Interspecies Hormonal Control of Host Root Morphology by Parasitic Plants

Interspecies hormonal control of host root morphology by parasitic plants

Thomas 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 for Research Advancement, Tokyo University of Science, Noda, Chiba 278-8510, Japan; eInstitute for Research Initiatives, Division for Research Strategy, Nara Institute 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 for review November 17, 2016) Parasitic plants share a common anatomical feature, the hausto- (3–5), but the biological relevance for this movement is not clear. rium. Haustoria enable both infection and nutrient transfer, which Beyond parasitic plants, various plant-pathogenic microbes, in- often leads to growth penalties for host plants and yield reduction sects, and nematodes produce compounds that move into the in crop species. Haustoria also reciprocally transfer substances, host and contribute to their virulence, including the plant hor- such as RNA and proteins, from parasite to host, but the biological mone cytokinin (6–8). relevance for such movement remains unknown. Here, we studied Cytokinins participate in many physiological and developmental such interspecies transport by using the hemiparasitic plant plant processes such as cell division, growth, vascular develop- Phtheirospermum japonicum Arabidopsis thali- during infection of ment, senescence, photosynthesis, and nutrient allocation (9). ana . Tracer experiments revealed a rapid and efficient transfer of Cytokinins are isoprenoid substituted adenines and in plants, carboxyfluorescein diacetate (CFDA) from host to parasite upon isoprenoid transfer by isopentenyltransferases (IPTs) is the rate- formation of vascular connections. In addition, Phtheirospermum limiting and crucial step for producing various types of cytoki- induced hypertrophy in host roots at the site of infection, a form nins including cis-zeatin (cZ), N6-(Δ2-isopentenyl)-adenine (iP), of enhanced secondary growth that is commonly observed during various parasitic plant–host interactions. The plant hormone cyto- trans-zeatin (tZ), and dihydrozeatin (DZ) (10). tZ is the most kinin is important for secondary growth, and we observed in- abundant and the most potent cytokinin in Arabidopsis (9). Cy- creases in cytokinin and its response during infection in both tokinins are further metabolized and inactivated through con- host and parasite. Phtheirospermum-induced host hypertrophy re- jugation to sugars or through cleavage by cytokinin oxidases quired cytokinin signaling genes (AHK3,4) but not cytokinin bio- (CKXs) (11). Cytokinins act at the site of biosynthesis and are synthesis genes (IPT1,3,5,7) in the host. Furthermore, expression of also mobile within the plant vascular system (12, 13). Grafting a cytokinin-degrading enzyme in Phtheirospermum prevented experiments with Arabidopsis ipt1,3,5,7 mutants demonstrated host hypertrophy. Wild-type hosts with hypertrophy were smaller root-to-shoot movement of tZ-type cytokinins and an opposing than ahk3,4 mutant hosts resistant to hypertrophy, suggesting hypertrophy improves the efficiency of parasitism. Taken to- Significance gether, these results demonstrate that the interspecies movement of a parasite-derived hormone modified both host root morphol- Parasitic plants are pests of many plants, including major crop ogy and fitness. Several microbial and animal plant pathogens use species. An important step toward creating resistance to par- cytokinins during infections, highlighting the central role of this asitic plants is gaining a better understanding of how these growth hormone during the establishment of plant diseases and pathogens control the physiology and development of their revealing a common strategy for parasite infections of plants. hosts. We combined genetic, cell-biological, and biochemical methods to identify the plant hormone cytokinin as a mobile cytokinin | transport | hypertrophy | parasitism | Arabidopsis signal between the hemiparasitic plant Phtheirospermum japonicum and the host Arabidopsis thaliana. Transport of arasitic plants are widespread agricultural pests and account parasite-derived cytokinins induced morphological changes in Pfor ∼1% of known flowering plants species (1). Parasitism host roots, revealing insights into how parasitic plants ma- ranges from holoparasites, which depend entirely on nutrient nipulate host development and laying the foundation for fu- supply from host plants, to hemiparasites, which obtain nutrients ture explorations for bioactive molecule transfer from parasitic PLANT BIOLOGY via their own photosynthesis and from their hosts (1). Many plants to hosts. hemiparasites do not depend on parasitism but often parasitize when conditions are suitable. These hemiparasitic plants include Author contributions: T.S., C.W.M., and K.S. designed research; T.S., C.W.M., T.W., J.Z., parasitic plants such as the commonly studied Orobanchaceae 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. species Rhinanthus minor, Triphysaria versicolor, and Phtheir- ospermum japonicum. Both hemiparasites and holoparasites The authors declare no conflict of interest. form specialized organs called haustoria that undergo a de- This article is a PNAS Direct Submission. J.J.K. is a guest editor invited by the Editorial Board. velopmental transition from proto-haustoria to mature haustoria 1T.S. and C.W.M. contributed equally to this work. during the penetration and infection of host tissues to acquire 2 Striga To whom correspondence may be addressed. Email: [email protected] or thomas. nutrients and water (2). Some parasitic plants such as or [email protected]. Rhinanthus form vascular connections exclusively to host xylem 3Present address: Department of Plant Biology, Swedish University of Agricultural Sci- via xylem bridges (xylem-feeding), whereas haustoria of other ences, Almas allé 5, 756 51, Uppsala, Sweden. plants such as Cuscuta or Orobanche also form symplastic 4Present address: Institute of Biotechnology, University of Helsinki, 00014, Helsinki, phloem-to-phloem connections to host plants (phloem-feeding) Finland. (1). In addition to water and nutrients, other small substances This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are transferred across haustoria, including RNAs and proteins 1073/pnas.1619078114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1619078114 PNAS | May 16, 2017 | vol. 114 | no. 20 | 5283–5288 Downloaded by guest on September 27, 2021 shoot-to-root movement of iP-type cytokinins (13), consistent with 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 downstream responses (14), including the transcriptional induction of A-type ARABIDOPSIS RESPONSE REGULATOR (ARR) genes such as ARR5 (15). Although Arabidopsis cytokinin receptors act largely redundantly, combinations of ahk double and triple mutants 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 the source of the cytokinins and the biological relevance were unknown (16, 17). Here, we demonstrate the parasitic plant Phtheirospermum increases its cytokinin levels upon infection, and these cytokinin species move into the host Arabidopsis.We further demonstrate these cytokinin species are bioactive in Arabidopsis roots and induce changes in gene response, cell division, and cell differentiation that leads to modifications in host root morphology and impacts host fitness. Results Phtheirospermum Parasitizes Arabidopsis. Phtheirospermum infects a 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 not absolutely depend on parasitism. As previously described, haustorium development occurs when Phtheirospermum comes in contact with its host on water-agar with no additional nutrients (18, 19). We used a similar water-only setup, but substituted Whatman filter paper and nylon membrane for agar to anchor the plants, similar to an experimental setup used for Arabidopsis Fig. 1. Phtheirospermum parasitizes Arabidopsis.(A) Phtheirospermum grafting (20). This low nutrient environment allowed efficient in- growing alone (Pj-) or infecting (Pj+) Arabidopsis (At+) show increased fection, consistent with observations that low levels of nitrogen are Phtheirospermum size and decreased Arabidopsis size compared with un- beneficial for infection by other parasitic plants such as Striga infected controls (At-) at 30 dpi. (B) Detail image of Phtheirospermum-infecting hermonthica (21). Under these conditions, Phtheirospermum for- Arabidopsis with Phtheirospermum haustorium (haust.) attachment site (HA). med haustoria on Arabidopsis roots within 3 d (Fig. 1 A and B). At (C) Images of Safranin-O stained proto- and mature haustoria show differences in xylem bridge (XB) formation. (D) CFDA transport ability of 11 dpi haustoria 7 d postinfection (dpi), infected and noninfected controls were were assayed 90 min after application of CFDA onto host leaves (asterisk). moved to 1/2 strength Murashige and Skoog (MS) growth medium (E) Ratios of mature haustoria (brown bar) and haustoria with CFDA transport to assess the long-term effects of Phtheirospermum parasitism on ability (green bar) (n = 35–86) were quantified for the indicated time points Arabidopsis in the absence of low nutrient stress. Infected and (Fisher Exact Test, P < 0.001). (F) A fluorescent image of a single optical plane of noninfected plants were of similar size at 7 dpi, but infected the haustorium 90 min after CFDA application onto a host leaf. CFDA fluores- Arabidopsis developed poorly in contrast to uninfected controls cence is green, and cell walls were stained with propidium iodide in magenta. and showed a clear reduction in size at 30 dpi (Fig. 1A and Movie (G) Schematic representation of F with indicated optical section of the haus- S1). Meanwhile, Phtheirospermum-infecting Arabidopsis grew larger torium (H), the host root above (I)orbelow(J) the HA. (Scale bars: A, B,andD, μ μ than Phtheirospermum growing solitarily (Fig. 1A and Movie S1). 1mm;C, F,andG,50 m; H and I,25 m.) Safranin-O staining of haustoria confirmed the formation of xylem bridges from Phtheirospermum to Arabidopsis during transition – surrounding the xylem bridges within the Phtheirospermum haus- from protohaustoria to mature haustoria, typically 3 4dpi,con- torium (Fig. 1 F and H). The fluorescence signal decreased sistent with normal haustorial development (Fig. 1 C and E) (18). gradually toward the proximal haustorial tissue (Fig. 1 F–H). At To characterize the functionality of nascent haustoria, we the same time, fluorescence in host roots above the haustorium monitored haustorial transport activity by using the vascular transport dye carboxyfluorescein diacetate (CFDA) (22). After attachment site was stronger than below, indicating preferential CFDA application to host roots or shoots, fluorescent signals CFDA uptake from the host vasculature via the haustorium (Fig. 1 I rapidly propagated from host vasculature into Phtheirospermum and J). To investigate the possibility of transport via phloem con- haustoria and shoots, indicating efficient transport from host to nections, we infected Phtheirospermum to Arabidopsis pSUC2::GFP parasite as early as 15 min after CFDA application (Fig. 1D, Fig. plants that express mobile GFP in the phloem (23). GFP was not S1 A and B,andMovie S2). To investigate the role of xylem detected in Phtheirospermum haustoria or root tips, whereas GFP bridges for CFDA transport, we compared the ratio of CFDA was efficiently unloaded in root tips of infected Arabidopsis uptake to the ratio of mature haustoria with xylem bridges at pSUC2::GFP plants (Fig. S1 C–E). Furthermore, no gradient in different time points (Fig. 1E). More than 50% of Phtheir- fluorescence intensities along haustoria was detected for phloem- ospermum developed haustoria with at least one xylem bridge at mobile GFP in infected Arabidopsis pSUC2::GFP roots (Fig. S1C). 4 dpi, whereas less than 10% of Phtheirospermum transported CFDA at this time point (Fig. 1E). The percentage of Phtheir- Phtheirospermum Induces Hypertrophy and Cytokinin Responses in ospermum transporting CFDA gradually increased at 6 and 11 dpi Host Plants. After Phtheirospermum established xylem connections when the majority of haustoria reached maturity (Fig. 1E). A to Arabidopsis, we observed a swelling of Arabidopsis tissue above strong fluorescent signal was detected in mature haustorial tissues haustorium attachment sites (Figs. 1B and 2A), a phenomenon

5284 | www.pnas.org/cgi/doi/10.1073/pnas.1619078114 Spallek et al. Downloaded by guest on September 27, 2021 referred to as hypertrophy (1). Hypertrophy resulted from an in- xylem bridge (84 hpi) (Fig. 2D and Fig. S4H). Hairy roots of crease in vascular cell size (hypertrophy) and cell number (hyper- Phtheirospermum resembled haustoria of nontransgenic Phtheir- plasia) that enlarged the vascular and xylem area in host tissue ospermum roots in development and transcriptional induction of above haustoria attachment sites compared with host tissue below pARR5::GFP in host plants (Fig. 1E and Fig. S4). the haustoria or uninfected controls (Fig. 2 B and C). Hypertrophy was not specific to Arabidopsis because a similar effect also occurred Phtheirospermum-Induced Host Hypertrophy Depends on Host in tomato (Solanum lycopersicum)infectedbyPhtheirospermum Cytokinin Signaling but Not on Host Cytokinin Biosynthesis Genes. (Fig. S2 A and B). Increases in vascular size were already detected The spatial and temporal overlap between host cytokinin re- above the haustoria at 11 dpi (Fig. S3 A and B), and by 20 dpi, sponses and hypertrophy suggested that they might be directly quantifications of root sections prepared above and below the linked. To test this possibility, we infected various Arabidopsis haustorium confirmed a further increase in vascular and xylem di- cytokinin signaling and biosynthesis mutants. Infected Col-0 wild ameter and cell number above haustorium attachment sites, whereas type showed more secondary growth including increased vascular no significant differences in uninfected plants were detected (Fig. diameter and xylem number at 20 dpi (Fig. 3 A and B and Fig. 2C). The vascular and xylem areas above haustorium attachment S5A) and developed roots with twofold larger diameters above sites were ∼4 times greater than in uninfected plants (Fig. S3C), haustoria compared with diameters below haustoria at 30 dpi (Fig. which was substantial considering that infected Arabidopsis showed 3C and Fig. S5B). Roots of infected Col-0 were even larger than an overall growth reduction compared with uninfected plants of the uninfected roots of the same age, despite their reduced shoot same age (Movie S1). The alteration in morphology suggested that growth (Fig. 1A and Fig. S5B). Hypertrophy was blocked in the Phtheirospermum locally induced secondary growth in the host root, cytokinin signaling mutants ahk2,3 and ahk3,4, but cytokinin bio- a phenomenon that resembles other cases of host hypertrophy synthesis mutants ipt3,5,7 and ipt1,3,5,7 had hypertrophy levels caused by parasitic plants such as mistletoes (Viscum album) similar to Col-0, thus partially rescuing the mutant root phenotype infecting crabapple trees (Malus toringoides)(Fig. S2 C and D). (Fig. 3 and Fig. S5). Consistent with these observations, transverse Root secondary growth depends on and is promoted by the plant sections above the haustoria at 20 dpi showed an increase in hormone cytokinin (13). To test whether infection altered cytokinin vascular diameter, vascular area, xylem cell number, and xylem signaling, we monitored cytokinin response in Arabidopsis by using area for Col-0 and ipt1,3,5,7, but not for ahk3,4 and only mar- the transcriptional reporter line pARR5::GFP (24). Infected plants ginally for ahk2,3 (Fig. 3 A and B and Fig. S5A). Hypertrophy in showed a substantial increase in GFP fluorescence at 3–4 dpi and, ipt1,3,5,7 was more variable at 20 dpi with some plants showing by 6 dpi, most host plants showed high GFP fluorescence (Fig. no macroscopic signs of hypertrophy. Increased cytokinin re- S4A).TheincreaseinGFP expression was restricted to tissue sponse occurred throughout the plant because endogenous ARR5 above haustoria attachment sites (Fig. S4B). Spatial localization of transcripts levels were also up-regulated at 11 dpi in shoots of fluorescence at 11 dpi revealed pARR5::GFP expression over- infected Col-0, ipt3,5,7,andipt1,3,5,7, but were not up- lapped with host tissues undergoing hypertrophic growth (Fig. S4 regulated in infected ahk2,3 and ahk3,4 (Fig. S6A). Likewise, B–E). Temporally, increased cytokinin response occurred during genes downstream of cytokinin signaling including NRT1.7 and hypertrophic growth because pARR5::GFP expression remained NRT1.5 showed substantial changes upon infection in Col- high even after 20 dpi (Fig. S4 F and G).Thesedatawerecon- 0 that were not observed in ahk3,4 at 11 dpi (Fig. S6B)(26). sistent with an increase in cytokinin response occurring in tissues undergoing increased cell division and xylem differentiation. Phtheirospermum Elevates Host Cytokinin Levels Independently of To address the source of this response, we generated transgenic Host Cytokinin Biosynthesis Genes. Our data demonstrated that Phtheirospermum hairy roots containing the cytokinin responsive an induction of host cytokinin responses was independent of promotor sequence of the Two Component signaling Sensor new several host IPT cytokinin biosynthesis genes, but it remained (pTCSn) fused to a triple mCherry-NLS (pTCSn::3xmCherry-NLS) unknown whether increased cytokinin response resulted from an (25). We then followed cytokinin responses simultaneously in increase of cytokinin levels in host plants during infection. To Phtheirospermum expressing pTCSn::3xmCherry-NLS infecting address this question, we quantified cytokinin species in host Arabidopsis expressing pARR5::GFP. No cytokinin responses in roots (including hypocotyls) and parasite roots above haustoria host and parasite were detected during penetration of host tissue at 11 dpi in Col-0 and ipt1,3,5,7 and compared the resulting cy- (10–60 h postinfection, hpi), but during the transition from proto- tokinin profiles to those of uninfected controls. Uninfected to mature haustoria (72–84 hpi) a parallel increase of cytokinin ipt1,3,5,7 were greatly depleted in tZ-type cytokinins compared response in parasite and host preceded the formation of the first with uninfected Col-0, consistent with previous findings (13) PLANT BIOLOGY

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

Spallek et al. PNAS | May 16, 2017 | vol. 114 | no. 20 | 5285 Downloaded by guest on September 27, 2021 medium, or low levels of pMAS::AtCKX3 and of control hairy roots expressing pMAS::GFP (Fig. 3 and Fig. S8 A and B). Whereas Phtheirospermum roots expressing pMAS::GFP induced similar levels of host hypertrophy compared with nontransgenic roots at 28 dpi (Figs. 3C and 5 A and B and Fig. S5B), Arabidopsis hypertrophy decreasedwithincreasingAtCKX3 expression in Phtheirospermum (Fig. 5). No hypertrophy was observed for Phtheirospermum hairy roots with the highest AtCKX3 expression. Hairy roots expressing pMAS::AtCKX3 had similar morphology and ability to develop haustoria compared with hairy roots transformed with pMAS::GFP; however, hairy roots expressing high levels of pMAS::AtCKX3 induced lower levels of pARR5::GFP expression in the host compared with pMAS::GFP controls (Fig. 5A and Fig. S8C).

Hypertrophy Correlates with Reduced Host Biomass and Increased Haustoria Density. Because Phtheirospermum infection caused Arabidopsis to retard growth and reduce biomass (Fig. 1A and Movie S1), we tested whether hypertrophy contributed to this phenomenon by infecting Col-0 and ipt1,3,5,7 that showed hypertrophy along with ahk3,4 that is hypertrophy resistant (Fig. 3). At 30 dpi, infection reduced Col-0 shoot weight by 66% compared with uninfected plants, whereas infected plants reduced ahk3,4 shoot weights by only 48% compared with uninfected plants, resulting in significantly heavier plants (Fig. 6A). Shoot weights of infected ipt1,3,5,7 showed an even lower reduction of 28% compared with uninfected controls (Fig. 6A), likely due to the smaller shoot size of uninfected ipt1,3,5,7 or possibly because parasite-derived cytokinins partially rescued the ipt1,3,5,7 phenotype. Conversely, Phtheirospermum shoot weights increased ∼2.8 fold at 30 dpi when infecting any of the three tested genotypes. Significant Phtheirospermum weight differences were only observed when it infected ipt1,3,5,7 compared with slightly lower weights when it in- Fig. 3. Phtheirospermum-induced hypertrophy depends on host cytokinin fected ahk3,4 (Fig. 6B). However, Phtheirospermum produced more signaling. (A) Ruthenium red-stained transverse sections of Arabidopsis (At) haustoria on ahk3,4 than on either Col-0 or ipt1,3,5,7 (Fig. 6C). roots were used to quantify (B) control (blue) and Phtheirospermum (Pj) infected (orange) vasculature diameters and xylem cell numbers above Discussion haustorium attachment sites (HA) at 20 dpi. “+” and “–” indicate ipt1,3,5,7 sub-populations with (+) and (-) without hypertrophy (n = 6–33, mean Combining the two model species Arabidopsis and Phtheir- values ± SE, ANOVA, P < 0.01). (C) Safranin-O staining of infected Arabidopsis ospermum created an experimental framework to study parasitic roots at 30 dpi (a, above; b, below HA). (Scale bars: A,25μm, C,200μm.) plants. Infection of Arabidopsis by Phtheirospermum was thereby truly parasitic because it caused growth benefits for the parasite and growth penalties for the host (Figs. 1 and 6). Consistent with (Fig. 4A, Fig. S7, and Dataset S1). Upon infection, both infected a redistribution of nutrients, the infection modified the source- Col-0 and infected ipt1,3,5,7 showed significant increases of sink movement of molecules including CFDA dye that was ef- tZ-type cytokinins. Compared with uninfected plants, tZ-type cy- ficiently transported from the Arabidopsis shoot to Phtheir- tokinins increased 8-fold in Col-0 and 29-fold in ipt1,3,5,7 (Fig. ospermum (Fig. 1D), but inefficiently transported to the 4A). Detailed profiling of cytokinins showed an increase of all Arabidopsis root below the haustorium attachment site (Fig. 1F). detected species of tZ-type cytokinins including bioactive tZ and Transport of CFDA occurred after differentiation of the xylem xylem mobile precursor tZ-riboside (tZR, Fig. S7 and Dataset bridge, suggesting CFDA may be transported via the xylem. S1). tZ-type cytokinins also increased in Phtheirospermum upon infection, however, the fold change was lower than compared with tZ-type cytokinin accumulation in host plants (Fig. 4B). No significant changes during infection were detected for cZ-, iP-, or DZ-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-type cytokinins in infected ipt1,3,5,7 similartolevelsfoundinCol-0(Fig. 4A) suggested that host tZ-type cytokinins were derived from Phtheirospermum. To address this possibility, we expressed an Arabidopsis cytokinin-degrading enzyme, AtCKX3,inhairyrootsof Phtheirospermum (Fig. 5). Overexpression of AtCKX3 reduces cytokinin levels in a distant relative of Phtheirospermum, Nicotiana Fig. 4. Cytokinin accumulation in the host is independent of host cytokinin tabacum (27), and caused various cytokinin-deficient phenotypes biosynthesis. (A) Different species of cytokinin were quantified by UPLC- in Arabidopsis and N. tabacum (11, 28). Thus, we reasoned that tandem mass spectrometry and categorized to different types (t.) from tissue of uninfected (open) and infected (hatched) Col-0 (gray) or ipt1,3,5,7 overexpressing AtCKX3 in Phtheirospermum would also reduce (white) above haustoria at 11 dpi. (B) Cytokinin quantifications in solitary parasite cytokinin levels. We quantified host hypertrophy during grown Phtheirospermum (Pj) (black) or Pj infecting Col-0 (gray) or ipt1,3,5,7 infection of Phtheirospermum hairy roots expressing either high, (hatched). Bars show mean values ± SE (n = 4, ANOVA, P < 0.05).

5286 | www.pnas.org/cgi/doi/10.1073/pnas.1619078114 Spallek et al. Downloaded by guest on September 27, 2021 Successful parasites uptake water and nutrients from their hosts, but less is known about the role of substance transport from parasite to host. Here, we demonstrate that upon infection, Phtheir- ospermum increases cytokinin levels and transports these across the haustorium to the Arabidopsis host. These results are analogous to the phloem-mediated, bidirectional exchange of proteins, RNAs, and viruses across haustoria (1, 5). However, the biological relevance of RNA and protein movement is unclear and might result from bulk flow transport of RNAs and proteins normally found in the parasite phloem or host phloem (5). Conversely, the increase of cytokinin levels in the parasite upon infection suggests an active process related to parasitism, and indeed, we observed morphological changes including hypertrophic root growth in the host that depended on the host cytokinin-signaling pathway. Thus, these data describe molecular movement from parasitic plant to host that has a clear physiological and developmental effect. Cytokinin responses in both parasite and host were detected several hours after successful penetration of host tissue, but also several hours before xylem bridges were fully formed in haustoria (Fig. 2D). This early detection of host cytokinin responses triggered by proto-haustoria rather than mature haustoria suggests a transport 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 the Fig. 5. Host hypertrophy depends on Phtheirospermum-derived cytoki- xylem, in particular, tZ-type species are typically found in the xylem nins. (A) Manually reassembled microscopy images show transgenic (12). We detected mainly transport of tZ-type species, consistent Phtheirospermum (Pj) roots expressing pMAS::GFP (Control) and high, with the idea that Phtheirospermum is a xylem feeder, and at least medium (med.), and low levels of pMAS::AtCKX3. These transformed hairy some of the tZ-type species detected could be moving through roots infected Arabidopsis (At) plants and were stained at 28 dpi with Safranin-O. (B) The amount of hypertrophy in hairy roots was quantified the xylem bridge after haustoria maturation. Notably, Arabi- by measuring root diameters above haustoria relative to the root diameter dopsis cytokinin receptor mutants formed normal haustoria (Fig. below haustoria (n = 2–3). (C) Relative AtCKX3 transgene expression in 3), whereas Phtheirospermum hairy roots overexpressing AtCKX3 Phtheirospermum hairy roots was determined by real-time quantitative could also form haustoria (Fig. 5), suggesting that cytokinins play PCR (RT-qPCR) using the expression of PjUBC2 as reference. (B and C)Bars a role in plant parasitism after haustorium formation. represent mean ± SE (*P < 0.05, **P < 0.01 Student’s t test). (Scale bars: Parasitic plants actively manipulate host physiology (1), and we 100 μm.) propose that one mechanism for this manipulation is by actively transporting cytokinins to the host. One visible physiological change that occurs during parasitism is hypertrophic root or stem growth Similar transport routes between parasitic plants and host plants that occurs in a variety of hosts infected by Alectra vogelii,mistle- Striga were proposed for xylem-feeding parasites such as (29). toes, or Cuscuta japonica (Fig. S2 C and D) (1, 16). Phtheir- Consistent with Phtheirospermum acting as a xylem-feeding para- ospermum induced such symptoms on Arabidopsis and tomato site, we did not observe uptake of phloem-mobile GFP from host suggesting that cytokinin transport could be a widely conserved to parasite. This absence of uptake contrasts with haustoria of mechanism used by parasitic plants. It is likely that other parasitic the phloem-feeding parasite Orobanche aegyptiaca, which forms plants in addition to Phtheirospermum produce cytokinins at the phloem-to-phloem connections and uptakes GFP during parasit- haustorial infection site. A gene homologous to IPT1 was tran- ism of tomato plants expressing pSUC2::GFP (3). However, we scriptionally induced in haustoria of sandalwood (Santalum album) cannot completely exclude the possibility of phloem or symplastic (30), and cytokinin responsive genes showed elevated levels in rice connections because CFDA moves in both the xylem and phloem infected with Striga hermonthica (31). We propose that hypertrophy (20), and its small size could allow it to move more easily through makes the parasite a more efficient sink to uptake nutrients from plasmodesmata than the larger GFP protein. the host because host biomass partially depended on hypertrophy PLANT BIOLOGY

Fig. 6. Hypertrophy is linked to host biomass reduction in host plants. (A and B) Shoot fresh weights (FW) of different Arabidopsis genotypes and Phtheirospermum 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 mean values ± SE (ANOVA P < 0.05).

Spallek et al. PNAS | May 16, 2017 | vol. 114 | no. 20 | 5287 Downloaded by guest on September 27, 2021 (Fig. 6A). This increase in host vascular size could lead to increased Materials and Methods sink strength at haustorium attachment sites, which could improve Plant Material. Phtheirospermum and its transformation was described in ref. nutrient withdrawal by parasitic plants. Indeed, Phtheirospermum 19. Arabidopsis ahk2-2,3–3; ahk3-3,4 (cre1-12) (32); ipt3,5,7 and ipt1,3,5,7 produced additional haustoria on hypertrophy-deficient ahk3,4,and (10), Col-0 pSUC2::GFP (23), and Ws pARR5::GFP (24) marker lines were this increase might have improved sink strength by partially com- described previously. Detailed protocol descriptions are available as SI pensating for the absence of hypertrophy (Fig. 6C). However, we Materials and Methods and Dataset S2. did not observe substantial differences in Phtheirospermum weights upon infecting hypertrophic or nonhypertrophic Arabidopsis geno- Cytokinin Quantification. Extraction and quantification of cytokinins from 11 dpi types (Fig. 6B). It remains to be shown whether this effect on hypocotyl and root segments above the first haustorium of Phtheirospermum biomass is conserved or stronger in different parasitic plant–host and Arabidopsis and corresponding tissues of uninfected plants were performed systems and whether such sink-source relationships become more with ultra-performance liquid chromatography (UPLC)-tandem mass spectrom- important in different environmental conditions. etry (AQUITY UPLC System/XEVO-TQS; Waters) with an ODS column (AQUITY

Cytokinin production appears to be a widespread strategy used UPLC BEH C18,1.7μm, 2.1 × 100 mm, Waters) as described (33). by a variety of different plant pathogens. Well-known plant pathogens that induce cytokinin production include the crown ACKNOWLEDGMENTS. We thank Ruth Stadler for providing pSUC2::GFP gall-inducing bacterium Agrobacterium tumefaciens (6), the fun- seeds, Mikiko Kojima and Yumiko Takebayshi for cytokinin quantification, gal rice blast pathogen Magnaporthe oryzae (7), and phytopath- Simon Saucet for technical help, and Nicola Patron for sharing the Modular ogenic nematodes such as Heterodera schachtii (8). Interspecies Cloning Toolkit. This work is partially supported by Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grants 24228008 and cytokinin transport thus may be a widely used mechanism for 15H05959 (to K.S.), 25114521, 25711019, and 25128716 (to S.Y.), and infectivity, and in parasitic plants, we suggest a scenario whereby 15H05962 (to S.M.); Japan Society for the Promotion of Science (JSPS) Post- multiple transport routes at the haustorium contribute to the doctoral Fellowship (to T.S.); JSPS Research Fellowship for Young Scientist (to bidirectional transfer of molecules that affects both host and T.W.); the RIKEN Special Postdoctoral Researchers Program (to T.S.); and parasite physiology. Gatsby Charitable Trust Grants GAT3272/C and GAT3273-PR1 (to C.W.M).

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