Cytokinin Biosynthesis Promotes Cortical Cell Responses during Nodule Development1[OPEN] Dugald Reid,a Marcin Nadzieja,a Ondrej Novák,b Anne B. Heckmann,a,2 Niels Sandal,a and Jens Stougaard a,3 a

Centre for Carbohydrate Recognition and Signaling, Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark b Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, CZ-78371 Olomouc, Czech Republic ORCID IDs: 0000-0001-9291-9775 (D.R.); 0000-0003-3452-0154 (O.N.); 0000-0002-5230-2532 (N.S.); 0000-0002-9312-2685 (J.S.).

Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone of Lotus japonicus roots. LjIpt2 and LjLog4 were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation of LjIpt2 and LjLog4 transcripts occurs independent of the LjLhk1 receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates that LjIpt2 and LjLog4 contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor, TCSn, showed that this response occurs in cortical cells before spreading to the epidermis in L. japonicus. While mutant analysis identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.

Symbiotic nodule development in legumes requires the coordination of infection by compatible rhizobia and host root organogenesis events. Following the perception of lipochitooligosaccharide Nod factors (NF) by the plant, the nodule organogenesis and infection pathways are initiated and coordinated to converge on the development of an infected and nitrogen-fixing nodule (Madsen et al., 2010). Cytokinin perception is required for nodule organogenesis, as mutations in the Lotus japonicus Lhk1 receptor cause impaired symbiotic events and nodulation is abolished in the L. japonicus lhk1 lhk1a 1

This work was supported by the Danish National Research Foundation (grant no. DNRF79), ENSA, Engineering Nitrogen Symbiosis for Africa, and the Ministry of Education, Youth, and Sports of the Czech Republic (NPU I program project no. LO1204). 2 Current address: Arla Foods Ingredients, Sønderhøj 10, 8260 Viby J, Denmark. 3 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jens Stougaard ([email protected]). J.S., D.R., and A.B.H. conceived the research plan; D.R., M.N., and O.N. conducted experiments; D.R., A.B.H., and N.S. identified and backcrossed mutants; D.R. and M.N. performed microscopy; O.N. performed cytokinin measurements; D.R., M.N., and J.S. prepared the figures and wrote the article with input from all authors. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00832

lhk3 triple mutant (Murray et al., 2007; Held et al., 2014). Exogenous cytokinin application and gain-of-function mutations in LjLhk1 have demonstrated that activation of cytokinin signaling in the root induces cortical cell divisions and spontaneous nodule formation (Cooper and Long, 1994; Bauer et al., 1996; Fang and Hirsch, 1998; Tirichine et al., 2007; Heckmann et al., 2011). Legumes forming indeterminate nodules share the requirement for cytokinin perception, which in Medicago truncatula is controlled primarily by the MtCRE1/CHK1 receptor together with some redundant activity of MtCHK2, MtCHK3, and MtCHK4 (Gonzalez-Rizzo et al., 2006; Plet et al., 2011; Boivin et al., 2016). In contrast to organogenesis, cytokinin signaling plays a negative regulatory role in rhizobia infection, as the Ljlhk1-1 mutant exhibits hyperinfection while mutations in LjCkx3, which cause increased root cytokinin, reduce infection (Murray et al., 2007; Reid et al., 2016). Therefore, cytokinin signaling is central to both pathways (Miri et al., 2016). Genetic screens in model legumes have helped to elucidate components of the signal transduction pathways and many of the transcriptional regulators required for organogenesis and/or infection. Initially, calcium-spiking events must be decoded by CALCIUM AND CALMODULIN-DEPENDENT KINASE (CCaMK; Lévy et al., 2004; Tirichine et al., 2006; Sieberer et al., 2009) and its phosphorylation target CYCLOPS (Singh et al., 2014). In the model legumes

Plant PhysiologyÒ, September 2017, Vol. 175, pp. 361–375, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved.

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L. japonicus and M. truncatula, a single copy or duplicated copies, respectively, of the ERF REQUIRED FOR NODULATION (LjERN1, MtERN1, and ERN2) transcription factors are required for the activation of both epidermal and cortical transcriptional responses (Cerri et al., 2012, 2016, 2017; Kawaharada et al., 2017; Yano et al., 2017). Downstream of cytokinin perception, nodule organogenesis in L. japonicus and M. truncatula requires Nodule Inception (Nin; Schauser et al., 1999; Marsh et al., 2007) and the GRAS transcription factors Nsp1 and Nsp2 (Kaló et al., 2005; Smit et al., 2005; Heckmann et al., 2006; Murakami et al., 2006; Hirsch et al., 2009). These transcription factors also are required in the rhizobia infection pathway. Activation of LjNin is a central function of cytokinin activity (Tirichine et al., 2007; Heckmann et al., 2011), and LjNin overexpression also is sufficient for the spontaneous initiation of cell divisions, which is dependent on the NUCLEAR FACTOR Y transcriptional activators NF-YA1 and NF-YB1 in L. japonicus (Soyano et al., 2013). Nin may play distinct roles in the cortex and epidermis, being required for both infection and organogenesis but inducing a negative feedback loop to restrict further infection (Soyano et al., 2014; Yoro et al., 2014). Expression of MtNin under the control of an epidermal enhanced promoter is sufficient to instigate nodule organogenesis and a positive feedback circuit of Nin and Cre1 cytokinin receptor expression in the cortical cells (Vernié et al., 2015). Cytokinin signaling responses are orchestrated by response regulators, which are induced during nodulation (Lohar et al., 2004, 2006; Gonzalez-Rizzo et al., 2006; Tirichine et al., 2007; Op den Camp et al., 2011). Response regulator signaling output as determined with the synthetic two-component signaling sensor (TCS; Müller and Sheen, 2008) has been shown in cortical and pericycle cells in response to NF in M. truncatula (van Zeijl et al., 2015). In L. japonicus, TCS activity occurred first in the dividing cells of the developing nodule and later in the epidermis (Held et al., 2014). Epidermal cytokinin responses to NF also have been reported in M. truncatula hairy roots using the more recently developed TCS new (TCSn) reporter (Zürcher et al., 2013; Jardinaud et al., 2016). It is likely that both cytokinin accumulation and receptor availability modulate these cytokinin response domains. Changes in root cytokinin levels have been detected in response to rhizobia and purified NF (van Zeijl et al., 2015; Reid et al., 2016). The early nodulation signaling pathway is required for this response, as it was not detected in the Mtdmi3 (ccamk) background (van Zeijl et al., 2015). In plants, cytokinins are adenine-derived molecules of either aromatic or isoprenoid type. Isoprenoid cytokinins are the most abundant and have the best defined biosynthetic pathway (Sakakibara, 2006). The biosynthesis of isopentenyl adenine (iP), trans-zeatin (tZ), and dihydrozeatin isoprenoid cytokinins from adenosine 59 phosphates first requires the action of isopentenyl transferase (IPT) to produce the iP nucleotides (Kakimoto, 2001; Takei et al., 2001). Cytochrome P450 362

monooxygenases of the CYP735A family then convert iP nucleotides to tZ nucleotides (Takei et al., 2004). LONELY GUYs (LOG) catalyze the production of cytokinin bases from their nucleotide precursors to directly produce the most active cytokinin forms (Kurakawa et al., 2007). In legumes, two M. truncatula Log genes were identified in RNA interference studies as contributing to nodule development (Mortier et al., 2014). Several additional M. truncatula biosynthetic genes responding to NF treatment also were identified in the Ipt, Cyp735a, and Log families (van Zeijl et al., 2015). Cytokinin metabolism genes also were identified in laser-dissected epidermis following NF treatment (Jardinaud et al., 2016). Although LjIpt3 was suggested to contribute to nodule organogenesis in an RNA interference study (Chen et al., 2014), mutant analysis indicated that LjIpt3 in fact plays a shoot-dependent negative regulatory role in nodulation (Sasaki et al., 2014). The precise timing of cytokinin synthesis and responses in the epidermal and cortical cells remains obscure and are key questions to resolve in the early stages of infection and nodule initiation. Previous studies have focused on biosynthesis genes active after the first cell divisions and, therefore, not contributing to the first rapid cytokinin accumulation that occurs (Mortier et al., 2014) or those activated by NF (van Zeijl et al., 2015). Another question is whether purified NF treatment reproduces the timing and patterns of cytokinin accumulation that occur during plant-rhizobia interaction, as the root is stimulated in all cells simultaneously, which may not reproduce the rhizobia response. We aimed to clarify the genetics, timing, and tissue specificity of cytokinin biosynthesis and response during the earliest stages of rhizobia-inoculated L. japonicus roots and later primordia formation. Several genes encoding components of the cytokinin biosynthesis pathway active during early stages of nodule development were identified, and the TCSn reporter was studied in a stable L. japonicus line. We found that activation of cytokinin biosynthesis localizes the initial cortical cytokinin response required to initiate nodule primordia. However, elevated epidermal cytokinin biosynthesis or response during this early stage was not detected. Overexpression of several cytokinin biosynthesis pathway genes leads to spontaneous nodule formation. This suggests that multiple cytokinin biosynthetic steps are limiting to nodule development. RESULTS Cytokinin Biosynthesis Genes Show Distinct Early and Late Expression Patterns during Nodule Development

To identify the biosynthesis genes contributing to the early accumulation of cytokinin in response to rhizobia, we searched publicly available and in-house Affymetrix and RNA sequencing expression data. We identified six members of the Ipt, Cyp735a, and Log gene families as candidates due to their expression during the early phases of NF response and nodule development (Fig. 1A; Plant Physiol. Vol. 175, 2017

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Supplemental Table S1). Detailed transcript-level analysis of these genes was focused on the area of the root susceptible to rhizobia infection and nodule development at the time of inoculation. This region occurs in and below emerging root hairs at the maturation zone of the root meristem. To capture a developmental time series of nodule initiation, a 2-cm root section corresponding to the symbiotic nodule susceptible zone that was susceptible at the time of inoculation was collected over a 10-d nodule development series (Fig. 1B). Confocal microscopy of plants grown in the same conditions indicated that the first foci of cortical cell divisions are present at 3 dpi, while significant infection and nodule primordia initiation are apparent at 5 dpi (Fig. 2). We

observed red, nitrogen-fixing nodules at 10 dpi in these conditions. qRT-PCR analysis of the cytokinin biosynthesis genes was conducted in this series (Fig. 1C). LjIpt2, LjIpt4, LjLog1, and LjLog4 transcript levels were responsive to inoculation from the earliest time point, 24 h after inoculation. The relative levels of LjIpt2 and LjLog4 within the susceptible zone of inoculated roots then declined over the course of nodule development. LjIpt4 transcript abundance showed a similar pattern, although its relative induction was not as great as that of LjIpt2. In contrast to these early responses, LjIpt3 and LjLog1 showed the largest relative increases when nodules were present 10 dpi. In the case of LjIpt3, no change was detected during the early stage of nodule development.

Figure 1. Transcript levels of cytokinin biosynthesis genes during nodule development. A, Simplified cytokinin biosynthesis pathway. Enzymes (uppercase) and cytokinin species (italics) are shown. B, The symbiotic nodule susceptible zone harvested for quantitative reverse transcription (qRT)-PCR analysis is indicated, including nodules at 10 d post inoculation (dpi). C, Relative transcript levels of the indicated biosynthesis genes in the sliding symbiotic nodule susceptible zone following inoculation with M. loti R7A. Values are relative to two housekeeping genes and indicate means 6 SE for n = 5 biological replicates. P values were calculated using Wilcoxon ranksum testing between mock and treatment groups and are indicated by asterisks: *, P , 0.05 and **, P , 0.01.

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LjCyp735a, which in Arabidopsis (Arabidopsis thaliana) is expressed in response to cytokinin accumulation (Takei et al., 2004), was enhanced significantly at all stages of nodule development, showing the highest transcript abundance once nodules were present after 10 d. By comparison, the expression of LjNin, which shows a cytokinin-dependent expression pattern (Heckmann et al., 2011), followed a pattern like LjIpt2 in this series (Supplemental Fig. S1). Regulation of Cytokinin Biosynthesis by NF and Cytokinin

To identify whether the early cytokinin biosynthesis responses to rhizobia were induced by NF signaling, we treated roots with 1028 M Mesorhizobium loti R7A NF for 8 and 24 h and conducted qRT-PCR (Fig. 3A). This analysis showed that after a 24-h NF treatment, LjIpt2, LjLog1, LjLog4, and LjCyp735a transcript levels were enhanced significantly. At the earlier 8-h treatment point, LjIpt2 and LjCyp735a showed slightly, although significantly, reduced transcript abundance, while LjIpt3 transcripts were increased significantly. LjIpt3 and LjIpt4 showed no response to NF when treatment was maintained for 24 h. In M. truncatula, two Log genes exhibit Cre1-dependent transcript increases during nodulation (Mortier et al., 2014). To identify whether the two most responsive early biosynthesis genes in L. japonicus, LjIpt2 and LjLog4, were responding to existing cytokinin pools or contributing to them, we treated roots with 5 mM tZ in a time series. qRT-PCR analysis indicated that LjIpt2 and LjLog4 transcript abundance is reduced rapidly by cytokinin treatment relative to untreated roots (Fig. 3B). The Cytokinin Response Increases Rapidly in the Cortex following Rhizobia Inoculation

Figure 2. Activity of the TCSn cytokinin reporter during nodule development. Spatiotemporal activity of the synthetic TCSn promoter in the symbiotic nodule susceptible zone was determined in an L. japonicus TCSn:YFPnls stable line using confocal microscopy (nucleus localized; green). A, Uninoculated. B to F, Inoculated with M. loti expressing DsRed (magenta) at 1 dpi (B), 2 dpi (C), 3 dpi (D), 4 dpi (E), and 5 dpi (F). G, Symbiotic nodule susceptible zone of uninoculated root. H, 364

Following the expression patterns of TCSn in the consistent genetic background of a stable line would facilitate the identification of the cell and tissue types in which the first cytokinin responses to rhizobia occur. Therefore, we created a TCSn:YFPnls stable line (Thykjær et al., 1998) to characterize these responses in L. japonicus. We used nucleus-localized YFP to allow clear differentiation from autofluorescence. Treatment with cytokinin (1 mM 6-Benzylaminopurine, BAP) confirmed that this reporter was functional in all cell types, including epidermal cells, in the susceptible zone of the root (Supplemental Fig. S2). To determine the cytokinin response in a symbiotic context, we inoculated TCSn: YFPnls plants with M. loti MAFF303099 DsRed and

Symbiotic nodule susceptible zone 1 dpi with M. loti. A to F show cleared whole mounts and were imaged using identical parameters. G and H are uncleared cross sections. Bars = 100 mm. Plant Physiol. Vol. 175, 2017

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observed increased epidermal activity from 2 dpi (Fig. 2C), which was increased further by 5 dpi (Fig. 2F). We did not observe TCSn:YFPnls activity in root hairs that contained infection threads (ITs). As ITs progressed to the cortex, we observed reduced TCSn: YFPnls activity in the cells containing penetrating ITs (Fig. 4A; Supplemental Video S1). The expression of pLog4:tYFPnls was reminiscent of the TCSn activity at the same age, showing greater activity in dividing cells than in neighboring infected cells (Fig. 4B). LjIpt2 and LjLog4 Expression Is Dependent on Common Symbiotic Transcription Factors But Independent of LjNin

Figure 3. Effects of exogenous NF application or cytokinin (tZ) on cytokinin biosynthesis gene mRNA levels. A, Relative transcript abundance after 8 and 24 h of 1028 M M. loti R7A NF treatment. B, Relative transcript abundance following treatment with 5 mM tZ. Values are relative to two housekeeping genes and indicate means 6 SE for n = 5 biological replicates in A and n = 3 biological replicates in B. P values were calculated in A using Wilcoxon rank-sum testing between mock and treatment groups and are indicated by asterisks: *, P , 0.05 and **, P , 0.01.

harvested roots at 24-h intervals over the following 5 d. Fixation and fluorescence-compatible clearing were performed to allow the imaging of deep cell layers of the root. To identify changes in cytokinin response, we selected imaging parameters with low levels of nucleus-localized fluorescence in the uninoculated root susceptible zone (Fig. 2A). From the earliest point, 24 h post inoculation, we found a broad area in the root cortex with distinct nucleus-localized fluorescence activity (Fig. 2B). We did not detect epidermal cytokinin responses at this early stage, which was confirmed by transverse sections of uncleared roots (Fig. 2, G and H). Over the following time points, the intensity of TCSn: YFPnls activity increased and became increasingly localized to the foci of dividing cortical cells corresponding to nodule primordia (Fig. 2, C–F). We first Plant Physiol. Vol. 175, 2017

To identify the genetic dependency of early cytokinin biosynthesis in L. japonicus during nodule initiation, we harvested the root susceptible zone 3 d after M. loti R7A inoculation in a collection of L. japonicus symbiotic mutants. Rhizobia-dependent transcript increases in LjIpt2 (Fig. 5A) and LjLog4 (Fig. 5B) showed genetic dependencies similar to each other. Both biosynthesis genes require LjErn1, LjCCaMK, LjNsp2, and LjCyclops for rhizobia-dependent transcriptional responses. Changes in LjLog4 but not LjIpt2 transcript levels require LjNsp1. Both biosynthesis genes were activated independently of LjNin and showed greater transcript levels in the nin-2 mutant than in wild-type Gifu. We also found that LjIpt2 and LjLog4 transcript abundance was significantly further elevated in the lhk1-1 mutant compared with wild-type Gifu. We also analyzed LjLhk1 transcript levels in the mutant series (Fig. 5C). The LjLhk1 transcript level showed genetic dependency consistent with the two analyzed cytokinin biosynthesis genes and was independent of NIN, increasing in the Ljnin-2 mutant following inoculation. Activity of Cytokinin Biosynthesis Gene Promoter Regions

Aiming to identify whether the spatial expression pattern of cytokinin biosynthesis genes correlates with the initial cortical TCSn response, we cloned promoters from the six cytokinin biosynthesis genes showing transcriptional changes during nodule development. We cloned upstream regions of 1.5 to 2.5 kb depending on available genomic sequence and fused them to GUS or tYFPnls reporter genes (for full sequences, see Supplemental Table S2). The activity of pIpt2:GUS in uninoculated roots was observed in lateral root primordia but did not show strong staining in the susceptible root zone (Fig. 6A; Supplemental Fig. S3). Twenty-four hours after inoculation with M. loti, pIpt2: GUS staining was observed in the rhizobia susceptible root zone, which sectioning revealed was localized in the middle and outer cortical cell layers (Supplemental Fig. S3). We continued to observe cortical pIpt2:GUS activity at 3 and 14 dpi (Fig. 6, B and C) but did not observe significant activity in nodules (Supplemental Fig. S3). The activity of pIpt4:GUS was observed in vascular tissue in uninoculated plants, while M. loti R7A inoculation 365

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Figure 4. TCSn cytokinin reporter and pLog4:tYFPnls activity in cortical cells surrounding ITs. A, L. japonicus TCSn:YFPnls (nucleus localized; green) stable line. B, pLog4:tYFPnls (nucleus localized; green) in A. rhizogenes-induced transgenic roots. Both images were acquired using confocal microscopy 6 dpi with M. loti MAFF303099 DsRed (magenta). Images are cleared whole mounts in maximum intensity projection. Bars = 100 mm.

similar to the early rhizobia-induced nodule primordia observed on wild-type plants (Fig. 7, B, D, and G). The expression of LjIpt3 did not induce nodule primordia or nodules under the same conditions. Given the ability of LjLog4 expression to induce nodule primordia, we also expressed LjLog4 using cortex (pCORTEX) and epidermal (pEXT1) enhanced promoters (Gavrilovic et al., 2016). Neither cortex- nor epidermis-enhanced expression of LjLog4 was sufficient to produce nodule primordia (Fig. 7A). Given that the LjLog4-induced nodules often failed to pass the nodule primordia stage, we asked whether the coexpression of multiple cytokinin biosynthesis genes could increase the efficiency, number, or size of nodule primordia. To address this, we cloned LjUbi:LjIpt3, LjUbi: LjLog4, and LjUbi:LjCyp735a in the same construct. This

induced significant cortical expression at 3 dpi and later activity in nodule vasculature (Fig. 6, D–F). We detected pLog4:GUS staining in vascular tissue independent of inoculation, but in contrast to the qRT-PCR analysis of Log4, we did not observe enhanced promoter activity at 3 dpi (Fig. 6, G and H). At later times (6 dpi), we detected significant expression of pLog4:tYFPnls in the dividing cells of nodule primordia (Fig. 4B). pLog4:GUS showed strong staining in the vascular tissue at the base of nodules 14 dpi (Fig. 6I). The promoters of LjIpt3, LjLog1, and LjCyp735a did not show inoculation-responsive changes until nodules were visible on the roots. pCyp735a:GUS staining was not detected in uninoculated roots but was present in nodules (Fig. 6, J and K). pIpt3:GUS activity was weak in the root vasculature prior to inoculation but showed increased intensity at the base and vasculature of nodules (Fig. 6, L and M). pLog1: GUS showed staining in vasculature, lateral roots, and mature nodules (Fig. 6, N and O). Cytokinin Biosynthesis Gene Overexpression Induces Spontaneous Nodule Development

To determine whether the elevation of cytokinin biosynthesis is sufficient to induce nodule organogenesis, we overexpressed several biosynthesis genes. In order to avoid rhizobia-induced nodules and aid in the determination of nodules relative to lateral root primordia, we used the nodule-specific pNin:GUS marker in the Ljnfr5 mutant background (Radutoiu et al., 2003). Using the LjUbiquitin promoter, we expressed LjIpt3 or LjLog4 in Agrobacterium rhizogenes-induced transgenic roots. The expression of LjLog4 in these conditions induced a small number of nodules or nodule primordia (approximately one spontaneous nodule per plant), with most failing to develop beyond the primordia stage, even after 7 weeks of hairy root growth (Fig. 7, A and D). The LjUbi:Log4-induced nodule primordia showed pNin:GUS activity and were morphologically 366

Figure 5. Transcript levels of LjLog4, LjIpt2, and LjLhk1 in symbiotic mutants. The susceptible zone (ZON) of the root was harvested 3 dpi with M. loti R7A in the indicated symbiotic mutants for qRT-PCR analysis. Values for Ipt2 (A), Log4 (B) and Lhk1 (C) are relative to two housekeeping genes and indicate means 6 SE for n = 5 biological replicates. P values were calculated using ANOVA followed by Tukey’s posthoc testing. Groups indicated by the same letter are not significantly different at P , 0.05. Plant Physiol. Vol. 175, 2017

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Figure 6. Activity of cytokinin biosynthesis gene promoters during nodule development. Spatiotemporal expression driven by the indicated promoter was determined in hairy roots using light microscopy for promoter:GUS reporter constructs (blue). A to C, pIpt2:GUS. A, Uninoculated. B, M. loti R7A at 3 dpi. C, M. loti R7A at 14 dpi. D to F, pIpt4:GUS. D, Uninoculated. E, M. loti R7A at 3 dpi. F, M. loti R7A at 14 dpi. G to I, pLog4:GUS. G, Uninoculated. H, M. loti R7A at 3 dpi. I, M. loti R7A at 14 dpi. J and K, pCyp735a:GUS. J, Uninoculated. K, M. loti R7A at 14 dpi. L and M, pIpt3:GUS. L, Uninoculated. M, M. loti R7A at 14 dpi. N and O, pLog1:GUS. N, Uninoculated. O, M. loti R7A at 14 dpi. All images are longitudinal sections. Bars = 100 mm.

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Figure 7. Spontaneous nodule formation in hairy roots expressing cytokinin biosynthesis genes. A, Nodules and pNin:GUS-positive nodule primordia were scored in hairy roots produced on an nfr5 pNin:GUS line. Constitutive expression was conducted using LjUbiquitin or the tissue-enhanced expression of pCORTEX and pEXT (epidermis) promoters together with the indicated biosynthesis genes. B, Indicative image of pNin:GUS in the wildtype Gifu background 5 dpi with M. loti R7A. C, Spontaneous nodules formed on LjUbi:Ipt3 LjUbi:Cyp735a LjUbi:Log4 hairy roots. D, Indicative image 368

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levels were similar in Ljipt3-2 Ljipt4-1 double mutant and wild-type roots, with the mutant maintaining the rhizobia-dependent cytokinin increase reported previously (Reid et al., 2016). Cytokinin measurements of single mutants confirmed that both Ljipt3 and Ljipt4 single mutants also maintained the inoculation-dependent cytokinin increase (Supplemental Fig. S5).

construct produced many spontaneous nodules, and frequently, fused nodules showing large areas of cell division activation were observed (Fig. 7, A and C). In these constructs, smaller nodule primordia expressed pNin:GUS, which also was detected at the periphery of the large spontaneous growths after sectioning (Fig. 7F). GUS expression was undetectable in whole mounts of large spontaneous nodules (Fig. 7C). Thick sections were cut to identify the nature of vascular initiations, revealing that multiple vascular tissues were present in the nodule-like structures (Fig. 7E). To quantify the effect of the biosynthesis gene overexpression, we conducted cytokinin measurements on the hairy roots. Consistent with the significant morphological changes, the roots with coexpression of LjUbi:LjIpt3, LjUbi:LjLog4, and LjUbi:LjCyp735a had the most significant effects on cytokinin content (Fig. 7H). These roots showed significant increases in total cytokinin, cytokinin bases, iP ribosides, and tZ ribosides. LjUbi:LjLog4 also showed significant effects on the cytokinin pool, reducing the levels of the iPR and tZR substrates. In contrast, LjUbi: LjIpt3 and the tissue-enhanced expression of LjLog4 did not significantly alter the active cytokinin profile of the roots. The coexpression of LjUbi:LjIpt3, LjUbi:LjLog4, and LjUbi:LjCyp735a also produced significant levels of O- and N-glucosides and dihydrozeatin metabolites, while the expression of Ipt3 or Log4 alone did not significantly alter these pools (Supplemental Table S3). qRT-PCR analysis revealed that robust overexpression was achieved for all target genes in these constructs except for pEXT1:Log4, where only minor increased expression was achieved (Supplemental Fig. S4).

To identify whether the mutants showed nodulation phenotypes, we grew the LORE1 lines together with two Ljipt4 TILLING alleles (Supplemental Table S4) in boxes and inoculated them with M. loti R7A (Supplemental Fig. S6). Eight weeks after inoculation, both Ljipt4 LORE1 insertion alleles and a TILLING allele (Ljipt4-3) that is altered in a well-conserved amino acid showed significantly reduced nodulation. The ipt4-4 allele, which contains a mutation in a nonconserved amino acid, showed a wild-type phenotype. In contrast to previous reports using the same alleles (Sasaki et al., 2014), Ljipt3 single mutants maintained wild-type nodulation in our conditions. However, our analysis of the Ljipt3-3 Ljipt4-1 double mutant suggests that, in the absence of an early LjIpt4mediated cytokinin bust, LjIpt3-mediated cytokinin accumulation negatively influences nodule number. One hypothesis is that elevated LjIpt3 expression in developing nodules maintains nodules as sink tissues that, in the absence of LjIpt4 activity, suppress the formation of foci located nearby.

Identification of Cytokinin Biosynthesis Mutants

DISCUSSION

Where available, we obtained LORE1 mutants (Fukai et al., 2012; Urba nski et al., 2012; Małolepszy et al., 2016) in the L. japonicus cytokinin biosynthesis genes identified as regulated during nodule initiation and development. We obtained exonic Ljipt3, Ljipt4, and Ljcyp735a insertion mutants, while mutations in LjIpt2, LjLog1, and LjLog4 were not available (Supplemental Table S1). From these LORE1 lines, we produced an Ljipt3-2 Ljipt4-1 double mutant. We compared the cytokinin content of wild-type Gifu and Ljipt3-2 Ljipt4-1 roots 3 d after inoculation with M. loti R7A, the NF-deficient M. loti R7A nodC mutant, or uninoculated plants (Fig. 8). Consistent with LjIpt2 playing the major role in inoculation-dependent cytokinin biosynthesis, we found that total cytokinin and iP-type cytokinin

In this work, we found that the regulation of cytokinin biosynthesis can produce the required cytokinin buildup to initiate nodule organogenesis. Using a TCSn reporter line, we could identify cortical signaling as the earliest detectable cytokinin response during the L. japonicus-M. loti interaction. Cytokinin signaling is central to both nodule organogenesis and infection but plays contrasting roles in each process. In epidermal cells of L. japonicus, Lhk1-dependent cytokinin signaling restricts infection while cortical cytokinin responses are critical for the initiation of organogenesis (Murray et al., 2007). Much of the work underlying our understanding of the role of cytokinin in organogenesis has relied on a detailed analysis of both loss- and gain-of-function mutations in legume cytokinin receptors (Murray

LjIpt4 Contributes to Nodule Development in L. japonicus

Figure 7. (Continued.) of the spontaneous nodule primordia formed on LjUbi:Log4. E, Thick sections showing vasculature (v) formation in spontaneous nodules of LjUbi:Ipt3 LjUbi:Cyp735a LjUbi:Log4 hairy roots. F, Longitudinal section showing pNin:GUS activity in LjUbi:Ipt3 LjUbi:Cyp735a LjUbi:Log4 spontaneous nodules. G, Transverse section of LjUbi:Log4-induced nodule primordia. H, Cytokinin content determined in hairy roots induced with the indicated constructs and grown for 4 weeks. P values were calculated using Wilcoxon rank-sum testing between control and expression constructs and are indicated by asterisks: *, P , 0.05. FW, Fresh weight. Images in B to G are whole mounts (B–D) or sections (E–G) of roots stained for pNin:GUS activity. Bars = 1 mm in B to D and 100 mm in E to G. Black points in H represent values falling outside 1.5 * Interquartile Range. Plant Physiol. Vol. 175, 2017

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Figure 8. Cytokinin (CK) levels in L. japonicus wild-type (Gifu) and ipt3-2 ipt4-1 mutant roots. Total cytokinin (A) and iP-type cytokinin (B) levels are shown at 3 dpi with M. loti R7A. Values are means 6 SE for n = 4 biological replicates. P values were calculated using Wilcoxon rank-sum testing between mock and treatment groups and are indicated by the asterisk: *, P , 0.05. FW, Fresh weight.

et al., 2007; Tirichine et al., 2007; Plet et al., 2011; Held et al., 2014; Boivin et al., 2016). Because measurements of cytokinin in the root following rhizobia or NF treatment identified increases in response to either treatment (van Zeijl et al., 2015; Reid et al., 2016), we focused on the role that cytokinin biosynthesis plays in nodule initiation. Nodule organogenesis requires the stimulation of cell divisions in deeper cell layers in response to epidermal rhizobia perception. The nature of the non-cellautonomous signal activating these responses has remained obscure, with suggestions that transcription factors such as NIN (Vernié et al., 2015) or cytokinin itself may act as this signal (Hayashi et al., 2014). In L. japonicus, TCS readout and Lhk1 promoter activity occur first in the cortex before extending to epidermal cells (Held et al., 2014). In M. truncatula, contrasting results have been reported from TCSn and TCS reporter studies following NF application (van Zeijl et al., 2015; Jardinaud et al., 2016). TCSn activity was detected in the epidermis and supported by expression analysis of laser-capture microdissected cells, while TCS activity was found in cortical cells in response to NF application. The sensitivity of TCSn compared with TCS was suggested as a possible reason for these differences. The inherent variation and hormonal conditions in A. rhizogenes-induced transgenic roots also may play roles in these differences. There is little doubt that cytokinin responses occur in both tissues during nodulation, but understanding the relative timing is of key importance, as it can help identify whether cytokinin is likely to initiate epidermis-cortex signaling or occurs first in the cortex in response to epidermis-derived signals. Our analysis in the L. japonicus stable transformed line showed that TCSn is responsive to cytokinin application in both epidermal and cortical cells of the susceptible zone. Our finding that the initial TCSn response to rhizobia occurs in cortical cells, therefore, is not due to the unresponsiveness of the marker in epidermal cells, as is the case for TCS in Arabidopsis root hairs (Zürcher et al., 2013). Only after cortical signaling has initiated are epidermal responses evident in conjunction with the increased intensity in foci of cell 370

divisions. The absence of the TCSn response in root hairs with ITs is further evidence that successful infection is mutually exclusive with high cytokinin signaling in the epidermis. Cytokinin also may regulate cellular infection in the cortex, as we found both decreased Log4 expression and TCSn activity in the cells containing ITs. Our findings with TCSn are consistent with the model proposed by Held and associates (2014), where cortical cytokinin signaling initiates primordia formation and later epidermal activity, which occurs exclusively through LjLhk1, regulates infection. The increased responsiveness of TCSn does not seem sufficient to explain the difference observed between M. truncatula and L. japonicus responses. An inherent difference in indeterminate and determinate nodulating species or in the NF-triggered response compared with rhizobia may account for these differences. It is interesting that the cortical cytokinin response occurs at such an early stage in legume-rhizobia interaction, well before visible ITs are present. Our analysis of genetic dependencies suggests that key differences do exist in the cytokinin response in L. japonicus and M. truncatula, as the genetic requirement for NIN for Lhk1/Cre1 expression is different. In M. truncatula, elevated transcript levels of MtCre1 and cytokinin response regulator signaling are dependent on NIN (Vernié et al., 2015). This dependency was not observed in L. japonicus. Additionally, first nodule cell divisions in M. truncatula occur in the pericycle and inner cortex, while in L. japonicus, they occur in the middle and outer cortex (van Spronsen et al., 2001; Xiao et al., 2014). Our analysis of nodule developmental progression showed that LjIpt2 and LjLog4 are prime candidates for producing the cortical cytokinin response. LjIpt2 and LjLog4 transcripts were highest in the earliest phase of the interaction between L. japonicus and M. loti, when we could only observe cortical TCSn responses. Promoter analysis of LjIpt2 was consistent with this, as it was active in the middle and outer cortical layers 24 h after inoculation. Our promoter analysis of LjLog4 was less conclusive, as it did not show a consistent inoculation response at this early time. The transcript abundance of LjIpt2 and LjLog4 also was reduced rapidly by cytokinin treatment, indicating that it contributes to, rather than responds to, the cytokinin pool. Based on our findings, we propose a tissue and genetic model where cytokinin biosynthesis in the cortex, derived predominantly from the activity of LjIPT2, LjIPT4, and LjLOG4 but with some redundancy in both families, first activates the accumulation in cortical cells (Fig. 9). Following this initiation of biosynthesis and the resulting signaling, back signaling to the epidermis, likely through both diffusion of cytokinin and later epidermal biosynthesis, regulates infection. Failure to regulate the cortical cytokinin increase, as occurs in Ljckx3 mutants, increases the negative feedback on rhizobia infection further, confirming the tight regulation requirement between events in these two tissues. In M. truncatula, NF-dependent cytokinin accumulation is Ccamk/Dmi3 dependent and partially dependent Plant Physiol. Vol. 175, 2017

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Figure 9. Proposed model of spatial and genetic determinants of cytokinin (CK) responses during L. japonicus nodule development. Following NF perception (1), Ipt2/Ipt4 and Log4 expression in the cortex (Co) leads to a local accumulation of active cytokinin (2). Cytokinin oxidase/dehydrogenase3 (CKX3) negatively regulates this cytokinin pool to restrict the cytokinin signaling domains. Cytokinin perception and signaling mediated by the LHK receptors and a positive feedback response with NIN localize sites of cytokinin activation and cell division activation (3). Cytokinin diffusion to the epidermis (Ep) and later epidermal biosynthesis activate LHK1-dependent repression of ITs in the epidermis (4).

on Nsp1 and Nsp2 (van Zeijl et al., 2015). In addition to LjCcamk, we found that LjCyclops, LjErn1, and LjNsp2 are required for increased LjIpt2 and LjLog4 transcript accumulation. Consistent with the partial requirement of MtNsp1 and MtNsp2 in NF-induced cytokinin accumulation, we found that LjNsp1 was not required for LjIpt2 abundance but is required for the normal LjLog4 response. Supporting the idea that cytokinin accumulation is a key target of the common symbiosis pathway, we found several biosynthesis genes with NF-responsive expression. Different magnitudes and directions of some responses after 8- or 24-h treatments highlight the importance of timing, concentration, and conditions in comparisons of inoculation and NF treatment experiments. While we found that a 24-h NF treatment or rhizobia inoculation produced similar transcript increases for LjIpt2, LjLog1, LjLog4, and LjCyp735a, NF treatment did not reproduce the early LjIpt4 transcript response to rhizobia. Conversely, an 8-h NF treatment did not appear to reproduce the observed inoculation responses, with minor repressive effects on LjIpt2 and LjCyp735a and a minor increase in LjIpt3. It is possible that epidermal cells require a higher threshold for NF-dependent cytokinin biosynthesis that can be reached by high concentrations of NF stimulation but that only occur later in an inoculation scenario. By focusing on the enriched signal in the root susceptible zone and following nodule development, we also identified a later increase in cytokinin biosynthesis gene expression. Once nodules are present, LjIpt3, LjLog1, and LjCyp735a are strongly induced while LjIpt2 and LjLog4 are no longer significant. The later expression of LjLog1 indicates that it may be related to the Log Plant Physiol. Vol. 175, 2017

genes reported at later stages of M. truncatula nodule development (Mortier et al., 2014). The distinct timing suggests divergent genetic networks activating cytokinin responses at different developmental stages and the requirement for ongoing cytokinin production during nodule growth and nitrogen fixation. The subtle phenotypes we found in Ljipt4 mutants and our lack of concordance with previously reported Ljipt3 phenotypes (Sasaki et al., 2014) also illustrate the redundancy present in cytokinin biosynthetic pathways and the importance of growth conditions in analyzing hormone effects. While the lack of mutations in LjIpt2 and LjLog4 prevents their unequivocal identification as essential regulators of nodule initiation, our cytokinin measurements in ipt3 ipt4 double mutants are consistent with Ipt2 being the major player in elevated cytokinin responses from the IPT family. Following cytokinin accumulation in the cortex, which may be considered a general stimulatory signal, responses must be localized to specific sites for primordia initiation. The interaction of cytokinin and ethylene signaling (Heckmann et al., 2011; Mohd-Radzman et al., 2016) and the increased nodulation of mutants in the EIN2 ortholog, sickle (Penmetsa et al., 2008), indicate that ethylene restriction of cytokinin signaling domains plays a role in this. Positive feedback regulation between MtNin and MtCre1 also plays a role in establishing nodule signaling locations (Vernié et al., 2015). In contrast to this cortical positive feedback, our finding that LjIpt2, LjLog4, and LjLhk1 levels are increased independently of LjNin indicates that a factor upstream of Nin is required to activate the cortical response in L. japonicus. It remains possible that different legume lineages have evolved some variation in the transcriptional activation networks of cytokinin signaling to account for the different cell types of the first cell divisions. The rapid symbiotic up-regulation of cytokinin biosynthesis places these genes among the first targets of the presumed epidermal-to-cortical signal and, therefore, would provide a basis to investigate signals activating their induction. Significant negative regulation of cytokinin biosynthesis occurs in an Lhk1-dependent manner to counter the effects of high cytokinin on root growth and infection. Cytokinin biosynthesis may be a target in restricting the size and progression of nodule primordia, as occurs in Arabidopsis lateral roots (Chang et al., 2015). The importance of tightly restricted cytokinin signaling domains is further illustrated by our findings with cytokinin biosynthesis overexpression. Overexpression of a complete biosynthetic pathway produced apparently uncontrolled cell divisions in a broad zone. This is consistent with the negative effects on root growth and infections observed in cytokinin overaccumulation or gain-of-function mutants (Tirichine et al., 2007; Reid et al., 2016). The absence of spontaneous nodules (Ipt overexpression) or primordia proliferation without full nodule development (Log overexpression) indicates that increases in IPT or LOG activity alone are insufficient to trigger the sufficient 371

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cytokinin increase or cell divisions required for normal nodule organogenesis. Despite robust overexpression, significant increases in cytokinin content were not observed when single genes were expressed, confirming the requirement for the expression of multiple biosynthesis genes to achieve nodule organogenesis. That pUBI:Log4 activity significantly reduces the substrate pool (iPR and tZR) without significantly increasing the cytokinin free bases is further indication that active cytokinins are tightly regulated in response to increased supply. The failure of the tissue-enhanced expression of Log4 or ubiquitous Ipt3 expression to influence the cytokinin pool, despite elevated levels of the target transcripts, also indicates the importance of the coexpression of multiple enzymes to achieve necessary cytokinin changes. Overexpression of AtIpt3 in Arabidopsis results in elevated rosette cytokinin levels, while the CKX activity remains unchanged (Galichet et al., 2008). Irreversible cytokinin degradation that regulates root cytokinin levels in L. japonicus (Reid et al., 2016) and negative feedback on endogenous biosynthesis gene expression may account for this difference. The absence of major phenotypic and metabolic changes in single biosynthesis gene mutants further supports the finely balanced nature of cytokinin metabolism. Future studies combining several of the biosynthetic enzymes under these, or alternative, tissueenhanced promoters could help to resolve this question further. The large areas of cell division activation in IptLog-Cyp735a overexpression roots relative to Log4 overexpression, snf2 plants, or BAP application show that nodule development requires increased cytokinin relative to primordia initiation (Tirichine et al., 2007; Heckmann et al., 2011). This may explain the second group of biosynthesis genes expressed at the later time (LjIpt3, LjLog1, and LjCyp735a) when a nodule is growing and a different set of transcriptional regulators may be present in the nodule. This is exemplified by the lack of pNin:GUS expression we observed in large spontaneous nodules. Nitrogen fixation also may be regulated by cytokinin at these later times, as phenotypes have been observed at the fixation stage in receptor and metabolism mutants (Boivin et al., 2016; Reid et al., 2016). In conclusion, we identified cytokinin biosynthesis and early cortical cytokinin responses as important steps in nodule organogenesis. Our data suggest that cytokinin biosynthesis is a central target of early symbiotic signaling to establish nodule initiation sites. Further understanding of the nature of transcription factor-hormone feedback and the mechanisms restricting the localization of cytokinin responses also will be important next steps in understanding nodule development.

MATERIALS AND METHODS Plant and Bacteria Genotypes Lotus japonicus ecotype Gifu was used in all experiments (Handberg and Stougaard, 1992). Homozygous LORE1 inserts were genotyped with allelespecific primers in combination with the P2 internal LORE1 primer as described 372

(Urba nski et al., 2012). Primer sequences were obtained from the LORE1 resource page (Małolepszy et al., 2016) or designed in the same region if amplification was unsuccessful (primers are listed in Supplemental Table S1). Mesorhizobium loti R7A and the NF-defective nodC variant (Rodpothong et al., 2009) were diluted to an inoculum density of OD600 = 0.01. For microscopy, the M. loti MAFF303099 strain carrying chromosomal DsRed insertion was used (Maekawa et al., 2009).

Plant and Bacteria Growth Conditions For nodulation phenotyping, 3-d-old seedlings were transferred to plastic boxes with lids that enable gas exchange (Combiness Microbox) containing a 1:4 leca:vermiculite mix and watered with nitrate-free one-quarter-strength B&D nutrients (Broughton and Dilworth, 1971). NF and cytokinin treatments were carried out in a bath of 1028 M M. loti R7A NF diluted in one-quarter-strength B&D nutrients used for mock treatment. Hairy roots were induced by infection of 6-d-old seedlings growing on vertical 0.8% (w/v) Phytagel (Sigma) plates with one-half-strength B5 salts and vitamins as described (Stougaard et al., 1987; Hansen et al., 1989; Stougaard, 1995). Three weeks after infection, primary roots were removed and the chimeric plants were transferred to plastic boxes containing a 1:4 leca:vermiculite mix. GUS staining on transformed hairy roots was performed by vacuum infiltration of GUS buffer (50 mM phosphate buffer, pH 7, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 0.05% (v/v) Triton X-100, and 0.5 mg mL21 5-bromo-4-chloro-3-indolyl-b-glucuronic acid) followed by incubation at room temperature overnight.

Bioinformatics Gene families and LORE1 inserts (Supplemental Fig. S7) were identified by BLAST in the L. japonicus genome using published IPT sequences (Chen et al., 2014; Supplemental Fig. S8) or from Arabidopsis (Arabidopsis thaliana) CYP735A (Supplemental Fig. S9) and LOG (Supplemental Fig. S10) protein sequences. Microarray or in-house RNA sequencing data were analyzed using the Lotus japonicus Gene Expression Atlas (Verdier et al., 2013) and LotusBase (lotus.au.dk; (Mun et al., 2016).

Cloning Promoter and genomic clones including introns were designed based on the L. japonicus 3.0 genome sequence (Sato et al., 2008; Małolepszy et al., 2016) and were either cloned or synthesized in GoldenGate-compatible vectors with BsaI and BpiI sites replaced with silent mutations in coding sequences (Engler et al., 2009). Overhangs were compatible with defined standards for plant synthetic biology (Patron et al., 2015). Amplification was carried out from L. japonicus MG-20 genomic DNA, and sequence-confirmed GoldenGate modules including four-base overhangs are listed (Supplemental Table S2). Promoter, coding sequence, and terminator modules were subsequently assembled and cloned into a pIV10 vector (Stougaard, 1995) modified to accept GoldenGate clones.

qRT-PCR For expression analysis, mRNA was isolated using the NucleoSpin RNA Plant kit (Macherey-Nagel). RevertAid Reverse Transcriptase (Thermo) was used for cDNA synthesis according to the manufacturer’s protocols. All cDNA samples were tested for genomic DNA contamination using primers specific for the NIN gene promoter (Lohmann et al., 2010). A LightCycler480 instrument and LightCycler480 SYBR Green I master (Roche Diagnostics) were used for the real-time quantitative PCR. ATP synthase and ubiquitin-conjugating enzyme were used as reference genes (Czechowski et al., 2005). The cDNA starting concentration for each gene was calculated using per amplicon PCR efficiency calculations calculated using LinRegPCR (Ramakers et al., 2003). Target genes were compared with the geometric mean of the housekeeping genes for each of five biological repetitions (each consisting of five to 10 plants). At least two technical repetitions were performed in each analysis. All primer sequences and determined efficiencies are listed in Supplemental Table S1.

Microscopy Confocal microscopy was performed with a Zeiss LSM510 or LSM780 Meta Confocal Microscope. Objective lenses were Zeiss Plan-Neofluar 103/0.3 and 203/0.5. Laser excitation was at 488 nm for YFP and 543 nm for DsRed, and emission filters were 505 to 550 nm for YFP and 585 to 615 nm for DsRed. For Plant Physiol. Vol. 175, 2017

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confocal sections, live roots were embedded in 3% (w/v) agarose and cut to 80- to 100-mm sections using a Leica VT 1000 S vibratome before imaging with the confocal microscope. Fluorescence-compatible clearing was conducted essentially as described (Warner et al., 2014) with the following modification: roots were harvested and fixed overnight in fixing solution (4% [w/v] p-formaldehyde and 80 mM PIPES, pH 7). Fixed samples were washed three times (5 min in 80 mM PIPES, pH 7, with shaking). Samples were cleared for 10 d in clearing solution (6 M urea, 30% [v/v] glycerol, 0.01% [v/v] Triton X-100, and 40 mM PIPES, pH 7). For TCSn sections, images were obtained using spectral imaging (Lambda mode) to unmix YFP fluorescence using a reference spectrum and epidermal cell wall autofluorescence. Images were processed using Zen software (Zeiss), and maximum intensity projections were produced using ImageJ (Rasband, 2016). Light microscopy was performed with a Zeiss Axioplan 2 microscope with a Zeiss Plan-Neofluar 103/0.3 objective lens. For light microscopy sections, roots were embedded in 4% (w/v) agarose and cut to 100-mm sections using a Leica VT 1000 S vibratome.

Supplemental Figure S6. Nodulation phenotypes of L. japonicus cytokinin biosynthesis mutants.

Endogenous Cytokinin Measurements

Supplemental Table S4. TILLING mutations in IPT4.

For cytokinin analysis, plants were grown on filter paper-covered agar slants as described previously (Reid et al., 2016). Ten-day-old plants were inoculated before whole roots were harvested at 72 h after treatment. For Agrobacterium rhizogenes-induced roots, plants were grown in a 1:4 leca:vermiculite mix for 4 weeks before harvesting and pooling roots for cytokinin analysis. Parallel samples also were used for gene expression analysis. Prepared biological quadruplicates were extracted and purified using the method published previously (Novák et al., 2008) with some minor modifications. Ten to 20 mg fresh weight was extracted in 1 mL of modified Bieleski buffer (60% [v/v] methanol, 10% formic acid [v/v], and 30% [v/v] water) together with a cocktail of 23 stable isotope-labeled cytokinin internal standards (0.5 pmol of cytokinin bases, ribosides, and N-glucosides and 1 pmol of O-glucosides and nucleotides) to check the recovery during purification and to validate the determination. The samples were purified using a combination of C18 (100 mg mL21) and MCX (30 mg mL21) cartridges and immunoaffinity chromatography based on widerange specific monoclonal antibodies against cytokinins (Faiss et al., 1997). The eluates from the immunoaffinity chromatography columns were evaporated to dryness and dissolved in 20 mL of the mobile phase used for quantitative analysis. The samples were analyzed by a liquid chromatography-tandem mass spectrometry system consisting of the Acquity UPLC System (Waters) and a Xevo TQ-S (Waters) triple quadrupole mass spectrometer. Quantification was obtained using the multiple reaction-monitoring mode of selected precursor ions and the appropriate product ion.

Supplemental Video S1. Activity of the TCSn cytokinin reporter in cortical cells surrounding ITs.

Statistical Analysis Statistical analysis was carried out using R software (R Core Team, 2015). Comparison of multiple groups included ANOVA followed by Tukey’s posthoc testing to determine statistical significance, as indicated by different letter annotations. Student’s t test or the Wilcoxon rank-sum test was used as indicated, depending on sample size when making single comparisons. All data are plotted as means with SE for the indicated number of biological replicates.

Accession Numbers L. japonicus 3.0 gene identifiers are listed in Supplemental Table S1.

Supplemental Data The following supplemental materials are available. Supplemental Figure S1. NIN transcript abundance in nodule development series. Supplemental Figure S2. Activity of the TCSn cytokinin reporter following cytokinin treatment. Supplemental Figure S3. Ipt2 promoter activity. Supplemental Figure S4. Transcript abundance of target genes in transgenic hairy roots. Supplemental Figure S5. Total cytokinin content of ipt3 and ipt4 single mutants. Plant Physiol. Vol. 175, 2017

Supplemental Figure S7. Gene structure and mutation positions for L. japonicus cytokinin biosynthesis genes. Supplemental Figure S8. Gene annotation information for L. japonicus IPT genes. Supplemental Figure S9. Gene annotation information for L. japonicus CYP735A. Supplemental Figure S10. Gene annotation information for L. japonicus LOG genes. Supplemental Table S1. Gene identifiers and primers used in this study. Supplemental Table S2. Sequences of GoldenGate modules. Supplemental Table S3. Cytokinin metabolite profile in transgenic hairy roots.

ACKNOWLEDGMENTS We thank Finn Pedersen and Karina Kristensen for glasshouse assistance, Anna Jurkiewicz for microscopy assistance, Clive Ronson, John Sullivan, and Nicolai Maolanon for purified Nod factor, Eva Hirnerová and Michaela Glosová for technical assistance in cytokinin analysis, Yasuyuki Kawaharada for ern1 seeds, and Mette H. Asmussen for production of the L. japonicus stable line. Received July 6, 2017; accepted July 18, 2017; published July 21, 2017.

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