PLANT SCIENCE

A single fungal MAP kinase controlsplant cell-to-cell invasion by therice blast fungusWasin Sakulkoo,1 Miriam Osés-Ruiz,1 Ely Oliveira Garcia,2 Darren M. Soanes,1

George R. Littlejohn,1* Christian Hacker,1 Ana Correia,1

Barbara Valent,2 Nicholas J. Talbot1†

Blast disease destroys up to 30% of the rice crop annually and threatens global food security.The blast fungusMagnaporthe oryzae invades plant tissue with hyphae that proliferate andgrow from cell to cell, often through pit fields, where plasmodesmata cluster.We showed thatchemical genetic inhibition of a single fungal mitogen-activated protein (MAP) kinase, Pmk1,preventsM. oryzae from infectingadjacent plant cells, leaving the fungus trappedwithin a singleplant cell. Pmk1 regulates expression of secreted fungal effector proteins implicated insuppression of host immune defenses, preventing reactive oxygen species generation andexcessive callose deposition at plasmodesmata. Furthermore, Pmk1 controls the hyphalconstriction required for fungal growth from one rice cell to the neighboring cell, enabling hosttissue colonization and blast disease.

Blast diseases of cereals are caused by thefilamentous fungusMagnaporthe oryzae(synonym of Pyricularia oryzae), de-stroying sufficient rice each year to feed60millionpeople (1), andwheat blast disease

now threatenswheat production in South Americaand, most recently, Asia (2). Plant infection re-quires an infection cell, called an appressorium,which uses a pressure-drivenmechanism to breachthe tough cuticle of the leaf (3, 4). Once insideplant tissue, the fungus elaborates pseudohypha-like invasive hyphae that rapidly colonize livinghost cells, secreting effectormolecules to suppresshost immunity and facilitate infection (5).M.oryzaeeffectors are delivered into host cytoplasm bymeans of a biotrophic interfacial complex (BIC),a plant-derived membrane-rich structure in whicheffectors accumulate during transit to the host(5–8). Hyphae then appear to locate pit fields,composed of plasmodesmata, which are traversedby constricted, narrowhyphae, enabling the spreadof the fungus to adjacent host cells (9). The fungusrapidly colonizes host tissue, and disease lesionsappear within 4 to 5 days of initial infection byspores.In this study, we investigated how M. oryzae

colonizes host tissue and, in particular, how itspreads from one plant cell to the next. We firstperformed ultrastructural analysis of rice sheathcells infected with the pathogenic strain Guy11.This analysis confirmed constriction of hyphaefrom an average diameter of 5.0 mm to 0.6 mmduring traversal of rice cells (Fig. 1, A and B, and

fig. S1). The rice plasmamembrane in the secondinvaded cell remained intact as an electron-denselining near the rice cell wall, continuous with theplant plasmamembrane around hyphae (Fig. 1B)(5, 8, 9). By contrast, the rice plasma membranein the first invaded cell lost integrity upon exitof the fungus to the next rice cell (Fig. 1, B and E)(7, 9, 10).One of the plant’s defenses against infection is

to close intercellular plasmodesma channels bydeposition of callose (11, 12). We visualized cal-lose using aniline blue staining of rice cells (Fig. 1Cand fig. S3). Callose papillae often form at appres-soriumpenetration sites, but no callose occlusionswere initially observed at plasmodesmata duringinfection of the first rice cell at 27 hours post-inoculation (hpi) (fig. S3). Later callose depositionwas observed at plasmodesmata by 30 hpi, con-sistent with the onset of cell death among ini-tially invaded rice cells (figs. S1 and S2). Callosecollars then formed around the base of invasivehyphae after they invaded adjacent cells, at 34 hpi(Fig. 1C and fig. S3). These observations suggestthat M. oryzae is able to clear plasmodesmal oc-clusion materials before penetrating pit fields(Fig. 1D). We also observed a switch from polar-ized to isotropic fungal growth by invasive hy-phae at rice cell junctions. The polarisomemarkerSpa2–green fluorescent protein (Spa2-GFP) local-ized to hyphal tips and then disappeared as hy-phal tips swelled upon contact with the host cellwall (fig. S2) (5). Spa2-GFP then appeared againat hyphal tips in newly colonized cells (fig. S2).Cell wall crossing was accompanied by reorgani-zation of fungal septins and F-actin into anhourglass shape at the point of maximumhyphalconstriction (Fig. 1F, figs. S2 and S10, andmovie S1)(3, 5, 13).Plasmodesmata are dynamic structures through

which proteins diffuse between plant cells (11).To test whether the blast fungus can manipulate

plasmodesmata by increasing their size exclu-sion limit to facilitate effector diffusion to ad-jacent plant cells, we bombarded rice tissueinfected withM. oryzae at 24 hpi with single-and double-sized mCherry expression vectors.In uninfected rice tissue, a 28.8-kDa singlemCherry protein moved into neighboring ricecells but a 57.6-kDa double mCherry fusionprotein generally did not, owing to its larger size(Fig. 1, G and H). By contrast, in blast-infectedtissue, double mCherry protein diffused to ad-jacent rice cells. The gating limit of rice plas-modesmata is therefore relaxed during earlyM. oryzae infection. We then carried out time-lapse imaging of the fungal apoplastic effectorBas4 (biotrophy-associated secreted protein 4)–GFP (8), expressed under its native promoter,during plant infection (fig. S2 and movie S2).We observed that upon exit of the fungus toan adjacent cell, Bas4-GFP leaked into the hostcytoplasm of initially colonized rice cells. How-ever, fluorescence did not diffuse into the newlycolonized cells. Plasmodesmata therefore remainopen at early stages of infection (24 to 27 hpi) butare closed at later stages, consistent with theincrease in plasmodesmal callose deposition at30 to 34 hpi (fig. S3) (7, 14), suggesting that theblast fungus is able to overcome callose deposi-tion at pit fields to invade neighboring cells.Plasmodesmata may lose their structural integ-rity after this time, as initially infected rice cellslose their viability.To study regulatory mechanisms controlling

invasive growth, we characterized Pmk1, a fungalmitogen-activated protein kinase (MAPK) essen-tial for appressorium development and pathoge-nicity that is conserved in many plant-pathogenicfungi (15). Pmk1 null mutants ofM. oryzae cannotinfect rice plants even when mutants are inocu-lated onto wounded leaves (15). We decided toconditionally inactivate the Pmk1 MAPK usinga chemical genetic approach. We generated ananalog-sensitive (AS) allele of PMK1 (pmk1AS) bymutating the gatekeeper residue of the kinaseadenosine triphosphate (ATP)–binding site into asmall amino acid residue, glycine. The equivalent(Shokat) mutation previously reported in yeastfus3-as1 confers susceptibility to the ATP analog1-naphthyl-PP1 (1NA-PP1) (16). Expression ofthe pmk1AS allele, under control of its nativepromoter, restored pathogenicity to a Dpmk1mutant (fig. S4). Addition of 1NA-PP1 selectivelyinhibited the function of Pmk1AS mutants, pre-venting appressorium development (fig. S4), aresult that was identical to the effects of PMK1deletion or expression of a kinase-inactive allele(17). We also observed that inhibiting Pmk1 afterappressorium formation blocked cuticle penetra-tion by preventing assembly of the septin ring atthe appressorium pore (fig. S5).To test the role of Pmk1 in tissue invasion, we

allowed a pmk1AS mutant to invade the first riceepidermal cell before adding 1NA-PP1 at 26 hpi.This treatment blocked invasion of adjacent epi-dermal cells, resulting in the first infected cellsbecoming filled with fungal hyphae (Fig. 2, A andB). Pmk1 inactivation did not affect the structure

RESEARCH

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1School of Biosciences, University of Exeter, Exeter EX4 4QD,UK. 2Department of Plant Pathology, Kansas State University,4024 Throckmorton Plant Sciences Center, Manhattan, KS66506-5502, USA.*Present address: School of Biological and Marine Sciences,Plymouth University, Portland Square Building, Drake Circus,Plymouth PL4 8AA, UK.†Corresponding author. Email: n.j.talbot@exeter.ac.uk

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of BICs or the morphology of invasive hyphae(fig. S6A). In the presence of 1NA-PP1, hyphae ofthe pmk1AS mutant still formed terminal swell-ings at host wall contact points (Fig. 2C) (9) butcould not breach adjacent cells. Inhibition of Pmk1also blocked rice cell invasion in a second ricecultivar, Mokoto, and in barley (fig. S6, B and C).Pmk1 inhibition was accompanied by rapid de-repression of reactive oxygen species (ROS) gen-eration, a keyplant defense response, and increasedROS-dependent callose deposition (Fig. 2, D andE, and fig. S6). To confirm the role of Pmk1 in cellwall crossing, we performed live-cell imaging of afunctional Pmk1-GFP protein, which showed ex-pression of the protein during appressorium-dependent cuticle penetration (Fig. 2F) (17) butalso upon contact of invasive hyphae with ricecell walls just before invasion of the neighboringcell (Fig. 2G and movie S4). M. oryzae has twoadditional MAPKs: Osm1, which is dispensablefor virulence (18), and Mps1, which regulates cellintegrity and is necessary for fungal infection(19). We therefore generated an analog-sensitive

mps1 (mps1 AS) mutant and found that selec-tive inactivation of Mps1 enhanced host de-fenses (20) but did not block cell-to-cell invasion(fig. S7).To investigate how Pmk1 regulates invasive

growth,we performedRNA sequencing (RNA-seq)analysis to compare M. oryzae gene expressionlevels during infection with the pmk1ASmutantin the presence and absence of 1NA-PP1. Using anadjusted P value of ≤0.05 for differential geneexpression,we found 1457 fungal geneswith alteredexpression during Pmk1 inhibition, accountingfor 11.5%of total protein-encoding genes. Of these,715 fungal genes were up-regulated and 742 weredown-regulated (table S2). A subset of effectorgenes implicated in plant immunity suppressionwere positively regulated by Pmk1, including genesfor Avr-Pita1 (6); Slp1, which suppresses chitin-triggered immunity (21); Avr-Pik; and several Baseffectors, including Bas2 and Bas3 effectors (8)that putatively function at cell wall crossing sites(Fig. 3A and fig. S8). We expressed Bas2-GFP andBas3-GFP in the pmk1AS mutant and found that

they were not expressed in the presence of 1NA-PP1 (Fig. 3, B andC).We also generated a pmk1AS

strain expressing cytosolic GFP under controlof the Bas3 promoter (Fig. 3, D to F). The ad-dition of 1NA-PP1 inhibited GFP expressionduring appressorium-mediated infection (Fig. 3D)and during invasive growth (Fig 3, E and F).Localization of other fungal effectors was notaffected by Pmk1 inactivation (fig. S8), andPmk1 therefore affects expression of a subsetof fungal effectors involved in suppression ofhost immunity.To test whether suppression of host immunity,

particularly at plasmodesmata, could explain therole of Pmk1 in cell invasion by M. oryzae, wesuppressed host immune reactions simulta-neouslywithPmk1 inhibition.We found that chem-ical suppression of plant ROS or disruption ofsalicylic acid regulation did not reverse the effectsof Pmk1 inactivation (fig. S9). To suppress hostimmunity completely, we therefore killed planttissue by ethanol treatment before rehydrationand inoculationwith the pmk1ASmutant. In the

Sakulkoo et al., Science 359, 1399–1403 (2018) 23 March 2018 2 of 5

Fig. 1. Cell-to-cell invasion and plasmodesmal manipulation byM. oryzae. (A) Transmission electron micrograph of an invasive hypha(IH) traversing a rice cell wall (RCW) at 42 hpi. Scale bar, 0.5 mm.(B) High-magnification view of the crossing site. The rice plasma membrane(RPM), fungal plasma membrane (FPM), and fungal cell wall (FCW),and extrainvasive hyphal membrane (EIHM) are indicated. Scale bar, 20 nm.(C) Callose deposition in an infected rice cell at 34 hpi, shown by anilineblue staining. Arrowheads indicate plasmodesmal callose deposition.Arrows indicate callose collars that form around hyphae after they enteradjacent cells (asterisks). Scale bar, 5 mm. (D) Hyphae traversing thecell wall at a pit field (arrow). Scale bar, 0.5 mm. (E) Difference in host

cytoplasmic contents between the first and second invaded cells(for a larger image, see fig. S1G). VC, vacuole. Scale bar, 1 mm.(F) Localization of a septin Sep5-GFP collar at rice cell crossing points(arrow and high-magnification inset) at 40 hpi. Scale bars, 10 mm(main panel); 5 mm (inset). (G) Diffusion of single and double mCherryfluorescent proteins in uninfected leaf tissue (left and middle panels,respectively) and in leaves infected with M. oryzae (right panel) at 24 hours(9). Asterisks indicate bombarded cells. Scale bars, 10 mm. (H) Percentagesof bombarded cells showing diffusion of mCherry proteins in blast-infectedand uninfected rice tissues (*P = 0.05; n = 100 cells; error barsindicate SE).

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presence of 1NA-PP1, the mutant still remainedtrapped in the first dead plant cell (Fig. 4A). Wetherefore hypothesized that Pmk1-dependenthyphal constriction must be critical for cell wallcrossing at pit fields. This relationship wouldcorrespond with the role of Pmk1 in septin-dependent appressorium repolarization (fig. S5)(3, 13). Consistent with this idea, RNA-seq anal-ysis revealed several morphogenetic regulatorsdown-regulated during Pmk1 inhibition. Genesfor Chm1, a homolog of Cla4 p21-activated pro-tein kinase that phosphorylates septins (22), anda putative F-actin cross-linking protein, alpha-actinin, for example, are among genes positivelyregulated by Pmk1 during plant infection (table S2).

We therefore investigated septin organizationduring Pmk1 inhibition. Sep5-GFP still accu-mulated at cell wall contact points but as a dis-organizedmass instead of the septin collars thatnormally form at cell wall crossing sites (Fig. 4,figs. S10 and S11, andmovies S3 and S5). Finally,we investigated the ability of septin mutantsto invade plant tissue. Septin mutants do notpenetrate the rice cuticle efficiently becauseof the roles of septins in appressorium repolar-ization and the development of penetrationhyphae (3). However, a small proportion ofpenetration events are successful. In these rareinstances, the Dsep6mutant, in particular, showeda reduction in its ability to spread between rice

cells, consistent with a role for septins in cellinvasion (fig. S12).Taken together, our results demonstrate that

the Pmk1 MAPK pathway controls plant tissueinvasion by controlling the constriction of inva-sive hyphae to traverse pit fields in order to in-vade new rice cells while maintaining the cellularintegrity of the host. To accomplish this feat, theMAPK also regulates expression of a battery ofeffectors to suppress plant immunity, thereby pre-venting plasmodesmal closure until the fungushas invaded neighboring cells. Plant tissue inva-sion by the blast fungus is therefore orchestrated,rapid, and necessary for the devastating conse-quences of the disease.

Sakulkoo et al., Science 359, 1399–1403 (2018) 23 March 2018 3 of 5

Fig. 2. Pmk1 MAPK-dependent regulation of cell-to-cell spread byM. oryzae. (A and B) Effect of Pmk1 inhibition on host colonization at48 hpi. Infected rice tissues were treated with 5 mM 1NA-PP1 at 26 hpi.Asterisks indicate appressorium penetration sites. Error bars in(B) indicate SE. (C) Formation of swollen hyphae (arrows) by the pmk1AS

mutant upon contact with the host cell wall in the presence of 1NA-PP1,imaged at 40 hpi. (D and E) Induction of ROS production, shown by

3,3′-diaminobenzidine (DAB) staining, after the addition of 1NA-PP1 at26 hpi. Images were taken at 48 hpi. n = 300 infection sites; *P < 0.05,**P < 0.01, ***P < 0.001, unpaired Student’s t test. Error bars in(E) denote SE. Scale bars in (A), (C), and (D), 20 mm. (F) Transientaccumulation of Pmk1-GFP in an appressorium during emergenceof a penetration hypha. (G) Transient accumulation of Pmk1-GFP inhyphae at rice cell crossing points (asterisks).

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Fig. 3. Pmk1-dependent regulation of effectorgene expression during biotrophic growth.(A) Bar chart showing relative expression levels[fragments per kilobase of transcript per millionmapped reads (FPKM)] of known effector genesdifferentially regulated during Pmk1 inhibition inRNA-seq analysis. (B and C) Localization ofBas2-GFP and Bas3-GFP effectors in a pmk1AS

mutant with and without 1NA-PP1. BICs areindicated by arrows. Scale bars in (B), (C), and(E), 20 mm. (D to F) Expression of cytosolicGFP driven by the BAS3 promoter (Bas3p:GFP)in a pmk1AS mutant. (D) Strong GFP expressionat 24 hpi in appressoria undergoing infectionand lack of GFP expression in appressoriatreated with 1NA-PP1 at 8 hpi. Scale bar,10 mm. (E) GFP expression in pmk1AS hyphae exposedto 1NA-PP1 at 26 hpi and imagedat 30 hpi. (F) Dot plot of Bas3p:GFP fluorescencein a pmk1AS mutant treated with 5 mM 1NA-PP1at 26 hpi or left untreated. Images were takenat 30 hpi, and background was subtractedbefore measurement of fluorescence levels.****P < 0.0001, unpaired Student’s t test; threebiological replicates.

Fig. 4. Pmk1 controls septin-dependentmorphogenesis of narrow invasive hyphaetraversing cell walls. (A) Micrographsshowing that the pmk1AS mutant, growingin ethanol-killed rice tissue treated with1NA-PP1 at 14 hpi, remained trapped insideinitially invaded rice cells in all 200 infectionsites examined at 48 hpi. Asterisksindicate appressorium penetration sites.Scale bar, 20 mm. (B) Confocal micrographsshowing localization of Sep5-GFP ina pmk1AS mutant at 42 hpi after the additionof 1NA-PP1 (5 mM) at 26 hpi or in theabsence of 1NA-PP1. Arrows indicateaccumulation of Sep5-GFP at rice cell contactpoints. Scale bars, 10 mm. Red arrows inhigh-magnification panels show line scans usedto generate corresponding fluorescence intensitydistribution graphs.

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ACKNOWLEDGMENTS

We thank S. Yoshida (RIKEN, Japan) for providing Nipponbare andNahG rice lines. Funding: This work was funded by a HalpinScholarship for rice blast research to W.S., a European ResearchCouncil (ERC) Advanced Investigator Award to N.J.T. under theEuropean Union’s Seventh Framework Programme (FP7/2007-2013)and ERC grant no. 294702 GENBLAST, and Agriculture and FoodResearch Initiative Competitive grant no. 2012-67013-19291 toB.V. from the U.S. Department of Agriculture National Institute of

Food and Agriculture. This is contribution number 18-174-J fromthe Kansas Agricultural Experiment Station. Author contributions:W.S., B.V., and N.J.T. conceived of the project. W.S., M.O.-R.,and E.O.G. performed experimental work. D.M.S. providedbioinformatic data analysis. G.R.L. carried out laser confocalimaging. C.H. and A.C. performed ultrastructural analysis. W.S.,M.O.-R., B.V., and N.J.T. wrote the paper with the assistance and inputof all coauthors. Competing interests: The authors declare nocompeting interests. Data and materials availability: All data areavailable in the article or in the supplementary materials. Differentialgene expression analysis data can be accessed at Gene ExpressionOmnibus (www.ncbi.nlm.nih.gov/geo/) under accession numberGSE106845.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/359/6382/1399/suppl/DC1Materials and MethodsFigs. S1 to S13Tables S1 and S2References (23–40)Movies S1 to S5

7 October 2017; accepted 24 January 201810.1126/science.aaq0892

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A single fungal MAP kinase controls plant cell-to-cell invasion by the rice blast fungus

Correia, Barbara Valent and Nicholas J. TalbotWasin Sakulkoo, Miriam Osés-Ruiz, Ely Oliveira Garcia, Darren M. Soanes, George R. Littlejohn, Christian Hacker, Ana

DOI: 10.1126/science.aaq0892 (6382), 1399-1403.359Science 

, this issue p. 1399Sciencesame time, Pmk1 regulated the fungus's hyphal constriction, which allows movement into new host cells.genes involved in suppression of host immunity, allowing the fungus to manipulate plasmodesmal conductance. At the fungus. Inhibition of Pmk1 trapped the fungus within a rice cell. Pmk1 regulated the expression of a suite of effectorchemical genetic approach to selectively inhibit a single MAP (mitogen-activated protein) kinase, Pmk1, in the blast

used aet al.The fungus then moves to adjacent cells via plasmodesmata, the plant's intercellular channels. Sakulkoo When the rice blast fungus enters a rice cell, the plasma membrane stays intact, so the rice cell remains viable.

Keeping the channels open

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MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/03/21/359.6382.1399.DC1

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