2018-Sakulkoo-A single fungal MAP kinase contr.pdf

PLANT SCIENCEA single fungal MAP kinase controlsplant cell-to-cell invasion by therice blast fungusWasin Sakulkoo,1Miriam Oss-Ruiz,1Ely Oliveira Garcia,2Darren M.Soanes,1George R.Littlejohn,1*Christian Hacker,1Ana Correia,1Barbara Valent,2Nicholas J.Talbot1Blast disease destroys up to 30%of the rice crop annually and threatens global food security.The blast fungus Magnaporthe 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.oryzaefrominfectingadjacentplantcells,leavingthefungustrappedwithinasingleplant 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 fungus Magnaporthe oryzae(synonym of Pyricularia oryzae),de-stroying sufficient rice each year to feed60 millionpeople(1),andwheatblastdiseasenowthreatenswheatproductioninSouthAmericaand,most recently,Asia(2).Plant infection re-quires an infection cell,called an appressorium,whichusesapressure-drivenmechanismtobreachthe tough cuticle of the leaf(3,4).Once insideplanttissue,the fungus elaborates pseudohypha-like invasive hyphae that rapidly colonize livinghostcells,secretingeffectormoleculestosuppresshostimmunityandfacilitateinfection(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(58).Hyphae then appear to locate pit fields,composedofplasmodesmata,whicharetraversedbyconstricted,narrowhyphae,enablingthespreadofthefungustoadjacenthostcells(9).Thefungusrapidly colonizes host tissue,and disease lesionsappear within 4 to 5 days of initial infection byspores.In this study,we investigated how M.oryzaecolonizes 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,andfig.S1).The riceplasmamembraneinthesecondinvaded cell remained intact as an electron-denseliningnearthericecellwall,continuouswiththeplantplasmamembranearoundhyphae(Fig.1B)(5,8,9).By contrast,the rice plasma membranein the first invaded cell lost integrity upon exitofthefungustothenextricecell(Fig.1,BandE)(7,9,10).One of the plants defenses against infection isto close intercellular plasmodesma channels bydeposition of callose(11,12).We visualized cal-loseusinganilinebluestainingofricecells(Fig.1Cand fig.S3).Callose papillae often form at appres-soriumpenetrationsites,butnocalloseocclusionswere initially observed at plasmodesmata duringinfection of the first rice cell at 27 hours post-inoculation(hpi)(fig.S3).Latercallose 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 invasivehyphaeaftertheyinvadedadjacentcells,at34hpi(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-phaeatricecelljunctions.ThepolarisomemarkerSpa2green 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 anhourglassshapeatthepointofmaximumhyphalconstriction(Fig.1F,figs.S2andS10,andmovieS1)(3,5,13).Plasmodesmataaredynamicstructuresthroughwhich proteins diffuse between plant cells(11).To test whether the blast fungus can manipulateplasmodesmata by increasing their size exclu-sion limit to facilitate effector diffusion to ad-jacent plant cells,we bombarded rice tissueinfected with M.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 fusionproteingenerallydidnot,owingtoitslargersize(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 newlycolonizedcells.Plasmodesmatathereforeremainopenatearlystagesofinfection(24 to27hpi)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 controllinginvasivegrowth,wecharacterizedPmk1,afungalmitogen-activated protein kinase(MAPK)essen-tial for appressorium development and pathoge-nicity that is conserved in many plant-pathogenicfungi(15).Pmk1nullmutantsofM.oryzaecannotinfect 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)alleleofPMK1(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 pmk1ASallele,under control of its nativepromoter,restored pathogenicity to a Dpmk1mutant(fig.S4).Additionof1NA-PP1 selectivelyinhibited the function of Pmk1ASmutants,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 afterappressoriumformationblockedcuticlepenetra-tion by preventing assembly of the septin ring atthe appressorium pore(fig.S5).To test the role of Pmk1 in tissue invasion,weallowed a pmk1ASmutant 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 cellsbecomingfilledwithfungalhyphae(Fig.2,AandB).Pmk1 inactivation did not affect the structureRESEARCHSakulkoo et al.,Science 359,13991403(2018)23 March 20181 of 51School 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.talbotexeter.ac.ukon December 28,2018 http:/science.sciencemag.org/Downloaded from of BICs or the morphology of invasive hyphae(fig.S6A).In the presence of 1NA-PP1,hyphae ofthe pmk1ASmutant still formed terminal swell-ings at host wall contact points(Fig.2C)(9)butcouldnotbreachadjacentcells.InhibitionofPmk1also 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,akeyplantdefenseresponse,andincreasedROS-dependent callose deposition(Fig.2,D andE,andfig.S6).ToconfirmtheroleofPmk1incellwallcrossing,weperformedlive-cellimagingofafunctional 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-sensitivemps1(mps1AS)mutant and found that selec-tive inactivation of Mps1 enhanced host de-fenses(20)butdidnotblockcell-to-cellinvasion(fig.S7).To investigate how Pmk1 regulates invasivegrowth,weperformedRNAsequencing(RNA-seq)analysis to compare M.oryzae gene expressionlevels during infection with the pmk1ASmutantinthepresenceandabsenceof1NA-PP1.Usinganadjusted P value of 0.05 for differential geneexpression,wefound1457fungalgeneswithalteredexpression during Pmk1 inhibition,accountingfor11.5%oftotalprotein-encodinggenes.Ofthese,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,includinggenesfor 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.3Aandfig.S8).WeexpressedBas2-GFPandBas3-GFP in the pmk1ASmutant and found thatthey were not expressed in the presence of 1NA-PP1(Fig.3,BandC).Wealsogeneratedapmk1ASstrain expressing cytosolic GFP under controlof the Bas3 promoter(Fig.3,D to F).The ad-dition of 1NA-PP1 inhibited GFP expressionduringappressorium-mediatedinfection(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.Totestwhethersuppressionofhostimmunity,particularly at plasmodesmata,could explain therole of Pmk1 in cell invasion by M.oryzae,wesuppressed host immune reactions simulta-neouslywithPmk1inhibition.Wefoundthatchem-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 rehydrationandinoculationwiththepmk1ASmutant.IntheSakulkoo et al.,Science 359,13991403(2018)23 March 20182 of 5Fig.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 hostcytoplasmic 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).RESEARCH|REPORTon December 28,2018 http:/science.sciencemag.org/Downloaded from 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 positivelyregulatedbyPmk1duringplantinfection(tableS2).We therefore investigated septin organizationduring Pmk1 inhibition.Sep5-GFP still accu-mulated at cell wall contact points but as a dis-organizedmassinsteadoftheseptincollarsthatnormally form at cell wall crossing sites(Fig.4,figs.S10and S11,and moviesS3andS5).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,theDsep6mutant,inparticular,showeda reduction in its ability to spread between ricecells,consistent with a role for septins in cellinvasion(fig.S12).Taken together,our results demonstrate thatthe 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 359,13991403(2018)23 March 20183 of 5Fig.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 pmk1ASmutant 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 by3,3-diaminobenzidine(DAB)staining,after the addition of 1NA-PP1 at26 hpi.Image