2018-Sakulkoo-AsinglefungalMAPkinasecontr

PLANT SCIENCE A single fungal MAP kinase controls plant cell-to-cell invasion by the rice blast fungus Wasin Sakulkoo,1Miriam Oss-Ruiz,1Ely Oliveira Garcia,2Darren M. Soanes,1 George R. Littlejohn,1* Christian Hacker,1Ana Correia,1 Barbara Valent,2Nicholas J. Talbot1 Blast 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 and grow from cell to cell, often through pit fields, where plasmodesmata cluster.We showed that chemical genetic inhibition of a single fungal mitogen-activated protein (MAP) kinase, Pmk1, preventsM.oryzaefrominfectingadjacentplantcells,leavingthefungustrappedwithinasingle plant cell. Pmk1 regulates expression of secreted fungal effector proteins implicated in suppression of host immune defenses, preventing reactive oxygen species generation and excessive callose deposition at plasmodesmata. Furthermore, Pmk1 controls the hyphal constriction required for fungal growth from one rice cell to the neighboring cell, enabling host tissue colonization and blast disease. B last diseases of cereals are caused by the filamentous fungus Magnaporthe oryzae (synonym of Pyricularia oryzae), de- stroying sufficient rice each year to feed 60 millionpeople(1),andwheatblastdisease nowthreatenswheatproductioninSouthAmerica and, most recently, Asia (2). Plant infection re- quires an infection cell, called an appressorium, whichusesapressure-drivenmechanismtobreach the tough cuticle of the leaf (3, 4). Once inside planttissue,the fungus elaborates pseudohypha- like invasive hyphae that rapidly colonize living hostcells,secretingeffectormoleculestosuppress hostimmunityandfacilitateinfection(5).M.oryzae effectors are delivered into host cytoplasm by means of a biotrophic interfacial complex (BIC), a plant-derived membrane-rich structure in which effectors accumulate during transit to the host (58). Hyphae then appear to locate pit fields, composedofplasmodesmata,whicharetraversed byconstricted,narrowhyphae,enablingthespread ofthefungustoadjacenthostcells(9).Thefungus rapidly colonizes host tissue, and disease lesions appear within 4 to 5 days of initial infection by spores. In this study, we investigated how M. oryzae colonizes host tissue and, in particular, how it spreads from one plant cell to the next. We first performed ultrastructural analysis of rice sheath cells infected with the pathogenic strain Guy11. This analysis confirmed constriction of hyphae from an average diameter of 5.0 mm to 0.6 mm during traversal of rice cells (Fig. 1, A and B, and fig.S1).The riceplasmamembraneinthesecond invaded cell remained intact as an electron-dense liningnearthericecellwall,continuouswiththe plantplasmamembranearoundhyphae(Fig.1B) (5, 8, 9). By contrast, the rice plasma membrane in the first invaded cell lost integrity upon exit ofthefungustothenextricecell(Fig.1,BandE) (7, 9, 10). One of the plants defenses against infection is to close intercellular plasmodesma channels by deposition of callose (11, 12). We visualized cal- loseusinganilinebluestainingofricecells(Fig.1C and fig. S3). Callose papillae often form at appres- soriumpenetrationsites,butnocalloseocclusions were initially observed at plasmodesmata during infection of the first rice cell at 27 hours post- inoculation(hpi)(fig. S3).Latercallose deposition was observed at plasmodesmata by 30 hpi, con- sistent with the onset of cell death among ini- tially invaded rice cells (figs. S1 and S2). Callose collars then formed around the base of invasive hyphaeaftertheyinvadedadjacentcells,at34hpi (Fig. 1C and fig. S3). These observations suggest that 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.Thepolarisomemarker Spa2green fluorescent protein (Spa2-GFP) local- ized to hyphal tips and then disappeared as hy- phal tips swelled upon contact with the host cell wall (fig. S2) (5). Spa2-GFP then appeared again at hyphal tips in newly colonized cells (fig. S2). Cell wall crossing was accompanied by reorgani- zation of fungal septins and F-actin into an hourglassshapeatthepointofmaximumhyphal constriction(Fig.1F,figs.S2andS10,andmovieS1) (3, 5, 13). Plasmodesmataaredynamicstructuresthrough 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 tissue infected with M. oryzae at 24 hpi with single- and double-sized mCherry expression vectors. In uninfected rice tissue, a 28.8-kDa single mCherry protein moved into neighboring rice cells but a 57.6-kDa double mCherry fusion proteingenerallydidnot,owingtoitslargersize (Fig. 1, G and H). By contrast, in blast-infected tissue, double mCherry protein diffused to ad- jacent rice cells. The gating limit of rice plas- modesmata is therefore relaxed during early M. oryzae infection. We then carried out time- lapse imaging of the fungal apoplastic effector Bas4 (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 to an adjacent cell, Bas4-GFP leaked into the host cytoplasm of initially colonized rice cells. How- ever, fluorescence did not diffuse into the newly colonizedcells.Plasmodesmatathereforeremain openatearlystagesofinfection(24 to27hpi)but are closed at later stages, consistent with the increase in plasmodesmal callose deposition at 30 to 34 hpi (fig. S3) (7, 14), suggesting that the blast 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 cells lose their viability. To study regulatory mechanisms controlling invasivegrowth,wecharacterizedPmk1,afungal mitogen-activated protein kinase (MAPK) essen- tial for appressorium development and pathoge- nicity that is conserved in many plant-pathogenic fungi(15).Pmk1nullmutantsofM.oryzaecannot infect rice plants even when mutants are inocu- lated onto wounded leaves (15). We decided to conditionally inactivate the Pmk1 MAPK using a chemical genetic approach. We generated an analog-sensitive (AS)alleleofPMK1(pmk1AS)by mutating the gatekeeper residue of the kinase adenosine triphosphate (ATP)binding site into a small amino acid residue, glycine. The equivalent (Shokat) mutation previously reported in yeast fus3-as1 confers susceptibility to the ATP analog 1-naphthyl-PP1 (1NA-PP1) (16). Expression of the pmk1ASallele, under control of its native promoter, restored pathogenicity to a Dpmk1 mutant(fig. S4).Additionof1NA-PP1 selectively inhibited the function of Pmk1ASmutants, pre- venting appressorium development (fig. S4), a result that was identical to the effects of PMK1 deletion or expression of a kinase-inactive allele (17). We also observed that inhibiting Pmk1 after appressoriumformationblockedcuticlepenetra- tion by preventing assembly of the septin ring at the appressorium pore (fig. S5). To test the role of Pmk1 in tissue invasion, we allowed a pmk1ASmutant to invade the first rice epidermal cell before adding 1NA-PP1 at 26 hpi. This treatment blocked invasion of adjacent epi- dermal cells, resulting in the first infected cells becomingfilledwithfungalhyphae(Fig.2,Aand B). Pmk1 inactivation did not affect the structure RESEARCH Sakulkoo et al., Science 359, 13991403 (2018)23 March 20181 of 5 1School of Biosciences, University of Exeter, Exeter EX4 4QD, UK. 2Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506-5502, 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.uk on December 28, 2018 /Downloaded from of BICs or the morphology of invasive hyphae (fig. S6A). In the presence of 1NA-PP1, hyphae of the pmk1ASmutant still formed terminal swell- ings at host wall contact points (Fig. 2C) (9) but couldnotbreachadjacentcells.InhibitionofPmk1 also blocked rice cell invasion in a second rice cultivar, 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,andincreased ROS-dependent callose deposition (Fig. 2, D and E,andfig.S6).ToconfirmtheroleofPmk1incell wallcrossing,weperformedlive-cellimagingofa functional Pmk1-GFP protein, which showed ex- pression of the protein during appressorium- dependent cuticle penetration (Fig. 2F) (17) but also upon contact of invasive hyphae with rice cell walls just before invasion of the neighboring cell (Fig. 2G and movie S4). M. oryzae has two additional MAPKs: Osm1, which is dispensable for virulence (18), and Mps1, which regulates cell integrity and is necessary for fungal infection (19). We therefore generated an analog-sensitive mps1 (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 invasive growth,weperformedRNAsequencing(RNA-seq) analysis to compare M. oryzae gene expression levels during infection with the pmk1ASmutant inthepresenceandabsenceof1NA-PP1.Usingan adjusted P value of 0.05 for differential gene expression,wefound1457fungalgeneswithaltered expression during Pmk1 inhibition, accounting for11.5%oftotalprotein-encodinggenes.Ofthese, 715 fungal genes were up-regulated and 742 were down-regulated (table S2). A subset of effector genes implicated in plant immunity suppression were positively regulated by Pmk1, includinggenes for Avr-Pita1 (6); Slp1, which suppresses chitin- triggered immunity (21); Avr-Pik; and several Bas effectors, including Bas2 and Bas3 effectors (8) that putatively function at cell wall crossing sites (Fig.3Aandfig.S8).WeexpressedBas2-GFPand Bas3-GFP in the pmk1ASmutant and found that they were not expressed in the presence of 1NA- PP1(Fig.3,BandC).Wealsogeneratedapmk1AS strain expressing cytosolic GFP under control of the Bas3 promoter (Fig. 3, D to F). The ad- dition of 1NA-PP1 inhibited GFP expression duringappressorium-mediatedinfection(Fig.3D) and during invasive growth (Fig 3, E and F). Localization of other fungal effectors was not affected by Pmk1 inactivation (fig. S8), and Pmk1 therefore affects expression of a subset of fungal effectors involved in suppression of host immunity. Totestwhethersuppressionofhostimmunity, particularly at plasmodesmata, could explain the role of Pmk1 in cell invasion by M. oryzae, we suppressed host immune reactions simulta- neouslywithPmk1inhibition.Wefoundthatchem- ical suppression of plant ROS or disruption of salicylic acid regulation did not reverse the effects of Pmk1 inactivation (fig. S9). To suppress host immunity completely, we therefore killed plant tissue by ethanol treatment before rehydration andinoculationwiththepmk1ASmutant.Inthe Sakulkoo et al., Science 359, 13991403 (2018)23 March 20182 of 5 Fig. 1. Cell-to-cell invasion and plasmodesmal manipulation by M. 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 aniline blue staining. Arrowheads indicate plasmodesmal callose deposition. Arrows indicate callose collars that form around hyphae after they enter adjacent cells (asterisks). Scale bar, 5 mm. (D) Hyphae traversing the cell 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 mCherry fluorescent 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) Percentages of bombarded cells showing diffusion of mCherry proteins in blast-infected and uninfected rice tissues (*P = 0.05; n = 100 cells; error bars indicate SE). RESEARCH|REPORT on December 28, 2018 /Downloaded from presence of 1NA-PP1, the mutant still remained trapped in the first dead plant cell (Fig. 4A). We therefore hypothesized that Pmk1-dependent hyphal constriction must be critical for cell wall crossing at pit fields. This relationship would correspond 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 regulators down-regulated during Pmk1 inhibition. Genes for Chm1, a homolog of Cla4 p21-activated pro- tein kinase that phosphorylates septins (22), and a putative F-actin cross-linking protein, alpha- actinin, for example, are among genes positively regulatedbyPmk1duringplantinfection(tableS2). We therefore investigated septin organization during Pmk1 inhibition. Sep5-GFP still accu- mulated at cell wall contact points but as a dis- organizedmassinsteadoftheseptincollarsthat normally form at cell wall crossing sites (Fig. 4, figs.S10and S11,and moviesS3andS5).Finally, we investigated the ability of septin mutants to invade plant tissue. Septin mutants do not penetrate the rice cuticle efficiently because of the roles of septins in appressorium repolar- ization and the development of penetration hyphae (3). However, a small proportion of penetration events are successful. In these rare instances,theDsep6mutant,inparticular,showed a reduction in its ability to spread between rice cells, consistent with a role for septins in cell invasion (fig. S12). Taken together, our results demonstrate that the Pmk1 MAPK pathway controls plant tissue invasion by controlling the constriction of inva- sive hyphae to traverse pit fields in order to in- vade new rice cells while maintaining the cellular integrity of the host. To accomplish this feat, the MAPK also regulates expression of a battery of effectors to suppress plant immunity, thereby pre- venting plasmodesmal closure until the fungus has 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 5 Fig. 2. Pmk1 MAPK-dependent regulation of cell-to-cell spread by M. oryzae. (A and B) Effect of Pmk1 inhibition on host colonization at 48 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 at 26 hpi. Images were taken at 48 hpi. n = 300 infection sites; *P 0.05, *P 0.01, *P 0.001, unpaired Students t test. Error bars in (E) denote SE. Scale bars in (A), (C), and (D), 20 mm. (F) Transient accumulation of Pmk1-GFP in an appressorium during emergence of a penetration hypha. (G) Transient accumulation of Pmk1-GFP in hyphae at rice cell crossing points (asterisks). RESEARCH|REPORT on December 28, 2018 /Downloaded from Sakulkoo et al., Science 359, 13991403 (2018)23 March 20184 of 5 Fig. 3. Pmk1-dependent regulation of effector gene expression during biotrophic growth. (A) Bar chart showing relative expression levels fragments per kilobase of transcript per million mapped reads (FPKM) of known effector genes differentially regulated during Pmk1 inhibition in RNA-seq analysis. (B and C) Localization of Bas2-GFP and Bas3-GFP effectors in a pmk1AS mutant with and without 1NA-PP1. BICs are indicated by arrows. Scale bars in (B), (C), and (E), 20 mm. (D to F) Expression of cytosolic GFP driven by the BAS3 promoter (Bas3p:GFP) in a pmk1ASmutant. (D) Strong GFP expression at 24 hpi in appressoria undergoing infection and lack of GFP expression in appressoria treated with 1NA-PP1 at 8 hpi. Scale bar, 10 mm. (E) GFP expression in pmk1AShyphae exposed to 1NA-PP1 at 26 hpi and imaged at 30 hpi. (F) Dot plot of Bas3p:GFP fluorescence in a pmk1ASmutant treated with 5 mM 1NA-PP1 at 26 hpi or left untreated. Images were taken at 30 hpi, and background was subtracted before measurement of fluorescence levels. *P 0.0001, unpaired Students t test; three biological replicates. Fig. 4. Pmk1 controls septin-dependent morphogenesis of narrow invasive hyphae traversing cell walls. (A) Micrographs showing that the pmk1ASmutant, growing in ethanol-killed rice tissue treated with 1NA-PP1 at 14 hpi, remained trapped inside initially invaded rice cells in all 200 infection sites examined at 48 hpi. Asterisks indicate appressorium penetration sites. Scale bar, 20 mm. (B) Confocal micrographs showing localization of Sep5-GFP in a pmk1ASmutant at 42 hpi after the addition of 1NA-PP1 (5 mM) at 26 hpi or in the absence of 1NA-PP1. Arrows indicate accumulation of Sep5-GFP at rice cell contact points. Scale bars, 10 mm. Red arrows in high-magnification panels show line scans used to genera