Evolutionary Conservation of a Phosphate Transporter in the Arbuscular Mycorrhizal Symbiosis
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Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis Vladimir Karandashov*†,Re´ ka Nagy*, Sarah Wegmu¨ ller*, Nikolaus Amrhein‡, and Marcel Bucher*§ *Federal Institute of Technology (ETH) Zurich, Institute of Plant Sciences, Experimental Station Eschikon 33, 8315 Lindau, Switzerland; †K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia; and ‡Federal Institute of Technology (ETH) Zurich, Institute of Plant Sciences, Universita¨tsstrasse 2, 8092 Zurich, Switzerland Edited by Robert L. Metzenberg, University of California, Los Angeles, CA, and approved February 23, 2004 (received for review September 22, 2003) Arbuscular mycorrhizae are ancient symbioses that are thought to mosseae (BEG76), Glomus spurcum (W4217), and Paraglomus have originated >400 million years ago in the roots of plants, brasilianum (W4121). Pathogenic fungi used were Rhizopus pioneering the colonization of terrestrial habitats. In these asso- stolonifer var. stolonifer (MUCL20416), Nectria ventricosa ciations, a key process is the transfer of phosphorus as inorganic (MUCL33), Rhizoctonia solani (MUCL30282), and the oomy- phosphate to the host plant across the fungus–plant interface. cete Phytophthora infestans (CRA208). The dark septate root Mycorrhiza-specific phosphate transporter genes and their regu- endophyte Phialocephala fortinii, the binucleate Rhizoctonia sp., lation are conserved in phylogenetically distant plant species, and which is a root parasite that does not cause root-disease symp- they are activated selectively by fungal species from the phylum toms, and the plant growth stimulating root endophyte Pirifor- Glomeromycota. The potato phosphate transporter gene StPT3 is mospora indica (DSM11827) (11, 12) were used as well. expressed in a temporally defined manner in root cells harboring G. caledonium (BEG15) was maintained in monoxenic culture various mycorrhizal structures, including thick-coiled hyphae. The on carrot hairy roots (13), and all other AMF isolates were results highlight the role of different symbiotic structures in maintained on P. lanceolata grown in a soil–quartz sand mixture phosphorus transfer, and they indicate that cell–cell contact be- (1:10) fertilized with quarter-strength Hoagland solution (14) tween the symbiotic partners is required to induce phosphate containing 5 MNH4H2PO4 by using drop irrigation. Sterile transport. cultures of fungi other than AMF were maintained on potato dextrose agar (binucleate Rhizoctonia sp., N. ventricosa, P. indica, PLANT BIOLOGY rbuscular mycorrhizal fungi (AMF) are important biotro- P. infestans, and R. solani), malt 2% yeast extract agar (R. Aphic organisms, which live in symbiosis with Ϸ80% of land stolonifer), and Melin–Norkrans nutrient media (P. fortinii). plants, forming a mycorrhiza (i.e., a root colonized by a symbi- otic fungus). AMF affect plant biodiversity, as well as the Plant Growth Conditions. Stock cultures of wild-type and trans- variability and productivity of ecosystems (1, 2). As few as Ϸ150 genic potato plants were maintained in tissue culture by using fungal species are known to form arbuscular mycorrhizae (AM) MS medium (15) supplemented with 2% sucrose. In pot culture, in the roots of a vast number of plant species. No evidence for inoculation of potato plants with all AMF isolates except G. recombination has been found in the fungi, suggesting that they margarita was performed as described (16). For G. margarita, reproduce clonally and have been asexual for the entire period potato plantlets were cocultivated with sunflower, which served of their association with plants (3). Recently, AMF were placed as nurse plants. Each pot, containing the potato–sunflower into a new monophyletic group, the phylum Glomeromycota, combination, was inoculated with 15–20 spores of G. margarita. which probably originated from the same ancestral group as the Composite plants of L. japonicus and M. truncatula (see below) Ascomycota and Basidiomycota (4) Ϸ1,400–1,200 million years were introduced to already-established mycorrhizae of G. mos- ago and is much older than the earliest land plants, which seae on P. lanceolata, growing in a soil–quartz sand mixture. appeared Ϸ800 million years ago and whose primitive root Plants were harvested 2–6 weeks after inoculation. Freshly systems were associated with ancestral AMF. AMF may, thus, collected potato roots were frozen in liquid N2 and stored at Ϫ have played a crucial role in facilitating the colonization of land 80°C for subsequent RNA isolation, or they were immediately  by plants (5–8). It is also assumed that the ancient signaling stained for -glucuronidase (GUS) activity and for visualization pathways evolved in AM symbiosis were recruited subsequently of intraradical fungal mycelium (17). for the establishment of the evolutionary younger legume– Rhizobia nodulation symbiosis (9). Today, despite the large RT-PCR. RT-PCR was performed as described (18) by using number of plant species forming AM associations worldwide, gene-specific primers, as described in Supporting Methods, which only two major morphological types have been defined: the is published as supporting information on the PNAS web site. Arum and Paris types, respectively. In Arum-type mycorrhizae, GenBank accession nos. for StPT3, StPT1, MtPT4, MtPT1, and fungal hyphae spread between cortical cells and form short-lived PHT2;1 are AJ318822, AF156695, AY116210, AF000354, and heavily branched symbiotic structures (arbuscules) within cells. AF533081, respectively. In Paris-type mycorrhizae, cortical cells are colonized by intra- cellularly growing thick coiled hyphae, which occasionally form Plant Hairy-Root Cultures. Agrobacterium rhizogenes were trans- fine arbuscule-like ramifications (10). formed with pBin19 carrying StPT3 promoter-GUS (16), MtPT4 Materials and Methods This paper was submitted directly (Track II) to the PNAS office. Plant and Fungal Material. The plant material used was Daucus Abbreviations: AM, arbuscular mycorrhiza; AMF, arbuscular mycorrhizal fungus͞fungi; carota (carrot), Lotus japonicus cv. Gifu, Medicago truncatula cv. GUS, -glucuronidase. Jemalong, Petunia ϫ hybrida (petunia), Plantago lanceolata Data deposition: The sequences reported in this paper have been deposited in the GenBank (plantain), and Solanum tuberosum cv. De´sire´e (potato). AMF database [accession nos. AJ318822 (StPT3), AF156695 (StPT1), AY116211 and AY116210 isolates used were Acaulospora delicata (JJ1094), Archaeospora (MtPT4), AF000354 (MtPT1), AF533081 (PHT2;1), AY134608 and AY134609 (MtGst1), nicolsonii (W4147), Gigaspora margarita (BEG34), Glomus cale- At5g43340 (ARAth;Pht1;6), and At1g20860 (ARAth;Pht1;8)]. donium (BEG15 and BEG20), Glomus etunicatum (CZ), Glomus §To whom correspondence should be addressed. E-mail: [email protected]. geosporum (BEG11), Glomus intraradices (BEG75), Glomus © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306074101 PNAS ͉ April 20, 2004 ͉ vol. 101 ͉ no. 16 ͉ 6285–6290 Downloaded by guest on October 1, 2021 promoter-GUS, OsPT11 promoter-GUS, and StPT3 promoter- Phylogenetic Footprinting. The phylogenetic relationship between Fluorescent Timer chimeric genes, respectively, by means of the coding sequence of the Pht1 family genes StPT3 (AJ318822), electroporation. To initiate hairy-root formation, plant tissues LePTh (tomato; Lycopersicon esculentum; Avraham A. Levy, were inoculated with overnight cultures of A. rhizogenes grown Weizmann Institute of Sciences, Rehovot, Israel, personal com- on solid YEB medium. Potato and carrot hairy roots were munication), MtPT4 (GenBank accession no. AY116211), generated as described (19) by using tubers and tap roots, OsPT11 (GenBank accession no. AF536971), ARAth;Pht1;6 respectively. Sterile potato plantlets were also used for the (At5g43340), and ARAth;Pht1;8 (GenBank accession no. generation of hairy roots. The apical part of the plantlet was At1g20860) (Arabidopsis thaliana; ref. 23), and the AMF- excised and the sectioned stem surface was inoculated with A. inducible glutathione S-transferase MtGst1 (M. truncatula; Gen- rhizogenes. Inoculation of L. japonicus and M. truncatula sec- Bank accession nos. AY134608 and AY134609; ref. 24) was tioned seedling radicles with A. rhizogenes as described (20) determined by alignment with ClustalW. The corresponding allowed generation of vigorous composite plants consisting of promoter regions up to 1 kb upstream of the respective initiator wild-type shoots and hairy roots harboring appropriate con- ATGs were analyzed for conserved motifs by using FOOT- PRINTER 2.0 Web serve (available at http:͞͞bio.cs.washing- structs, which developed from the inoculation site. All inocu- ͞ lated plant material was kept in a growth chamber at low light ton.edu software.htm) (25), a computer algorithm designed to at 23°C until hairy roots appeared. Formed hairy roots were identify conserved motifs in noncoding regions of orthologous transferred aseptically to nutrient medium, containing 100 mg͞ genes from many species. Motifs were identified by using a motif liter ampicillin. We selected 10–15 independently transformed size of 8–10 with a maximal parsimony score of 0–2ina hairy roots of each plant and inoculated them with AMF (see subregion of 1,000, allowing for motif losses. The promoter sequences were then scanned manually for variations of the six below). Mycorrhizal hairy roots exhibiting strong reporter gene resulting