplasm mediating a signal cascade in response to environmental stress 13 . Glycogen synthase kinase 3 (GSK3) is a unique kinase that contains well-conserved protein structures and phosphorylation properties but with varied roles in mediating a wide range of signal pathways in eukaryotic organisms 14 . GSK3 was initially identified as a kinase that phosphorylates glycogen synthase and causes its inhibition in rabbit cells 15 . A growing body of literatures have stated that GSK3 is able to phosphorylate various targeting proteins in regulation of several cellular events and subjected to phosphorylation by other kinases in response to different signals 16 . One example is the phosphorylation of GSK3 at the N-terminal serine residue by the MAPK cascade and caused its inhibition in response to insulin-like growth factors 17 , subsequently leading to the de-phosphorylation and activation of the GSK3 downstream targeting proteins. In facts, as a ubiquitous kinase, the inhibition of GSK3 that unleashes the activities of the downstream targeting proteins, underpins its regulatory function 16 . One of the main roles for GSK3 is in regulation of cellular response to the extracellular stimuli. In S. cerevisiae, four homologs to the mammalian GSK3 were identified including the MCK1 that is responsible for the activation of the stress-responsive transcriptional activator MSN2 under various environmental stresses 18 . MCK1 is also a key regulator of the cell cycle progression promoting the timing degradation of the CDC6p as essential component of the pre-replicative complex (pre-RC) 19 . In the plant pathogen Fusarium graminearum, one GSK3 homolog Fgk3 was identified and proven to be required for proper growth, conidiogenesis, sexual production and pathogenesis 20 . The multiple phenotypic effects of the Δfgk3 mutant suggest that Fgk3 is a conservative regulator involved in various aspects of fungal development. The study also showed Fgk3 physically interacts with MSN2 and controls the expression of stress response-related genes under cold, heat, and salt stresses.
In an initial screen for possible downstream cytoplasmic targets of the Mps1 MAP kinase pathway in M. oryzae, we found the hydrophobin gene MPG1 and an uncharacterized glycogen synthase kinase 3 (GSK3) homolog MoGSK1 were differentially expressed in the absence of MPS1 and in response to environmental stresses. Here, we studied the functions of the GSK3 homolog in M. oryzae (MoGsk1) transcriptionally regulated by the MPS1 MAPK pathway and revealed its critical role in fungal development and plant infection. Deletion of MoGSK1 causes remarkable reduction of mycelial growth and loss of asexual spore production necessary for normal spread and infection. Moreover, inoculation using the mycelial plugs of Δmogsk1 showed a failure of appressorium-like structure penetration and the mutant is unable to infect barley or rice host plants. The complementation by the Fusarium gramienearum homolog FGK3 fully restores the growth and pathogenicity of Δmogsk1, and the gene product shows the same cytosolic localization as MoGsk1. This indicates that the two GSK3 homologs in M. oryzae and F. graminearum shares highly conserved functions. Overexpression of MoGSK1 caused deformed appressoriain, possibly due to misregulation of morphogenesis checkpoint. Taken together, our results proves that MoGsk1 is a central signal regulator involved in the stress-responsive mechanism and controls multiple aspects of development in M. oryzae, from growth, asexual production to pathogenesis. was up-regulated in the Δmps1 mutant in CM medium compared to Guy11. The transcriptional induction of MoGSK1 appeared to be much more prominent when mycelia were exposed to acute and chronic salt stress in the Δmps1 mutant compared to Guy11 (Fig. 1B). Interestingly, the MoGSK1 transcript was hardly detectable in Guy11 under all in vitro conditions tested (Fig. 1B), suggesting a suppressing effect of Mps1 on the MoGSK1 transcription in the mycelial stage of the wild type strain. The importance of hydrophobin Mpg1 on infection-related development in M. oryzae has been well described 21,22 , here we decided to focus on functional characterization of MoGSK1.

Identification of MPG1 and
To determine the phylogenetic relationship of MoGsk1 with other fungal members of GSK3, protein alignments were performed using both identified and predicted fungal GSK3 members from S. cerevisiae, F. graminearum, Colletotrichum graminicola and Ustilago maydis. MoGsk1 is 41% identical to the S. cerevisiae Mck1, 91% identical to the F. graminearum Fgk3, 92% identical to the predicted C. graminicola CgGsk1 and 69% identical to the predicted Ustilago maydis UmGsk1 (Fig. S1). Previous studies showed disruption of the S. cerevisiae MCK1 causes increased cold sensitivity 23 . To test whether MoGsk1 is a functional GSK3 homolog, we expressed the MoGSK1 under the S. cerevisiae galactose-inducible GAL1 promoter in the yeast Δmck1 mutant. Under the SCiEnTiFiC RepoRTs | 7:945 | DOI:10.1038/s41598-017-01006-w induction of galactose as the carbon source, cold tolerance of the Δmck1 mutant was recovered by MoGsk1 when grown at 12 °C for 14 days (Fig. 2).

MoGsk1 is essential for normal mycelial growth and conidiation. To functionally characterize
MoGsk1, we performed targeted gene replacement of MoGSK1 in the wild type M. oryzae Ku80 strain (Fig. S2). The Δmogsk1 mutant displayed remarkable reduction of growth on CM medium (Fig. 3A). Indeed, Δmogsk1 showed a remarkable reduction of growth rate compared to Ku80. No conidia were detected in Δmogsk1 when cultured on oat media under constant light exposure (Fig. 3B). The lactophenol aniline blue was used to detect the fungal conidiophore that resists the staining. The staining of fungal aerial structures showed only aerial hyphae were detected and lack of conidiophore in Δmogsk1, while the conidiophore stalks in Ku80 remained grey (Fig. 3C). However, defects in mycelial growth and conidiation were fully recovered in the complementation strain Δmogsk1/MoGSK1 when the MoGSK1 gene was expressed in Δmogsk1 under the native promoter. There results suggest that, similar to the GSK3 homologs in S. cerevisiae and F. graminearum, MoGsk1 plays a critical role in fungal growth and conidiation 20,24 .  MoGsk1 is required for hypha-driven appressorium mediated pathogenesis. It has been revealed that mycelial tips can form appressorium-like structures able to penetrate plant surface in M. oryzae 25 . To test the role of MoGsk1 for hypha-driven appressorium development, three day-old mycelial plugs from CM medium were sliced and incubated on hydrophobic cover slips to induce appressorium-like structures. After 48 hr induction, comparable domed and melanized appressorium-like structures were induced at mycelial tips in Ku80 and Δmogsk1 (Fig. 4A). The appressorium-like structures were also observed by inoculating mycelial plugs on barley leaves in both Ku80 and Δmogsk1 for 48 hr (Fig. 4B). However, appressorium-like structures developed by Δmogsk1 failed to penetrate the barley leaf surface while extensive primary invasive hyphae were detected on Ku80-inoculated barley leaf surface (Fig. 4B). The pathogenicity assay was conducted on detached rice and barley leaves using mycelial plug inoculation. After 5-day inoculation, blast disease lesions were observed on Ku80-inoculated leaf surface but absent on Δmogsk1-inoculated leaf surface ( Fig. 5C and D). Even after abrasion of barley leaf surface, Δmogsk1 was still not virulent (Fig. 5D). Importantly, pathogenicity was completely recovered in the complementation strain Δmogsk1/MoGSK1 inoculated on rice seedlings under the same conditions (Fig. 4C).
Lithium has been shown as a direct inhibitor of GSK3 activity in mammalian cells 26 . Since no conidia were detected in the Δmogsk1 mutant, application of LiCl with increase concentrations (3-10 mM) was performed on wild type Guy11 conidia during the germination on hydrophobic cover slips for 16 hr to test the role of GSK3 activity on appressorium development. A gradual inhibition of appressorium formation was found with a total inhibition at 10 mM without affecting the conidial germination (Fig. S3), indicating an important role of MoGsk1 activity in appressorium development.

Subcellular localization of MoGsk1 and functional conservation with Fgk3.
To examine the subcellular localization, an expression construct of MoGSK1 directed by the native promoter and fused with GFP at the C-terminus was created and transformed into the Δmogsk1 mutant. The phenotypic analysis showed that mycelial growth, conidiogenesis, pathogenicity in the transformant Δmogsk1/MoGSK1-GFP was rescued (Fig. 5A  and B), suggesting the expression construct is functional. Epifluorescence microscopic examination of conidia showed cytoplasmic presence of MoGsk1-GFP but absence in the nuclei (Fig. 5C), which was consistent with the finding of Fgk3 mainly localizing to the cytoplasm at the conidial stage 20 . To reveal the functional relatedness between MoGsk1 and Fgk3, we expressed FGK3 under the MoGSK1 native promoter fused with GFP at C-terminus in Δmogsk1. As expected, FGK3 complemented MoGSK1, and the growth, conidogenesis and pathogenic deficiencies in Δmogsk1 were recovered as observed in Δmogsk1/MoGSK1-eGFP ( Fig. 5A and B). The subcellular observation showed that Fgk3 localized in conidial cytoplasm (Fig. 5C). These results indicated that the GSK3 homologs in phytopathogenic fungi were functionally conserved.
The effect of the MoGSK1 overexpression on appressorium morphology. The regulatory function of MoGsk1 is strictly associated with its spatial and temporal pattern 27 . Here, we aimed to explore the effect of the overexpressing MoGSK1 in appressorium development and pathogenicity. MPG1 has been shown to be highly induced during appressorium maturation 28 , therefore MoGSK1 was expressed under the MPG1 promoter in the Guy11 strain. The expression level of MoGSK1 in transformant P MPG1 :MoGSK1 was highly upregulated compared to Guy11 (Fig. 6A). Interestingly, the MoGSK1 overexpression resulted in production of elongated appressoria on hydrophobic cover slips (Fig. 6B). However, these appressoria were fully capable of penetrating the plant surface and differentiate into invasive hyphae that displayed similar aggression as the Guy11 strain (Fig. 5C).

Discussion
The SLT2 MAP kinase-dependent cell wall integrity signaling pathway in S. cerevisiae is involved in coordination of environmental stresses on the cell surface to cellular responses and development 29 . Upon activation, SLT2-mediated signaling can target at transcriptional factors like Rlm1 and Swi6p that further control the expression of genes in charge of cell wall compound synthesis 30,31 . In M. oryzae, Mps1 as the functional homolog of SLT2 controls cell wall integrity 10 . The Δmps1 mutant develops cell walls sensitive to degradation enzymes, produces less conidia and forms appressoria incapable of penetrating the plant surface. Both Rlm1 and Swi6 are identified as nuclear targets of Mps1 signaling pathway in regulation of cell wall integrity, cellular response and pathogenicity in M. oryzae 11,32 . Here, we showed two genes (MoGSK1 and MPG1) with cytoplasmic location of their encoding products were targeted by the Mps1 signaling pathway in the Northern blot-based differential expression-screening assay (Fig. 1). Unexpectedly, higher expression of MoGSK1 was detected in the Δmps1 mutant particularly responding to hyper-osmotic stress, indicating an unusual role of MoGSK1 in the signaling transduction. In other organisms, besides nuclear targets, cell wall integrity signaling pathways have cytoplasmic targets such as calcium channels, MAP kinase phosphatases and tyrosine phosphatases 13 . However, no cytoplasmic targets have been characterized in the cell wall integrity signaling pathway in M. oryzae.
GSK3 is well-known for its varied functions in regulation of cellular events in eukaryotic organisms 14 . A recent study in F. graminearum showed the sole GSK3 homolog Fgk3 had pleiotropic effects in fungal development and pathogenesis 20 . Fgk3 is involved in stress response via physical interaction with the stress-responsive transcriptional activator Msn2. After sensing the environmental cues, signaling cascades in plant pathogens are triggered and promote the differentiation of infection-related structures on plant surface 33 . Appressorium formation and penetration are nesessary for infection of M. oryzae. Gene targeted replacement of MoGSK1 caused the production of non-functional hypha-driven appressoria, resembling the effect of MPS1 deletion on appressorium development (Fig. 4). As a constitutively active kinase, regulatory role of GSK3 is usually through its inactivation after being subject to phosphorylation by upstream kinases 16 . This suggests that expression of MoGSK1 may be targeted and suppressed by the Mps1 signaling pathway following the recognition of environmental cues, causing the subsequent unleash of GSK3 downstream targets and activation of genes related to cell wall biogenesis in matured appressoria. On the contrary, overexpression of MoGSK1 produced elongated appressoria, which also supports the timely regulated MoGSK1 signaling output is critical for proper appressorium development (Fig. 6).
As a central regulator in various cellular events, MoGsk1 has extensive roles in growth and development of M. oryzae. Deletion of MoGSK1 significantly reduces the mycelial growth and abolishes fungal conidiation (Fig. 3), which are more severe compared to phenotypes observed on the Δmps1 mutant 10 . This suggests that MoGsk1 is also involved in other signaling pathways more than the Mps1-mediated pathway. Pleiotropic effects of GSK3 in fungal life cycle has been observed in F. graminearum 20 . Expression of Fgk3 in Δmogsk1 has comparable effect with MoGSK1 in the complementation assays and shows the similar cytoplasmic localization in conidia (Fig. 5). Together with the cold-sensitivity rescue effect of MoGSK1 in S. cerevisiae Δmck1 (Fig. 2), these results suggest GSK3 members in fungi are structurally conserved and share fundamental functionalities across related species. Thus, the role of MoGsk1 is more about the regulation of the cell wall integrity pathway. In budding yeast S. cerevisiae, Mck1 was also identified as a component of the Ca 2+ signaling pathway that down-regulates Hsl1, a morphogenetic checkpoint protein kinase controlling proper timing of septin collar formation at mother-bud neck 34,35 . In M. oryzae, a septin-forming ring at the appressorium base has been observed to scaffold a toroidal F-actin network that is necessary for penetration of leaf cuticles 36 . However, both septin ring and F-actin network were absent in Δmps1, contributing to the penetration defect in Δmps1 36 . This may indicate that MoGsk1 mediates another signaling pathway to facilitate timing and establishment of appressorium morphogenesis. Elongated appressoria detected in the MoGSK1 over-expression strain also suggests a link between the morphogenesis checkpoint and MoGsk1 (Fig. 6).
Another possible role of MoGsk1 is upon on its regulation of glycogen metabolism that is important for pathogenic progression in M. oryzae 37 . In F. graminearum, conidia of Δfgk3 accumulates more glycogen than the wild type strain 20 , suggesting an unbalanced glycogen homeostasis. Given the close relatedness between two GSK3 members of M. oryzae and F. graminearum (Fig. 5), it is plausible that MoGsk1 is involved in glycogen metabolism. It is noted that, rather than serving as a nutrient source, glucose and its derivates play roles as signal molecules in mediation of the invasive growth of M. oryzae via the modulation of the NADPH-dependent genetic switch 37,38 . The protein kinase MoYAK1 responsible for modulating signal pathways triggered in response to surface hydrophobicity, glycogen status and cell wall integrity exert profound impacts on fungal growth, conidiation and infection-related development in M. oryzae 39 . This confirms that protein kinases functioning in transmitting extracellular stimuli are able to regulate a diverse array of downstream pathways. Therefore, the MoGsk1-centring regulatory network involved in infection-related development in M. oryzae remains to be clarified. In the future, it will be important to determine the physical downstream targets of MoGsk1 and reveal their roles in different aspects of fungal development and pathogenesis.

Methods
Fungal strains and growth condition. Media composition, fungal growth, nucleic acid extraction and DNA-mediated transformation were all carried out as described previously 40 . Restriction digests, gel electrophoresis, and DNA and RNA gel blot hybridizations were all carried out using standard procedures 41 .
Complementation of S. cerevisiae Δmck1 with MoGSK1. The 1.2 kb MoGSK1 cDNA was amplified using the Thermo Start Master Mix (Thermo Fisher Scientific), and ligated into the pYES2 vector (Invitrogen) using primers (GSK1YESF and GSK1YESR) resulting in the construct pYES-MoGSK1. Positive clones were confirmed by restriction digest and transformed into the S. cerevisiae Δmck1 strain (BY4741; Mata; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YNL307c::kanMX4) (EUROSCARF). The transformation of S. cerevisiae was carried out as described by 42 using the standard high efficiency transformation protocol.

MoGSK1 gene targeted replacement and complementation.
In order to knock out MoGSK1, a 1142 bp upstream fragment of the MoGSK1 ORF in M. oryzae genome was amplified using primers GSK11F and GSK11R, and cloned into the KpnI and HindIII sites on pCX62 generating the resulting construct pCX63. Then a 1267 bp fragment at the downstream of MoGSK1 ORF was amplified using primers GSK12F and GSK12R, and then cloned into SpeI and SacI sites on pCX63 resulting in the MoGSK1 ORF replacement construct pCX64, which contained the hygromycin phosphotransferase gene (hph) as the selective marker flanked by two MoGSK1 ORF flanking sequences. pCX64 was then transformed into Ku80 protoplast. Transformants with hygromycin resistance were screened by PCR with primers GSK13F and GSK13R to detect MoGSK1 ORF, and GSK14F and GSK14R to detect the fragment spanning over hph gene and 5′ end flanking sequence of MoGSK1 ORF. To rescue the Δmogsk1 phenotypes, a 7253 bp DNA fragment containing the native promoter, MoGSK1 ORF and 3′ end UTR was amplified using primers GSK15F and GSK15R. The complementary strain was generated by co-transforming the PCR fragment with the neomycin-resistant gene (neo) containing vector pKNTG into Δmogsk1 protoplast. Transformants were screened on neomycin containing plates and confirmed by Southern blot analysis.
Characterization of the Δmogsk1 mutant. Mycelial plugs of Ku80 and Δmogsk1 were inoculated on CM medium in 9 cm diameter petri dish, the diameter of fungal colonies were measured after 3, 5, 7 and 10 days inoculation with three replicates. The capability to produce conidia was measured by calculating conidia collected in 10-day-old cultures on oatmeal agar media with consistent exposure to light per pertri dish plate. The observation on conidial development from conidiophores was performed as follows. The mycelia on 5-day-old oat meal cultures were pushed down with a glass slide. A 5 mm thick oat meal agar block was out from the edge of the colony and placed on a glass slide and incubated in a moisture chamber exposed to continuous light for 48 hours. Development of conidia was then examined by a light microscope (Zeiss Axioplan). In order to stain the conidiophores, an aqueous of oat meal agar was placed on a sterile glass slide, following the inoculation of fungal strains on oat meal agar. The glass slides were kept in a moist chamber under continuous light for 4 days. The agar block was then removed, and stained using a drop of 0.1% lactophenol aniline blue for 3 minutes before a cover slip was applied on. The slide was observed under a light microscope (Zeiss Axioplan).
Infection assays with rice and barley leaves. Equal amounts of mycelial plugs (1 cm in diameter) were sliced out from the edge of 3-day-old colony on CM medium culture, and then inoculated on 7-day-old susceptible barley seedlings (CDC Silky) and 15-day-old rice seedlings (CO39). Lesion formation was examined after 5 days' inoculation. To observe the appressorium penetration and invasive hyphae growth, 48 hours old inoculated barley leaves were sampled and fixed in a fixation solution (60% methanol, 30% chloroform, 10% acetic acid) for decolorization. Fixed samples were then rehydrated with reduced gradient ethanol (95%, 75%, 50% and 25%) and distilled water. Appressoria and invasive hyphae were then examined by a light microscope (Zeiss Axioplan).
SCiEnTiFiC RepoRTs | 7:945 | DOI:10.1038/s41598-017-01006-w Subcellular localization and complementation of MoGsk1 and Fgk3. To construct MoGSK1-GFP, a DNA fragment containing the 5 kb promoter region, and 1.6 kb MoGSK1 ORF were PCR-amplified using primers (MoGSK1/NF and MoGSK1-GFP/R) from Ku80. To construct Fgk3-GFP, the 5 kb MoGSK1 promoter region was amplified using primers (MoGSK1/NF and MoGSK1/NR) from Ku80. The 1.2 kb Fgk3 ORF region was amplified using primers (Fgk3/OF and Fgk3-GFP/R) from wild type F. graminearum PH-1 cDNA and ligated at 3′ end of the MoGSK1 promoter region via overlap PCR approach. Both PCR-amplified fragments of MoGSK1 and Fgk3 under the same MoGSK1 promoter were cloned into KpnI and HindIII double-digested GFP-contained pKNTG by an in vitro recombination approach using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China). The resulting construct pKNTG-MoGSK1-GFP and pKNTG-Fgk3-GFP were transferred into Δmogsk1 protoplasts, respectively.
Creation of MoGSK1 over-expression strain P MPG1 :MoGSK1. The MoGSK1 over-expression vector was created by fusing a 1.3 kb fragment of the MPG1 promoter 43 with the MoGSK1 gene. A 1.9 kb genomic MoGSK1 fragment was amplified using a forward primer GSK1OEF designed to span the ATG translation initiation codon and a reverse primer GSK1OER after the stop codon. The 1.3 kb MPG1 promoter had been previously amplified and cloned into pGEM-5Z ® , to form pNJT190 44 . The 1.9 kb genomic fragment spanning the MoGSK1 locus was amplified by PCR using Thermo Start Master Mix (Thermo Fisher Scientific) and ligated into pNJT190. The complete 3.2 kb P MPG1 :MoGSK1 construct was removed from pNJT190 and ligated into pCB1004. The resulting construct pCB1004-P MPG1 :MoGSK1 was transformed into Guy11 protoplasts and screened on hygromycin B-containing plates. The expression of MoGSK1 was tested by Northern blot analysis using a 1 kb MoGSK1 fragment as the probes amplified from Poly (A)+ purified cDNA.
All primers are listed in Table S1.