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Mol Plant Pathol. 2024;25:e13493.
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1 of 17
https://doi.org/10.1111/mpp.13493
wileyonlinelibrary.com/journal/mpp
Received:26Januar y2024
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Revised:24J une2024
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Accepted :25June2024
DOI : 10.1111/mp p.13 493
ORIGINAL ARTICLE
A novel MAP kinase- interacting protein MoSmi1 regulates
development and pathogenicity in Magnaporthe oryzae
Yu Wang1,2 | Xinyue Cui1,2 | Junlian Xiao1,2 | Xiaoru Kang1,2 | Jinmei Hu1,2 |
Zhicheng Huang3 | Na Li1,2 | Chuyu Yang1,2 | Yuemin Pan1,2 | Shulin Zhang1,2
1Depar tment of Plant Pat holog y, College
ofPlantProtection,AnhuiAgricultural
University, Hefei, China
2AnhuiProvinceKeyL aboratoryofCrop
IntegratedPestM anagem ent,Anhui
AgriculturalUniversity,Hefei,China
3StateKeyLaboratoryforManagingBiotic
and Chem ical Threats to the Quality an d
SafetyofAgro- Produc ts,CollegeofLife
Science s, Zhejiang Universit y, Hangzhou,
China
Correspondence
Yuemin Pan and Shulin Zhang,
Depar tment of Plant Pat holog y, College
ofPlantProtection,AnhuiAgricultural
University, Hefei 230036, China.
Email: panyuemin2008@163.com and
zhangsl80h@ahau.edu.cn
Funding information
TheNaturalScienceFoun dationofA nhui
Higher Education Institutions, Grant/
AwardNumber:K J2020A0102;TheTalent
ResearchProjec tofAnhuiAgri cultur al
University,Gra nt/AwardNumber:
rc3420 01; The Nat ional Natural S cience
Foundat ionofChina,Gra nt/Award
Number: 32202253
Abstract
The cell wall is the first barrier against external adversity and plays roles in maintain-
ing normal physiological functions of fungi. Previously, we reported a nucleosome
assembly protein, MoNap1, in Magnaporthe oryzae that plays a role in cell wall integ-
rity (CWI), stress response, and pathogenicity. Moreover, MoNap1 negatively regu-
lates the expression of MoSM I1 encoded by MGG_03970. Here, we demonstrated
that deletion of MoSMI1 resulted in a significant defect in appressorium function,
CWI, cell morphology, and pathogenicity. Further investigation revealed that MoSmi1
interacted with MoOsm1 and MoMps1 and affected the phosphorylation levels of
MoOsm1, MoMps1, and MoPmk1, suggesting that MoSmi1 regulates biological
functionsby mediatingmitogen-activated proteinkinase(MAPK) signallingpathway
in M. oryzae. In addition, transcriptome data revealed that MoSmi1 regulates many
infection-relatedprocessesinM. oryzae,suchasmembrane-relatedpathwayandoxi-
dation reduction process. In conclusion, our study demonstrated that MoSmi1 regu-
latesCWIbymediatingtheMAPKpathwaytoaffectdevelopmentandpathogenicity
of M. oryzae.
KEYWORDS
appressorium formation, cell wall integrity, pathogenicity, regulation, rice blast fungus
1 | INTRODUC TION
Rice blast, caused by Magnaporthe oryzae, is one of the most de-
structive fungal diseases that decrease rice yield and seriously
threaten food security (Dean et al., 2012). M. oryzae infection be-
ginswithathree-celledconidium.Theconidiumattachestothehost
leafsurfaceanddevelopsintoagermtubewithin2 h.Subsequently,
a domed-shaped structure called the appressorium differentiates
from the tip of the germ tube (Hamer et al., 1988; Hamer & Talbot,
1998; Howard & Valent, 1996). With the accumulation of high con-
centrations of glycerol, the appressorium generates sufficient tur-
gor pressure to form an infection peg and penetrates the host cell
(Howard et al., 1991). Subse quently, typi cal necrotic s pots of rice
blast appear on plant sur facesaf ter about5 days. Finally,theinva-
sive hyphae spread within plant cells, resulting in t ypical lesions, and
the secondary conidia spread the disease to adjacent plants (Gupt a
et al., 2021). In the process by which the rice blast fungus infects the
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provide d the original wor k is properly cited.
©2024TheAut hor(s).Molecular Plant Pathologypub lishedbyBritis hSociet yforPlantPathologya ndJohnW iley&SonsLtd.
YuWang,Xiny ueCuian dJunli anXiaoco ntrib utedeq uallytot hiswork .
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host, numerous signal transduction pathways receive and transduce
extracellular signals to regulate grow th, development, and pathoge-
nicity of M. oryzae(Lietal.,2012).
Mitogen-activated protein kinase (MAPK)-mediated path-
ways have been shown to regulate appressorium development,
appressorium-mediated penetration, cell wall integrity (CWI)
and response to stress in M. oryzae (Cai et al., 2022; Tucker &
Tal b ot, 2001). Appressorium-mediated penetration isimpor tant
for pathogenicity of M. oryzae. During appressorium formation,
Gprotein-coupledreceptors(GPCRs)recognizehydrophobicsur-
face signals and activate the G protein signalling pathway, which
controls crosstalk between the cAMP-PKA and Pmk1 MAPK sig-
nalling pathways in M. oryzae (Ebbole, 2007;Kou &Naqvi, 2016;
Lietal., 2012; McDonough & Rodriguez, 2011).The CWIMAPK
cascade is composed of MoMck1 (Bck1 homologue), MoMkk1
(Mkk1/2 homologue), and MoMps1 (Slt2/Mpk1 homologue). To
maintain cell morphology, MoMck1 delivers signals to MAPK
kinase, MoMkk1, which in turn activates the MAPK MoMps1
through protein phosphorylation. MoMps1 phosphorylates down-
stream transcription factors to regulate the nuclear expression
of genes involved in cell wall biosynthesis and cell cycle progres-
sion (Jeon et al., 2008; Xu et al., 1998; Y in et al., 2 016). Cell wall
remodelling is important for maintaining fungal growth and de-
velopment. In M. oryzae, MoMCK1 regulates cell wall remodelling
and resists plant defences (Jeon et al., 2008). T he Osm1 MAPK
pathway consists of MoSsk2, MoPbs2, MoOsm1, and an adaptor
protein MoMst50, which play essential roles in response to hyper-
osmotic stress (Dixon et al., 1999;Lietal.,2017 ).
We previously reported a nucleosome assembly protein, MoNap1,
that regulates appressorium formation, response to cell wall stresses,
cytoplasmic division, and virulence (Zhang, Wang, et al., 2022).Based
on transcriptome data previously, we screened for differentially ex-
pressedgenes (DEGs) between the wild-type Guy11 and ΔMonap1
mutant and identified MGG_03970.Bioinformaticsanalysisrevealed
that the protein product encoded by MGG_03970 is homologous to
Smi1. Smi1, also k nown as Knr4, is an in trinsicall y disordered pr o-
tein (IDP) conserved in many fungi (Martin-Yken et al., 2016). In
Saccharomyces cerevisiae, Smi1 acts as a hub that physically interacts
withthe keycomponentsoft wopathways: RhoGTPase-proteinki-
naseC-MAPkinaseintheCWIpathwayandcalcineurinphosphatase
in the calcium-calcineurin pathway (Dagkessamanskaia, Durand,
et al., 2010;Dagkessamanskaia,ElAzzouzi,etal.,2010; M a r t i n - Y k e n
et al., 2016).K nr4hasdi versebiologi calfunctions,includi ngcel lcycl e
progression, CWI, morphogenesis, and response to heat and cell wall
stress,byregulatingassociatedtranscriptionalprogrammes(Lagorce
et al., 2003;Martin-Ykenetal.,2002; Penacho et al., 2012). In S. cer-
evisiae,Knr4participates inCln3-Cdc28-dependentgenetranscrip-
tionwithBck2attheG1/Stransition(Kuravietal.,2011; M a r t i n - Y k e n
et al., 2002) . A d d i t i o n a l l y , l o s s o f K n r 4 r e s u l t s i n t h e d i s o r d e r e d f u n c t i o n
of at least two cell cycle checkpoints: the morphogenesis checkpoint,
which combines cell division with bud grow th, and the mechanism
controlling daughter cell size during cytokinesis (Dagkessamanskaia,
Durand, et al., 2010; Dagkessamanskaia, El Azzouzi, et al., 2010;
Harrison et al., 20 01; Miyakawa & Mizunuma, 2007; Mizunuma
et al., 2001). In the human pathogen Candida albicans, Smi1 expres-
sion is induced in the pathogenic hyphal cells; the sm i1Δ/smi1Δ mu-
tant shows reduced cell wall β-glu cansynthe sisandb io fi lmformation
and reduced biofilm-associated fluconazole resistance, which sug-
gests a positive effect on the CWI pathway (Harcus et al., 2004; Nett
et al., 2011). In Fusarium asiaticum,FaSmi1isakeyproteinrequired
for the vegetative development, asexual reproduction, deoxyniva-
lenol (DON) produc tion and virulence (Zhang, Chen, et al., 2022).
In addition, KNR4 contributes to a significantly increased release of
polysaccharides and mannoproteins into the culture medium, which
is of spec ial int e res tin o eno l ogi c alf erm e ntat ion p roc ess e s(G o nza l ez-
Ramos et al., 2008).
AlthoughthereissomeevidencethatSmi1isinvolvedintheCWI
pathway,howSmi1regulatesMAPKcascadesandthepathogenicity
of the blast fungus remains unclear. Here, we evaluated the functions
of MoSmi1 and revealed that MoSmi1 regulates vegetative growth,
conidiation, cell morphology, appressorium formation, host reactive
oxygen species (ROS) scavenging, CWI, and pathogenicit y. In addi-
tion, MoSmi1 interacts with MoOsm1 and MoMps1, which are key
compone nts of the Osm1 and CW I MAPK pathways, re spectively.
Phosphorylation levels of MoOsm1, MoMps1, and MoPmk1 were in-
creased in ΔMosmi1. Our study reveals that MoSmi1 affects the bio-
logical f unctions of M. oryzaebyregulatingMAPKsignallingcascades.
2 | RESULTS
2.1 | Identification and analysis of MoSmi1 in M.
oryzae
Through R NA sequencing ( RNA-seq) analysis, we p reviously foun d
that the gene MGG_03970 was upregulated in the ΔMonap1 mutant
(Zhang, Chen, et al., 2022; Zhang, Wang, et al., 2022). To verify the
reliability of transcriptome data, we assessed the transcription lev-
els of MGG_03970 in Guy11 and ΔMonap1 mutant using reverse
transcription-quantitative PCR (RT-qPCR). The results showed that
the transcription level of MGG_03970 was upregulated approximately
1.6 times in the ΔMonap1mutantco mparedwit ht hatinth ewild-t yp e
strain Guy11 (Figure S1).Th isgen ee nc od esa571-aminoac id(aa )pro-
teinwith the Smi1_K nr4domain.Therefore,we namedMGG_03970
as MoS MI1 in M. oryzae. Phylogenetic analysis revealed that Smi1 is
conserved in the fungal kingdom, and MoSmi1 has the highest homol-
ogy with Smi1 of Colletotrichum graminicola (Figure S2a). Domain pre-
dictionanalysisrevealed that Smi1containsaSmi1_Knr4 domain in
various fungi (Figure S2b). We predicted three intrinsically disordered
regions (ID Rs) in the amino a cid sequence of M oSmi1 (Figure S2c).
In addition, MoSmi1 is closely related to proteins from other fungi.
TheresultsfrommultiplesequencealignmentindicatedthatMoSmi1
shares 58% amino acid identity to that of Fusarium graminearum,
52% to Trichoderma reesei, 56% to Neurospora crassa, and 33% to
Schizosaccharomyces pombe (Figure S2d). Taken together, these re-
sults revealed that the Smi1 proteins are conserved in fungi.
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2.2 | Subcellular localization of MoSmi1 at
different developmental stages in M. oryzae
To determine the subcellular localization of MoSmi1 at different
developmental stages in M. oryzae, aMoSmi1-green fluorescence
protein (GFP) construct under the control of its native promoter
wastransformedintothewild-type strainGuy11.GFPsignalswere
examined in mycelia, conidia, appressoria, and invasive hypha using
confocal m icroscopy. We observed t hat MoSmi1-GFP was mai nly
distributed throughout the cytoplasm in all tested stages and vacu-
oles at the a ppressorium fo rmation stage. In a ddition, punc tate green
fluorescence was observed in the conidia (Figure S3A). We further
stainedappressoriawith7-amino-4-chloromethylcoumarin(CMAC)
andverified thatMoSmi1-GFPwas also localizedinvacuolesatthe
appressorium formation stages (Figure S3B). These results suggest
that MoSmi1 plays an important role in all developmental and plant
infection processes of M. oryzae.
2.3 | MoSmi1 functions in vegetative growth,
conidiation and morphogenesis in M. oryzae
To investigate the biological functions of MoSMI1 in M. oryzae,
we generated the MoSMI1 deleti on mutant in t he wild-type st rain
Guy11 background using a homologous recombination strategy
(Figure S4a). The putative Mo SMI1 deletion mutant transformants
wereverifiedbyPCR ,RT-PCRandSouther nblotting(Figure S4b–d).
We named the M oSMI1 deletion mutant as ΔMosmi1. In addition, the
genetic complementation strain for the ΔMosmi1 mutant was gener-
ated by introducing the MoS MI1 gene with its native promoter into
the ΔMos mi1 mutant (#2), namely, ΔMosmi1/MoSMI1 (HB strain),
and verified by RT-PCR analysis (Figure S4d).The wild-type strain
Guy11, ΔMosm i1 mutant, and the complemented strain ΔMosmi1 /
MoSMI1 were used for phenotypic analyses.
To investigate the contribution of MoSMI1 in veget ative grow th
of M. oryzae,weculturedthewild-t ypestrainGuy11,ΔM osmi1 mu-
tant, and the complemented strain ΔMosmi1/MoSMI1 on complete
medium (CM) plates for 7 days at 28°C in darkness. Thecolony di-
ameter of the ΔMos mi1 mutant was significantly smaller than that of
thewild-typestrainGuy11andthecomplementedstrainΔMosm i1/
MoSMI1 (Figure 1a,b). In addition, we assessed the morphology of
the vegetative hyphae in all tested strains. Results from this assay
indicated that the vegetative hyphae of the ΔMosmi1 mutant
showed a cur ved morpholog y, suggesting MoSMI1 is important for
the maintenance of polarized grow th (Figure 1c). Given that conidia
are the primary infection propagules of M. oryzae, we also examined
whether Mo SMI1 deletion affects sporulation capacity and conidial
development.AsshowninFigure 1d, the number of spores on the
conidiophores of the ΔMosmi1 mutant was signific antly lower than
that on the c onidiophore s of the wild-type st rain Guy11, and t he
complemented strain ΔMosmi1/MoSMI1. We counted the number of
conidia and found that conidiation of theΔMosm i1 mutant was sig-
nificantly decreased (Figure 1e). Moreover, calcofluor white (CF W)
staining assay indicated that approximately 50% of the ΔMosm i1
mutantconidiawereone-ortwo-celled(Figure 1f,g). Taken together,
these results suggest that MoSM I1 is involved in vegetative grow th,
conidiation, and conidial development of M. oryzae.
2.4 | Deletion of MoSMI1 disrupts microtubule
structure and nuclear distribution in hyphae
Asshownintheaboveresult, deletionofM oSMI1 affected the po-
larized growth of vegetative hyphae. We inferred that MoSM I1 may
play a role in regulation of microtubule dynamics. To assess the role
of MoSMI1 in microtubule, we examined the sensitivity of all tested
strains to the microtubule inhibitor benomyl (Hoyt et al., 1991). As
shown in Figure 2a,b, the relative inhibition rate of the ΔMo sm i1 mu-
tantwashigherthanthatofwi ld-t ypestra in Gu y11,andthecomple -
mented strain ΔMosmi1/MoSMI1, suggesting that MoSmi1 affects
microtubule function. Fur thermore, we expressed a β-tubulin-red
fluorescence protein (RFP) fusion protein in the wild-type strain
Guy11, ΔMosmi1 mutant and the complemented strain ΔMosmi1/
MoSMI1 background. Microscopic examination showed that β-
tubulin-RFP of the wild-t ype strain Guy11 and the complemented
strain ΔMosmi1/MoSMI1 background were parallel to their grow th
axes and formed a linear structure in the cytoplasm. In contrast, in
the ΔMos mi1 mutant background, the linear structure of β-tubulin-
RFP was disrupted and the RFP signal exhibited a dispersed state
(Figure 2c). In addition,we treated the wild-t ype strain Guy11 ex-
pressing β-tubulin- RFP with 0. 4 μg/mL benomyl for 6 h an d found
that the linear RFP signal gradually dispersed (Figure S5), which was
similar to the ΔMosmi1 mutant, fur ther indicating that MoSM I1 af-
fects microtubule formation. Microtubules are important for normal
cell cycle progression (Hoyt et al., 1991). Therefore, we expressed
Histone1(H1-RFP)inthewild-t ypestrainGuy11andΔMosm i1 mu-
tant and stained hyphae using CFW to determine whether MoSMI1
deletion affects nuclear distribution. We found that in wild-type
Guy11, there was only one nucleus in most hyphal cells (92%),
However, the number of nuclei was abnormal (no or more than one)
in 38% hyphal cells of the ΔM osmi1 mutant, suggesting that MoSmi1
is involved in regulating cytokinesis of M. oryzae (Figure 2d,e).
Overall, these results indicated that MoSmi1 regulates the microtu-
bule structure and nuclear distribution in M. oryzae.
2.5 | MoSmi1 is required for pathogenicity of
M. oryzae
To determine whether deletion of MoSMI1 affects the pathogenicity
of M. oryzae,wetestedthevirulenceofthewild-t ypestrainGuy11,
ΔMos mi1 mutant, and the complemented strain ΔMosmi1/MoSMI1
on the susceptible barley cv. Golden Promise and susceptible rice
seedlings (Oryzae sativa ‘CO39’). First, mycelial plugs or conidial sus-
pensions(5 × 104 conidia/mL)ofalltestedstrainswereinoculatedon
detachedbarleyleaves.At5 dayspost-inoculation(dpi),theΔMosm i1
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FIGURE 1 MoSM I1 is important for vegetative growth, mycelial morphology, conidiation, and conidial morphology of Magnaporthe
oryzae.(a)Coloniesofthewild-typestrainGuy11(W T),ΔM osmi1 mutant, and the complemented strain ΔMosmi1/MoSMI1 on complete
medium(CM)plateswereobservedandcapturedafter7 daysat28°C.(b)Colonydiametersweremeasuredandstatisticallyanalysed.For
each strain, three independent biological experiments were performed with four replicates each time. Error bars represent SD and asterisks
indicatesignificantdifferencesbetweentheWTstrainGuy11and∆Mos mi1 mutant estimated using Student's t test (**p < 0.01).(c)The
hyphalmorphologyofalltestedstrains.AllthetestedstrainswereculturedinliquidCMfor48 handphotographedunderaninverted
fluorescentmicroscope.Bar,20 μm.(d)Allthestrainswereincubatedonanartificialhydrophobicsurfacefor24 hat28°C.Conidiaand
conidiophoreformationwereobservedandphotographedusinganinvertedfluorescentmicroscope.Bar,50 μm. (e) Statistical analysis of the
conidiation of all tested strains. For each strain, three independent biological experiments with four replicates were per formed each time.
Error bars represent SDandasterisksindicatesignificantdifferencesbetweenthewild-typestrainGuy11,ΔMosmi1 mutant estimated using
Student's t test (**p < 0.01).(f)Conidialmorphologyofthetestedstrains.ConidiacollectedfromtheWTstrainGuy11,ΔMosmi1 mutant
and complemented strain ΔMosmi1/MoSMI1 were stained with calcofluor white (CF W) and photographed under an inver ted fluorescent
microscope.Bar,20 μm. (g) Propor tion of each conidial type. One hundred conidia were counted for each strain and three experiments were
performed. Error bars represent SD and asterisks indicate significant differences (*p < 0.05).
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mutant caused more restricted lesions in both intact and wounded
leavesthanthewild-typestrainGuy11andthecomplementedstrain
ΔMosmi1/MoSMI1 (Figure 3a–d). Furthermore, conidial suspensions
(5 × 104 conidia/mL)ofalltestedstrainsweresprayedontosuscepti-
bleCO39riceseedlings.At7dpi,thewild-typestrainGuy11andthe
complemented strain ΔMosmi1/MoSMI1 produced typic al lesions on
rice leaves, whereas the ΔMo smi1 mutant produced smaller lesions
(Figure 3e,f).Basedontheseresults,weconcludethatMoSMI1 plays
an important role in the pathogenicity of M. oryzae.
2.6 | MoSmi1 affects appressorium formation,
invasive hyphal expansion and host ROS scavenging
To explore the cause of the pathogenicity defect in the ΔMos mi1 mu-
tant, we first determined whether deletion of MoSM I1 affects appres-
sorium formation in M. oryzae.Conidial suspensions (5 × 10 4 conidia/
mL)ofallthetestedstrainswereinoculatedonhydrophobiccoverslips,
and appressorium formation was observed at different time points (6,
12, and 24 h). We obser ved less than 10% of t he ΔMosmi1 mutant
conidiaformeda ppressoria,incontrasttomorethan90%ofthewild-
type s train Guy11 and th e complemented s train ΔMosmi1/MoSMI1 co-
nidia at all time points (Figure 4a,b). This result indicated that MoSMI1
is important for appressorium formation in M. oryzae. Furthermore,
weper formeda penetration assay usingthe wild-typestrain Guy11,
ΔMosmi1 mutant, and the complemented strain ΔMosmi1/MoSMI1 on
the barley epidermis. Invasive hyphae (IH) were classified into four
types: type I (no hyphal penetration), type II (IH with one branch), type
III (IH with at least two branches, but not extending to the neighbour-
ing cells), and t ype IV (IH that has numerous branches and extends to
the neigh bouring cel ls). At 24 hp ost-inocu lation (hpi), t he wild-t ype
strain Guy11 and the complemented strain ΔMo smi1/MoSMI1 formed
approximately 35% IH as type II and approximately 5% IH as type III,
whereas the ΔMosm i1mutantproducedmorethan90%astypeI.At
36 hpi, more than 85% IH were t ype I in the ΔMosmi1 mutant, com-
pared with approximately 5% type I, 30% type II, 50% t ype III, and
FIGURE 2 MoSM I1isrequiredfortheorganizationofmicrotubuleandcytoplasmicdivisioninMagnaporthe oryzae. (a) Colonies of the
wild-t ypestrainGuy11,ΔMosmi1 mutant, and complemented strain ΔMosmi1/MoSMI1 were cultured in complete medium (CM) plates
containing15 μg/mLbenomylindarknessat28°Cfor7 days.(b)Statisticalanalysisoftherelativeinhibitionrate(%)ofthetestedstrains.
For each strain, three independent biological experiment s with four replicates were performed each time. Error bars represent SD, and
asterisksabovethecolumnsindicatesignificantdifferencesbetweenthewild-typestrainGuy11,ΔMo sm i1 mutant estimated by Student's t
test (**p < 0.01).(c)Subcellularlocalizationofβ-tubulin-RFPinthewild-typestrainGuy11,andtheΔM os mi1 mutant, and the complemented
strain ΔMosmi1/MoSMI1invegetativehyphaestage.Bar,10 μm.(d)SubcellularlocalizationofH1-RFPinthewild-typestrainGuy11and
ΔMos mi1mutantinvegetativehyphae.Bar,10 μm. Cell wall was visualized using calcofluor white (CFW). The fluorescence signals were
observed using a laser scanning confocal microscope. (e) Statistical analysis of the proportion of abnormal number of cell nuclei in a single
cell.Atleast100hyphalcellswerecountedineachstrain.Threeexperimentswereperformed.ErrorbarsrepresentSD and asterisks indicate
significant differences (**p < 0.01).
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15% type IV i n the wild-t ype stra in Guy11 and the comp lemented
strain ΔMosmi1/MoSMI1. Even if at 48 hpi, the ΔMosmi1 mutant
formed more than 80% type I IH. However, more than 50% IH were
typeIVandtypeInol ongere xistedint hewild-ty pestr ainGuy11and
the complemented strain ΔMosmi1/MoSMI1 (Figure 4c,d). These data
indicate that MoSmi1 regulates IH expansion. Taken together, these
results demonstrate that MoS MI1 deletion delays appressorium for-
mation and restricts IH expansion.
Given the deficient invasive grow th of ΔMosm i1, we specu-
lated that host ROS inhibit the extensionof IH during infection. A
3,3′-diaminobenzidine(DAB)stainingexperimentwasperformedto
detectROSaccumulationinbarleyleafcells.At30hpi,onlyapprox-
imately25%plantcellsinfectedwiththewild-typestrainGuy11and
the complemented strain ΔMosmi1/MoSMI1 were stained, whereas
approximately 65% of plant cells infected by the ΔM osmi1 mutant
were stained with DAB (Figure 4e,f). These results suggest that
ROS accumulation increased in barley leaf cells infec ted with the
ΔMos mi1 mutant . We hypothesized that this was due to the limited
ROS scavenging abilit y of the ΔMosmi1 mutant. Furthermore, di-
phenyleneiodonium (DPI) was used to treat barley epidermal cells.
Wefoundthatat30hpiwhentreatedwith0.5 μM DPI, the invasive
growth was greatly enhanced compared with the control dimethyl
sulfoxide (DMSO) treatment (Figure 4g). In addition, we measured
the expr ession level of 10 ROS detoxif ication-related gen es (Guo
et al., 2010; Huang et al., 2011; Ren et al., 2022; Yi et al., 2009) in
the wild-type strain Guy11andthe ΔMosm i1 mutant. Asshown in
Figure 4h, APX2, PR X1, TPX1, CCP1, LHS1, and KAR2 were signifi-
cantly downregulated in ΔMosmi1 compared with the wild-t ype
strain Guy11. The relative expression levels of HYR1 and TRX2
were slightly upregulated, and there was no significant difference
in ATF1 and N MO1 in the ΔMo sm i1mutantcomparedwiththewild-
type strain Guy11. Taken together, our data indicate that MoSmi1 is
involved in scavenging host ROS and that the defective pathogenic-
ity of ΔMosm i1 was partly due to the accumulation of ROS.
2.7 | MoSmi1 affects the organization of the septin
ring in M. oryzae
Anormalappressoriumiskeyforpathogenpenetration,whichrelies
ontherecruitmentandorganizationofseptin-dependentcytoskel-
etalcomponents(Lietal.,2021). Previous study demonstrated that
septin proteins bind phosphatidylinositol phosphates at the appres-
sorium pore membrane to assemble into a ring, promoting the for-
mationofapenetrationpegthatisrequiredforhostinfectionbyM.
oryzae (Dagdas et al., 2012). Considering there is delayed appresso-
rium formation and restricted penetration by the ΔMosm i1 mutant,
we speculated that the septin ring structure may be abnormal in the
FIGURE 3 MoSMI1isrequiredforpathogenicityofMagnaporthe oryzae. (a) Pathogenicity on barley leaves. Mycelial agar plugs of all tested
strainswereinoculatedon7-day-oldbarleyleavesandphotographedat5 dayspost-inoculation(dpi).U,unwounded(intact)leaf;W,wounded
leaf.Bar,10 mm.(b)StatisticalanalysisofthelesionareaofalltestedstrainsonbarleyleavesusingImageJsoftware.Threeexperimentswere
performed. Error bars represent SD and asterisks indicate significant differences (**p < 0.01).(c)Pathogenicityonbarleyleaves.Conidial
suspensions(5 × 104 conidia/mL)ofalltestedstrainsweredroppedon7-day-oldbarleyleavesandphotographedat5dpi.Bar,10 mm.(d)
Statistical analysis of the lesion area of all tested strains on barley leaves using ImageJ software. Three experiments were performed. Error
bars represent SD and asterisks indicate significant differences (**p < 0.01).(e)Pathogenicit yonriceseedlings.Conidialsuspensions(5 × 10 4
conidia/mLina0.2%wt/volgelatinsolution)fromeachtestedstrainweresprayedonto14-day-oldriceseedlingsandphotographedat5dpi.
Bar,10 mm.(f)Lesionnumberswerecountedwithina5 cmlengthofleaffromeachstrain,andaminimumofthreeleaveswereassessedfor
each strain. Three experiments were per formed. Error bars represent SD and asterisks indicate significant differences (**p < 0.01).
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ΔMos mi1 mutant. Totest this, we expressed Sep3-GFP and Sep5-
GFPinthewild-typestrainGuy11andΔMosm i1 mutant.At24 hpi,
bothSep3-GFP and Sep5-GFPexhibited aringstructure intheap-
pressoriumcentreinthewild-typeGuy11background,whereasthe
GFP fluorescence signal was disordered in the ΔMosm i1 mutant
(Figure 5a,b). In summary, our results suggest that MoSmi1 is in-
volved in septin ring formation in M. oryzae.
2.8 | MoSmi1 is required for cell wall integrity and
stress response
To investigate the contribution of Mo SMI1 to the CWI of M. oryzae,
we observed the mycelial growth of all tested strains on CM sup-
plemente d with cell wall s tress agents (600 μg /mL Congo red [CR],
200 μg/mL CF W,and 0.004% sodium dodecylsulphate[SDS]). At 7
dpi, the colony diameters of all tested strains were measured. The re-
sults showed that the relative inhibition rate of the ΔM osmi1 mutant
wassignificantly higherthan that of the wild-type strainGuy11and
the complemented strainΔMosmi1/MoSMI1 under cell wall stress con-
ditions (Figure 6a,b). In addition, we performed protoplast release as-
sayswithcellwall-lysingenzymetoexaminewhetherMoSMI1 plays a
crucial role in the maintenance of CWI. When the mycelia of all tested
strainswere treatedwith cellwall-lysing enzyme,fewer protoplasts
were generated in the ΔMosmi1mutantthaninthewild-typestrain
Guy11 and the complemented strain ΔMosmi1/MoSMI1 after incuba-
tionfor30,60,and90 min(Figure 6c,d). Taken together, these results
indicate that MoSmi1 is involved in maintaining CWI in M. oryzae.
To investigate the contribution of Mo SMI1 in environmental
stress tolerance in M. oryzae, we observed the mycelia growth of
FIGURE 4 MoSmi1affectsappressoriumformation,invasivehyphae(IH)expansion,andhostreac tiveoxygenspecies(ROS)scavenging.
(a)Conidialsuspensions(5 × 104 conidia/mL)ofallthetestedstrainswereinoculatedonanartificialhydrophobicsurfaceandviewedat6,
12,and24 hpost-inoculation(hpi).Bar,20 μm.(b)Statisticalanalysisofappressoriumformationrate(%)ofalltestedstrains.Aminimumof
100 conidia were obser ved and counted in each strain. Three experiments were performed. Error bars represent SD and asterisks indicate
significant differences (**p < 0.01).(c)Conidialsuspensions(5 × 104 conidia/mL)ofalltestedstrainsweredroppedonthebackofbarley
leaves,andbarleyepidermalcellswereobservedat24,36,and48 hpi.TypeI,onlypenetrationpegwithoutinvasivehypha;TypeII,only
one single invasive hypha without branches; Type III, more than one branch but restricted to one host cell; Type IV, more than one branch
andextendedtoneighbouringhostcells.Bar,20 μm.(d)StatisticalanalysisoffourtypesofIH.Atleast100penetrationsiteswerecounted
for each strain. Three experiments were performed. Error bars represent SD. (e) Conidial suspensions of all tested strains were inoculated
ontobarleyleavesfor30 handstainedwith3,3′-diaminobenzidine(DAB)solution.Bar,25 μm. (f) Statistical analysis of the proportion of
infectedcellsstainedbyDAB.Foreachstrain,atleast100invadingcellswereobservedandthenumberofstainedcellswascounted.Error
bars represent SD and asterisks indicate significant differences (**p <0.01).(g)Barleyleaveswereinoculatedwithconidialsuspensionsofall
testedstrainstreatedwithdiphenyleneiodonium(DPI),andIHgrow thwasobservedat30 hpi.Dimethylsulphoxide(DMSO)treatmentwasa
controlthatwasusedtodissolveDPI.Bar,25 μm.(h)Relativeexpressionof10ROSdetoxification-relatedgenesinthewild-t ypeGuy11and
ΔMos mi1 mutant . The β- tubulin gene (MGG_00604) was used as the reference gene. Three independent biological experiments with three
replicates were performed. Error bars represent SD and asterisk represents significant differences (*p < 0.05,**p < 0.01,NS,p > 0.05).
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WANG et al.
all tested strains on CM supplemented with osmotic stress agents
(0.7 MNaCl,1 Msorbitol,and0.6 MKCl)andoxidativestressagents
(5 mMand10 mMH2O2).At7dpi,thecolonydiametersofalltested
strains were measured. We found that the ΔM osmi1 mutant was
more sens itivity to 0.7 M NaC l, whereas it was mor e resistant to
0.6 M KCl, but not 1 M sorbitol (Figure 6e,f), and sensitive to both
5 mMand10 mMH2O2 concentrations (Figure 6g,h). Our results in-
dicate that Mo SMI1 plays an important role in stress response.
2.9 | MoSmi1 interacts with MoOsm1 and
MoMps1, and regulates their phosphorylation level in
M. oryzae
To explore the underlying mechanism of the function of MoS MI1, we
performed immunoprecipitation combined with mass spectrometry
analysis (IP-MS) to identify the proteins that interact withMoSmi1
(Table S1). To confir m the interacti on between MoSm i1 and MoOsm1
orMoMps1,theyeasttwo-hybrid(Y2H)assaywasperformed,which
demonstrated that MoSmi1 interacts with MoOsm1 but not with
MoMps1(Figure 7a,b, Figure S6). We inferred that Y2H may not be
sufficiently sensitive to detect the interaction between MoSmi1 and
MoMps1.ThedifferentdomainsofKnr4havebeendemonstratedto
play diverse roles in protein–protein interactions (Dagkessamanskaia,
Durand, et al., 2010). To explore whether MoSmi1 domains have
different effects on the interaction with MoOsm1, we generated
six MoSmi1 derivative constructs and paired them with MoOsm1,
which were t hen co-tran sformed into the ye ast strain Y2H Go ld.
These results showed that all MoSmi1 derivatives interacted with
MoOsm1. We also observed that the MoSmi1–MoOsm1 interaction
wasgreatly enhancedwhentheC-terminaldomainofMoSmi1was
absent, indicating that the C-terminal domain could partially sup-
press the MoSmi1–MoOsm1 interaction (Figure 7a,b).Additionally,
the interaction between MoSmi1 with MoOsm1 and MoMps1 was
detected using co-immunoprecipitation (Co-IP) and bimolecular
fluorescence complementation (BiFC). The results indicated that
MoSmi1 interacted with MoOsm1 and MoMps1 (Figure 7c ,d).
Given that MoSmi1 interacts with the protein kinase MoOsm1
and MoMps1, we wondered whether the activity of MoSmi1 may be
regulated by MoOsm1 or MoMps1 through protein phosphor ylation.
Therefore, we determined the phosphorylation level of MoSmi1 by
Mn2+-Phos-tagSDS-PAGE.Theputative MoMPS1 deletion mutants
wereverifiedbyPCRandRT-qPCR,andwerenamedtheΔMomps1
mutant (Figure S7). The MoSMI1- GFP constructs were transferred
into the wil d-typ e strain Gu y11,ΔM oosm1 and ΔM om ps1 mutant.
The MoSmi1-GFP protein was extracted from the wild-t ype strain
Guy11, ΔMoos m1 and ΔMomps1 mutants, and treated with phos-
phatase or phosphatase inhibitor. Protein samples were separated
by Mn2+-Phos-tagSDS-PAGEanddetectedbyimmunoblottingwith
ananti-GFPantibody.Themobilit yofMoSmi1-GFPwassimilarinall
samples (Figure S8), indicating that MoOsm1 or MoMps1 could not
regulate MoSmi1 through protein phosphorylation.
In M. oryzae, MAPK signalling pathways regulate development,
appressorium formation, appressorium-mediated penetration,
stress resistance, and pathogenicity (Cai et al., 2022; Tucker &
Tal b ot, 2001). Among t hese MAPK sign alling pathway s, the Pmk1
FIGURE 5 MoSM I1 affects septin ring
formation in Magnaporthe oryzae. (a, b)
Theconidialsuspensions(5 × 10 4 conidia/
mL)ofthewild-typeGuy11andΔMosmi1
mutantexpressingSep3-GFPorSep5-
GFP were inoculated on an artificial
hydrophobic surface and the appressoria
wereobser vedat24 hpost-inoculation
under a laser scanning confocal
microscope. The distribution of the
fluorescence signal in a transverse section
(indicated by the white dotted line) was
analysedusingImageJsoft ware.Bar,5 μm.
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WANG et al.
MAPkinase pathwayis important forappressoriumformation and
plant infection (Xu & Hamer, 1996 ; Zhao et al., 2007). The Mps1
MAP kinasepathway regulates CWI, penetration, and infection(Li
et al., 2012). The Osm1MAPkinase pathwayis mainly responsible
for the osm otic stress r esponse (Li e t al., 2012). As sh own by the
above results, the ΔMos mi1 mutant exhibited defects in appressoria
formation, CWI, osmotic response, and penetration. Therefore, we
performed western blotting to determine the phosphorylation lev-
elsofPmk1,Mps1andOsm1.Comparedwiththewild-typeGuy11,
the phosphorylation levels of Pmk1, Mps1 and Osm1 were higher
in the ΔMo sm i1 mutant (Figure 7e,f ), suggesting that MoSmi1 medi-
ates appressoria formation, CWI, osmotic response, and penetration
by regulating the phosphorylation levels of Pmk1, Mps1 and Osm1.
Taken together, these results indicate that MoSmi1 interacts with
the protein kinase MoOsm1 and MoMps1, and regulates their phos-
phorylation levels in M. oryzae.
2.10 | MoSMI1 regulates various metabolic
pathways in M. oryzae
To further investigate the potential regulatory mechanism of
MoSMI1, we performed transcriptome analysis of the wild-type
strain Guy11 and ΔMosm i1mutantmyceliausingRNA-seq.Atotalof
610differentiallyexpressedgenes(DEGs)(falsediscoveryrate[FDR]
<0.05 and log2(foldchange[FC])>1) were identified in the ΔMos mi1
mutant compared to the wild type Guy11, 278 of which were up-
regulated genes and 332 downregulated (Figure 8a, Table S2). The
MoSmi1-encoding gene MGG_03970 was significantly downregu-
lated, sug gesting the RN A-seq data we re reliable (FDR = 2.3e−24,
log2FC = −15;Table S2).KyotoEncyclopediaofGenesandGenomes
(KEGG)andGeneOntology(GO)enrichmentanalysesindic atedthat
various pathways or biological processes, such as metabolic path-
way, membrane -relate d pathway and oxidat ion–reduction p rocess
were regulated by MoSMI1 (Figure 8b,c). To confirm the authenticit y
of the RNA-seq re sults, 15 DEGs we re randoml y selected f or RT-
qPCR anal ysis, and the res ults were consis tent with those of th e
transcriptome analysis (Table S3).
3 | DISCUSSION
Adaptationtoenvironmentalstressisimportanceforthesur vival
and colonization of pathogen. The cell wall is the first barrier be-
tween the cell and the external environment. Disordered CWI
contributestomultiplefungalphenotypicdefects.Knr4hasbeen
identified as a hub protein conserved among fungi and response
to cell wall stresses. In Candida albicans, the Δsmi1 mutant shows
FIGURE 6 MoSmi1isrequiredforcellwallintegrityandstressresponse.(a)Colonymorphologyofallthetestedstrainsoncomplete
medium(CM)platessupplementedwith200 μg/mLcalcofluorwhite(CFW),600 μg/mLCongored(CR)or0.004%sodiumdodecylsulphate
(SDS). (b) Statistical analysis of the relative inhibition rate (%) of the tested strains. (c) Protoplasts of all tested strains were obser ved and
photographedaftertreatmentwithcellwall-degradingenzymesfor60 minat30°C.Bar,25 μm. (d) Statistical analysis of the protoplast
number.Protoplastnumberswascalculatedat30,60and90 min.(e)ColonymorphologyofallthetestedstrainsonCMplatessupplemented
with1 Msorbitol,0.7 MNaCl,or0.6 MKCl.Thecoloniesweremeasuredandphotographedat7 dayspost-inoculation(dpi).(f)St atistical
analysis of the relative inhibition rate (%) of the tested strains. (g) Colony morphology of all the tested strains on CM plates supplemented
with5 mMH2O2or10 mMH2O2.Thecoloniesweremeasuredandphotographedat7 dpi.(h)Statisticalanalysisoftherelativeinhibitionrate
(%) of the tested strains. For each strain, three independent biological experiments were performed with four replicates. Error bars represent
SD,andasterisksabovethecolumnsindicatesignificantdifferencesbetweenthewild-type(WT)Guy11andΔMosmi1 mutant estimated by
Student's t test (*p < 0.05,**p < 0.01)andNSindicatesnon-significantdifferencesbetweenthewild-typeGuy11andΔMos mi1 mutant.
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a clear hypersensitivity to CFW or SDS treatment and affects the
cell wall β-glucansynthesis(Nettetal.,2011). In S. cerevisiae, cell
wall biosynthesis is important for cell structure and morphology
(Cabib et al., 2001; Orlean, 2012). In S. cerevisiae, KNR4 is not es-
sential for growth under standard laboratory conditions (30°C,
rich medium). However, its deletion leads to growth defec ts under
numerous stresses, such as elevated temperature, SDS, caffeine,
andvarious cell wall disrupting agents(Martin-Ykenet al.,2016).
In this study, we found that MoSmi1 regulated the CWI pathway,
conidiation and morphogenesis of conidia and hyphae.
Evidence has suggested that microtubules play a role in cell
cycle progression and nuclear division (Hughes et al., 2008; Jansen
et al., 2023). Nuclear positioning through microtubule dynam-
ics is an essential process for many types of cells (Gundersen &
Worman, 2013). In fission yeast cells, microtubules are used to repo-
sitionnuclei(Bellingham-Johnstunetal.,2023). In Arabidopsis thali-
ana,transportproteinparticleII(TR APPII)tetheringfactorsinteract
withthemicrotubule-associatedproteinsofthePLEIADE/AtMAP65
family,whicharerequiredtocoordinatecytokinesiswiththenuclear
division cycle (Steiner et al., 2016). Microtubules also play an import-
ant role in cell morphology and material transportation. In mammals,
thestabilityofdisk-likeseptinsdependsonintactmicrotubules(Sellin
et al., 2011). In filamentous fungi, cytoplasmic microtubules serve as
highwaysforthelong-distancebidirectionaltransportoforganelles,
mRNA,andothersubcellularcargos (Abenzaetal.,20 09). In shor t,
microtubules play roles in cell cycle regulation, cell morphology, and
material trafficking. Here, we determined that MoSmi1 plays a vital
role in microtubule organization and cytoplasmic division, which are
key factors of cell morphogenesis in M. oryzae. Moreover, the dele-
tion of Mo SMI1 resulted in a significantly decreased appressorium
formation rate, which led to a lower penetration rate and weakened
pathogenicity. We also found that MoSmi1 participates in the func-
tional septin ring organization during the appressoria maturation.
Cell morphogenesis in eukaryotes is a complex process that
requiresperfect coordinationofseveral differentregulation and
signal transduction pathways. MAPK is a family of protein ki-
nases that regulate proliferation, gene expression, differentiation,
mitosis, survival, and apoptosis in a wide variet y of organisms
(Avru ch, 2007; Manning et al., 2002). In S. cerevisiae, Knr4 was
identified as a member of the PKC1-mediated MAPK cascade
signalling pathway and is involved in the regulation of cell cycle
progressionby cooperationwithBck2(Martin-Ykenet al., 2002).
Inaddition,Knr4interacts with the Slt 2MAPkinase,a key com-
ponent of the CWI pathway, which has been shown to affect the
transcriptionaloutputsofCWIpathway(Martin-Ykenetal.,2003,
2016).Moreover,theMAPkinaseHog1isactivatedinsmi1Δ cells
FIGURE 7 MoSmi1interac tswithMoOsm1andMoMps1,andregulatestheirphosphorylationinMagnaporthe oryzae. (a) Domain map
ofMoSmi1.I,full-lengthofMoSmi1;II,deletionN-terminaldomain;III,deletionSMI1_KNR4domain;IV,deletionC-terminaldomain;V,
deletionSMI1_KNR4andC-terminaldomains;VI,deletionofN-terminalandC-terminaldomains;VII,deletionofN-terminalandSMI1_KNR4
domains.ThedomainpredictionofMoSmi1wasperformedwiththeSMARTanalysis.(b)Yeasttwo-hybridassayofsevenMoSmi1variants
andMoOsm1.PairsofdifferentcombinationsofthetruncatedconstructsofMoSmi1andMoOsm1wereco-transformedintoyeaststrain
Y2HGoldandculturedinSD−Leu−TrpandSD−Ade−His−Leu−TrpmediumaddedwithX-α-gal.(c)Co-immunoprecipitationassayforthe
interactionsbetweenMoSmi1andMoOsm1/MoMps1.MoSmi1-GFPandMoOsm1-3 × FLAG/MoMps1-3 × FLAGwereexpressedinthe
wild-t ypestrainGuy11.Theexperimentwasperformedwithanti-GFPbeads,andtheelutedproteinwasanalysedbywesternblotting
usinganti-FLAGandanti-GFPantibodies.(d)BimolecularfluorescencecomplementationassayforinteractionsbetweenMoSmi1and
MoOsm1/MoMps1.YFPsignalswasdetectedinvegetativehyphaeexpressingMoSmi1-YFPC and YFPN- M o O s m 1 / Y F P N-MoMps1.The
strains expressing YFPC and YFPN-MoOsm1,YFPC and YFPN-MoMps1,MoSmi1-YFPC and YFPN, or YFPC and YFPN were used as negative
controls.Bar,10 μm.(e)AnalysisofPmk1andMps1phosphorylationlevels.Phosphor ylatedPmk1andMps1weredetectedusingantibodies
anti-Phospho-p44/42MAPKandanti-Phospho-p42antibody.(f)AnalysisofthephosphorylationlevelofOsm1.PhosphorylatedOsm1was
detectedusingp-p38MAPKantibodyandp38antibody.CBSindicatesCoomassiebrilliant-bluestaining.
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WANG et al.
and the activation of Hog1 induces the translocation of Msn2 into
the nucle us. The nuclear a ccumulation of Msn2 en hances Sir2-
mediated rDNA stabilityand affects yeast cell cycleprogression
(Hong & Huh, 2021). In M. oryzae, osmotic stress regulates the
nuclear localization pattern of MoHog1 and MoMsn2, activating
the transcription of the target genes in response to environmental
stresses(Bohner tetal.,2019; Zhang et al., 2014). In this study, we
found that MoSmi1 interacts with MoMps1 and MoOsm1, which
arecorecomponentsoftheMps1MAPKandOsm1MAPKsignal-
ling pathways in M. oryzae. In addition, the phosphorylation levels
of MoMps1, MoOsm1, and MoPmk1 were significantly increased
in the ΔMo sm i1 mutant, suggesting that MoSmi1 regulates growth,
development, appressorium maturation, and virulence by mediat-
ingtheMAPKsignallingpathway.
In S. cerevisiae, a role of Knr4 in ge ne express ion was firs t re-
vealed by its ability to simultaneously repress all three chitin syn-
thase gene s upon overexpression (Mart in et al., 1999). Smi1 was also
found to reg ulate the transcr iption of genes involve d in the cell cycle,
cell wall synthesis, morphogenesis, and transcriptional responses
toheatandcell wall stressindifferent fungi (Lagorce et al., 2003;
Martin-Yken et al.,2002; Penacho et al., 2012). Smi1, as a regula-
tor of glucan synthases and glucanases, mediates the trafficking,
stabil ity, and localiz ation of Bgs4, w hich is impor tant for grow th,
responsetostress,andcytoplasmic division(Longoetal.,2022). In
our study, transcriptome data suggested that MoSmi1 is involved in
regulationofmembrane-relatedpathwaysandoxidation–reduction
process, which is associated with the response to oxidative stress
and the partial localization of the membrane of MoSmi1.
Hub proteins are intrinsically disordered proteins (IDPs)
or contain IDRs, which allow them to be involved in multiple
interactions and various biological functions (Dunker et al., 2005;
Uversky, 2013). K nr4 contains a large I DR and is consider ed to
be an important hub of the yeast interactome (Martin-Yken
et al., 2016). The N-terminal d omain of Knr4 is re quired for th e
interaction of Knr4 with several partners (Dagkessamanskaia,
Durand, et al., 2010; Fino et al., 2010). Additionally,deletion of
the C-terminal domain greatly enhances the Knr4-Slt2 interac-
tion, and the N-terminal region is required for the interaction to
occur(Batistaetal.,2023). In this study, we predic ted three IDRs,
two of them positi oned in the C-te rminal doma in and the othe r
in the SMI1_ KNR4 domain. I n addition , we verified t hat the de-
letion of any one domain did not prevent the Smi1–Osm1 inter-
action andthattheloss ofthe C-terminaldomain couldpromote
the Smi1–Osm1 interaction. Furthermore, IDRs has been shown
tomediateproteinphaseseparationinseveralstudies(Lindstrom
et al., 2022; Majumdar et al., 2019 ; Saito et al., 2019; Schuster
et al., 2020; Wang et al., 2018). Phase separation can prompt
non-membrane-bound components to formcompartments, such
as stress granules (SGs),P-body (PB), and nucleolus, toseparate
from the surrounding environment, and the components within it
can diffuse freely so that chemical reactions can take place inside
(Hyman et al., 2014). In S. cerevisiae,liquid–liquidphaseseparation
was identified to promote formation of foci under stress, which
provides valuable clues for understanding the mechanisms under-
lyingSGformationandSG-associatedhumandiseases(Lindstrom
et al., 2022). The IDRs of MoSmi1 may be related to its ability to
respond to stress, which may be mediated by phase separation.
In future, we may provide novel strategies to control rice blast by
connecting phase separation with the pathogenic mechanism of
M. oryzae.
FIGURE 8 (a)Globalviewofexpressedgenesinthewild-typestrainGuy11andΔMo sm i1 mutant strain. (b) Comparison of upregulated
anddownregulateddifferentiallyexpressedgenes(DEGs)betweenthewild-typestrainGuy11andΔMosmi1 mutant strains. (c) Top 20
pathwaysfromKEGGpathwayandGOpathwayenrichmentanalysisofsignificantlyupregulatedanddownregulatedgenes.
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WANG et al.
Taken together, our study revealed that MoSmi1 regulates CWI
bymodulatingMAPKsignallingpathwaystoaffectcellmorphology,
cytoskeletal dynamics, cell cycle progression, stress response, and
pathogenicity of M. oryzae.
4 | EXPERIMENTAL PROCEDURES
4.1 | Strains and culture conditions
WeusedGuy11asthewild-type(WT)straininthisstudy.Allstrains
were cultu red on complet e medium (CM) (10 g d-gl ucose, 2 g pep-
tone, 1 gyeast extract,1 g casamino acids,50 mL20× nitrate salt s,
1 mLvitamin,1 mLtraceelements,15 gagar,andwateraddedto1 L)
at28°Cunderdark conditions. For DNA ,RNA, and proteinextrac-
tion,alltestedstrainsweregrown inliquidCMin arot atoryshaker
at120 rpm,and28°Cfor48 h.
4.2 | Plasmid construction and genetic
transformation
Togeneratethetarget gene deletion mutants, the 1.2 kbupstream
fragment and downstream fragment of the target gene were am-
plified fromthe genomicDNAofthewild-type strain Guy11using
primers (Table S4). The hygromycin resistance gene (HPH) fragment
was amplif ied from pFGL 821 using primer s HF/HR. All fragm ents
werecloned intotheHindIII/XbaI-linearized vectorpKO1Businga
one-stepcloningkit(Vazyme)toobtainagene-knockoutvector.The
knockout vector was transformed into the wild-type strain Guy11
using Agrobacterium tumefaciens-media ted transfor mation (ATMT).
Transformants werescree ned usingCM plates containing 250 μg/
mLhygromycinBandfur therverifiedbyPCRandRT-PCRusingthe
pairs listed in Table S4. Positive transformants were fur ther con-
firmedbySouthernblot tingorRT-qPCR.Twoindependentmutants
with similar phenot ypes were obtained and one was selected for
further experiments.
For the complemented strain ΔMosmi1/MoSMI1, an approxi-
mately1.5 kbsequencecontainingthenativepromoterandopen
reading frame (ORF ) of MoSMI1 without a stop codon was ampli-
fiedfromthegenomicDNAofthewild-typestrainGuy11using
primer pairs listed in Table S4. The PCR products were cloned
into yeast s train XK1-25 with Xho I-linear ized vector pYF11 to
obtain a complementedvec tor pYF11-MoSmi1. Theconstruc t
was transformed into ΔMosmi1 mutant strain using polyeth-
ylene glycol (PEG)-mediated transformation. Transformants
were screened using CM plates containing 60 μg/mL bleomy-
cin and fur ther verified by fluorescence. The positive transfor-
mantswerefurtherverifiedbyRT-PCRusingprimerpairslisted
in Table S4. The phenotypes of all transformants recovered
to those of th e wild-typ e strain Guy 11,a nd one of them was
selected for further experiments. The same method was used
to obtain t he wild-t ype stra in Guy11 and ΔMo sm i1 expressing
β-tubulin-pYF11,Sep3-pYF11,Sep5-pYF11,respectively.Toob-
serve the localization of β-tubulin,anapproximately 1.5 kb se-
quencecontainingthenativepromoterandopenreadingframe
(ORF) of the β- tubulin gene without a stop codon was amplified
from the genomic DNA of the wild-type strain Guy11 using
primer pairs listed in Table S4. PCR products were cloned into
KpnI/XhoI-linearizedpFGL820(fused withRFP). The construct
was transformed into the wild-type strain Guy11, ΔMosm i1
mutant strain, and complemented strain ΔMosmi1/MoSMI1
usingATMTmethod.Transformantswerescreenedonmedium
(1.7 gyeast nitrogen basewithoutamino acids, 10 g d-glucose,
2 gasparagine, 1 g NH4NO3, 15 g agar, and wateradded to 1 L)
containing40 μg/mLchlorimuron-ethyl andfurther verified by
fluorescence.
4.3 | RT- qPCR analysis
TotalRNAofthetestedstrainswasisolatedfromfreshmycelialpel-
lets using RNeasyMiniKit(Qiagen).ForcDNAsynthesis,5 μg total
RNAfromeachstrainwasreverse-transcribed usingHiScript II1st
Strand cD NA Synthesis K it (+gDNA wiper) ( Vazyme). RT-P CR was
performed to confirm the deletion and complementation of the tar-
geted gene using thegene-specific primers listed inTable S4. The
ACTIN gene (MGG_03982) was used as an endogenous reference.
qPCR was pe rformed with C hamQ Universal S YBR qPCR Master
Mix(Vazyme)usingCFXConnectReal-Time PCRDetectionSystem
(Bio-Rad). The β-tubulingene(MGG_00604) was used as an endog-
enous reference. The experiment was repeated three times with
three replicates each time.Allprimersusedintheassaysarelisted
in Table S4.
4.4 | Assays for vegetative growth, conidiation,
appressorium formation, and stress agents
For growth assay, mycelial blocks of the wild-t ype strain Gu y11,
ΔMos mi1 mutant, and complemented strain ΔMosmi1/MoSMI1 were
culturedon CMplates at28°C underdarkconditions.After 7 days,
the colony diameter was measured, and all tested strains were
photographed using a camera (Nikon). For conidiation analysis, the
wild-t ypestrainGuy11,ΔMo smi1 mutant, and complemented strain
ΔMosmi1/MoSMI1 were cultured on rice decoc tion and cornmeal
(RDC)medium at28°C for5 daysinthedark,followed by3 daysof
continuousilluminationunderfluorescentlight.Subsequently,10 mL
water was used to collec t conidia, and the conidial suspensions were
condensed to 1 mL. The number of conidia was counted with a
haemocytometer under a microscope. For appressorium formation
analysis, conidial suspensions of all tested strains were induced on
artif icial hydroph obic surf aces at 28°C in th e dark. The a ppresso-
riumformationratewasdetectedafter6,12,and24 h.Imagesofap-
pressorium formation were obtained using an inverted fluorescence
microscope (Nikon).
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Todeterminestrains'responsetovariousstressagents,thewild-
type strain Guy11, ΔMosmi1 mutant, and the complemented strain
ΔMosmi1/MoSMI1 were cultured on CM plates supplemented with
15 μg/mLbenomyl,1 Msorbitol,0.7 MNaCl,0.6 MKCl,5 mMH2O2,
10 mM H2O2, 200 μg/mL CF W,6 00 μg/mL CR, or 0. 004% SDS at
28°C in dar k. Subseque ntly, the colonies wer e photographe d and
the diame ter of the colonies wa s measured after 7 d ays. Relative
inhibitionrate = (diameter of the untreated strain − diameter of the
straintreatedwithchemicals)/(diameteroftheuntreatedstrain).All
experiments were repeated three times with three replicates each
time.
4.5 | Staining assay
Too b s er vet h evac u o l elo c a l izat i o nof M oSm i 1- G FPd u r inga p p res-
sorium formation, the appressoria from the wild type expressing
MoSmi1-GFPwerestainedwith10 mMCMAC(Invitrogen)atroom
temperaturefor30 minunderdarkconditionsandobservedunder
a laser scanning confocal microscope. To observe the morphology
ofconidia, conidia (5 × 104 conidia/mL) of all tested strains were
stained with 10 μg/mL CF W (Sigma-Aldri ch) solution for 10 min
and photographed under a laser scanning confoc al microscope
(Nikon). For the ROS staining assay, barley leaves infected with
aconidial suspensionof all tested strains at 30 hpi were stained
with1 mg/mLDAB(Sigma-Aldrich) solution (pH 3.8)for12 h,and
thendestainedwithethanolfor4 honashakerat 28°C.ForROS
inhibitionassay,0.5 μMDPI (Sigma-Aldrich) (dissolved in DMSO)
was added to a conidial suspension of all tested strains to inhibit
hostROS.DMSOwasusedascontrol.Barleyep ide rmalcellswere
observed under an inverted fluorescence microscope (Nikon). To
determinethecytoplasmicdivisionofthewild-typestrainGuy11
and the ΔMosmi1 m utant, the myce lia of strains e xpressing H1-
RFPwerestainedwith10 μg/mLCFW(Sigma-Aldrich)solutionfor
10 minandphotographedunder a laserscanning confocal micro-
scope (Nikon).
4.6 | Virulence assays
For the virulence study, the rice cultivar CO39 and the barley cul-
tivar Golden Promise were used. The mycelial blocks or conidial
suspensions(5 × 104 conidia/mL)ofalltestedstrainswere inocu-
lated on 7-day-old isolated barleyleaves and keptin abiological
incubatorat28°Cwith90%humidityinthedarkforthefirst24 h,
followed bya 12/12 hlight/darkc ycle.Thelesion spotswere ob-
served and captured at 5 dpi. The lesion areas of all tested strains
were analy sed using Image J software . Approximatel y 15 mL co-
nidial suspensions (5 × 104 conidia/mL in a 0.2% wt/vol gelatin
solutio n) from each tes ted strain wer e sprayed onto 14-day-ol d
riceseedlings and kept in a biological incubator at28° with 90%
humidityinthedarkforthefirst24 h,followedbya12/12 hlight/
dark cycle. The disease spots were observed and photographed
at 5 dpi. Thr ee leaves from ea ch strain wer e used for stat istical
analysis of the number of lesions.
To observe the expansion of invasive hyphae (IH), conidial sus-
pensions(5 × 104conidia/mL)ofthetestedstrainsweredroppedon
thebackof barleyleavesandkept inabiologicalincubatorat28°C
with90%humidityunderthedark.Barleyepidermalcellswereob-
servedat24,36, and 48 hpiusing an inverted fluorescence micro-
scope(Nikon).Alltheaboveexperimentswererepeatedthreetimes
with three replicates each time.
4.7 | Protoplast release assay
AllthetestedstrainswereculturedinliquidCMat 28°Cfor48 h.
Then, 0. 2 g of mycelia were collected and treated with 0.01 g/
mL cellulase, 0.01 g/mL pectinase, and 0.0035 g/mL driselase
(dissolved i n 10 mL 0.7 M NaCl solution) f or 30, 60, and 90 mi n
at30°C. Theprotoplasts were then observed underan inverted
fluorescence microscope (Nikon) and counted using a haemocy-
tometer. This experiment was repeated three times with three
replicates each time.
4.8 | Fluorescent microscopic observation
For the subcellular localization assay, the fluorescence signals of
conidia,appressoriumformation(6,12,and24 hpi),andIH(24 hpi)
were observed using a laser scanning confocal microscope (Nikon).
Toanalyse the morphology of the microtubulesin the wild-type
strain Guy11, ΔMosm i1 mutant, and the complemented strain
ΔMosmi1/MoSMI1, the fluorescence signals of mycelia were ob-
served using a laser scanning confocal microscope (Nikon). To
detect the morphology of the septin ring in the wild-type strain
Guy11 and ΔMosm i1 mutant, the fluorescence signals of appres-
soria(24 hpi)wereobservedusingalaserscanningconfocalmicro-
scope (Nikon).
4.9 | Western blot assay
Total protein was ex tracted f rom mycelia inocula ted in liquid CM
for 48 h, as p reviously de scribed (Zh ang et al., 2017). The protein
sample s were detect ed by anti-GFP antib ody, anti-FL AG antibo dy
(Abmart), Phospho-p44/42 MAPK antibody, p44/42 MAPK anti-
body (Cell SignallingTechnology), Phospho-p-p38 MAPKantibody,
orp38MAPKantibody(SantaCruzBiotechnolog y).
4.10 | Affinity purification and mass
spectrometry analysis
The pYF11-MoSmi1 vector was transformed into the wild-
type strain Guy11 through PEG-mediated transformation. The
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WANG et al.
bleomycin -resist ant transfo rmants were ex amined unde r a laser
scanning confocal microscope (Nikon), and confirmed by western
blotting with anti-GFP antibody. The total protein of the posi-
tive transformants was ex tracted and incubated with anti-GFP
nanobody agarose beads (AlpalifeBio) for 4 h at 4°C.The bound
proteinswereelutedfromtheGFP-beadsandelectrophoresedin
10%SDS-PAGEuntilproteinswereconcentratedintothesepara-
tion gel. The gel containing the protein sample was stained with
Coomassie brilliant blue and sent to APTBIO (Shanghai, China)
for mass spectrometry analysis as previously described (Zhang
et al., 2017).
4.11 | Yeast- two hybrid assays
The cDNA fragment s of full-length Mo SMI1 and it s domain de-
rivatives, including the N-terminal deletion region (deletion 1–161
aminoacid),SMI1_KNR4deletion region(deletion 162–347amino
acid),C-terminal deletionregion(deletion348–571aminoacid),N-
terminaldomain(remaining1–161aminoacid),SMI1_KNR4domain
(remaining 162–347 amino acid), and C-terminal domain (remain-
ing 348–571 amino ac id) were amplifi ed using cDNA temp late of
the wild-typ e strain Guy11 and cl oned into pGADT7 a s the prey
constructs. The cDNA fragments of Mo OSM1 (MGG_01822) and
MoMPS1 (MGG_04943) were amplified using cDNA template of
the wild-type strain Guy11 and cloned into pGBKT7 as the bait
constructs. Corresponding primers are listed in Table S4.Both the
prey const ruct and bait con structs we re co-transfo rmed into the
yeast strain Y2HGold, according to the manufac turer's instruc-
tions (Matc hmaker Gold Yeast Two-Hy brid System). The t ransfor-
mants were grown on a synthetic-definedmedium lacking leucine
and tryptophan (SD−Leu−Trp) and then transferred to synthetic
defined medium lacking adenine, histidine, leucine, and tryptophan
(SD−Ade−His−Leu−Trp).Th epo sit ivecontrolwastheinterac tionb e-
tweenpGBKT7-53andpGADT7-T,andthenegativecontrolwasthe
interactionbet weenpGBKT 7-LamandpGADT7-T.
4.12 | Bimolecular fluorescence
complementation assay
For the BiFC assay, the MoSMI1 gene fragment without a stop
codon wascloned into BamHI-linearized pKD2-YFPCTF (hygromy-
cin B resist ance) and Mo OSM1 or MoMPS1 gene fragments with-
out a stop co don were cloned into X baI-line arized pKD5-YFPNTF
(chlorimuron-ethylresistance)usingaone-stepcloningkit(Vazyme).
Corresponding primers are listed in Table S4.Subsequently,pKD2-
M o S m i 1 - Y F P CTFw as c o - t r a n s f o r me d i n t o t h ew i l d - t y p e s t r a i nG u y 1 1
w i t h p K D 5 - M o O s m 1 - Y F P NTF a n d p K D 5 - M o M p s 1 -Y F P NTF using the
ATMT method. The transformants were screened using medium
(1.7 gyeast nitrogenbase without aminoacids, 10 g d-glucose,2 g
asparag ine, 1 g NH4NO3, 15 g agar, and water a dded to 1 L) con-
taining 250 μg/mL hygromycin B an d 40 μg/mLchlorimuron-ethyl
and further verified by fluorescence. Fluorescence signals were ob-
served using mycelia under a laser scanning confocal microscope
(Nikon).
4.13 | Co- immunoprecipitation assay
ToconfirmtheinteractionbetweenMoSmi1-MoOsm1andMoSmi1-
MoMps1invivousingCo-IP,theORFsofMoOSM1 or MoMPS1 with-
outastop codonwereamplified and clonedinto HindIII-linearized
pKNRP27Flag (3 × FLAG tag) vector (neomycin resistance) using
a one-step clo ning kit (Vaz yme). Corresp onding prim ers are listed
in Table S4.Subsequently,MoOsm1-RP27-3 × FLAGand MoMps1-
R P 2 7 - 3 × FLAG were transferred into the wild-t ype strain Guy11
expressingpYF11-MoSmi1usingPEG-mediatedprotoplasttransfor-
mation method. The transformant s were screened on CM containing
200 mg/mL G418andverifiedbywesternblot ting.Thetotalprotein
was incubated with GFPagarosebeads for4 hat4°C. Bound pro-
teinswereelutedfromGFP-Trapbeadsforwesternblottinganalysis.
Elutedprotein samplesweredetectedusinganti-GFPor anti-FL AG
antibodies(Abmart).
4.14 | Phosphorylation analysis through Phos- tag
gel electrophoresis
Toa nalyse the ph osphory lation level of MoS mi1i n the wild-t ype
strain Guy11 and ΔMomps1 or ΔMoosm1 mutants,pYF11-MoSmi1
was trans formed into th e wild-typ e strain Guy11, ΔM oo sm1, and
ΔMomps1 mutants using PEG-mediated protoplasttransformation
method.TransformantswerescreenedusingCMcontaining60 μg/
mL bleomyci n and furth er verified by wes tern blott ing. The total
proteins ex trac ted from mycelia of the tested strains were treated
with phosphatase or phosphatase inhibitors. Subsequently, the
treated protein samples were electrophoresed in 8% SDS-PAGE
containing 50 mM acrylamide-dependent Phos-tag ligand and
100 mM MnCl2. Gel electrophoresis was performed at constant
voltage(80 V)for3–4 h.Afterelectrophoresis,thegelswereequili-
bratedtwiceintransferbufferwith5 mMEDTAfor20 min,followed
bytransferbufferwithoutEDTAforanother20 min.Afterwards,the
protein was transferred from gels to a PVDF membrane, which was
performedforabout30 hat80 Vat4°C .ThePVDFmembranewas
analysedbywesternblottingusingananti-GFPantibody(Abmart).
4.15 | Transcriptome analysis
Mycelia of the w ild-ty pe strai n Guy11 and ΔMosmi1 mutant were
collectedfromliquidCMandsenttoGENEDENOVO(Guangdong,
China)forRNAextractionandRNA-seq.Threebiologicalreplicates
were used for each strain. DEGs were analysed by the DESeq2
package, and expression with log2FC > |1| and FDR < 0.05 were
definedas DEGs (Love etal., 2014). Ontology enrichment analysis
|
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WANG et al.
was performed using the topGO R package, and p < 0.05werecon-
sidered significantly enriched by DEGs (Young et al., 2010).KEGG
enrichment analysis was per formed using clusterProfiler R package
to test the s tatistic al enrichmen t of DEGs in KEGG pathways ( Yu
et al., 2012).
4.16 | Statistical analysis
Allresultsof the statistical analysesarepresentedasmean ± SD of
three independent biological replications, along with at least three
technical replicates. Statistical significance was determined using a
two-sampleStudent'st test, performed with Microsoft Office Excel
software. The p value was used to assess the statistical significance
of the results (NS, p > 0.05,*p < 0.05,**p < 0.01).
ACKNOWLEDGEMENTS
This study was supported by the National Natural Science
Foundation of China (32202253), the Natural Science Foundation of
AnhuiHigher EducationInstitutions (KJ2020A0102)andtheTalent
ResearchProjectofAnhuiAgriculturalUniversity(rc342001).Weare
grateful to Professor Jianping Lu ofZhejiang University, Professor
Xiaolin Chen of Huazhong Agricultural University, Professor
Haifeng Zhang of Nanjing Agriculture University for providing
plasmid pKO1B,pKD2-YFPCTF, p K D 5 - Y F P NTF, pKN- RP27FlAG , and
ΔMoosm1 mutant.
CONFLICT OF INTEREST STATEMENT
The authors have no competing financial interest and solely respon-
sible for the experimental designs and data analysis.
DATA AVA ILAB ILITY STATE MEN T
All data s upport ing the find ings of the cu rrent stud y are availabl e
within figures and Suppor ting Information. All strains generated
during this study are available from the corresponding author upon
reasonablerequest.
ORCID
Shulin Zhang https://orcid.org/0000-0003-0211-6448
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SUPPORTING INFORMATION
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Suppor ting Information section at the end of this article.
How to cite this article: Wang,Y.,Cui,X .,Xiao,J.,Kang,X .,Hu,
J.,Huang,Z.etal.(2024)AnovelMAPkinase-interacting
protein MoSmi1 regulates development and pathogenicity in
Magnaporthe oryzae. Molecular Plant Pathology, 25, e13493.
Availablefrom:ht tps://doi.org/10 .1111/m pp.13493
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