the mtdmi2-mtpub2 negative feedback loop plays a role in ... · (venkateshwaran et al., 2015)....

The MtDMI2-MtPUB2 Negative Feedback Loop Plays aRole in Nodulation Homeostasis1[OPEN]

Jiaxing Liu,a,2 Jie Deng,a,2 Fugui Zhu,a Yuan Li,b Zheng Lu,c Peibin Qin,d Tao Wang,a,3,4 and Jiangli Donga,3,4

aState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University,Beijing, 100193, ChinabState Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China AgriculturalUniversity, Beijing, 100193, ChinacUniversity of Wyoming, Department of Atmospheric Science, Laramie, WyomingdShanghai AB Sciex Analytical Instrument Trading Co., Ltd., Chaoyang District, Beijing, 100015, China

ORCID IDs: 0000-0002-7652-7365 (J.L.); 0000-0003-2979-0803 (Y.L.); 0000-0002-8331-9265 (P.Q.); 0000-0002-4001-9828 (T.W.);0000-0003-2643-6358 (J.D.).

DOES NOT MAKE INFECTION 2 (MtDMI2) is a Leu rich repeat-type receptor kinase required for signal transduction in theMedicago truncatula/Sinorhizobium meliloti symbiosis pathway. However, the mechanisms through which MtDMI2 participates innodulation homeostasis are poorly understood. In this study, we identified MtPUB2—a novel plant U-box (PUB)–type E3 ligase—and showed that it interacts with MtDMI2. MtDMI2 and MtPUB2 accumulation were shown to be similar in various tissues.Roots of plants in which MtPUB2 was silenced by RNAi (MtPUB2-RNAi plants) exhibited impaired infection threads, fewernodules, and shorter primary root lengths compared to those of control plants transformed with empty vector. Using liquidchromatography-tandem mass spectrometry, we showed that MtDMI2 phosphorylates MtPUB2 at Ser-316, Ser-421, and Thr-488residues. When MtPUB2-RNAi plants were transformed with MtPUB2S421D, which mimics the phosphorylated state, MtDMI2was persistently ubiquitinated and degraded by MtPUB2S421D, resulting in fewer nodules than observed in MtPUB2/MtPUB2-RNAi-complemented plants. However, MtPUB2S421A/MtPUB2-RNAi-complemented plants showed no MtPUB2 ubiquitinationactivity, and their nodulation phenotype was similar to that of MtPUB2-RNAi plants transformed with empty vector. Furtherstudies demonstrated that these proteins form a negative feedback loop of the prey (MtDMI2)-predator (MtPUB2) type. Ourresults suggest that the MtDMI2-MtPUB2 negative feedback loop, which displays crosstalk with the long-distanceautoregulation of nodulation via MtNIN, plays an important role in nodulation homeostasis.

Legumes possess the capacity for symbiotic nitrogenfixation, in contrast to nonleguminous plants such asrice (Oryza sativa), maize (Zea mays), and wheat (Triti-cum aestivum; Vijn et al., 1993). Biological nitrogen

fixation by legumes is estimated to have contributedapproximately 90% of the 100 to 140 teragrams (Tg) ofannual nitrogen fixation that occurred on earth prior toagricultural activity (Gage, 2004). Leguminous plantsuse specific organs, namely nodules, to accommodatesymbiotic rhizobia and provide a compatible environ-ment for nitrogen fixation (Desbrosses and Stougaard,2011). The host plant provides rhizobia with photo-synthetic products and controls nodulation during thesymbiotic process (Magori et al., 2009; Desbrosses andStougaard, 2011), and in conjunction with this, rhizobiasupply the host with ammonia (Magori et al., 2009).Nodulation must be regulated, because nitrogen fixa-tion consumes a large amount of energy (Fergusonet al., 2010; Mortier et al., 2012). Nitrogen and carbonmetabolism and photosynthesis are also in homeostasisin plants (Huppe and Turpin, 1994). As noted above,nodulation homeostasis might be maintained throughmany mechanisms.

Previous studies have proposed a model for the au-toregulation of nodulation (AON) involving root-to-shoot communication to maintain an optimal numberof nodules by systemically reducing nodulation (Searleet al., 2003; Soyano et al., 2014). In another study, nodfactor (NF)-induced ethylene production was shown to

1 This work was financially supported by grants from the NationalNatural Science Foundation of China (31772658 and 31571587) andthe Project for Extramural Scientists of State Key Laboratory of Agro-biotechnology (2018SKLAB6-16).

2 These authors contributed equally to this work.3 These authors contributed equally to this work.4 Address correspondence to [email protected] and wangt@cau.

edu.cn.The authors responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) are:Jiangli Dong ([email protected]) and Tao Wang ([email protected]).

J.L. and J. Deng performed the main experiments and analyzed thedata together; F.Z. participated in the modification of some con-structs; Y.L. participated in the in vitro phosphorylation assays;Z.L. performed the mathematical modelling and calculations; P.Q.performed the LC-MS/MS assays; J. Dong and T.W. led the studyand revised the manuscript; all authors read and approved the finalarticle.

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Plant Physiology, April 2018, Vol. 176, pp. 3003–3026, www.plantphysiol.org 2018 American Society of Plant Biologists. All Rights Reserved. 3003

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http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0003-2643-6358

http://orcid.org/0000-0002-7652-7365

http://orcid.org/0000-0003-2979-0803

http://orcid.org/0000-0002-8331-9265

http://orcid.org/0000-0002-4001-9828

http://orcid.org/0000-0003-2643-6358

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inhibit nodulation, suggesting a local equilibriummechanism that controls nodule numbers (Oldroydet al., 2001). It is very likely that more than one mech-anism plays a critical role in maintaining this equilib-rium. If such a mechanism exists, which molecule is theregulatory target?

The DOES NOT MAKE INFECTION 2 (MtDMI2)-MtDMI1-MtDMI3 signaling pathway plays a vital rolein the Medicago truncatula/Sinorhizobium meliloti symbi-otic process (Oláh et al., 2005). MtDMI1 (the ortholog ofLotus japonicus CASTOR and POLLUX) is an ion chan-nel that controls calcium oscillations, which are re-quired for symbiosis in legumes (Ané et al., 2004; Rielyet al., 2007; Charpentier et al., 2008). MtDMI3 (theortholog of L. japonicus CCaMK) is a Ca2+/calmodulin-dependent protein kinase that acts downstream of cal-cium spiking and is required for nodulation signaltransduction (Lévy et al., 2004; Mitra et al., 2004).MtIPD3 (the ortholog of L. japonicus CYCLOPS) inter-acts withMtDMI3 and is also required for the symbioticsignaling pathway (Horváth et al., 2011). Recent studieshave indicated that LjCYCLOPS is a phosphorylationsubstrate of LjCCaMK and that it can bind the promoterof the nodule inception gene (LjNIN) to initiate symbi-otic root nodule development (Singh et al., 2014).MtDMI2 (AJ418369.1, also known as SYMBIOSIS RE-CEPTORKINASE [SymRK] in L. japonicus) is a Leu-richrepeat-type receptor kinase with an extracellular do-main containing three Leu-rich motifs, a malectin-likedomain in the extracellular domain, and a Ser/Thr-typekinase domain in the intracellular domain (Endre et al.,2002; Antolín-Llovera et al., 2014). MtDMI2 actsdownstream ofNODFACTORPERCEPTION (MtNFP)and LYS MOTIF-CONTAINING RECEPTOR-LIKEKINASE 3 (MtLYK3; Endre et al., 2002; Antolín-Llovera et al., 2014).

Because of the importance of MtDMI2 in the rhizhobiaand Arbuscular mycorrhiza symbiosis signaling pathway,previous studies have identified a number of its inter-acting proteins. MtPUB1, a plant U-box (PUB)-type E3ligase, interacts with MtLYK3 and negatively regulatesnodulation (Mbengue et al., 2010). A recent study sug-gests that MtDMI2 can interact with and phosphorylateMtPUB1 in the symbiosis pathway (Vernié et al., 2016).However, in these two cases, the targets of MtPUB1and the molecular mechanisms remain unknown.MtHMGR1, a 3-hydroxy-3-methylglutaryl CoA reduc-tase 1 in the mevalonate pathway, is involved in nod-ulation, interacts with MtDMI2, and is necessary fornodulation in M. truncatula (Kevei et al., 2007). Fur-thermore, research indicates that mevalonate is the di-rect product of MtHMGR1 and that it might be anovel second messenger in the nodulation pathway(Venkateshwaran et al., 2015). Similarly, LjSymRK-INTERACTING PROTEIN 2, a type of MAPKK fromL. japonicus, interacts with LjSymRK to regulate noduleorganogenesis (Chen et al., 2012). LjSymRK-interactingE3 ubiquitin ligase (LjSIE3) is a REALLY INTERESTINGNEWGENE finger-type E3 ligase that can use LjSymRKas a substrate for ubiquitination. LjSIE3 is a positive

regulator involved in nodulation. Moreover, SEVEN INABSENTIA 4 (LjSINA4) has been reported to be involvedin the turnover of LjSymRK, and their interaction is spa-tiotemporally controlled in L. japonicus during nodulation(Den Herder et al., 2012); however, the exact mechanismsthrough which LjSIE3 and LjSINA4 regulate LjSymRKremain unclear (Den Herder et al., 2012; Yuan et al., 2012).

As mentioned above, although several proteins in-teract with MtDMI2 and LjSymRK, the molecularmechanisms contributing to nodulation homeostasisvia phosphorylation and ubiquitination are poorlyunderstood. To determine the direct regulator ofMtDMI2, we used a yeast two-hybrid (Y2H) approachand identified a novel PUB-type E3 ubiquitin ligase thatinteracts with MtDMI2, which we named MtPUB2. Bi-ochemical functional assays of MtPUB2 showed that ithas E3 ubiquitin ligase activity, and the conservedamino acid Val-274 in the U-box domain is required forE3 activity. MtDMI2 activates MtPUB2 via phospho-rylation at Ser-421, and activated MtPUB2 directly tar-gets MtDMI2 for ubiquitination-mediated degradation.MtPUB2-RNAi roots demonstrate impaired infectionthreads, fewer nodules, shorter root length, and lowernodule densities. Biological and biochemical assaysshow that MtPUB2 and MtDMI2 exhibit similar tissueexpression, and they each show a wave-shaped curveduring nodulation. We adopted the numerical solu-tions of Lotka-Volterra equations (LV equations; Lotka,1910) to describe the protein levels of MtDMI2 andMtPUB2 during nodulation from 24 h after inoculationto 21 d after inoculation (DAI). Thus, the purpose of thenegative feedback loop between MtDMI2 and MtPUB2is to achieve a stable root nodulation system.

RESULTS

MtPUB2 Is a New PUB-Type E3 Ligase That IsHomologous to AtPUB13 and OsSPL11

To identify proteins that interact with MtDMI2, weperformedY2H screening using a cDNA library. The rootsof M. truncatula genotype cv Jemalog A17 plants weretreated with S. meliloti strain 1021 at different time points.MtDMI2IR (intracellular region, amino acids 543–919) wascloned into pGBKT7 and used as the bait. The screeningdata also includedpreviously identifiedMtHMGR1 (Keveiet al., 2007; Supplemental Fig. S1). We then selectedMtPUB2 (KU285617.1) for further testing. Full-lengthMtPUB2 DNA contains 4,531 bp and includes four exons(Supplemental Fig. S2A). Full-length MtPUB2 cDNAcontains an open reading frame of 1,989 nucleotides thatencodes apolypeptide of 662 amino acids.MtPUB2has thestructure of a UND-PUB-ARM protein and contains oneU-box domain followed by a region containing six AR-MADILLO (ARM) repeats (Supplemental Fig. S2B). Pro-tein domains were identified with the SMART (http://smart.embl-heidelberg.de/) database. A phylogeneticanalysis (MEGA4.0) showed that the closest homologs ofMtPUB2 are AtPUB13 (AT3G46510; Lu et al., 2011) andOsSPL11 (AAT94161; Zeng et al., 2004), which are

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distantly related to MtPUB1 (DAA33939) (SupplementalFig. S3). The protein sequence of MtPUB2 shares 66.77%identity with AtPUB13 and 62% identity with AtPUB12and OsSPL11.We selected two additional PUB-type proteins,

namely Medtr1g094021.1 and Medtr4g028960.1, to de-termine whether they would interact with MtDMI2IR.They did not interact with MtDMI2IR in yeast cells(Supplemental Fig. S4).MtPUB2 is associated with the plasma membrane in

onion (Allium cepa) epidermal peels (Fig. 1A). This

result was further confirmed with a cell fractionationassay using specific antibodies against H+-ATPase andcFBPase (cytosolic Fru-1,6-bisphosphatase) to validatethemembrane and cytosolic fractions, respectively (Fig.1C). MtDMI2 was also localized to the cell plasmamembrane in M. truncatula (Riely et al., 2013). Thisfinding suggested thatMtPUB2 andMtDMI2 localize tothe same subcellular compartment.

To analyze the function of MtPUB2, an in vitroubiquitination assay was performed (Fig. 1B). Val-272of OsSPL11 and Val-273 of AtPUB13 are the key amino

Figure 1. MtPUB2 is a membrane-associated protein and a novel PUB-type E3 ubiquitin ligase. A, Subcellular localizationanalysis of GFP in onion epidermal peels following particle gun-mediated transformation with Pro-35S:MtPUB2-cGFP and thevector alone (control). The peels were imaged by epifluorescence using identical exposure settings. Plasmolysis occurred 5 minafter treatmentwith 4%NaCl solution. The scale bars indicate 60mm. B (right), In vitro ubiquitination assayswith aMBP-MtPUB2andMBP-MtPUB2V274R in the presence of E1 (UBE1), E2 (UbcH5c), and Arabidopsis ubiquitin. Multiple HMWbands indicate thepoly-ubiquitination of MBP-MtPUB2 in the presence of E1 and E2 enzymes, ubiquitin, and ATP. Reactions with various com-ponents omitted (-) and MBP-MtPUB2V274R were used as controls. The MBP-MtPUB2 protein is approximately 112 kD. The MBPtag is approximately 40 kD. Both anti-MBP and anti-Ubi antibodies were used on individual immunoblots (top and bottom,respectively). M, represents protein marker. C (bottom left), Cellular fractionation assays of transient transgenic N. benthamianaleaves expressing MtPUB2-HA. Anti-cFBPase and anti-H+ATPase (PM) were used as cytoplasm and membrane markers, respec-tively.

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MtDMI2-MtPUB2 Function in Nodulation Homeostasis






acids for maintaining the ubiquitination ability of theseE3 ligases. This residue is highly conserved in differentU-box proteins andwas demonstrated to be vital for thebiological and biochemical functions of U-box proteins(Zeng et al., 2004; Liu et al., 2012). Therefore, Val-274 inMtPUB2 was substituted with Arg to further study itsrole in ubiquitination. MtPUB2 and MtPUB2V274R wereboth tagged with a maltose-binding protein (MBP-MtPUB2 and MBP-MtPUB2V274R), expressed in Esche-richia coli strain BL21, and purified using amylose resin.Mutant MBP-MtPUB2V274R did not self-ubiquitinate(Fig. 1B, lane 1). MtPUB2 was converted to a mixtureof high-Mr ubiquitinated protein products in the pres-ence of E1 (Ub-activating), E2 (Ub-conjugating), ubiq-uitin, and ATP, suggesting that MtPUB2 is capable ofself-ubiquitination (Fig. 1B, lane 2). The high-molecular-weight (HMW) bands were not observed inreactions lacking ubiquitin, E1, E2, or MtPUB2 (Fig. 1B,lanes 3–6). Antibodies recognizing MBP and ubiquitinwere both used in immunoblot analysis (Fig. 1B).However, there were some unknown proteins, recog-nized by the anti-Ubi antibody in lanes 1, 3, 4, 5, and 6.These bands may be E1 or E2 with ubiquitin (Fig. 1B,anti-Ubi; Lu et al., 2011).

In summary, these results indicate that MtPUB2, ahomolog of AtPUB13/PUB12 and OsSPL11, is a novelPUB-type E3 ligase in M. truncatula.

MtPUB2 Affects the Number of Infection Threadsand Nodules

Mtpub2 mutants were not available in the fast-neutron (Rogers et al., 2009) or transposable element oftobacco (Nicotiana tabacum) cell type 1 insertion collection(Noble Foundation; Cheng et al., 2011). Therefore, tofunctionally characterize MtPUB2, we created Agro-bacterium rhizogenes-mediated RNAi hairy root culturesand stable Agrobacterium tumefaciens-mediated RNAilines.

The A. rhizogenes-mediated RNAi plants were plan-ted on square petri dishes (130 3 130 mm) with Fåh-raeus medium lacking nitrogen (pH 6.5) and inoculatedwith S. meliloti strain 1021 after 7 d of growth. The RNAiefficiency in A. rhizogenes-mediated hairy root cultureswas approximately 72.8% in the test transgenic samples(Fig. 2B). Infection threads (ITs) were imaged afterstaining of hairy root cultures with 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside acid (X-gal) and countedin the control (empty vector [EV], GUS-RNAi) andMtPUB2-RNAi lines at 5 DAI. In both GUS-RNAi andMtPUB2-RNAi cultures, two types of ITs exist: ITs inthe outer cortical cells and ITs in the inner cortex cells(Fig. 2C; Supplemental Fig. S5). However, the averagetotal number of ITs was decreased approximately 2- to4-fold inMtPUB2-RNAi root cultures compared to thatin control root cultures (Fig. 2D).

We scored the average nodule numbers in GUS-RNAi andMtPUB2-RNAi hairy root cultures at 21 DAI.The average number of nodules in the MtPUB2-RNAi

lines was decreased compared with the control GUS-RNAi lines. Approximately 83% fewer nodules formedon average in the MtPUB2-RNAi root cultures than incontrol root cultures (Fig. 2, A and E).

Taken together, our data indicate that MtPUB2 par-ticipates in the development of ITs and nodules. Pre-vious studies revealed that MtDMI2 knock-downblocks the release of rhizobia in the nodule, and the ITsof dmi2-1 plants are aberrant (Limpens et al., 2005).Therefore, MtPUB2 andMtDMI2 both participate in thedevelopment of ITs and nodulation formation.

MtPUB2 Affects Primary Root Length, Nodule Densities,and Nodule Development in M. truncatula

We analyzed the nodulation phenotypes of stable-transgenic MtPUB2-RNAi lines to further verify thebiological function ofMtPUB2. The RNAi efficiencies instable transgenic MtPUB2-RNAi lines (T3 generation)were 0.60, 0.65, and 0.70 in lines 32, 115, and 119,respectively (Fig. 3E). We also tested the relative ex-pression of the other PUB gene, Medtr1g094025.1, instable-transgenic MtPUB2-RNAi lines; the relative ex-pression of this gene was not reduced in the roots ofMtPUB2-RNAi plants compared to that in controlplants (Supplemental Fig. S6). This result indicates theefficient and specific down-regulation of MtPUB2.

An RNAi efficiency of 0.95 caused severe reactions,such as delayed plant growth, sterility, and death.For the nodulation phenotype assays, the seeds ofEV-transformed plants and stable-transgenic MtPUB2-RNAi plants (32, 115, and 119) were planted on Fåh-raeus medium without nitrogen (pH 6.5) and inocu-lated with S. meliloti strain 1021 after 7 d of growth.The nodule number, primary root length (cm), andnodule density (No. cm21) were measured in theEV-transformed plants and stable-transgenic MtPUB2-RNAi plants at 21 DAI.

An analysis of the distribution of nodule numbers perplant indicated that approximately 63.3% of MtPUB2-RNAi (line 32, line 115, and line 119) roots producedzero or one nodule, whereas 20% of roots showed twonodules and 13.3% of roots had three nodules (n = 15,three biological repeats/line; Fig. 3, A and H; Qiu et al.,2015). All three stable-transgenicMtPUB2-RNAi plantsexhibited retarded growth (Fig. 3, A and C). Con-versely, 100% of the EV-transformed plants developed5 to 10 nodules (Fig. 3, A and H). Moreover, theMtPUB2-RNAi plants exhibited a significant reductionin primary root lengths (5.8–7 cm, Fig. 3, A and G) andnodule densities (0.5–0.8 cm21 No. root length; Fig. 3, Aand F) comparedwith EV-transformed plants (7.8–8 cmroot length and 1.5 No. cm21 root length; Fig. 3, A, G,and F). These results demonstrated that the reducednodule numbers were not caused by the short rootlength. As shown in Figure 3, B and D, the nodules onEV-transformed plants and stable-transgenic MtPUB2-RNAi plants were both pink at the time of harvest(21 DAI). At 21 DAI, although all appeared pink in


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color, the maturation of nodules was different (Fig. 3B).We further investigated the role of MtPUB2 in nodulesenescence. Lugol staining is used to indicate thepresence of amyloplasts, the accumulation of which is

linked to premature nodule senescence as shown inseveral studies using bacteria defective in nitrogen fix-ation (Lodwig et al., 2003; Harrison et al., 2005). Lugolstaining was performed in 21-DAI and 42-DAI nodules.

Figure 2. Inhibition of nodulation and infection threads by knock-down of MtPUB2 in A. rhizogenes-mediated MtPUB2-RNAihairy root cultures. A, Nodulation in GUS-RNAi and MtPUB2-RNAi hairy root cultures generated by A. rhizogenes-mediatedtransformation at 21 DAI. The black arrows indicate enlarged photos of the roots and nodules. B, The MtPUB2-RNAi constructefficiently down-regulates MtPUB2 gene expression. MtPUB2 mRNA quantification was normalized against two internal ref-erence genes (Mtactin andMtEF1a) in each sample. The SDs between samples are shown as error bars. C, ITs inGUS-RNAi roots(top) and MtPUB2-RNAi roots (bottom) at 5 DAI imaged by staining with X-gal. ITs are labeled by black arrows. The scale barsindicate 100 mm. D, Quantification of ITs depicted in C. Shown are mean values 6 SE calculated from the three independentexperiments (** indicates significant differences betweenGUS-RNAi andMtPUB2-RNAi, P# 0.01, Student’s t test, n = 20, threerepeats). E, Quantification of nodulation depicted in A. Nodule numbers were counted in GUS-RNAi and MtPUB2-RNAi hairyroot cultures at 21 DAI. Depicted are mean values 6 SE calculated from the three independent experiments (** indicates sig-nificant differences between GUS-RNAi and MtPUB2-RNAi, P # 0.01, Student’s t test, n = 20, three repeats).





Figure 3. Inhibition of nodulation, root length, and development by knock-down of MtPUB2 in A. tumefaciens-mediatedMtPUB2-RNAi and EV stable-transformed lines. A, Whole plants of EV-transformed lines andMtPUB2-RNAi lines (lines 115 and119) at 21 DAI. Left, EV-transformed lines; right, MtPUB2-RNAi lines 115 and 119. B, Close-up stereomicroscope view of theroots and nodules at 21 and 42 DAI. Bar = 5 mm. C, The development of 49-d-old plants of MtPUB2-RNAi lines and


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At 21 DAI, the nodules of EV-transformed plants weremature (Fig. 3D); however, the nodules of MtPUB2-RNAi plants were immature (Fig. 3D). At 42 DAI, moresenescent cells were observed in the nodules ofEV-transformed plants than in those of MtPUB2-RNAiplants (Fig. 3D). In summary, the down-regulation ofMtPUB2 can induce a significant delay in nodule de-velopment.Stable-overexpression transgenic plants were also

created using a pCAMBIA1302-MtPUB2 vector along-side control plants transformed with pCAMBIA1302 asthe EV. The plants were also inoculated with S. melilotistrain 1021 after 7 d of growth on square petri dishes(130 3 130 mm). The nodule numbers were counted21 DAI. However, there was no difference in nodulenumber or primary root length (cm) between theMtPUB2-overexpressing plants and the EV-transformedplants (Supplemental Fig. S7, A and C). Reverse tran-scription quantitative-PCR (RT-qPCR) was used tomeasure the relative expression of MtPUB2 in theoverexpressing lines (Supplemental Fig. S7B).Thus, MtPUB2 not only is involved in the develop-

ment of plants, but also participates in the formationand development of nodules.

MtPUB2 Shows Similar Tissue Expression Patternsas MtDMI2

To assess the tissue expression pattern of MtPUB2, a2.3-kb promoter region of MtPUB2 was cloned andinserted into the pkGWFS7 vector (Huault et al., 2014)carrying a downstreamGUS fusion construct. StableM.truncatula transformants were prepared. In non-inoculated plants, proMtPUB2-GUS expression wasobserved in the leaves, stems, and roots (Fig. 4, A–C)and particularly in the cortex, endodermis, pericycle,and phloem of the roots (Fig. 4, C and G). proMtPUB2-GUS was expressed in the primordial of nodules at5 DAI with S. meliloti strain 1021 (Fig. 4F). At 21 DAIwith S. meliloti strain 1021, proMtPUB2-GUS wasexpressed in different parts of the nodules, such as thedistal, apical, and persistent meristem (zone I) and inzones of increasing cell age, comprising an infection zone(zone II), an interzone (zone II-III), and a nitrogen-fixation

zone (zone III; Fig. 4, D and H). RT-qPCR assays con-firmed the expression of MtPUB2 in the roots, stems,leaves, and nodules (Fig. 4E). The results showed thatMtPUB2 is primarily expressed in the roots and nod-ules (Fig. 4, A–E).

The expression pattern of MtDMI2 has been studiedpreviously (Bersoult et al., 2005), and is similar to that ofMtPUB2 in the roots and nodules of M. truncatula.

MtPUB2 and MtDMI2 Interact in Vitro and in Planta

To confirm that MtPUB2 and MtDMI2 interact, aY2H assay, a glutathione S-transferase (GST) pull-down assay, and a bimolecular fluorescence comple-mentation (BiFC) assay were performed. The results ofthe Y2H assay showed that in yeast, MtPUB2 specifi-cally interacts with MtDMI2. COMPACT ROOT AR-CHITECTURE 2 (MtCRA2) andMtLYK3, which do notinteract with MtPUB2 in yeast (Fig. 5A), were used ascontrols. The truncated domain of MtPUB2 was clonedinto pGBKT7, and the sequence encoding the proteinkinase domain ofMtDMI2was cloned into pGADT7. Inaddition, this interaction occurred in the ARM repeatsdomain but not in the U-box domain of MtPUB2(Supplemental Fig. S2C). The interaction was alsotested through a GST pull-down assay. Purified His-MtPUB2 and GST-MtDMI2IR proteins were employedin GST pull-down assays (Fig. 5B). The 76-kD His-MtPUB2 protein was found to associate with the71-kD GST-MtDMI2IR protein, whereas the controlGST protein (27 kD) did not display a positive interac-tion.

Furthermore, a BiFC assay was performed by coex-pressing the split yellow fluorescent protein (YFP) paircombinations in onion epidermal peels. The C-terminaldomain of YFP was fused to the C terminus of MtDMI2(MtDMI2-Yc), whereas the N-terminal domain of YFPwas fused to the N terminus of MtPUB2 (Yn-MtPUB2).YFP fluorescence (green) was detected in onion epi-dermal cells 16 h after excitation in the transient BiFCassay. The results showed interaction at the plasmamembrane of the cells (Fig. 5C).

In summary, these results show that MtDMI2 inter-acts with MtPUB2 in vitro and in planta.

Figure 3. (Continued.)EV-transformed lines in soil. D, Lugol staining of the nodules of EV-transformed lines and MtPUB2-RNAi line 119 at 21 and42 DAI. Bar = 200 mm. E, RT-qPCR analysis of relativeMtPUB2 expression in the stableMtPUB2-RNAi lines 32, 115, and 119 (T3

generation). Shown are mean values 6 SE calculated from three independent experiments (** indicates significant differencebetween EV-transformed lines andMtPUB2-RNAi lines, P# 0.01, Student’s t test). F, Nodule density (No. cm21 root length) assayin EV-transformed lines and MtPUB2-RNAi lines (lines 32, 115, and 119). Shown are mean values 6 SE calculated from threeindependent experiments (** indicates significant difference between EV-transformed lines and MtPUB2-RNAi lines, P # 0.01,Student’s t test, n = 10, three repeats). G, Root length (cm) of 4-week-old plants of EV-transformed lines andMtPUB2-RNAi lines(lines 32, 115, and 119). Shown are mean values 6 SE calculated from three independent experiments (** indicates significantdifference between EV-transformed lines andMtPUB2-RNAi lines, P# 0.01, Student’s t test, n= 10, three repeats). H, The numberof nodules on each of the transgenic root systems of each plant transformed with the EV or pANDA-MtPUB2 (lines 32, 115, and119, n = 15, three biological repeats/line). The x axis represents the nodule numbers, while the y axis represents the number ofplants.








Figure 4. Histochemical localization ofproMtPUB2-GUS in M. truncatula. Theexpression pattern of MtPUB2 was testedusing its 2.3-kb promoter region. M. trun-catula plants transformed by A. tumefa-ciens were used for proMtPUB2-GUSassays during nodulation using X-gluc (bluein A—H). A to C, Expression of proMt-PUB2-GUS in the leaves, stems, and roots,respectively, of 4-week-old M. truncatulaplants. The scale bars indicate 200 mm in Aand B, and 100 mm in C. D, Expression ofproMtPUB2-GUS in nodules of M. trunca-tula roots at 21 DAI. The scale bar indicates200 mm. E, RT-qPCR analysis of relativeMtPUB2 gene expression in the root, stem,leaves, and nodules of 4-week-old wild-type R108 plants (internal reference geneswere MtEF1a and Mtactin). Error bars inthis panel represent the SDs calculatedfrom 15 samples. F, Expression of proMt-PUB2-GUS in the primordia of nodules at5 DAI (black arrows indicate the primordiaof nodules). The scale bar indicates200 mm. G, Expression of proMtPUB2-GUS in the cortex, inner cortex, pericycle,and the phloem of the roots. The scale barindicates 100 mm. H, Expression ofproMtPUB2-GUS in the longitudinal sec-tion of 21-d-old nodules (section, 80 mmthick). I (Zone I, a distal, apical, and per-sistence meristem followed by zones ofincreasing cell age), II (Zone II, comprisingan infection zone), II to III (Zone II–III, aninterzone), III (Zone III, a nitrogen-fixationzone). The scale bar indicates 200 mm. Allimages were taken under a light micro-scope.


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Figure 5. Interaction assays between MtDMI2 and MtPUB2. A, Y2H assays of MtPUB2, MtDMI2IR, MtLYK3, and MtCRA2(intracellular region) in transformed Saccharomyces cerevisiae AH109 cells grown on SD/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trpmedium. pGBKT7-p53/pGADT7-RecTand pGBKT7/pGADT7were used as positive and negative controls, respectively. An a-Galassay was performed on SD/-Ade/-His/-Leu/-Trp medium. B, GST-pull down assay of MtPUB2 and MtDMI2IR. His-MtPUB2 waspulled down by GST-MtDMI2IR protein but not by the GST protein alone. The GST-MtDMI2IR fusion protein is approximately71 kD. The GST tag is 27 kD. The His-MtPUB2 fusion protein is approximately 76 kD. Themolecular weights of these proteins areindicated on the left of the blot. C, A BiFC analysiswas performed by coexpressing split YFP pair combinations in onion epidermalpeels. The C-terminal domain of YFP was fused to the C terminus of MtDMI2 (MtDMI2-Yc), and the N-terminal domain of YFPwas fused to the N terminus of MtPUB2 (Yn-MtPUB2). YFP fluorescence (green) was imaged by epi-fluorescence using identicalexposure settings. The scale bar indicates 60 mm.





MtPUB2 Mediates MtDMI2 Ubiquitination and ItsAssociated 26S Proteasome-Dependent Degradation inVitro and in Planta

MtPUB2 has E3 ubiquitin ligase activity and interactswith MtDMI2. Thus, we performed in vitro ubiquiti-nation assays and in vivo degradation assays to testwhether MtDMI2 is the ubiquitination substrate ofMtPUB2. In vitro ubiquitination assays revealed thatMtPUB2 ubiquitinates MtDMI2 (Fig. 6A, lane 4).MtPUB2V274R did not ubiquitinateMtDMI2 in vitro (Fig.6A, lane 2). This result demonstrated that Val-274 is akey amino acid for E3 ubiquitin ligase activity, as ob-served in the self-ubiquitination assays. The HMWbands were not observed in reactions lacking ubiquitin,E1, E2, or MtPUB2. GST protein was also used as anegative control. Next, we examined these samples byimmunoblot analysis using antibodies that recognizeGST and ubiquitin. However, there were some un-known proteins recognized by the anti-Ubi antibody inlanes 1, 2, 6, 7, 8, and 9. This result might be caused byE1 or E2 with associated ubiquitin (Fig. 6A).

We then co-infiltrated agrobacterium host constructsexpressing MtPUB2-HA andMyc-MtDMI2 with controlsinto the same leaf areas of N. benthamiana to test in vivodegradation. Samples were collected to detect the proteinand RNA levels of the constructs. In this experiment,GFP-HA was used as an internal control to determinewhether equal amounts ofMtPUB2were expressed in thedifferent co-infiltrations. Quantitative immunoblot anal-ysis showed that MtDMI2 was degraded by the additionof MtPUB2 (Fig. 6B, lane 1), but this degradation wasprevented by the addition of (N-[(phenylmethoxy)-carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leu-cinamide (MG132; 26S proteasome inhibitor; Fig. 6B, lane2). The mutant MtPUB2V274R-HA did not mediate thedegradation of MtDMI2 in the presence or absence ofMG132 (Fig. 6B, lanes 3 and 4). RT-qPCR was used tofurther verify that this degradation occurred at the pro-tein level.NbEF1awas used as an internal reference gene.There were no obvious changes in MtDMI2 or NbEF1atranscript levels. These results showed that MtPUB2mediates the degradation of MtDMI2.

To determine whether the degradation of MtDMI2involved ubiquitination, we performed semi-in vivodegradation assays. Total proteins were extracted fromN. benthamiana expressing Myc-MtDMI2 or MtPUB2-HA. The protein extracts were then mixed at 4°C forthe indicated times (0–6 h). These samples were probedwith anti-HA and anti-Myc antibodies. In this experi-ment, GFP-HA was used as an internal control. To testwhether this degradation was mediated by the 26Sproteasome instead of another pathway, dimethylsulfoxide and MG132 were added to the samples. Theresults show that the degradation of MtDMI2 was en-hanced over time (0–6 h; Fig. 6C), this degradation wasprevented by the addition of MG132 (0–6 h, Fig. 6D).

In summary, MtPUB2 mediates the ATP-dependentdegradation ofMtDMI2 through ubiquitination and the26S proteasome pathway.

MtDMI2 Phosphorylates MtPUB2 in Vitro and in Vivo

Because MtDMI2 has a protein kinase domain(amino acids 595 to 867), we investigated whetherMtDMI2 kinase could phosphorylate MtPUB2. As ex-pected, strong MtDMI2IR auto-phosphorylation wasobserved (Fig. 7A, lane 4). In the presence of GST-MtDMI2IR, MBP-MtPUB2 was phosphorylated (Fig.7A, lane 3), whereas MBP protein alone showed weakphosphorylation (Fig. 7A, lane 8). Alignment of theMtDMI2 intracellular domain and LjSYMRK revealedthat Thr-762 in MtDMI2 is equivalent to Thr-760 inLjSYMRK (Yoshida, 2005). In vitro phosphorylationassays indicated that GST-MtDMI2IRT762A did not un-dergo auto-phosphorylation unlike GST-MtDMI2IR(Fig. 7B).

A phos-tag reagent (BOPPARD) was used in aphospho-protein mobility shift assay to detect thephosphorylation of MtPUB2 in vivo. Proteins were ac-quired from the roots and nodules of wild-type roots(M. truncatula cv Jemalong A17) treated with S. melilotistrain 1021 at 7, 14, and 21 DAI. Immunoblot analysisshowed that MtPUB2 is phosphorylated by MtDMI2 atthe three time points in vivo (Fig. 7, C and D). At21 DAI, the root proteins were treated with calf intes-tinal alkaline phosphatise (CIAP), and the resultsshowed that the phosphorylation bands of MtPUB2disappeared (Fig. 7C).

To explore whether MtPUB2 can be specificallyphosphorylated by MtDMI2 in vivo, we subsequentlyperformed phos-tag assays using nodulated 7-DAI,14-DAI, and 21-DAI wild-type A17 and dmi2-1 (TR25)roots. The results showed that the smear of MtPUB2phosphorylated bands was greatly diminished insamples prepared from dmi2-1 (TR25) roots comparedwith that from wild-type A17 roots, and the MtPUB2bands were also diminished (Fig. 7D). Moreover, im-munoblot analysis revealed more phosphorylatedforms in the nodulated wild-type A17 roots at 7 DAIthan at 14 DAI or 21 DAI (Fig. 7D). These results clearlysuggest that MtDMI2 has strong auto-phosphorylationactivity and that it can phosphorylate MtPUB2 in vitroand in vivo.

In summary, MtPUB2 can be phosphorylated byMtDMI2 in vivo, and the stability of MtPUB2 dependson its phosphorylation status.

MtPUB2 Can Be Phosphorylated by MtDMI2 at Ser-421,Which Appears to Enhance the E3 Ubiquitin LigaseActivity of MtPUB2

Weperformed a liquid chromatography-tandemmassspectrometry (LC-MS/MS, SCIEX) analysis to identifywhich amino acid of MtPUB2 is phosphorylated byMtDMI2. Purified GST-MtDMI2IR and His-MtPUB2proteins were used in the in vitro phosphorylation re-action (three biological repeats), and the phosphorylatedMtPUB2 was used for LC-MS/MS. The results suggestthat Ser-316, Ser-421, and Thr-488 are the potentialMtPUB2 phosphorylation sites (Supplemental Fig. S8,


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Figure 6. MtPUB2 targets MtDMI2 for ubiquitination and degradation via the 26S proteasome pathway. A, In vitro ubiquitinationof MtDMI2 by MtPUB2. The ubiquitination of GST-MtDMI2IR (MW = 71 kD) by MBP-MtPUB2 (MW = 112 kD) was detected byanti-GST and anti-Ubi antibodies in the presence of E1 (UBE1), E2 (UbcH5c), and Arabidopsis ubiquitin. MBP-MtPUB2V274R,MBP, and GST proteins were used as negative controls. B, Degradation of MtDMI2 promoted by MtPUB2 in N. benthamianaleaves. Either MtPUB2-HA or Myc-MtDMI2 was coexpressed with GFP-HA by agro-infiltration in N. benthamiana. Tissues wereharvested 3 d after infiltration. MG132 (50 mM) was added to the corresponding protein mixture samples to prevent proteindegradation by the 26S proteasome. Estimates of MtDMI2 protein levels relative to the GFP-HA protein are shown at the bottom.GFP-HA was expressed as an internal control. Expressed target genes and NbEF1a mRNA expression levels were analyzed byRT-PCR (bottom). TheMyc-MtDMI2 protein is 111.8 kD. TheMtPUB2-HA protein is 84 kD. The 63Myc tag is approximately 7.9kD. The 33 HA tag is 12 kD. C and D, MG132 was either added (C) or not added (D) to the corresponding protein mixturesamples. Ponceau S staining (bottom) of the Rubisco protein is shown as a loading control. Reactions with various componentsomitted (2) were used as controls.





Figure 7. Phosphorylation assays with MtDMI2 and MtPUB2 in vitro and in M. truncatula. A, Phosphorylation of MtPUB2 byactivated MtDMI2 in vitro. Purified GST-MtDMI2IR was incubated with MtPUB2-MBP in the presence of [g-32P]ATP and kinasereaction productswere resolved on SDS-PAGE gel. The star indicates the phosphorylation ofMtPUB2 byMtDMI2. Reactionswithvarious components omitted (2) were used as controls. The CBB showed the SDS-PAGE of GST-MtDMI2IR and MBP-MtPUB2proteins (8% SDS-PAGE gel). The red arrows represent MBP-MtPUB2, GST-MtDMI2IR, and GST proteins. M, protein marker. B,Auto-phosphorylation of MtDMI2 in vitro. Purified GST-MtDMI2IR and GST-MtDMI2IRT762A were incubated in the presence of[g-32P]ATP, and kinase reaction products were resolved on SDS-PAGE gel (12%). C, Phos-tag assays with MtPUB2. Proteins wereharvested from the roots of wild-type A17 roots inoculated with S. meliloti strain 1021 at 21 DAI. Samples were separated in aphos-tag SDS-PAGE gel. b-Actin was used as a loading control. Reactions with various components omitted (2) were used ascontrols. For CIAPassays, the proteins were treated with CIAPat 37°C for 30 min and then detected through immunoblot analysiswith anti-MtPUB2 polyclonal antibodies (BPI Co) and the antiactin antibodies (CWBio). D, Phos-tag mobility shift detection ofphosphorylated MtPUB2 in wild-type A17 roots and dmi2-1 (TR25) roots after inoculation with S. meliloti strain 1021 at 7 DAI,14 DAI, and 21 DAI. Protein extracts from the roots treated with S. meliloti strain 1021 for different times were separated in anSDS-PAGE gelwith phos-tag reagent.b-Actinwas used as a loading control. P in B, C, andD represents the phosphorylated bands,while np represents the nonphosphorylated bands.


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A–C). These sites were identified in the three biologicalreplicates, and we found that the prediction of phos-phorylation sites passed a 99% confidence level. Fur-thermore, Ser-421 and Thr-488 were located in theARM-repeats domain, and Ser-316 was in the U-boxdomain of MtPUB2. We subsequently only focused onthese three amino acids.We then performed the in vitro ubiquitination assays

to investigate which phosphorylation site is most im-portant. Unphosphorylated state protein mimics(MtPUB2S421A, MtPUB2T488A, and MtPUB2S316A) andphosphorylated state protein mimics (MtPUB2S421D,MtPUB2T488D, and MtPUB2S316D) were purified inE. coli for in vitro ubiquitination analysis. An immu-noblot analysis indicated that the self-ubiquitinationactivity of MtPUB2S421A was significantly reduced,whereas MtPUB2S421D exhibited enhanced activitycompared with MtPUB2 (Fig. 8A). The MBP-MtPUB2S421D protein was also tested for ubiquitinationin the presence or absence of E1, E2, and ubiquitin(Supplemental Fig. S9).In contrast, the self-ubiquitination activity of

MtPUB2S316A was comparable to that of MtPUB2, andMtPUB2S316D exhibited no self-ubiquitination activity(Fig. 8A). However, MtPUB2T488A and MtPUB2T488D

displayed activity comparable to that of MtPUB2. Wethen performed the in vitro ubiquitination assays usingMtDMI2 as the substrate. MtDMI2IR ubiquitination byMtPUB2S421A was greatly reduced compared withubiquitination by MtPUB2S421D or MtPUB2. These re-sults reveal that Ser-421, and not the Thr-488 site in theARM-repeat domain, is the key phosphorylation site ofMtPUB2 required for its activation (Fig. 8, A and B). Wealso performed in vitro ubiquitination assays withMtDMI2IR,MtPUB2S316A, andMtPUB2S316D. The resultsshowed that MtPUB2S316D could not poly-ubiquitinateMtDMI2, whereas MtPUB2S316A could use MtDMI2 as apoly-ubiquitinated substrate (Fig. 8C). The results in-dicate that the phosphorylation of Ser-316 inactivatesthe capability of E3 ubiquitin ligase; however, it is ac-tivated by the phosphorylation of Ser-421.Taking into account the involvement of MtPUB2 and

MtDMI2 proteins in nodulation, we decided to generatethe complemented plants and analyze their nodulationphenotypes. This study centers upon the biologicalfunction of Ser-421 during nodulation.

Ser-421 of MtPUB2 Plays an Important Role in Nodulation

We tested the biological functions of MtPUB2S421A andMtPUB2S421D during nodulation. MtPUB2S421A andMtPUB2S421D were overexpressed in stable-transgenic T3generation MtPUB2-RNAi lines 32 and 119, respectively,via the hairy-root transformationmethod. pCAMBIA2301-modified overexpression transformants were used as EVcontrols.Analysis of the distribution of nodule numbers per line

demonstrated that 98.6% to 100% of MtPUB2S421D

/MtPUB2-RNAi (line 119 and line 32) roots developed

zero to two nodules; by contrast, for EV/MtPUB2-RNAilines (line 119 and line 32), 90% to 94.3%of roots developedzero to two nodules and 5.7% to 10% of roots developedthree nodules (n=20–25per repeat, three biological repeatsper line; Qiu et al., 2015). Similarly, for MtPUB2S421A

/MtPUB2-RNAi lines (line 119 and line 32), 91.4% to 94.3%of roots developed zero to two nodules and 8.6% to 20%ofroots developed three tofive nodules (n=20–25per repeat,three biological repeats per line; Fig. 9, A–E; SupplementalFig. S10; Qiu et al., 2015). These data indicate that the rootsofMtPUB2S421D/MtPUB2-RNAi lines (line 119 and line 32)form fewer nodules than those of MtPUB2/MtPUB2-RNAi lines, demonstrating that the stability of MtDMI2 isnecessary to produce an appropriate nodule number (Fig.9, B and D; Supplemental Fig. S10). The nodule number inroots of MtPUB2S421A/MtPUB2-RNAi lines (line 119 andline 32) was comparable to that of EV/MtPUB2-RNAilines; they do not recover to the same level observed inMtPUB2/MtPUB2-RNAi lines (Fig. 9, B and D;Supplemental Fig. S10). MtPUB2/MtPUB2-RNAi lineswere used as the control; 35.7% to 37.1% of roots ofMtPUB2/MtPUB2-RNAi lines (line 119 and line 32) de-veloped zero to three nodules, whereas 62.9% to 64.3% ofroots developed six to eight nodules (n = 20–25 per repeat,three biological repeats per line; Fig. 9, B and D;Supplemental Fig. S10). The relative expression ofMtPUB2in the rescued roots was 1.0 in EV/MtPUB2-RNAi roots,approximately 75-fold up-regulated inMtPUB2/MtPUB2-RNAi roots, approximately 70-fold up-regulated inMtPUB2S421D/MtPUB2-RNAi roots, and approximately72-fold up-regulated in MtPUB2S421A/MtPUB2-RNAiroots (Fig. 9, C and E; Supplemental Fig. S10). Together,thesefindings clearlydemonstrate that thephosphorylatedformofMtPUB2 is essential for determining the number ofnodules. In summary, Ser-421 is not only necessary forMtPUB2 ubiquitin ligase activity but also critical for nod-ulation in M. truncatula.

Therefore, the results lead to the question of whethera negative feedback loop between MtPUB2 andMtDMI2 exists during nodulation to maintain homeo-stasis.

MtDMI2 and MtPUB2 Function in a Prey-PredatorRelationship during Nodulation

To directly test the above hypothesis, we performedquantitative immunoblot assays (with two biologicalrepeats, using actin protein as the loading control) inM. truncatula and analyzed the data using numericalsolutions in detail (Lev Bar-Or et al., 2000; Marcianoet al., 2016). Wild-type A17, dmi2-1 (TR25), andMtPUB2-RNAi plants were inoculated with S. meliloti1021 and sampled at different time points (0 h, 5 h,12 h, 24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, 21 DAI, and28 DAI) for immunoblot analysis. The results showedthat both MtDMI2 and MtPUB2 levels increased andthen decreased during the nodulation process in wild-type plants, whereas this trend was not observed indmi2-1 (TR25) or MtPUB2-RNAi plants (Fig. 10A;













Supplemental Fig. S11). We speculated that the inter-action between MtDMI2 and MtPUB2 follows a prey-predator relationship during nodulation in wild-typeplants.

To substantiate this hypothesis, we adopted LVequations, which are pairs of first-order, nonlinear,differential equations. Thismodel wasfirst proposed byLotka in 1910 (Lotka, 1910),

Figure 8. Identification of the phosphorylation sites of MtPUB2 and in vitro ubiquitin assays. A, In vitro ubiquitination assayswith MBP-tagged MtPUB2 (MBP-MtPUB2), MBP-MtPUB2S421A, MBP-MtPUB2S421D, MBP-MtPUB2S316A, MBP-MtPUB2S316D,MBP-MtPUB2T488A, and MBP-MtPUB2T488D in the presence of E1 (UBE1), E2 (UbcH5c), and Arabidopsis ubiquitin. MultipleHMW bands indicated the poly-ubiquitination of MBP-MtPUB2. The MBP-MtPUB2 protein is approximately 112 kD. TheMBP tag is approximately 40 kD. Immunoblots were incubated with anti-MBP (top) and anti-Ubi (bottom). B, In vitro ubiq-uitination of MtDMI2 by MtPUB2. The assays were performed with MBP-MtPUB2, MBP-MtPUB2S421A, MBP-MtPUB2S421D,MBP-MtPUB2T488A, and MBP-MtPUB2T488D. Poly-ubiquitination was detected with anti-Ubi antibody; anti-GSTand anti-MBPantibodies were also used in immunoblot analysis. The red boxes labels in A and B represent the in vitro ubiquitination assayswith MBP-MtPUB2S421A and MBP-MtPUB2S421D. C, In vitro assays for the ubiquitination of MtDMI2 by MtPUB2S316A andMtPUB2S316D. The ubiquitination of GST-MtDMI2IR (MW = 71 kD) by MBP-MtPUB2, MBP-MtPUB2S316A, and MBP-MtPUB2S316D (MW = 112 kD) were detected using anti-MBP, anti-GST, and anti-Ubi antibodies. The red arrows representMBP-MtPUB2 and GST-MtDMI2IR, while the red boxes labels represent the in vitro ubiquitination assays with MBP-MtPUB2S316A and MBP-MtPUB2S316D.


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Figure 9. Nodulation phenotypes of A. rhizogenes-transformed roots in rescued MtPUB2-RNAi lines. A, pCAMBIA2301-MtPUB2, pCAMBIA2301 (EV), pCAMBIA2301-MtPUB2S421A, and pCAMBIA2301-MtPUB2S421D were introduced into the stableMtPUB2-RNAi lines (lines 32 and 119), respectively. The nodulation phenotypeswere counted at 21DAI. B andD,Quantificationof nodule numbers at 21 DAI in EV/MtPUB2-RNAi lines, MtPUB2/MtPUB2-RNAi lines, MtPUB2S421A/MtPUB2-RNAi lines, and





dx=dt ¼ ax2by

dy=dt ¼ dxy2 gy

where X represents the number of prey (or MtDMI2 inour study); Y represents the number of predators (orMtPUB2); aX and dxy represent the increasing rates ofX and Y, respectively; and bXY and gY represent thedecreasing rates of X and Y, respectively. We appliedthe aforementioned method to designate repeat 1 andrepeat 2 and determined two sets of best-fit parameters(a = 0.7, b = 1.4, g = 1.2, d = 0.1, and a = 1.7, b = 2.6, g =1.0, d = 0.1).

These results verified our conjecture of the negativefeedback betweenMtDMI2 andMtPUB2 from 24 h afterinfection to 21 DAI (Fig. 10B). As shown in Figure 10, Cand D, the curves derived from LV equations perfectlydepict the MtDMI2 and MtPUB2 protein levels mea-sured at 24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, and 21 DAIin both repeat 1 and repeat 2. This negative feedbackloop was nonexistent in the inoculated dmi2-1 plantsand MtPUB2-RNAi lines (Supplemental Fig. S11).

The MtDMI2-MtPUB2 Negative Feedback Loop DisplaysCrosstalk with the AON via MtNIN

To investigate the relationship between the MtDMI2-MtPUB2 negative feedback loop and the AON, thenodulation marker genes MtNSP1, MtNIN, MtDMI1,and MtDMI3 were detected by RT-qPCR in stable-transformed MtPUB2-RNAi and wild-type roots inoc-ulated with S. meliloti strain 1021.

The relative expression of MtDMI1, MtDMI3,MtNSP1, andMtNINwas induced approximately 1.8-,1.6-, 10-, and 9-fold by inoculation with S. melilotistrain 1021 inMtPUB2-RNAi roots compared to that inwild-type roots at 7 DAI (Fig. 11, A–D). The relativeexpression of MtNIN was up-regulated throughoutthe experiment; for instance, it was up-regulated ap-proximately 9- to 16-fold in MtPUB2-RNAi rootscompared to wild-type roots at 5 and 7 DAI with S.meliloti strain 1021 (Fig. 11B). MtNIN not only is re-quired for nodule organogenesis (Marsh et al., 2007),but also plays a key role in the AON pathway (Soyanoet al., 2014). The up-regulation ofMtNINwill cause theup-regulation of CLAVATA3/ENDOSPERM SUR-ROUNDING REGION-related small peptides (CLEs;Soyano et al., 2014). We inferred that potential cross-talk exists between the MtDMI2-MtPUB2 negativefeedback loop and AON in M. truncatula.

Taken together, nodulation signals first triggered anincrease in MtDMI2 through MtLYK3 and MtNFP.MtDMI2 phosphorylates MtPUB2 on Ser-316 and Ser-421. Before 7 DAI, because the phosphorylation on Ser-316 inactivated MtPUB2, MtDMI2 was accumulatedand some unknown downstream genes were activated.From 7 DAI to 21 DAI, MtDMI2 was degraded be-cause of the activation of MtPUB2 caused by thephosphorylation on Ser-421. As a result, nodulationhomeostasis was achieved. After nodulation homeo-stasis was disrupted, we found that MtDMI2 was nolonger degraded, MtNIN expression was up-regulatedexcessively, and the number of nodules decreased. Inthe future, we will study in detail how MtPUB2Ser-316

and MtPUB2Ser-421 cooperate during nodulation(Fig. 12).

DISCUSSION

The MtDMI2-MtDMI1-MtDMI3 symbiotic signalingpathway transfers nodulation signals, and the roots ofdmi2, dmi1, and dmi3mutants display no nodules (Waiset al., 2000; Endre et al., 2002; Lévy et al., 2004; Mitraet al., 2004; Riely et al., 2007). The dmi2-1mutant (TR25)shows swollen root hair tips, but does not exhibitbranching and has a nonnodulation phenotype(Esseling et al., 2004). The down-regulation ofMtDMI2is associated with various phenotypes, including nu-merous bulbous infection threads in the central tissue ofthe nodule but without the release of bacteria (Limpenset al., 2005). This study also showed that the majority ofM. truncatula lines with down-regulated MtDMI2 didnot develop nodules (Limpens et al., 2005). Here, weidentified a new PUB-type E3 ubiquitin ligase that candegrade MtDMI2 via the 26S proteasome pathway. Weshowed that MtPUB2-RNAi roots exhibit impaired in-fection threads, fewer nodules, a shorter primary rootlength, and decreased nodule densities compared withEV-transformed roots (Fig. 3). Our results clearlyidentify a novel MtDMI2-MtPUB2 negative feedbackloop that depends on phosphorylation and ubiquiti-nation during nodulation. We speculate that thismechanism locally and tightly calibrates nodulationhomeostasis.

Regarding the negative feedback loop workingmodel, we analyzed the quantitative immunoblotanalysis data via numerical solutions (Lev Bar-Or et al.,2000; Marciano et al., 2016). As shown in Figure 10, thecurves derived from LV equations perfectly depictedthe MtDMI2 and MtPUB2 protein levels measured at24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, and 21 DAI in both

Figure 9. (Continued.)MtPUB2S421D/MtPUB2-RNAi lines based on lines 119 (B) and 32 (D). The nodules were counted by the grading statistic method(n = 20–25 per repeat, three biological repeats per line). C and E, RT-qPCR analysis of relativeMtPUB2 expression in the rescuedlines EV/MtPUB2-RNAi,MtPUB2/MtPUB2-RNAi,MtPUB2S421A/MtPUB2-RNAi, andMtPUB2S421D/MtPUB2-RNAi based on lines119 (C) and 32 (E). MtPUB2 mRNA quantification was normalized against two reference genes (Mtactin and MtEF1a) in eachsample. Shown are mean values 6 SE calculated from three independent experiments (***P # 0.001, Student’s t test).


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repeat 1 and repeat 2. The curves in the two repeatswere not completely identical due to discrepancies inplant development. The curves showed time pointsfrom 24 h to 21 DAI but not before 24 h, because the ITsdid not show any development from 0 to 24 h (Loharet al., 2006). We speculate that the negative feedbackbetween MtDMI2 and MtPUB2 is not yet establishedduring this period (Fig. 12). At 3 DAI, the number ofplants responding to rhizobia inoculation was signifi-cantly different compared to those subjected to mocktreatment (Bersoult et al., 2005). At 5 DAI, most of theplants exhibited developing nodules emerging from theroots (Bersoult et al., 2005). In addition, most nodule

development occurred from 7 to 21 DAI, and noduleageing occurred after 28 DAI (Puppo et al., 2005). Ourdata showed that the dynamics occurred within theperiod from 24 h to 21DAI, and once nodulation signalstriggered an increase inMtDMI2, the activatedMtPUB2degrades MtDMI2 and itself (Fig. 10).

This negative feedback loop between MtPUB2 andMtDMI2 was not established in the inoculated dmi2-1 plants and MtPUB2-RNAi plants (Supplemental Fig.S11). In the inoculated MtPUB2-RNAi roots (0 h, 5 h,12 h, 24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, 21 DAI,and 28 DAI), MtDMI2 was always present at a rela-tively higher level compared with that in inoculated

Figure 10. Quantitative immunoblot assays and the prey-predator relationship between MtDMI2 and MtPUB2. A, Quantitativeimmunoblot analysis of wild-type (A17) plants inoculated with S. meliloti strain 1021. The abundance of MtDMI2 (top) andMtPUB2 (bottom) was determined by quantitative immunoblot analysis using the whole root systems. The roots were collected atdifferent time points after inoculation (0 h, 5 h, 12 h, 24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, 21 DAI, and 28 DAI). Actin protein wasused as the loading control. The abundance of MtPUB2 and MtDMI2 was determined using anti-MtPUB2 and anti-MtDMI2polyclonal antibodies (BPI, Co. Ltd.) in experiments. Data from quantitative immunoblot bands were analyzed using ImageJsoftware. These experiments were repeated twice. B, The changes in MtPUB2 and MtDMI2 present as a wave-shaped curveduring the nodulation process. The red box shows the time points selected for solving the prey-predator model (24 h–21 DAI). C,The curves derived from LVequations. The equations were solved numerically using MATLAB. The blue curve shows the changesinMtDMI2, and the gray shows the changes inMtPUB2 (repeat 1). The initial x and ywere set to theMtDMI2 andMtPUB2 proteinlevels measured at 24 h. D, Similar to C, the orange curve shows the changes in MtDMI2, and the yellow shows the changes inMtPUB2 (repeat 2).







wild-type roots (Supplemental Fig. S11, A and B). Ad-ditionally, in the inoculated dmi2-1 (TR25) roots (0 h,5 h, 12 h, 24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, 21 DAI, and28 DAI), MtPUB2 was always found at a relativelylower level compared with that in inoculated wild-typeroots (Supplemental Fig. S11, C and D). The results ofphos-tag assays further confirmed that MtPUB2 phos-phorylation was decreased in dmi2-1 roots comparedwith that in wild-type roots (Fig. 7). Based on the resultsof the quantitative immunoblot and phos-tag analyses,we hypothesized that the stability of MtPUB2 was de-pendent on phosphorylation by MtDMI2.

To further test the working model of feedback be-tweenMtDMI2 andMtPUB2, we analyzed the rhizobialinduction of crucial gene expression of the CSP(MtDMI1, MtDMI3, MtNIN, and MtNSP1) pathway inwild-type andMtPUB2-RNAi roots.MtDMI1,MtDMI3,MtNIN, and MtNSP1 expression was required for aNF-activated signal transduction pathway (Catoiraet al., 2000; Gonzalez-Rizzo et al., 2006; Messinese et al.,2007; Lopez-Gomez et al., 2012). MtNSP1, MtNIN,MtDMI1, and MtDMI3 expression increased simulta-neously through the nodulation time course inMtPUB2-RNAi roots, suggesting that MtDMI2 was partlybeyond the control of MtPUB2 in MtPUB2-RNAiroots (Fig. 11, A–D). In particular, MtNIN expressionwas up-regulated in nodulated MtPUB2-RNAi rootscompared to that in wild-type roots during the entireexperiment (Fig. 11, A and B); however, the nodulenumbers of MtPUB2-RNAi roots were decreased (Figs.2 and 3). As we mentioned regarding AON, MtNINexpression can systemically suppress nodulation via

AON to control the number of nodules (Soyano et al.,2014). We assumed that the up-regulation of MtNINexpression, which could further lead to theup-regulation of CLEs, inhibited the nodulation byAON in our study. Thus, we hypothesized that theMtDMI2-MtPUB2 negative feedback loop, which dis-plays crosstalkwith the long-distance AONviaMtNIN,plays a vital role in nodulation homeostasis. Recently,Saha and DasGupta claimed that crosstalk may existbetween supernodulating null and SYMRK receptorsfor the activation as well as restriction of nodule de-velopment inM. truncatula (Saha and DasGupta, 2015).These data suggest that regulation of nodulation iscomplex.

After its phosphorylation, MtPUB2 gains the abilityto ubiquitinate MtDMI2; identifying the key amino acidthat MtDMI2 phosphorylates MtPUB2 via LC-MS/MSis very important (Supplemental Fig. S8). We selectedthree potential amino acids, namely Ser-421, Ser-316,and Thr-488, for further study. The results demon-strated that Ser-421 in the ARM-repeats domain is thekey phosphorylation site of MtPUB2 that affects itsubiquitination activity. Further study revealed thatMtPUB2S421D (mimicking the phosphorylated form)could ubiquitinate MtDMI2 in vitro, but MtPUB2S421A

had minimal activity (Fig. 8A). Furthermore, we per-formed rescue assays with MtPUB2S421D andMtPUB2S421A in stable MtPUB2-RNAi lines to test theirfunctions during nodulation. MtPUB2/MtPUB2-RNAilines and EV/MtPUB2-RNAi lines were used as con-trols in this experiment (Fig. 9). The results showed thatMtPUB2S421D/MtPUB2-RNAi lines formed the fewest

Figure 11. RT-qPCR analysis of nodu-lation marker gene expressions in thenodulated MtPUB2-RNAi roots andwild-type roots. A and D, RT-qPCRanalysis of relative MtNSP1, MtNIN,MtDMI1, and MtDMI3 expression innodulatedMtPUB2-RNAi and wild-typeroots (0 h, 5 h, 12 h, 24 h, 3 DAI, 5 DAI,7 DAI, 14 DAI, 21 DAI, and 28 DAI).mRNA quantification was normalizedagainst two internal reference genes(Mtactin and MtEF1a) in each sample.Shown are mean values 6 SE calculatedfrom three independent experiments.The gray box shows the MtPUB2-RNAiroots, and the black box shows the wild-type roots.


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nodules.MtPUB2S421A/MtPUB2-RNAi lines had almostthe same number of nodules as EV/MtPUB2-RNAilines, and MtPUB2/MtPUB2-RNAi lines formed simi-lar numbers of nodules as wild-type plants (Fig. 9, Band C). The reasons for the different phenotypes wereas follows: (1) In the MtPUB2S421D/MtPUB2-RNAilines, because of the mimicked sustained phosphoryl-ation of MtPUB2, MtDMI2 was ubiquitinated con-stantly, leading to the fewest nodules duringnodulation. (2) In the MtPUB2S421A/MtPUB2-RNAilines, MtPUB2 lacked ubiquitination activity, and thusthe nodulation phenotype was similar to that of EV/MtPUB2-RNAi lines (Fig. 9). We also assessed thefunction of Ser-316 by performing in vitro ubiquitina-tion assays with MtDMI2 and in vivo rescue assayswith MtPUB2S316D in stable MtPUB2-RNAi lines to testits function during nodulation (Fig. 8; SupplementalFigs. S12 and S13). Based on alignment of the U-boxdomain with AtPUB13, AtPUB12, OsSPL11, Gly-ma20g32340.1, Lj5g3v1796810.2, AtPUB14, MtPUB1,and AtPUB22, Ser-316 conservation is 100%(Supplemental Fig. S11). MtPUB2S316D cannot performself-ubiquitination; however, MtPUB2S316A retains self-ubiquitination capacity and transfers ubiquitin toMtDMI2 (Fig. 8C). In addition, the results of rescue

assays with MtPUB2S316D showed that MtPUB2S316D

/MtPUB2-RNAi lines had similar nodule numbers asEV/MtPUB2-RNAi lines (lines 115 and 119;Supplemental Fig. S13).

The quantitative immunoblot analysis results dem-onstrate that before 7 DAI, MtDMI2 andMtPUB2 levelsboth show a steady increasing trend in the two repeats(Figs. 10, C andD and 12). Thus, it is likely thatMtDMI2phosphorylated MtPUB2 at Ser-316; however, becauseMtPUB2S316D lost the capacity for self-ubiquitinationand the transfer of ubiquitin to MtDMI2 (Fig. 8C),MtPUB2might accumulate. At 7 DAI, the accumulationof MtDMI2 andMtPUB2S316D may ensure the activationto the downstream genes of MtDMI2. After 7 DAI, thephosphorylation of Ser-421 was greater than that of Ser-316, which enhanced self-ubiquitination and MtDMI2ubiquitination. From 7 DAI to 21 DAI, MtPUB2 causedthe degradation of MtDMI2 via ubiquitination, andMtPUB2 caused its own degradation via self-ubiquitination (Figs. 10, C and D, and 12). These dataalso substantiated the prey (MtDMI2)-predator(MtPUB2) working model during nodulation. Furtherstudies are still needed to fully understand the phos-phorylation changes of Ser-316 and Ser-421 and theircontributions to the nodulation phenotype.

Figure 12. Proposed MtDMI2-MtPUB2 negative feedback loop working model. M. truncatula perceives NFs signals via MtNFPand MtLYK3. The signals are then transferred to MtDMI2. MtDMI2 activates MtPUB2 by phosphorylation (potential Ser-316 andSer-421 sites ofMtPUB2). Before 7DAI, the phosphorylation on Ser-316 inactivatedMtPUB2,which induced the accumulation ofMtDMI2 and the activation of some unknown downstream genes. From 7 DAI to 21 DAI, the phosphorylation on Ser-421 causedthe activation of MtPUB2, which induced the degradation on MtDMI2 and maintained the nodulation homeostasis. Once thisbalance was broken, MtDMI2 was no longer degraded, MtNIN was up-regulated excessively, and the nodule numbers weredecreased. The graph shows the LVequations from the second repeat. The blue curve shows the changes inMtDMI2, and the grayshows the changes in MtPUB2. However, the details of how MtPUB2Ser-316 (in red italic font, Ser-316) and MtPUB2Ser-421 (in reditalic font, Ser-421) cooperate during nodulation require further study.









In conclusion, this study describes a novel dynamicmodel involving an E3 ubiquitin ligase and a proteinreceptor kinase during nodulation in M. truncatula.Moreover, this negative feedback model plays a criticalrole in controlling nodulation homeostasis in legumes.Therefore, we propose that there is crosstalk betweenthe AON and the MtPUB2-MtDMI2 negative feedbackloop during nodulation in M. truncatula.

MATERIALS AND METHODS

cDNA Library Construction

To generate a high-quality Medicago truncatula Y2H cDNA library, we usedthe roots of M. truncatula genotype cv Jemalog A17 plants inoculated with S.meliloti strain 1021 or mock-inoculated for different durations (0 h, 5 h, 12 h,24 h, 3 d, 5 d, 7 d, 14 d, 21 d, 28 d, and 35 d). The library was constructedaccording to the protocols supplied with the Mate & Plate kit (Clontech, cat. no.630490). The complexity of the cDNA library was 2.8 3 107, the concentrationwas 1,150 ng/mL, the average insertion size was 750 bp, and the recombinantefficiency was 90% (nonamplified transformant random check).

Plant Materials

M. truncatula cv Jemalog ecotype A17 and ecotype R108 were used forphenotypic and genotypic analyses. The homozygous mutant dmi2-1 (TR25,frame shift due to a 1-bp deletion, premature translational termination in theextracellular domain, Nod-Myc-) was acquired from Jean-Michel Ané (Endreet al., 2002), and the seeds were treated for germination as previously described(Pennycooke et al., 2008). The seeds were planted in a chamber under the fol-lowing environmental conditions: 14 h/10 h light/dark cycle, dim light(60 mmol photons s-1m22, Philips; TL5 Essential Super 80), 22°C/18°C day/night regime, and 70% relative humidity (RH).

Phylogenetic Tree Analysis

The PUB domains for each subfamily were used as an input for the phylo-genetic tree analysis. For the phylogenetic analysis of MtPUB2 and homologousPUBs inM. truncatula, Lotus japonicas, Arabidopsis (Arabidopsis thaliana),Glycinemax, andOryza sativa, sequences were obtained from Phytozome v11.0 (http://www.phytozome.net; Supplemental File S2). We analyzed the multiple se-quence alignment of PUB amino acids using ClustalW2 software and con-structed the phylogenetic tree using the neighbor-joiningmethod. The numberson the branches indicate bootstrap values based on 1,000 replicates.

Spatiotemporal Expression of MtPUB2

The 2.3-kb promoter of MtPUB2 was cloned from the DNA ofM. truncatulacv JemalogA17. The promoterwas initially cloned into the pTOPO-Entry vector(Invitrogen) using Gateway technology and then into the pkGWFS7 vector thatcarried a downstream GUS fusion construct (Huault et al., 2014). The finalproMtPUB2-GUS construct was transformed into M. truncatula via Agro-bacterium tumefaciens. The plants were then used for proMtPUB2-GUS assaysduring nodulation. The roots, nodules, leaves, and stems were immersed in aX-gluc solution at 37°C for approximately 18 h in a vacuum for 2 h. The colorwas removed using 75% ethanol. The samples were slicedwith a Leica VT1000Sand photographed using an Olympus BX50 microscope. The primers related tothis experiment are listed in Supplemental Table S2.

M. truncatula Transformation

An MtPUB2-specific RNAi construct was prepared to target a 450-bp frag-ment containing the first 450 bp of the MtPUB2 coding sequence (1–450 bpstarting from ATG). The primers used for PCR are listed in Supplemental TableS1. This 450-bp fragment was cloned into the pANDA vector (Clontech), whichis driven by the ubiquitin promoter, to create pANDA-MtPUB2. The stableoverexpression transgenic lines were prepared using the pCAMBIA1302 vector(pCAMBIA1302-MtPUB2) driven by the cauliflower mosaic virus 35S promoter

(CaMV35S). Hygromycin (10 mg/L, Roche) was used to select transformedplants. The regenerated identified T0 transformed plantlets were further culti-vated in soil. After 8 to 10 months, the seeds of the transgenic plants wereharvested for further analysis (www.noble.org/MedicagoHandbook/).

For the Agrobacterium rhizogenes-mediated hairy root transformation ofM. truncatula genotype R108 plants, germinated seeds were transformed withthe A. rhizogenes strain ARqual1, as described by Boisson-Dernier et al. (2001).For RNAi experiments, the PCR product was cloned into the pFRN-RNAivector (Gonzalez-Rizzo et al., 2006). For overexpression assays, the CDS ofMtPUB2 was cloned from A17 cDNA via PCR and inserted into the modifiedpCAMBIA2301 vector under the control of the dual CaMV35S to obtain therecombinant pCAMBIA2301-MtPUB2 vector. The transgenic seedlings werethen transferred to square petri dishes (130 3 130 mm) on Fåhraeus mediumwithout nitrogen (pH 6.5) after 4weeks of growth. Five to six plants were placedon each plate. All plates were incubated in the growth chamber for 2 weeksunder the following conditions: 16-h/8-h-light/-dark cycle, dim light (60 mmolphotons s21 m22), 24°C day/night regime, and 70% RH. The inoculation of S.meliloti strain 1021 grown to anOD600 of 0.5 was performed 7 d after transferringthe seedlings to the square petri dishes.

For the nodulation assay, roots of 1-week-old seedlingswere inoculatedwithrhizobial bacteria; plants were flood-inoculated with a total of 20 mL of aresuspended cell suspension (OD600 = 0.5) per plate. Nodulation phenotypeswere recorded at 21 DAI. Photographs of nodules were taken under a stereo-microscope (MZFLIII, Leica). All primers used in this experiment are listed inSupplemental Table S2.

RT-qPCR Analysis

Total RNA was extracted with TRIzol (Invitrogen) from the roots, leaves,stems, and nodules of M. truncatula plants (different treatments), and 1 mg oftotal RNA was used for reverse transcription. RT-qPCR reactions were per-formed using SYBR Premix Ex Taq (TaKaRa) on a Bio-Rad CFX-96 PCR system(Bio-Rad) according to the manufacturer’s instructions. The internal referencegenes were MtEF1a and Mtactin. Three independent biological replicates andthree independent technical replicates were performed for each conditiontested. We sampled the roots, leaves, stems, and nodules taken from threedifferent plants at the same time grown in different pots. Total RNA wasextracted from pooled roots, leaves, stems, and nodule tissues; there are threeplants/seedlings pooled per sample. Gene expression was calculated using the2-CTmethod. The primers used in this experiment are listed in SupplementalTable S2.

Infection Threads Assay and Lugol Staining of Nodules

Infection threads assays were performed 5 DAI. Rhizobia used in this assayare S. meliloti 1021 pXLD4 expressingHemA-lacZ. The roots and noduleswere ina vacuum for three cycles of 30 s with 2.5% glutaraldehyde in 0.1 M PIPES (pH7.2), fixed for 2 h, and rinsed in 0.1 M PIPES (pH 7.2) three times. Thewhole rootswere then incubated in staining solution containing 50 mM potassium ferro-cyanide, 50 mM potassium ferricyanide, 0.1 M PIPES (pH 7.2), and 0.08% X-galfor 16 h at 28°C. Finally, the stained roots were rinsed with 0.1 M PIPES (pH 7.2)with 2.4% sodium hypochlorite for 15 min before being photographed with anOlympus BX50 microscope.

For the amyloplast detection of nodules, 21-DAI and 42-DAI nodules werecollected and embedded in 6%agarose. The sampleswere then sliced into 80mmwith Leica VT1000S microtome. The amyloplasts were stained in the lugol so-lution (Sigma-Aldrich) for 5 min and then washed with water. The sectionswere photographed using an Olympus BX50 microscope.

Y2H Screen and Pairwise Interactions

The sequence encoding the intracellular region ofMtDMI2was amplified byPCR and cloned into the pGBKT7 (binding domain) vector (Clontech), whichwas transformed into the yeast AH109 strain using the LiAc-mediated yeasttransformation method (Gietz and Schiestl, 2007). The cDNA inserts of positiveclones were amplified by PCR from yeast cells and were then sequenced. Theprotein domains of MtPUB2 were cloned using primers specific to the U-box(258 to 321 aa), ARM (382 to 628 aa), an unnamed domain (1–257 aa), and thefull-length (1–662 aa). MtCRA2, MtLYK3, Medtr1g094021.1, andMedtr4g028960.1were cloned using specific primers. The truncated domains ofMtPUB2 were cloned into pGBKT7, and the sequence encoding the proteinkinase domain of MtDMI2 was cloned into pGADT7 (activation domain). Yeast


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transformants were screened on 4D selective medium (SD/-Ade/-His /-Leu/-Trp)supplied with 8 mM 3-amino-1,2,4-trazole to suppress self-activation and incubatedat 28°C for 4 to 5 d. The primers used in this experiment are listed in SupplementalTable S2.

Subcellular Localization and BiFC Experiments in OnionEpidermal Peels

The open reading frame of MtPUB2 without the termination codon wasinserted into the modified pE3025 plasmid containing yellow fluorescentprotein (YFP, green) instead of red fluorescent protein at the EcoRI and SalIsites to generate the Pro-35S:MtPUB2-cGFP vector. YFP fluorescence wasvisualized using a confocal laser-scanning microscope. BiFC experimentswere performed in a structural complementation assay using pSY735- andpSY736-derived plasmids driven by the CaMV35S (MtDMI2-Yc, Yn-MtPUB2,Yn-Medtr1g094021.1) for transient expression of the fusion proteins in onion(Allium cepa) epidermal peels. Relevant negative controls and positive con-trols were produced at the same time. YFP fluorescence (green) was moni-tored by excitation at 488 nm with an argon laser combined with a 505- to520-nm bandpass filter using a Nikon ECLIPSE TE2000-E inverted fluores-cence microscope equipped with a Nikon D-ECLIPSE C1 (Nikon) spectralconfocal laser-scanning system. All primers used in this experiment are listedin Supplemental Table S2 (pE3025-MtPUB2-F, pE3025-MtPUB2-R, pSY736-MtPUB2-F, pSY736-MtPUB2-R, pSY735-MtDMI2-F, pSY735-MtDMI2-R,pSY736-Medtr1g094021.1-F, pSY736-Medtr1g094021.1-R).

GST Pull-Down Assay

MtDMI2IR was cloned into pGEX-4T-1 as a GST fusion and then trans-formed into the Escherichia coli BL21 strain. The recombinant protein was pu-rified by GST-agarose affinity 610 chromatography, as described previously(Yang et al., 2011). MtPUB2 was cloned into pET-30a (+) as a His-fusion andthen transformed into the E. coli BL21 strain. The His-MtPUB2 protein waspurified by chromatography using Ni-NTA agarose (Invitrogen) following themanufacturer’s instructions. Immobilized GST-MtDMI2IR was obtained fromthe lysate by incubation with Glutathione Sepharose 4B agarose beads for 1 h at4°C (GE Healthcare Life Science). Unbound proteins were removed with lysisbuffer at 4°C for 5 min. Finally, the interaction complex was analyzed by im-munoblot analysis using anti-GST and anti-His antibodies (mouse monoclonal,Abcam). The primers used in this experiment are listed in Supplemental TableS2 (pET30a-MtPUB2-F, pET30a-MtPUB2-R, pGEX-4T-1-MtDMI2IR-F, pGEX-4T-1-MtDMI2IR-R).

In Vitro Ubiquitination Assay

The reactions contained 500 ng of substrate protein, 20 ng of E1 (UBE1,rabbit, Boston Biochem), 40 ng of E2 (UbcH5c, human recombinant, BostonBiochem), 5 mg of Arabidopsis ubiquitin (Boston Biochem), and 3 mg of pu-rifiedMBP-MtPUB2 (MtPUB2was cloned into pMAL-c2x, expressed in E. colistrain TB1 with an MBP-tag at the N terminus, and purified using amyloseresin) in ubiquitination buffer (0.1 M Tris-HCl pH 7.5, 25 mM MgCl2, 2.5 mM

dithiothreitol, and 10 mM ATP) in a final volume of 30 mL. The reactions wereincubated at 30°C for 2 h and were stopped by the addition of 43 SDS samplebuffer. The samples were detected by immunoblot analysis with anti-MBP,anti-GST, and anti-Ubi (mouse monoclonal, Abcam) antibodies.

The primers used in this experiment are listed in Supplemental Table S2.

In Vitro Phosphorylation Assay

For the protein kinase assays, approximately 5 mg of kinase and 50 mg ofthe substrate protein were incubated in 45 mL of 0.5 M HEPES, pH 7.4, 15 mM

MnCl2, 1 M dithiothreitol, 50 mM ATP, and 1 mCi of [g-32P]-ATP per reaction at28°C for 30 min. The reaction was stopped by the addition of 43 SDS loadingbuffer and boiling at 100°C for 5 min. The samples were then used directlyand separated through 10% SDS-PAGE. After electrophoresis, the gel wasdried, and kinase autophosphorylation activities were detected via autora-diography. The primers used in this experiment are listed in SupplementalTable S2 (pMAL-C2x-MtPUB2-F, pMAL-C2x-MtPUB2-R, pGEX-4T-1-MtDMI2IR-F, pGEX-4T-1-MtDMI2IR-R, pGEX-4T-1-MtDMI2IRT762A-F,pGEX-4T-1-MtDMI2IRT762A-R).

CIAP and Phos-Tag Assays in M. truncatula

Proteins were extracted with native extraction buffer 1 (NB1; 10 mM HEPES,pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100 and pro-tease inhibitor cocktail (Roche), and PMSF [AMRESCO]). For in vivo calf in-testinal (CIAP, New England Biology) assays, the proteins were extracted fromthe roots of wild-type Jemalong A17 and dmi2-1 (TR25) plants at 21 DAI. Theproteins were treated with CIAP at 37°C for 30 min and then detected with ananti-MtPUB2 (BGI, Co. Ltd.) polyclonal antibody and an antiactin (CWBio)antibody on immunoblots. For the phos-tag (BOPPARD) assay, proteins wereextracted from the roots of wild-type A17 plants at 7 DAI, 14 DAI, and 21 DAI,and phos-tag was added to SDS-PAGE gels at a concentration of 5.0 mM. Anti-MtPUB2 and antiactin antibodies were used in the immunoblot analysis.

Cellular Fractionation Assay

For the cellular fractionation assay, wild-typeN. benthamiana plants were usedas the host plants for the assays. Plantswere grown in a growth chamber at 22°C/18°C day/night regime, 70% RH under a 16-h/8-h-light/-dark photoperiod forapproximately 4 to 5 weeks before infiltration. Agrobacterium EHA105 strainscarryingMtPB2-HA and p19 expression constructs were co-infiltrated at differentratios. Three days after infiltration, the samples were collected for analysis. Thefusion proteins were extracted according to the protocols of Lei et al. (2015) andDuan et al. (2017). Then the fractions (1 mg) were separated by 10% SDS-PAGEand subjected to immunoblot analysis where a 1:5,000 dilution of H+ATPase(Agrisera; approximately 95 kD, plasma membrane marker antibody) and a1:5,000 dilution of cFBPase (Agrisera; approximately 37 kD, cytosolic fractionmarker antibody) were used as the marker antibodies.

In Vivo Degradation

Themethodsweremodified from those described by Liu et al. (2010).Wild-typeNicotiana benthamiana plants were used as the host plants for the assays. For thein vivo degradation experiments, Agrobacterium EHA105 strains carrying E3 ligase,substrate, p19 expression constructs, and internal control plasmids wereco-infiltrated at different ratios. Three days after infiltration, the samples were col-lected for analysis. MG132 (N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-for-myl-3-methylbutyl]-L-leucinamide, Sigma-Aldrich) dissolved in 10 mM MgCl2 at afinal concentration of 50mMwas infiltrated into the previous infiltrated region 12 h beforesample collection. The proteins were extractedwithNB1 (10mMHEPES, pH 7.5, 100mM

NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, protease inhibitor cocktail [Roche],andPMSF [AMRESCO]). The sampleswereused in immunoblot analysis. The expressionof MtDMI2 and NbEF1a were detected by RT-qPCR assays to confirm that the degra-dation ofMtDMI2wasmediatedbyMtPUB2at theprotein level. Theprimersused in thisexperiment are listed inSupplementalTableS2 (pCAM1300-Myc-MtDMI2-F,pCAM1300-Myc-MtDMI2-R, pCAM1300-HA-MtPUB2-F, pCAM1300-HA-MtPUB2-R).

Semi-in Vivo Degradation Assay

The degradation assay and semi-in vivo protein degradation analysis wereconducted bymodifying a previously reported method (Liu et al., 2010). For thesemi-in vivo protein degradation analysis, 3 d after infiltration, Myc-MtDMI2,MtPUB2-HA, MtPUB2V274R-HA, GFP-HA, and wild-type samples were har-vested. The samples were separately extracted in NB1. To preserve the functionof the 26S proteasome, ATP (Sigma-Aldrich) was added to the cell lysates at afinal concentration of 10 mM. The MtPUB2-HA/MtPUB2V274R-HA extract wasthen mixed with Myc-MtDMI2 or wild-type extract in a volume ratio of 1:1.MG132 was added to the corresponding mixtures to a final concentration of50 mM. The mixtures were incubated at 4°C with gentle shaking. The sampleswere used in immunoblot analysis and detected with anti-HA and anti-Mycantibodies. All primers used in this experiment are listed in Supplemental TableS2 (pCAM1300-Myc-MtDMI2-F, pCAM1300-Myc-MtDMI2-R, pCAM1300-HA-MtPUB2-F, pCAM1300-HA-MtPUB2-R).

LC-MS/MS Assay

To detect the sites of MtPUB2 that were phosphorylated by MtDMI2, weperformed an LC-MS/MS assay using in vitro-expressed proteins (MBP-MtPUB2 and GST-MtDMI2IR) in the E. coli BL21 strain. The samples wereseparated using 8% SDS-PAGE gels and digested with trypsin. The digestedpeptides were analyzed in a SCIEX TripleTOF 5600+ high-resolution mass
















spectrometer (AB SCIEX). The ion spray voltage was 2.4 kV. The time of flightmasses of time of flight MS and the product ion were 3,500 to 1,500 D and 100 to1,500 D, respectively. The time-of-flight masses (TOFMS) and the production ionwere 350 to 1,500 Da and 100 to 1,500 Da, respectively. In the LC-MS/MS assay,three biological replicates were performed for control and phosphorylated sam-ples. We sampled each specimen of cultures of unique E. coli BL21 strain trans-formants in three different biological replicates.

The data were analyzed using ProteinPilot 5.0 software and Paragon algo-rithm search parameters (AB SCIEX).

Quantitative Immunoblot Analysis and Dynamic Analysisin M. truncatula

In the quantitative immunoblot assay, theM. truncatula ecotypeA17was thewild-type material, and MtPUB2-RNAi roots and dmi2-1 (TR25) roots were the mutantmaterials. The roots of 7-d-oldplantswithout a nitrogen source (0 h)were treatedwithS.meliloti strain1021, and theentire root systemswereanalyzedatdifferent timepoints(0 h, 5 h, 12 h, 24 h, 3DAI, 5DAI, 7DAI, 14DAI, 21DAI, and 28DAI). The abundanceof actin protein was used as a loading control. The abundance of MtPUB2 andMtDMI2 in the total root systemswas assessed using anti-MtPUB2 and anti-MtDMI2polyclonal antibodies (BPI).

MtPUB2 (full length) and a truncated MtDMI2 (amino acids 543–919) wereexpressed in theE. coliBL21 strain using thepET-30a (+) vector andpGEX-4T-1 vector,respectively, purified, and used to produce anti-MtPUB2 and anti-MtDMI2 anti-bodies, respectively. The antibodieswereproducedbyBeijingProtein Innovation (BPI,http://www.proteomics.org.cn). The arterial blood serum was collected, and theantibodies were purified by Protein A Agarose (GE Healthcare; 17-0402-01).

Roots for quantitative immunoblot analysis were collected at 0 h, 5 h, 12 h,24 h, 3 DAI, 5 DAI, 7 DAI, 14 DAI, 21 DAI, and 28 DAI. The quantitative im-munoblot banddatawere analyzedwith ImageJ software. Finally, the datawereassessed as the MtPUB2/Mtactin and MtDMI2/Mtactin ratios.

To model the negative feedback loop between MtDMI2 and MtPUB2 acrossvarious time points, we adopted LV equations (better known as prey-predatorequations), which are a pair of first-order, nonlinear, differential equations(Lotka, 1910). These equations are widely applied to many natural systems,such as chemical reactions and aerosol-cloud-precipitation interactions (Lotka,1910; Koren and Feingold, 2011). The equations are expressed as follows:

dx=dt ¼ ax2by

dy=dt ¼ dxy2 gy

Therefore, a set of four positive parameters (a, b, d, and g) dictates the concentration ofMtDMI2andMtPUB2asa functionof time. In this study,we let theparametersa/b varyfrom0.1 to10and theparametersd/g vary from0.1 to1withan intervalof0.1.The initialvalues of x and ywere set to theMtDMI2 andMtPUB2 protein levels measured on day1 (24 h). The equations were solved numerically using the MATLAB program. The bestset of the a, b, d, and g parameters was determined using the least square fit for themodeled x andywith theMtDMI2 andMtPUB2protein levelsmeasured at 24 h, 3DAI,5 DAI, 7 DAI, 14 DAI, and 21 DAI. The program is shown in Supplemental File S1.

Rescue of the MtPUB2-RNAi and NodulationPhenotype Assays

For the A. rhizogenes-mediated hairy root transformation of M. truncatulagenotype R108 plants, germinated seeds were transformed with the A. rhizogenesstrain ARqual1, as described by Boisson-Dernier et al. (2001). For the rescue tests,the CDSs ofMtPUB2,MtPUB2S421A,MtPUB2S421D, andMtPUB2S316Dwere insertedinto the modified pCAMBIA2301 vector under the control of dual CaMV35S toobtain the recombinant pCAMBIA2301-MtPUB2 vector, pCAMBIA-MtPUB2S421A

vector, pCAMBIA-MtPUB2S421D vector, and pCAMBIA-MtPUB2S316D vector.pCAMBIA2301 was used as the empty vector control. The primers used in thisexperiment are listed in Supplemental Table S2.

Accession Numbers

Sequence data from this article can be found in the M. truncatula genomedatabase and the GenBank data library under accession numbersMedtr5g030920 (MtDMI2), Medtr8g043970 (MtDMI3), Medtr2g005870(MtDMI1), Medtr5g099060 (MtNIN), XM_003627246 (MtNSP1), AY372402(MtLYK3), XM_013606619 (MtCRA2), and KU285617.1 (MtPUB2).

SUPPLEMENTAL DATA

The following supplemental materials are available.

Supplemental Figure S1. Selection of MtDMI2-interacting proteins by Y2H.

Supplemental Figure S2. Structure analysis and phylogenetic tree analysisof MtPUB2.

Supplemental Figure S3. Phylogenetic tree of the homologous PUBs inMedicago truncatula (Medtr), Arabidopsis thaliana (At), Glycine max(Glyma), Oryza sativa (Os) and L. japonicus (Lj).

Supplemental Figure S4. Interaction assays between MtDMI2IR,Medtr1g094021.1, and Medtr4g028960.1 in yeast cells.

Supplemental Figure S5. Representative infection threads in GUS-RNAiand MtPUB2-RNAi roots.

Figure Figure S6. Relative expressions ofMtPUB2 andMedtr1g094025.1 in theGUS-RNAi lines and MtPUB2-RNAi lines.Supplemental Figure S7. Nodulation phenotypes of the MtPUB2-overex-

pressing lines.

Supplemental Fig. S8. MtPUB2 sites with high possibility of phosphoryl-ation based on LC-MS/MS assay.

Supplemental Figure S9. In vitro ubiquitination assays with MBP-MtPUB2S421D and GST-MtDMI2IR.

Supplemental Figure S10. Nodule quantification in transgenic lines.

Supplemental Figure S11. Quantitative immunoblot assays of the nodu-lated MtPUB2-RNAi roots and dmi2-1 (TR25) roots.

Supplemental Figure S12. Amino acid sequence and domain structure ofPUB members in M. truncatula (Medtr), Arabidopsis (At), soybean(Glyma), rice (Os), and L. japonicus (Lj).

Supplemental Figure S13. Nodulation phenotypes of A. rhizogenes-trans-formed roots in rescued MtPUB2-RNAi lines.

Supplemental Table S1. LC-MS/MS data of phosphorylation sites inMtPUB2.

Supplemental Table S2. Primers used in this study.

Supplemental File S1. MATLAB program.

Supplemental File S2. Text file of the amino acid sequence of PUB-typeproteins in M. truncatula (Medtr), Arabidopsis (At), soybean (Glyma),rice (Os), and L. japonicus (Lj) that were used as an input for the phylo-genetic tree analysis.

ACKNOWLEDGMENT

We thank Dr. Ton Bisseling (Wageningen University, The Netherlands) and Dr.Florian Frugier (CNRS, France) for their guidance regarding this work. We thank Dr.Jean Marie Prosperi and Dr. Magalie Delalande (Biological Resource Center for M.truncatula, UMR1097, INRA,Montpellier, France) for providing seeds ofM. truncatulacv. Jemalog A17 and R108, Dr. Jean-Michel Ané (Department of Agronomy, TheUniversity of Wisconsin, Madison, WI) for providing seeds of the dmi2-1 (TR25) mu-tant, and Dr. Mathias Brault (Institut des Sciences du Végétal, CNRS, France) forproviding S. meliloti strain 1021. We thank Dr. Qi Xie (Institute of Genetics and De-velopmental Biology, Chinese Academy of Sciences, China) for kindly providing thevectors used in this study.

Received November 3, 2017; accepted February 6, 2018; published February 13,2018.

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