Plant flotillins are required for infection by nitrogen-fixing bacteria Cara H. Haney and Sharon R. Long1 Department of Biology, Stanford University, Stanford, CA 94305-5020 Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved November 12, 2009 (received for review September 2, 2009) To establish compatible rhizobial-legume symbioses, plant roots ponents of the proposed NF entry receptor pathway are currently support bacterial infection via host-derived infection threads (ITs). limited to NIN and LYK3; how LYK3 signaling is perceived and Here, we report the requirement of plant flotillin-like genes (FLOTs) translated into IT initiation is unknown. in Sinorhizobium meliloti infection of its host legume Medicago trun- Sinorhizobium meliloti, the nitrogen-fixing symbiont of Medi- catula. Flotillins in other organisms have roles in viral pathogenesis, cago (the alfalfa genus), is a close relative of the animal pathogen fi endocytosis, and membrane shaping. We identi ed seven FLOT Brucella. Sinorhizobium and Brucella have common genes re- genes in the M. truncatula genome and show that two, FLOT2 and quired for infection and invasion of their hosts (9). Given these FLOT4, are strongly up-regulated during early symbiotic events. This common features, we asked whether eukaryotic host-cell factors up-regulation depends on bacterial Nod Factor and the plant's ability required for infection might also be shared across kingdoms. to perceive Nod Factor. Microscopy data suggest that M. truncatula Upon uptake into host cells, Brucella remains surrounded by a FLOT2 and FLOT4 localize to membrane microdomains. Upon rhizo- fl bial inoculation, FLOT4 uniquely becomes localized to the tips of host-derived membrane, which contains the protein otillin (10). elongating root hairs. Silencing FLOT2 and FLOT4 gene expression Flotillins [also called Reggies (11)] are often used as markers for reveals a nonredundant requirement for both genes in IT initiation cholesterol-rich, detergent-resistant, membrane microdomains and nodule formation. FLOT4 is uniquely required for IT elongation, called “lipid rafts” and are now known to define a clathrin-in- and FLOT4 localizes to IT membranes. This work reveals a critical role dependent, caveolin-independent endocytic pathway required for for plant flotillins in symbiotic bacterial infection. endocytosis of cholera toxin (12). By interacting with effectors that can bind actin, flotillins mediate membrane-shaping events PLANT BIOLOGY Medicago | plant membrane | Sinorhizobium | symbiosis including membrane budding, actin-mediated neuronal differ- entiation, and filopodia formation (13–15). ymbiotic nitrogen-fixing rhizobial bacteria live in association Because of the importance of flotillins in pathogenesis and Swith legume roots inside developmentally unique structures membrane shaping, we wondered whether plant flotillin-like called nodules. Bacteria penetrate nodules via plant-derived proteins might be involved in symbiotic events such as IT initiation infection threads (ITs). Invagination of the root hair plasma and elongation, and the final endocytosis of bacteria into plant membrane during IT initiation resembles a partial endocytosis cells. Plant flotillin-like proteins, identified by sequence similarity event where the membrane begins to bud but, instead of pinching to animal flotillins, are predicted to have many features of their fi off, elongates through the cell. As the IT nishes traversing the mammalian counterparts including a similar tertiary structure, a cell, bacteria release into the intercellular space. New membrane conserved palmitoylation site at Cys-35, and a C-terminal coiled- invagination and IT formation take place in the underlying cell coil domain (16). Many plant flotillin-like genes are annotated as layers. Eventually, the bacteria are released into host cells via an nodulins (a generic name for a gene that is expressed uniquely in endocytosis-like event where they remain surrounded by a host- nodules) because of the early identification of a plant flotillin-like derived membrane (1, 2). A bacterially produced lipochitooligosaccharide called “Nod gene (GmNod53b) that is induced in soybean nodules (17). Factor” (NF) is a species-specific rhizobial signal that is recognized GmNod53b-like peptides have been isolated from the peribacte- by the legume host. NF promotes nodule development (3) and via roid membranes in soybean and pea nodules (18, 19), and an signal transduction induces calcium spiking and transcriptional Arabidopsis flotillin-like protein (named AtFlot1) was found in changes (4, 5). Forward genetic studies in rhizobial-legume systems detergent-resistant membrane domains (20). These results imply have revealed genes required for host NF signal perception, signal that flotillins are conserved between plants and animals. The transduction, and transcriptional changes (see ref. 6 and references conserved subcellular localization suggests the possibility of con- therein). NF perception is mediated by LysM-family receptor-like served function(s). In this work, we employ a reverse genetics kinases NFP and LYK3 (in Medicago truncatula; NFR1/NFR5 in approach to investigate the role of the Medicago truncatula flo- Lotus japonicus). The NFP receptor induces calcium spiking via a tillin-like gene family (FLOTs) in symbiosis. We demonstrate that signaling pathway that includes a Leucine-rich repeat receptor-like two family members, FLOT2 and FLOT4, are required for early kinase and a putative ion channel. Calcium spiking appears to symbiotic events and have nonredundant functions. control activity of a calcium/calmodulin-dependent protein kinase and two downstream GRAS-family transcription factors, NSP1 and NSP2, which interact in the plant nucleus and directly bind the Author contributions: C.H.H. and S.R.L. designed research; C.H.H. performed research; promoters of nodulation genes (7). NSP1 and NSP2 are necessary C.H.H. contributed new reagents/analytic tools; C.H.H. and S.R.L. analyzed data; and for all known NF-dependent transcriptional changes (5). C.H.H. wrote the paper. A second “entry” pathway (controlling bacterial entry into root The authors declare no conflict of interest. hair cells) has been proposed that includes the high-stringency This article is a PNAS Direct Submission. receptor encoded by LYK3. Null lyk3 mutants undergo a small Freely available online through the PNAS open access option. number of cortical cell divisions and form no ITs. An additional Database deposition: The sequences reported in this paper have been deposited in the transcriptional regulator NIN is required for nodule organo- GenBank database accession nos. GU224278-GU224282. genesis and infection events but not NF-dependent transcriptional 1To whom correspondence should be addressed. E-mail: [email protected]. changes. Current evidence suggests that NIN may coordinate This article contains supporting information online at www.pnas.org/cgi/content/full/ signals through both the signaling and entry pathways (8). Com- 0910081107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0910081107 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Results and Fig. S3), suggesting that known cell-surface polysaccharides Identification of Flotillin-like Genes in M. truncatula. A search of the are not required. M. truncatula genome sequence yielded seven genomic regions Evidence suggests that NF effects are transduced via a “sig- − with high homology (E-value < 1e 151) to the GmNod53b amino naling” pathway and a distinct “entry” pathway (6). The most acid sequence, which we have designated FLOT1-7 (Table S1). downstream component of the signaling pathway, transcription All seven predicted ORFs have >85% identity on the nucleotide factor NSP2, is required for up-regulation of FLOT2 and FLOT4 level (Fig. S1A). FLOT1-5 are located within a 30-kb region of at 1 dpi (Fig. 1B). The parallel entry NF perception pathway, chromosome 3 (Fig. S1B ). Whereas all other plant flotillin-like defined by LYK3, is also required for up-regulation of FLOT2 and proteins described thus far have a conserved predicted palmi- FLOT4 at 1 dpi (Fig. 1B), as is the transcriptional regulator NIN, toylation site at Cys-35 (16), FLOT4 is predicted to have a Tyr which is common to both signaling and entry pathways (Fig. 1B). substitution at this residue (Fig. S1A). FLOT5 has no obvious However, ERN (BIT1), an ERF-like transcription factor required translational start site and lacks a two-exon, one-intron structure. for up-regulation of a subset of early nodulins but not for IT in- Only three FLOT genes are present in the Arabidopsis genome itiation (22), is not required (Fig. 1B). NF perception by both (16), implying an expansion of the FLOT family in M. truncatula. signaling and entry NF receptors and signal transduction via NSP2 and NIN are required for up-regulation of FLOT2 and FLOT2 and FLOT4 Show Nod Factor-Dependent Up-Regulation During FLOT4 at 1 dpi. Nodulation. We assayed expression of M. truncatula FLOTs during We asked whether FLOT2 and FLOT4 were up-regulated nodulation by quantitative RT-PCR at times corresponding to key specifically in the plant cells that become infected by S. meliloti. events in nodule development (21). FLOT2 and FLOT4 ex- Using transcriptional fusions consisting of FLOT promoters and pression increases early in symbiosis with the wildtype S. meliloti the GUS reporter gene, we found that in uninoculated roots, strain Rm1021 (Fig. 1A). FLOT2 is up-regulated for the entire 21- FLOT1-4 are primarily