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 down- stream 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 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 the source of the cytokinins and the biological relevance were un- known (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 di- vision, 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, haus- torium 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