Volume 22, Issue 23, Pages 2236-2241 (December 2012) A GRAS-Type Transcription Factor with a Specific Function in Mycorrhizal Signaling  Enrico Gobbato, John F. Marsh, Tatiana Vernié, Ertao Wang, Fabienne Maillet, Jiyoung Kim, J. Benjamin Miller, Jongho Sun, S. Asma Bano, Pascal Ratet, Kirankumar S. Mysore, Jean Dénarié, Michael Schultze, Giles E.D. Oldroyd  Current Biology  Volume 22, Issue 23, Pages 2236-2241 (December 2012) DOI: 10.1016/j.cub.2012.09.044 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 A Novel Locus Required for Mycorrhizal Colonization (A) M. truncatula ram1-1 plants developed normal nodules 3 weeks after inoculation with S. meliloti (upper panels), but showed greatly reduced infection by Glomus intraradices, 6 weeks postinoculation (lower panels), as assayed following ink staining. Scale bars represent 5 mm in upper panels and 40 μm in lower panels. (B) Quantification of G. intraradices 6 wpi indicates very low colonization in ram1-1 and ram1-2 mutants compared with the corresponding wild-type A17 and R108, respectively. Error bars represent standard errors, and the differences between the mutants and the respective wild-type (WT) are significant (p<0.01). (C) Quantification of G. intraradices hyphopodia formation on ram1-1 roots at 6 wpi. Error bars represent standard errors. WT: wild-type. ∗∗∗p < 0.01 in a t test. (D) Ink-stained roots showing an occasional hyphopodia on a ram1-1 root as compared with hyphopodia on wild-type or dmi3 roots. Scale bars represent 10 μm. Current Biology 2012 22, 2236-2241DOI: (10.1016/j.cub.2012.09.044) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 RAM1 Encodes a GRAS-Domain Transcription Factor (A and B) Wild-type (WT) and ram1-1 plants were transformed using the hairy-root technique with either a vector containing the RAM1 GRAS candidate gene (GenBank: JN572683) or the empty vector (EV) and analyzed 6 wpi with G. intraradices. (A) Representative ink-stained roots showing fungal colonization of ram1-1 roots transformed with the GRAS protein. Scale bar represents 0.2 mm. (B) Quantification of G. intraradices colonization levels. Each bar represents an independent Agrobacterium rhizogenes transformed root and the variability in the degree of complementation is likely due to the different expression levels of RAM1 in each of these independently transformed lines. Data shown are from one of three independent experiments, which gave analogous results. (C) An unrooted phylogenetic tree based on an amino-acid alignment among all known A. thaliana GRAS transcription factors, NSP1, NSP2, RAM1, and the closest RAM1 homologs in grapevine, poplar, cacao, castor oil, sorghum, rice, moss, spikemoss, Arabidopsis lyrata, and Brassica rapa (VvRAM1, PtRAM1, TcRAM1, RcRAM1, SbRAM1, OsGRAS2, PpRAM1, SmRAM1, AlXP 002887024.1, and BrAY928550.1, respectively). Bootstrap values can be found in Figure S4. Current Biology 2012 22, 2236-2241DOI: (10.1016/j.cub.2012.09.044) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 RAM1 Is a Mycorrhizal-Induced Transcription Factor that Regulates RAM2 (A) RAM1 is not present on the Medicago Affymetrix genechip, and therefore relative expression of RAM1 in different plant tissues and in roots uninoculated or inoculated with S. meliloti (Sm) or G. intraradices (mycorrhizal) was measured using qRT-PCR. The data is the average of three biological replicates with technical replicates. (B) RAM2 is present on the Medicago Affymetrix genechip, and therefore RAM2 expression across the same range of conditions and tissues was assessed from the Medicago truncatula Gene Expression Atlas [11]. (C) RAM2 expression in G. intraradices inoculated ram1-1 and wild-type (WT) plants at 20, 30, and 40 dpi, measured using qRT-PCR. (D) ChIP from mycorrhized wild-type and ram1-1 roots, using the αRAM1 antibody. Presence of portions of the RAM2 and NIN promoters in input fractions and immunoprecipitated fractions were monitored by PCR. Specific immunoprecipitation of a portion of the RAM2 promoter from wild-type but not ram1-1 indicates binding by RAM1. In contrast, RAM1 does not bind the rhizobial-induced NIN promoter. Error bars represent standard errors. Current Biology 2012 22, 2236-2241DOI: (10.1016/j.cub.2012.09.044) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 RAM1 Is Required for Myc Factor Signaling, Has No Function in Nod Factor Signaling, and Binds to NSP2 (A and B) Stimulation of root branching in (A) 10 nM nonsulphated Myc factor- (purple bars) and mock-treated plants (gray bars) and (B) 0.1 nM Nod factor-treated (orange bars) and mock-treated plants (gray bars) reveals that RAM1 is essential for Myc factor signaling, but redundant for Nod factor signaling. The significance of the difference between treated and mock treated plants: ∗∗∗p < 0.01, ∗∗p < 0.05. Error bars are standard errors. (C) Induction of pENOD11::GUS in ram1-1 plants treated with Nod factor (1 nM) as compared to wild-type plants. Scale bars represent 1 mm. (D) NIN induction in ram1-1 plants 24 hr after treatment with 10 nM Nod factor, as compared with wild-type plants, and as measured with qRT-PCR. This is the average of three biological replicates, with technical replicates. Error bars are standard error. (E) Interactions between RAM1 and NSP2 measured using BiFC. Confocal images of N. benthamiana epidermal cells transiently expressing YFPN:: RAM1 and YFPC:: NSP2. The insert shows the same interaction as observed in yeast two-hybrid assay. As a control, no interaction is observed between RAM1 and NSP1 in either BiFC or yeast. Combinations with different orientations of the YFPN and YFPC fluorophores gave identical results. Arrows indicate nuclei. (F) Competition assays. The percentage of nuclei displaying fluorescence when expressing RAM1:: YFPN and YFPC::NSP2 alone (control) or in combination with the expression of nontagged NSP1 (+NSP1) or RAM1 (+RAM1). ∗p < 0.05; ∗∗p < 0.01 (χ2). Error bars represent standard error. Current Biology 2012 22, 2236-2241DOI: (10.1016/j.cub.2012.09.044) Copyright © 2012 Elsevier Ltd Terms and Conditions