Interspecies hormonal control of host root morphologyby parasitic plantsThomas Spalleka,1,2, Charles W. Melnykb,1,3, Takanori Wakatakea,c, Jing Zhangb,4, Yuki Sakamotod, Takatoshi Kibaa,Satoko Yoshidae, Sachihiro Matsunagad,f, Hitoshi Sakakibaraa, and Ken Shirasua,c,2

aRIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan; bThe Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR,United Kingdom; cGraduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan; dImaging Frontier Center, Organization forResearch Advancement, Tokyo University of Science, Noda, Chiba 278-8510, Japan; eInstitute for Research Initiatives, Division for Research Strategy, NaraInstitute of Science and Technology, Ikoma, Nara, 630-0192, Japan; and fDepartment of Applied Biological Science, Faculty of Science and Technology,Tokyo University of Science, Noda, Chiba 278-8510, Japan

Edited by Joseph J. Kieber, University of North Carolina, Chapel Hill, NC, and accepted by Editorial Board Member Joseph R. Ecker April 3, 2017 (received forreview November 17, 2016)

Parasitic plants share a common anatomical feature, the hausto-rium. Haustoria enable both infection and nutrient transfer, whichoften leads to growth penalties for host plants and yield reductionin crop species. Haustoria also reciprocally transfer substances,such as RNA and proteins, from parasite to host, but the biologicalrelevance for such movement remains unknown. Here, we studiedsuch interspecies transport by using the hemiparasitic plantPhtheirospermum japonicum during infection of Arabidopsis thali-ana. Tracer experiments revealed a rapid and efficient transfer ofcarboxyfluorescein diacetate (CFDA) from host to parasite uponformation of vascular connections. In addition, Phtheirospermuminduced hypertrophy in host roots at the site of infection, a formof enhanced secondary growth that is commonly observed duringvarious parasitic plant–host interactions. The plant hormone cyto-kinin is important for secondary growth, and we observed in-creases in cytokinin and its response during infection in bothhost and parasite. Phtheirospermum-induced host hypertrophy re-quired cytokinin signaling genes (AHK3,4) but not cytokinin bio-synthesis genes (IPT1,3,5,7) in the host. Furthermore, expression ofa cytokinin-degrading enzyme in Phtheirospermum preventedhost hypertrophy. Wild-type hosts with hypertrophy were smallerthan ahk3,4 mutant hosts resistant to hypertrophy, suggestinghypertrophy improves the efficiency of parasitism. Taken to-gether, these results demonstrate that the interspecies movementof a parasite-derived hormone modified both host root morphol-ogy and fitness. Several microbial and animal plant pathogens usecytokinins during infections, highlighting the central role of thisgrowth hormone during the establishment of plant diseases andrevealing a common strategy for parasite infections of plants.

cytokinin | transport | hypertrophy | parasitism | Arabidopsis

Parasitic plants are widespread agricultural pests and accountfor ∼1% of known flowering plants species (1). Parasitism

ranges from holoparasites, which depend entirely on nutrientsupply from host plants, to hemiparasites, which obtain nutrientsvia their own photosynthesis and from their hosts (1). Manyhemiparasites do not depend on parasitism but often parasitizewhen conditions are suitable. These hemiparasitic plants includeparasitic plants such as the commonly studied Orobanchaceaespecies Rhinanthus minor, Triphysaria versicolor, and Phtheir-ospermum japonicum. Both hemiparasites and holoparasitesform specialized organs called haustoria that undergo a de-velopmental transition from proto-haustoria to mature haustoriaduring the penetration and infection of host tissues to acquirenutrients and water (2). Some parasitic plants such as Striga orRhinanthus form vascular connections exclusively to host xylemvia xylem bridges (xylem-feeding), whereas haustoria of otherplants such as Cuscuta or Orobanche also form symplasticphloem-to-phloem connections to host plants (phloem-feeding)(1). In addition to water and nutrients, other small substancesare transferred across haustoria, including RNAs and proteins

(3–5), but the biological relevance for this movement is not clear.Beyond parasitic plants, various plant-pathogenic microbes, in-sects, and nematodes produce compounds that move into thehost and contribute to their virulence, including the plant hor-mone cytokinin (6–8).Cytokinins participate in many physiological and developmental

plant processes such as cell division, growth, vascular develop-ment, senescence, photosynthesis, and nutrient allocation (9).Cytokinins are isoprenoid substituted adenines and in plants,isoprenoid transfer by isopentenyltransferases (IPTs) is the rate-limiting and crucial step for producing various types of cytoki-nins including cis-zeatin (cZ), N6-(Δ2-isopentenyl)-adenine (iP),trans-zeatin (tZ), and dihydrozeatin (DZ) (10). tZ is the mostabundant and the most potent cytokinin in Arabidopsis (9). Cy-tokinins are further metabolized and inactivated through con-jugation to sugars or through cleavage by cytokinin oxidases(CKXs) (11). Cytokinins act at the site of biosynthesis and arealso mobile within the plant vascular system (12, 13). Graftingexperiments with Arabidopsis ipt1,3,5,7 mutants demonstratedroot-to-shoot movement of tZ-type cytokinins and an opposing

Significance

Parasitic plants are pests of many plants, including major cropspecies. An important step toward creating resistance to par-asitic plants is gaining a better understanding of how thesepathogens control the physiology and development of theirhosts. We combined genetic, cell-biological, and biochemicalmethods to identify the plant hormone cytokinin as a mobilesignal between the hemiparasitic plant Phtheirospermumjaponicum and the host Arabidopsis thaliana. Transport ofparasite-derived cytokinins induced morphological changes inhost roots, revealing insights into how parasitic plants ma-nipulate host development and laying the foundation for fu-ture explorations for bioactive molecule transfer from parasiticplants to hosts.

Author contributions: T.S., C.W.M., and K.S. designed research; T.S., C.W.M., T.W., J.Z.,Y.S., and T.K. performed research; T.S., C.W.M., S.Y., S.M., H.S., and K.S. analyzed data;and T.S., C.W.M., and K.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.J.K. is a guest editor invited by the EditorialBoard.1T.S. and C.W.M. contributed equally to this work.2To whom correspondence may be addressed. Email: ken.shirasu@riken.jp or thomas.spallek@riken.jp.

3Present address: Department of Plant Biology, Swedish University of Agricultural Sci-ences, Almas allé 5, 756 51, Uppsala, Sweden.

4Present address: Institute of Biotechnology, University of Helsinki, 00014, Helsinki,Finland.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1619078114/-/DCSupplemental.

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shoot-to-root movement of iP-type cytokinins (13), consistentwith the idea that tZ-type cytokinins predominate in the xylem,whereas iP-types predominate in the phloem (13). ARABI-DOPSIS HISTIDINE KINASE 2, 3 and 4 (AHK2, AHK3,AHK4) receptors directly bind cytokinins and trigger down-stream responses (14), including the transcriptional induction ofA-type ARABIDOPSIS RESPONSE REGULATOR (ARR) genessuch as ARR5 (15). Although Arabidopsis cytokinin receptors actlargely redundantly, combinations of ahk double and triple mu-tants show various cytokinin-deficient phenotypes (14).Increased cytokinin levels were previously reported during

infection by parasitic plants such as Cuscuta spp., mistletoes,Santalum album, and several Orobanchaceae species, but thesource of the cytokinins and the biological relevance were un-known (16, 17). Here, we demonstrate the parasitic plantPhtheirospermum increases its cytokinin levels upon infection,and these cytokinin species move into the host Arabidopsis. Wefurther demonstrate these cytokinin species are bioactive inArabidopsis roots and induce changes in gene response, cell di-vision, and cell differentiation that leads to modifications in hostroot morphology and impacts host fitness.

ResultsPhtheirospermum Parasitizes Arabidopsis. Phtheirospermum infectsa variety of plant species including rice, maize, and Arabidopsis(18, 19). We sought to identify conditions that promote Arabi-dopsis infection because growth of Phtheirospermum does notabsolutely depend on parasitism. As previously described, haus-torium development occurs when Phtheirospermum comes incontact with its host on water-agar with no additional nutrients(18, 19). We used a similar water-only setup, but substitutedWhatman filter paper and nylon membrane for agar to anchor theplants, similar to an experimental setup used for Arabidopsisgrafting (20). This low nutrient environment allowed efficient in-fection, consistent with observations that low levels of nitrogen arebeneficial for infection by other parasitic plants such as Strigahermonthica (21). Under these conditions, Phtheirospermum for-med haustoria on Arabidopsis roots within 3 d (Fig. 1 A and B). At7 d postinfection (dpi), infected and noninfected controls weremoved to 1/2 strength Murashige and Skoog (MS) growth mediumto assess the long-term effects of Phtheirospermum parasitism onArabidopsis in the absence of low nutrient stress. Infected andnoninfected plants were of similar size at 7 dpi, but infectedArabidopsis developed poorly in contrast to uninfected controlsand showed a clear reduction in size at 30 dpi (Fig. 1A and MovieS1). Meanwhile, Phtheirospermum-infecting Arabidopsis grew largerthan Phtheirospermum growing solitarily (Fig. 1A and Movie S1).Safranin-O staining of haustoria confirmed the formation of xylembridges from Phtheirospermum to Arabidopsis during transitionfrom protohaustoria to mature haustoria, typically 3–4 dpi, con-sistent with normal haustorial development (Fig. 1 C and E) (18).To characterize the functionality of nascent haustoria, we

monitored haustorial transport activity by using the vasculartransport dye carboxyfluorescein diacetate (CFDA) (22). AfterCFDA application to host roots or shoots, fluorescent signalsrapidly propagated from host vasculature into Phtheirospermumhaustoria and shoots, indicating efficient transport from host toparasite as early as 15 min after CFDA application (Fig. 1D, Fig.S1 A and B, and Movie S2). To investigate the role of xylembridges for CFDA transport, we compared the ratio of CFDAuptake to the ratio of mature haustoria with xylem bridges atdifferent time points (Fig. 1E). More than 50% of Phtheir-ospermum developed haustoria with at least one xylem bridge at4 dpi, whereas less than 10% of Phtheirospermum transportedCFDA at this time point (Fig. 1E). The percentage of Phtheir-ospermum transporting CFDA gradually increased at 6 and 11 dpiwhen the majority of haustoria reached maturity (Fig. 1E). Astrong fluorescent signal was detected in mature haustorial tissues

surrounding the xylem bridges within the Phtheirospermum haus-torium (Fig. 1 F and H). The fluorescence signal decreasedgradually toward the proximal haustorial tissue (Fig. 1 F–H). Atthe same time, fluorescence in host roots above the haustoriumattachment site was stronger than below, indicating preferentialCFDA uptake from the host vasculature via the haustorium (Fig. 1 Iand J). To investigate the possibility of transport via phloem con-nections, we infected Phtheirospermum to Arabidopsis pSUC2::GFPplants that express mobile GFP in the phloem (23). GFP was notdetected in Phtheirospermum haustoria or root tips, whereas GFPwas efficiently unloaded in root tips of infected ArabidopsispSUC2::GFP plants (Fig. S1 C–E). Furthermore, no gradient influorescence intensities along haustoria was detected for phloem-mobile GFP in infected Arabidopsis pSUC2::GFP roots (Fig. S1C).

Phtheirospermum Induces Hypertrophy and Cytokinin Responses inHost Plants. After Phtheirospermum established xylem connectionsto Arabidopsis, we observed a swelling of Arabidopsis tissue abovehaustorium attachment sites (Figs. 1B and 2A), a phenomenon

Fig. 1. Phtheirospermum parasitizes Arabidopsis. (A) Phtheirospermumgrowing alone (Pj-) or infecting (Pj+) Arabidopsis (At+) show increasedPhtheirospermum size and decreased Arabidopsis size compared with un-infected controls (At-) at 30 dpi. (B) Detail image of Phtheirospermum-infectingArabidopsis with Phtheirospermum haustorium (haust.) attachment site (HA).(C) Images of Safranin-O stained proto- and mature haustoria show differencesin xylem bridge (XB) formation. (D) CFDA transport ability of 11 dpi haustoriawere assayed 90 min after application of CFDA onto host leaves (asterisk).(E) Ratios of mature haustoria (brown bar) and haustoria with CFDA transportability (green bar) (n = 35–86) were quantified for the indicated time points(Fisher Exact Test, P < 0.001). (F) A fluorescent image of a single optical plane ofthe haustorium 90 min after CFDA application onto a host leaf. CFDA fluores-cence is green, and cell walls were stained with propidium iodide in magenta.(G) Schematic representation of F with indicated optical section of the haus-torium (H), the host root above (I) or below (J) the HA. (Scale bars: A, B, and D,1 mm; C, F, and G, 50 μm; H and I, 25 μm.)

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referred to as hypertrophy (1). Hypertrophy resulted from an in-crease in vascular cell size (hypertrophy) and cell number (hyper-plasia) that enlarged the vascular and xylem area in host tissueabove haustoria attachment sites compared with host tissue belowthe haustoria or uninfected controls (Fig. 2 B and C). Hypertrophywas not specific to Arabidopsis because a similar effect also occurredin tomato (Solanum lycopersicum) infected by Phtheirospermum(Fig. S2 A and B). Increases in vascular size were already detectedabove the haustoria at 11 dpi (Fig. S3 A and B), and by 20 dpi,quantifications of root sections prepared above and below thehaustorium confirmed a further increase in vascular and xylem di-ameter and cell number above haustorium attachment sites, whereasno significant differences in uninfected plants were detected (Fig.2C). The vascular and xylem areas above haustorium attachmentsites were ∼4 times greater than in uninfected plants (Fig. S3C),which was substantial considering that infected Arabidopsis showedan overall growth reduction compared with uninfected plants of thesame age (Movie S1). The alteration in morphology suggested thatPhtheirospermum locally induced secondary growth in the host root,a phenomenon that resembles other cases of host hypertrophycaused by parasitic plants such as mistletoes (Viscum album)infecting crabapple trees (Malus toringoides) (Fig. S2 C and D).Root secondary growth depends on and is promoted by the plant

hormone cytokinin (13). To test whether infection altered cytokininsignaling, we monitored cytokinin response in Arabidopsis by usingthe transcriptional reporter line pARR5::GFP (24). Infected plantsshowed a substantial increase in GFP fluorescence at 3–4 dpi and,by 6 dpi, most host plants showed high GFP fluorescence (Fig.S4A). The increase in GFP expression was restricted to tissueabove haustoria attachment sites (Fig. S4B). Spatial localization offluorescence at 11 dpi revealed pARR5::GFP expression over-lapped with host tissues undergoing hypertrophic growth (Fig. S4B–E). Temporally, increased cytokinin response occurred duringhypertrophic growth because pARR5::GFP expression remainedhigh even after 20 dpi (Fig. S4 F and G). These data were con-sistent with an increase in cytokinin response occurring in tissuesundergoing increased cell division and xylem differentiation.To address the source of this response, we generated transgenic

Phtheirospermum hairy roots containing the cytokinin responsivepromotor sequence of the Two Component signaling Sensor new(pTCSn) fused to a triple mCherry-NLS (pTCSn::3xmCherry-NLS)(25). We then followed cytokinin responses simultaneously inPhtheirospermum expressing pTCSn::3xmCherry-NLS infectingArabidopsis expressing pARR5::GFP. No cytokinin responses inhost and parasite were detected during penetration of host tissue(10–60 h postinfection, hpi), but during the transition from proto-to mature haustoria (72–84 hpi) a parallel increase of cytokininresponse in parasite and host preceded the formation of the first

xylem bridge (84 hpi) (Fig. 2D and Fig. S4H). Hairy roots ofPhtheirospermum resembled haustoria of nontransgenic Phtheir-ospermum roots in development and transcriptional induction ofpARR5::GFP in host plants (Fig. 1E and Fig. S4).

Phtheirospermum-Induced Host Hypertrophy Depends on HostCytokinin Signaling but Not on Host Cytokinin Biosynthesis Genes.The spatial and temporal overlap between host cytokinin re-sponses and hypertrophy suggested that they might be directlylinked. To test this possibility, we infected various Arabidopsiscytokinin signaling and biosynthesis mutants. Infected Col-0 wildtype showed more secondary growth including increased vasculardiameter and xylem number at 20 dpi (Fig. 3 A and B and Fig.S5A) and developed roots with twofold larger diameters abovehaustoria compared with diameters below haustoria at 30 dpi (Fig.3C and Fig. S5B). Roots of infected Col-0 were even larger thanuninfected roots of the same age, despite their reduced shootgrowth (Fig. 1A and Fig. S5B). Hypertrophy was blocked in thecytokinin signaling mutants ahk2,3 and ahk3,4, but cytokinin bio-synthesis mutants ipt3,5,7 and ipt1,3,5,7 had hypertrophy levelssimilar to Col-0, thus partially rescuing the mutant root phenotype(Fig. 3 and Fig. S5). Consistent with these observations, transversesections above the haustoria at 20 dpi showed an increase invascular diameter, vascular area, xylem cell number, and xylemarea for Col-0 and ipt1,3,5,7, but not for ahk3,4 and only mar-ginally for ahk2,3 (Fig. 3 A and B and Fig. S5A). Hypertrophy inipt1,3,5,7 was more variable at 20 dpi with some plants showingno macroscopic signs of hypertrophy. Increased cytokinin re-sponse occurred throughout the plant because endogenous ARR5transcripts levels were also up-regulated at 11 dpi in shoots ofinfected Col-0, ipt3,5,7, and ipt1,3,5,7, but were not up-regulated in infected ahk2,3 and ahk3,4 (Fig. S6A). Likewise,genes downstream of cytokinin signaling including NRT1.7 andNRT1.5 showed substantial changes upon infection in Col-0 that were not observed in ahk3,4 at 11 dpi (Fig. S6B) (26).

Phtheirospermum Elevates Host Cytokinin Levels Independently ofHost Cytokinin Biosynthesis Genes. Our data demonstrated thatan induction of host cytokinin responses was independent ofseveral host IPT cytokinin biosynthesis genes, but it remainedunknown whether increased cytokinin response resulted from anincrease of cytokinin levels in host plants during infection. Toaddress this question, we quantified cytokinin species in hostroots (including hypocotyls) and parasite roots above haustoriaat 11 dpi in Col-0 and ipt1,3,5,7 and compared the resulting cy-tokinin profiles to those of uninfected controls. Uninfectedipt1,3,5,7 were greatly depleted in tZ-type cytokinins comparedwith uninfected Col-0, consistent with previous findings (13)

Fig. 2. Phtheirospermum induces hypertrophy inArabidopsis. (A) Safranin-O stained Phtheirospermum(Pj) infected Arabidopsis (At) Col-0 root with hausto-rium attachment site (HA) at 20 dpi. (B) Rutheniumred-stained transverse sections were taken 8 mm(1–3 mm above the HA) and 13 mm below thehypocotyl-root junction (2–4 mm below the HA) ofcontrol and infected Col-0 plants at 20 dpi to quan-tify vascular diameter and xylem cell number ofcontrol (blue) and infected Col-0 plants (orange) in C(mean ± SE, n = 6–20, ANOVA, P < 0.01). (D) Confocalimages were taken between 10 and 84 hpi of Pj hairyroots expressing pTCSn::3xmCherry-NLS (magenta)and host roots (At pARR5::GFP, green) before andafter xylem bridge (XB) formation. (Scale bars: A andD, 100 μm; B, 25 μm.)

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(Fig. 4A, Fig. S7, and Dataset S1). Upon infection, both infectedCol-0 and infected ipt1,3,5,7 showed significant increases oftZ-type cytokinins. Compared with uninfected plants, tZ-type cy-tokinins increased 8-fold in Col-0 and 29-fold in ipt1,3,5,7 (Fig.4A). Detailed profiling of cytokinins showed an increase of alldetected species of tZ-type cytokinins including bioactive tZ andxylem mobile precursor tZ-riboside (tZR, Fig. S7 and DatasetS1). tZ-type cytokinins also increased in Phtheirospermum uponinfection, however, the fold change was lower than comparedwith tZ-type cytokinin accumulation in host plants (Fig. 4B). Nosignificant changes during infection were detected for cZ-, iP-, orDZ-type of cytokinins in either host or parasite (Fig. 4).

Host Hypertrophy Inducing Cytokinins Are Derived from Phtheirospermum.The presence of hypertrophy (Fig. 3) and the increase of tZ-typecytokinins in infected ipt1,3,5,7 similar to levels found in Col-0 (Fig.4A) suggested that host tZ-type cytokinins were derived fromPhtheirospermum. To address this possibility, we expressed anArabidopsis cytokinin-degrading enzyme, AtCKX3, in hairy roots ofPhtheirospermum (Fig. 5). Overexpression of AtCKX3 reducescytokinin levels in a distant relative of Phtheirospermum, Nicotianatabacum (27), and caused various cytokinin-deficient phenotypesin Arabidopsis and N. tabacum (11, 28). Thus, we reasoned thatoverexpressing AtCKX3 in Phtheirospermum would also reduceparasite cytokinin levels. We quantified host hypertrophy duringinfection of Phtheirospermum hairy roots expressing either high,

medium, or low levels of pMAS::AtCKX3 and of control hairy rootsexpressing pMAS::GFP (Fig. 3 and Fig. S8 A and B). WhereasPhtheirospermum roots expressing pMAS::GFP induced similar lev-els of host hypertrophy compared with nontransgenic roots at 28 dpi(Figs. 3C and 5 A and B and Fig. S5B), Arabidopsis hypertrophydecreased with increasing AtCKX3 expression in Phtheirospermum(Fig. 5). No hypertrophy was observed for Phtheirospermum hairyroots with the highest AtCKX3 expression. Hairy roots expressingpMAS::AtCKX3 had similar morphology and ability to develophaustoria compared with hairy roots transformed with pMAS::GFP;however, hairy roots expressing high levels of pMAS::AtCKX3 in-duced lower levels of pARR5::GFP expression in the host comparedwith pMAS::GFP controls (Fig. 5A and Fig. S8C).

Hypertrophy Correlates with Reduced Host Biomass and IncreasedHaustoria Density. Because Phtheirospermum infection causedArabidopsis to retard growth and reduce biomass (Fig. 1A andMovie S1), we tested whether hypertrophy contributed to thisphenomenon by infecting Col-0 and ipt1,3,5,7 that showed hy-pertrophy along with ahk3,4 that is hypertrophy resistant (Fig. 3).At 30 dpi, infection reduced Col-0 shoot weight by 66% com-pared with uninfected plants, whereas infected plants reducedahk3,4 shoot weights by only 48% compared with uninfectedplants, resulting in significantly heavier plants (Fig. 6A). Shootweights of infected ipt1,3,5,7 showed an even lower reduction of28% compared with uninfected controls (Fig. 6A), likely due tothe smaller shoot size of uninfected ipt1,3,5,7 or possibly becauseparasite-derived cytokinins partially rescued the ipt1,3,5,7 phenotype.Conversely, Phtheirospermum shoot weights increased ∼2.8 fold at30 dpi when infecting any of the three tested genotypes. SignificantPhtheirospermum weight differences were only observed when it in-fected ipt1,3,5,7 compared with slightly lower weights when it in-fected ahk3,4 (Fig. 6B). However, Phtheirospermum produced morehaustoria on ahk3,4 than on either Col-0 or ipt1,3,5,7 (Fig. 6C).

DiscussionCombining the two model species Arabidopsis and Phtheir-ospermum created an experimental framework to study parasiticplants. Infection of Arabidopsis by Phtheirospermum was therebytruly parasitic because it caused growth benefits for the parasiteand growth penalties for the host (Figs. 1 and 6). Consistent witha redistribution of nutrients, the infection modified the source-sink movement of molecules including CFDA dye that was ef-ficiently transported from the Arabidopsis shoot to Phtheir-ospermum (Fig. 1D), but inefficiently transported to theArabidopsis root below the haustorium attachment site (Fig. 1F).Transport of CFDA occurred after differentiation of the xylembridge, suggesting CFDA may be transported via the xylem.

Fig. 3. Phtheirospermum-induced hypertrophy depends on host cytokininsignaling. (A) Ruthenium red-stained transverse sections of Arabidopsis (At)roots were used to quantify (B) control (blue) and Phtheirospermum (Pj)infected (orange) vasculature diameters and xylem cell numbers abovehaustorium attachment sites (HA) at 20 dpi. “+” and “–” indicate ipt1,3,5,7sub-populations with (+) and (-) without hypertrophy (n = 6–33, meanvalues ± SE, ANOVA, P < 0.01). (C) Safranin-O staining of infected Arabidopsisroots at 30 dpi (a, above; b, below HA). (Scale bars: A, 25 μm, C, 200 μm.)

Fig. 4. Cytokinin accumulation in the host is independent of host cytokininbiosynthesis. (A) Different species of cytokinin were quantified by UPLC-tandem mass spectrometry and categorized to different types (t.) from tis-sue of uninfected (open) and infected (hatched) Col-0 (gray) or ipt1,3,5,7(white) above haustoria at 11 dpi. (B) Cytokinin quantifications in solitarygrown Phtheirospermum (Pj) (black) or Pj infecting Col-0 (gray) or ipt1,3,5,7(hatched). Bars show mean values ± SE (n = 4, ANOVA, P < 0.05).

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Similar transport routes between parasitic plants and host plantswere proposed for xylem-feeding parasites such as Striga (29).Consistent with Phtheirospermum acting as a xylem-feeding para-site, we did not observe uptake of phloem-mobile GFP from hostto parasite. This absence of uptake contrasts with haustoria ofthe phloem-feeding parasite Orobanche aegyptiaca, which formsphloem-to-phloem connections and uptakes GFP during parasit-ism of tomato plants expressing pSUC2::GFP (3). However, wecannot completely exclude the possibility of phloem or symplasticconnections because CFDA moves in both the xylem and phloem(20), and its small size could allow it to move more easily throughplasmodesmata than the larger GFP protein.

Successful parasites uptake water and nutrients from their hosts,but less is known about the role of substance transport from par-asite to host. Here, we demonstrate that upon infection, Phtheir-ospermum increases cytokinin levels and transports these across thehaustorium to the Arabidopsis host. These results are analogous tothe phloem-mediated, bidirectional exchange of proteins, RNAs,and viruses across haustoria (1, 5). However, the biological rele-vance of RNA and protein movement is unclear and might resultfrom bulk flow transport of RNAs and proteins normally found inthe parasite phloem or host phloem (5). Conversely, the increase ofcytokinin levels in the parasite upon infection suggests an activeprocess related to parasitism, and indeed, we observed morpho-logical changes including hypertrophic root growth in the host thatdepended on the host cytokinin-signaling pathway. Thus, thesedata describe molecular movement from parasitic plant to host thathas a clear physiological and developmental effect.Cytokinin responses in both parasite and host were detected

several hours after successful penetration of host tissue, but alsoseveral hours before xylem bridges were fully formed in haustoria(Fig. 2D). This early detection of host cytokinin responses triggeredby proto-haustoria rather than mature haustoria suggests a trans-port mechanism that is initially independent of xylem bridges and,thus, uncoupled from bulk nutrient influxes from host to parasite.Different cytokinin species move in the phloem compared with thexylem, in particular, tZ-type species are typically found in the xylem(12). We detected mainly transport of tZ-type species, consistentwith the idea that Phtheirospermum is a xylem feeder, and at leastsome of the tZ-type species detected could be moving throughthe xylem bridge after haustoria maturation. Notably, Arabi-dopsis cytokinin receptor mutants formed normal haustoria (Fig.3), whereas Phtheirospermum hairy roots overexpressing AtCKX3could also form haustoria (Fig. 5), suggesting that cytokinins playa role in plant parasitism after haustorium formation.Parasitic plants actively manipulate host physiology (1), and we

propose that one mechanism for this manipulation is by activelytransporting cytokinins to the host. One visible physiological changethat occurs during parasitism is hypertrophic root or stem growththat occurs in a variety of hosts infected by Alectra vogelii, mistle-toes, or Cuscuta japonica (Fig. S2 C and D) (1, 16). Phtheir-ospermum induced such symptoms on Arabidopsis and tomatosuggesting that cytokinin transport could be a widely conservedmechanism used by parasitic plants. It is likely that other parasiticplants in addition to Phtheirospermum produce cytokinins at thehaustorial infection site. A gene homologous to IPT1 was tran-scriptionally induced in haustoria of sandalwood (Santalum album)(30), and cytokinin responsive genes showed elevated levels in riceinfected with Striga hermonthica (31). We propose that hypertrophymakes the parasite a more efficient sink to uptake nutrients fromthe host because host biomass partially depended on hypertrophy

Fig. 6. Hypertrophy is linked to host biomass reduction in host plants. (A and B) Shoot fresh weights (FW) of different Arabidopsis genotypes andPhtheirospermum were assessed from three independent experiments of plants growing next to Phtheirospermum (-) and of plants infected with Phtheir-ospermum (+) at 30 dpi (n = 35–47). (C) Average number of haustoria per Phtheirospermum (Pj) on corresponding genotypes at 30 dpi. Bars represent meanvalues ± SE (ANOVA P < 0.05).

Fig. 5. Host hypertrophy depends on Phtheirospermum-derived cytoki-nins. (A) Manually reassembled microscopy images show transgenicPhtheirospermum (Pj) roots expressing pMAS::GFP (Control) and high,medium (med.), and low levels of pMAS::AtCKX3. These transformed hairyroots infected Arabidopsis (At) plants and were stained at 28 dpi withSafranin-O. (B) The amount of hypertrophy in hairy roots was quantifiedby measuring root diameters above haustoria relative to the root diameterbelow haustoria (n = 2–3). (C ) Relative AtCKX3 transgene expression inPhtheirospermum hairy roots was determined by real-time quantitativePCR (RT-qPCR) using the expression of PjUBC2 as reference. (B and C) Barsrepresent mean ± SE (*P < 0.05, **P < 0.01 Student’s t test). (Scale bars:100 μm.)

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(Fig. 6A). This increase in host vascular size could lead to increasedsink strength at haustorium attachment sites, which could improvenutrient withdrawal by parasitic plants. Indeed, Phtheirospermumproduced additional haustoria on hypertrophy-deficient ahk3,4, andthis increase might have improved sink strength by partially com-pensating for the absence of hypertrophy (Fig. 6C). However, wedid not observe substantial differences in Phtheirospermum weightsupon infecting hypertrophic or nonhypertrophic Arabidopsis geno-types (Fig. 6B). It remains to be shown whether this effect onbiomass is conserved or stronger in different parasitic plant–hostsystems and whether such sink-source relationships become moreimportant in different environmental conditions.Cytokinin production appears to be a widespread strategy used

by a variety of different plant pathogens. Well-known plantpathogens that induce cytokinin production include the crowngall-inducing bacterium Agrobacterium tumefaciens (6), the fun-gal rice blast pathogen Magnaporthe oryzae (7), and phytopath-ogenic nematodes such as Heterodera schachtii (8). Interspeciescytokinin transport thus may be a widely used mechanism forinfectivity, and in parasitic plants, we suggest a scenario wherebymultiple transport routes at the haustorium contribute to thebidirectional transfer of molecules that affects both host andparasite physiology.

Materials and MethodsPlant Material. Phtheirospermum and its transformation was described in ref.19. Arabidopsis ahk2-2,3–3; ahk3-3,4 (cre1-12) (32); ipt3,5,7 and ipt1,3,5,7(10), Col-0 pSUC2::GFP (23), and Ws pARR5::GFP (24) marker lines weredescribed previously. Detailed protocol descriptions are available as SIMaterials and Methods and Dataset S2.

Cytokinin Quantification. Extraction and quantification of cytokinins from 11 dpihypocotyl and root segments above the first haustorium of Phtheirospermumand Arabidopsis and corresponding tissues of uninfected plants were performedwith ultra-performance liquid chromatography (UPLC)-tandem mass spectrom-etry (AQUITY UPLC System/XEVO-TQS; Waters) with an ODS column (AQUITYUPLC BEH C18, 1.7 μm, 2.1 × 100 mm, Waters) as described (33).

ACKNOWLEDGMENTS. We thank Ruth Stadler for providing pSUC2::GFPseeds, Mikiko Kojima and Yumiko Takebayshi for cytokinin quantification,Simon Saucet for technical help, and Nicola Patron for sharing the ModularCloning Toolkit. This work is partially supported by Ministry of Education,Culture, Sports, Science and Technology KAKENHI Grants 24228008 and15H05959 (to K.S.), 25114521, 25711019, and 25128716 (to S.Y.), and15H05962 (to S.M.); Japan Society for the Promotion of Science (JSPS) Post-doctoral Fellowship (to T.S.); JSPS Research Fellowship for Young Scientist (toT.W.); the RIKEN Special Postdoctoral Researchers Program (to T.S.); andGatsby Charitable Trust Grants GAT3272/C and GAT3273-PR1 (to C.W.M).

1. Heide-Jørgensen HS, Heide-Jorgensen H (2008) Parasitic Flowering Plants (Brill Aca-demic Publishers, Leiden, The Netherlands).

2. Yoshida S, Cui S, Ichihashi Y, Shirasu K (2016) The haustorium, a specialized invasiveorgan in parasitic plants. Annu Rev Plant Biol 67:643–667.

3. Aly R, et al. (2011) Movement of protein and macromolecules between host plantsand the parasitic weed Phelipanche aegyptiaca Pers. Plant Cell Rep 30:2233–2241.

4. Birschwilks M, Sauer N, Scheel D, Neumann S (2007) Arabidopsis thaliana is a sus-ceptible host plant for the holoparasite Cuscuta spec. Planta 226:1231–1241.

5. Kim G, LeBlanc ML, Wafula EK, dePamphilis CW, Westwood JH (2014) Plant science.Genomic-scale exchange of mRNA between a parasitic plant and its hosts. Science345:808–811.

6. Akiyoshi DE, Klee H, Amasino RM, Nester EW, Gordon MP (1984) T-DNA of Agro-bacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl AcadSci USA 81:5994–5998.

7. Chanclud E, et al. (2016) Cytokinin production by the rice blast fungus is a pivotalrequirement for full virulence Emilie. PLoS Pathog 12:e1005457.

8. Siddique S, et al. (2015) A parasitic nematode releases cytokinin that controls celldivision and orchestrates feeding site formation in host plants. Proc Natl Acad Sci USA112:12669–12674.

9. Kieber JJ, Schaller GE (2014) Cytokinins. Arabidopsis Book 12:e0168.10. Miyawaki K, et al. (2006) Roles of Arabidopsis ATP/ADP isopentenyltransferases and

tRNA isopentenyltransferases in cytokinin biosynthesis. Proc Natl Acad Sci USA 103:16598–16603.

11. Werner T, et al. (2003) Cytokinin-deficient transgenic Arabidopsis plants show mul-tiple developmental alterations indicating opposite functions of cytokinins in theregulation of shoot and root meristem activity. Plant Cell 15:2532–2550.

12. Hirose N, et al. (2008) Regulation of cytokinin biosynthesis, compartmentalizationand translocation. J Exp Bot 59:75–83.

13. Matsumoto-Kitano M, et al. (2008) Cytokinins are central regulators of cambial ac-tivity. Proc Natl Acad Sci USA 105:20027–20031.

14. Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokinin receptormutants reveal functions in shoot growth, leaf senescence, seed size, germination,root development, and cytokinin metabolism. Plant Cell 18:40–54.

15. Stolz A, et al. (2011) The specificity of cytokinin signalling in Arabidopsis thaliana ismediated by differing ligand affinities and expression profiles of the receptors. PlantJ 67:157–168.

16. Furuhashi T, et al. (2013) Morphological and plant hormonal changes during para-sitization by Cuscuta japonica on Momordica charantia. J Plant Interact 9:220–232.

17. Zhang X, et al. (2012) Endogenous hormone levels and anatomical characters ofhaustoria in Santalum album L. seedlings before and after attachment to the host.J Plant Physiol 169:859–866.

18. Cui S, et al. (2016) Haustorial hairs are specialized root hairs that support parasitism inthe facultative parasitic plant Phtheirospermum japonicum. Plant Physiol 170:1492–1503.

19. Ishida JK, Yoshida S, Ito M, Namba S, Shirasu K (2011) Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum. PLoSOne 6:e25802.

20. Melnyk CW, Schuster C, Leyser O, Meyerowitz EM (2015) A developmental frameworkfor graft formation and vascular reconnection in Arabidopsis thaliana. Curr Biol 25:1306–1318.

21. Cechin I, Press MC (1993) Nitrogen relations of the sorghum-Striga hermonthica host-parasite association: Germination, attachment and early growth. New Phytol 124:681–687.

22. Oparka KJ, Duckett CM, Prior OAM, Fisher DB (1994) Real-time imaging of phloemunloading in the root tip of Arabidopsis. Plant J 6:759–766.

23. Imlau A, Truernit E, Sauer N (1999) Cell-to-cell and long-distance trafficking of thegreen fluorescent protein in the phloem and symplastic unloading of the protein intosink tissues. Plant Cell 11:309–322.

24. Yanai O, et al. (2005) Arabidopsis KNOXI proteins activate cytokinin biosynthesis. CurrBiol 15:1566–1571.

25. Zürcher E, et al. (2013) A robust and sensitive synthetic sensor to monitor the tran-scriptional output of the cytokinin signaling network in planta. Plant Physiol 161:1066–1075.

26. Kiba T, Kudo T, Kojima M, Sakakibara H (2011) Hormonal control of nitrogen ac-quisition: Roles of auxin, abscisic acid, and cytokinin. J Exp Bot 62:1399–1409.

27. Polanská L, et al. (2007) Altered cytokinin metabolism affects cytokinin, auxin, andabscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure intransgenic tobacco. J Exp Bot 58:637–649.

28. Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth bycytokinin. Proc Natl Acad Sci USA 98:10487–10492.

29. Pageau K, Simier P, Le Bizec B, Robins RJ, Fer A (2003) Characterization of nitrogenrelationships between Sorghum bicolor and the root-hemiparasitic angiosperm Strigahermonthica (Del.) Benth. using K15 NO3 as isotopic tracer. J Exp Bot 54:789–799.

30. Zhang X, et al. (2015) RNA-Seq analysis identifies key genes associated with haustorialdevelopment in the root hemiparasite Santalum album. Front Plant Sci 6:661.

31. Swarbrick PJ, et al. (2008) Global patterns of gene expression in rice cultivars un-dergoing a susceptible or resistant interaction with the parasitic plant Striga her-monthica. New Phytol 179:515–529.

32. Higuchi M, et al. (2004) In planta functions of the Arabidopsis cytokinin receptorfamily. Proc Natl Acad Sci USA 101:8821–8826.

33. Kojima M, et al. (2009) Highly sensitive and high-throughput analysis of plant hor-mones using MS-probe modification and liquid chromatography-tandem mass spec-trometry: An application for hormone profiling in Oryza sativa. Plant Cell Physiol 50:1201–1214.

34. Engler C, et al. (2014) A golden gate modular cloning toolbox for plants. ACS SynthBiol 3:839–843.

35. Ishida JK, et al. (2016) Local auxin biosynthesis mediated by a YUCCA flavin mono-oxygenase regulates haustorium development in the parasitic plant Phtheir-ospermum japonicum. Plant Cell 28:1795–1814.

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