a ubiquitin ligase of symbiosis receptor kinase involved in nodule

A Ubiquitin Ligase of Symbiosis Receptor KinaseInvolved in Nodule Organogenesis1[C][W][OA]

Songli Yuan, Hui Zhu, Honglan Gou, Weiwei Fu, Lijing Liu, Tao Chen, Danxia Ke, Heng Kang, Qi Xie,Zonglie Hong, and Zhongming Zhang*

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070,China (S.Y., H.Z., H.G., W.F., T.C., D.K., H.K., Z.Z.); State Key Laboratory of Plant Genomics, National Centerfor Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,Datun Road, Beijing 100101, China (L.L., Q.X.); and Department of Plant, Soil, and Entomological Sciencesand Program of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho83844 (Z.H.)

The symbiosis receptor kinase (SymRK) is required for morphological changes of legume root hairs triggered by rhizobialinfection. How protein turnover of SymRK is regulated and how the nodulation factor signals are transduced downstream ofSymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase (SIE3) was shown to bind and ubiquitinateSymRK. The SIE3-SymRK interaction and the ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3represents a new class of plant-specific E3 ligases that contain a unique pattern of the conserved CTLH (for C-terminal to LisH),CRA (for CT11-RanBPM), and RING (for Really Interesting New Gene) domains. Expression of SIE3 was detected in all testedtissues of Lotus japonicus plants, and its transcript level in roots was enhanced by rhizobial infection. The SIE3 protein waslocalized to multiple subcellular locations including the nuclei and plasma membrane, where the SIE3-SymRK interaction tookplace. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas downregulation of SIE3 transcripts byRNA interference inhibited infection thread development and nodule organogenesis. These results suggest that SIE3 represents anew class of E3 ubiquitin ligase, acts as a regulator of SymRK, and is involved in rhizobial infection and nodulation in L.japonicus.

The formation of root nodules is initiated in re-sponse to rhizobia-derived nodulation factors (NFs), agroup of lipochitooligosaccharides with N-linked acylgroups and other host-specific decorations. NFs cantrigger a series of host responses, including root hairdeformation, cellular alkalinization, membrane po-tential depolarization, ion flux changes, early nodulingene expression, and nodule primordial formation(Ehrhardt et al., 1996; Felle et al., 1999; D’Haeze andHolsters, 2002; Wais et al., 2002). These responsesallow the rhizobia to enter the root epidermal cells

through infection threads (ITs). Concomitant with therhizobial infection, NFs stimulate distant cells of theroot pericycle layer to undergo cytoskeletal rear-rangement and develop nodule primordia (Timmerset al., 1999). Rhizobia are released from the end of ITsinto the cytoplasm of root cortical cells, where they areenclosed by the symbiosome membrane and reducethe atmospheric nitrogen into organic nitrogen com-pounds that are exported to the host in exchange forcarbohydrates.

A series of plant genes involved in perception ofrhizobial NF signals have recently been identified inLotus japonicus, Medicago truncatula, and other legumes(Catoira et al., 2000; Wais et al., 2000; Miwa et al., 2006;Madsen et al., 2010; Oldroyd et al., 2011). NFs areperceived by two receptor kinases, nodulation factorreceptor1 (NFR1) and NFR5, which belong to theLysin-motif receptor-like kinases (RLKs; Madsen et al.,2003; Radutoiu et al., 2003, 2007). Recognition of NFsby their receptors leads to activation of the commonsymbiosis pathway, which is required for establishingsymbiotic relationships with rhizobia and nutrition-improving mycorrhizal fungi (Kistner and Parniske,2002; Banba et al., 2008).

The symbiosis receptor kinase (SymRK) does notbind NFs directly, but acts in a key step downstream ofNFR1/NFR5. The SymRK gene is indispensable forroot endosymbiosis with rhizobia and arbuscular mycor-rhizal fungi in legumes, and may function at the junction

1 This work was supported by funds from the National Basic Re-search Program of China (973 program grant no. 2010CB126502), theNational Natural Science Foundation of China (grant no. 170224), theMinistry of Agriculture of China (grant no. 2009ZX08009–116B),the State Key Laboratory of Agricultural Microbiology (grant no.AMLKF200804), and the Graduate Education Innovation Fund ofHuazhong Agricultural University.

* Corresponding author; e-mail: [email protected] author 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) is:Zhongming Zhang ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

tion.www.plantphysiol.org/cgi/doi/10.1104/pp.112.199000

106 Plant Physiology�, September 2012, Vol. 160, pp. 106–117, www.plantphysiol.org � 2012 American Society of Plant Biologists. All Rights Reserved.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/160/1/106/6109734 by guest on 23 Septem

ber 2021

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.112.199000

of rhizobial and fungal signaling cascades (Endre et al.,2002; Stracke et al., 2002). The SymRK protein containsa signal peptide sequence for protein sorting, an ex-tracellular region for interacting with unidentifiedsignal molecules, a transmembrane segment for an-choring to the plasma membrane, and an intracellularprotein kinase domain (Endre et al., 2002; Stracke et al.,2002; Mitra et al., 2004; Capoen et al., 2005).Both the SymRK extracellular region and the kinase

domain are important for nodulation in L. japonicus. Arecent report has identified a mutation in the Gly-Asp-Pro-Cys motif of the SymRK extracellular region thatblocks the IT formation in root hairs (Kosuta et al.,2011). Previously, the SymRK kinase domain has beenshown to interact with 3-hydroxy-3-methylglutarylCoA reductase1 (HMGR1) from M. truncatula (Keveiet al., 2007), and an ARID-type DNA-binding protein(Zhu et al., 2008). HMGR1 catalyzes the formation ofmevalonic acid and is the rate-limiting step of themevalonic acid pathway, which is required for pro-duction of specific isoprenoid compounds such ascytokinins, phytosteroids, and isoprenoid-modifiedsignal proteins. Reducing the level of HMGR1 by in-hibitor lavastatin or RNA interference (RNAi) expres-sion results in a drastic decrease in nodulation in M.truncatula (Kevei et al., 2007). SIP1 binds the promoterof the NIN (for nodule inception) gene (Zhu et al., 2008),which is required for rhizobial recognition, IT forma-tion, and nodule primordial initiation (Schauser et al.,1999). These results suggest that SymRK may formprotein complex with key regulatory proteins ofdownstream cellular responses. Symbiotic Remorin1(SYMREM1) from M. truncatula has been shown tointeract with SymRK, and may act as a scaffold proteinfor assembly of signaling complexes involved in rhi-zobial infection (Lefebvre et al., 2010).In this study, we identified a SymRK-interacting E3

ligase (SIE3) as a regulator of rhizobial infection andnodulation in L. japonicus. SIE3 was shown to bind anduse SymRK as a substrate for ubiquitination in planta.Alteration of SIE3 expression levels in transgenic hairyroots via overexpression and RNAi exhibited drasticeffects on rhizobial infection and nodulation. Thus,SIE3 appears to represent a new class of plant-specificE3 ligases that are involved in symbiotic nitrogen fix-ation in legumes.

RESULTS

SIE3 Is a Novel E3 Ubiquitin Ligase

In search of interacting proteins of SymRK, we tookthe two-hybrid approach and screened an L. japonicuscomplementary DNA (cDNA) library constructed in ayeast (Saccharomyces cerevisiae) expression vector (Zhuet al., 2008). Several positive clones were isolated andshown to contain cDNA inserts representing the samegene. Because none of them appeared to contain a full-length coding region, we searched the Lotus database

and found two expressed sequence tag clones (Lj-G0027263 and Lj-G0032470). We designed primersaccording to the reconstituted full-length coding re-gion and amplified its cDNA using RNA isolated fromL. japonicus roots inoculated with Mesorhizobium loti.The 1,161-bp coding region encodes 387 amino acids,and its sequence has been deposited to GenBank(JQ009197).

The deduced peptide, containing a CTLH (for C-terminal to LisH) motif, a CRA (for CT11-RanBPM)domain, and a RING (for Really Interesting NewGene) finger domain (Fig. 1A), represents a novel classof the RING-type E3 ubiquitin ligase family. Thisprotein was designated as SIE3 for SymRK-interactingE3 ubiquitin ligase. E3 proteins containing the CTLH,CRA, and RING domains are conserved in higherplants with no known biological function (Fig. 1B;Supplemental Fig. S1). Homology modeling revealedthat SIE3 may exist as a reverse parallel homodimer(Supplemental Fig. S2A). In yeast cells, SIE3 interactedwith itself (Supplemental Fig. S2B), which is consistentwith the homodimer model. The C terminus of SIE3contains a RING finger domain, encompassing eighthighly conserved Cys/Ser/His residues that may co-ordinate two Zn2+ ions (Fig. 1C). The first Zn2+ ion iscoordinated by four Cys/Ser residues at positions 1, 2,5, and 6, and the second Zn2+ by Cys/His residues atpositions 3, 4, 7, and 8 (Lorick et al., 1999; Fang andWeissman, 2004). In SIE3 and some of its plant ho-mologs, the Cys residues at positions 2 and 6 are re-placed with Ser, Leu, or Thr (Fig. 1C), which disablesthe coordination of the first Zn2+ ion. The four residuescoordinating the second Zn2+ ion are highly conservedin the plant proteins (Fig. 1C). Despite the substitu-tions, the three-dimensional model of the degeneratedRING finger domain in SIE3 still resembles that of thecanonical RING (Supplemental Fig. S2C).

Dissection of Peptide Domains Involved in SIE3-SymRKInteraction in Yeast

To dissect domains required for the interaction be-tween SIE3 and SymRK, deletions of both SymRK andSIE3 cDNAs were made. The clone (pSIE3-DCTLH)isolated from the initial library screen contained theCRA and RING domains but lacked the CTLH do-main, suggesting that the CTLH domain is not essen-tial for the interaction (Fig. 2B). When the RINGdomain was removed (SIE3-DRING; Fig. 2A), the in-teraction became very weak, suggesting that RING isimportant for the interaction. Experiments usingSymRK deletions showed that both the extracellularregion (SymRK-EC) and the PK domain of SymRK(SymRK-PK) have very weak interaction with SIE3(Fig. 2B). Because of the weak interaction betweenSymRK and SIE3, we asked if SIE3 also interacts withother RLKs such as NFR1, NFR5, and L. japonicus Hiskinase1 (LHK1). Our results showed that SIE3 does notinteract with the PK domain of NFR1, NFR5, and

Plant Physiol. Vol. 160, 2012 107

A Ubiquitin Ligase of Symbiosis Receptor Kinase

Dow



ber 2021

http://www.plantphysiol.org/cgi/content/full/pp.112.199000/DC1




LHK1, or with the extracellular region of LHK1 (Fig.2C). Thus, we conclude that SIE3 can specifically in-teract with SymRK in yeast cells, and the full-lengthSIE3 can also interact with both SymRK-EC andSymRK-PK in yeast cells.

Interaction of SIE3 with SymRK in planta

We took the bimolecular fluorescence complemen-tation (BiFC) approach to investigate if the SIE3-SymRK interaction took place in tobacco leaf cellscoexpressing recombinant SIE3 and SymRK proteins.SymRK was fused to the split cyan fluorescent protein(CFP) C terminus (SymRK:SCC), while SIE3 was at-tached to the split CFP N terminus (SIE3:SCN). Inter-action of the two proteins would reconstitute the cyanfluorescent chrome, allowing for detection of cyanfluorescence (Waadt et al., 2008). Leaf epidermal cellswere analyzed 2 d after infiltration with Agrobacteriumtumefaciens harboring the constructs. Strong cyan fluo-rescence was observed in the plasma membrane ofleaf cells expressing the two proteins (Fig. 3A), sug-gesting an interaction at the plasma membrane. Thefluorescence was as strong as the positive control,which coexpressed Arabidopsis (Arabidopsis thaliana)calcineurin B-like (CBL) and CIPK24 (for CBL-inter-acting protein kinase24; Waadt et al., 2008). WhenSymRK:SCC and SIE3:SCN were expressed separately,no fluorescence signal was observed (Fig. 3A). Theexpression of recombined proteins in tobacco leaveswas confirmed by immunoblot analysis using anti-

hemagglutinin (HA) and anti-FLAG monoclonal an-tibodies (Fig. 3B). These results showed that SIE3interacts with SymRK at the plasma membrane inplant cells. The interactions between the full-lengthSIE3 and SymRK-EC or SymRK-PK were furtherconfirmed by the BiFC approach (Supplemental Fig.S3).

Co-IP of SIE3 with SymRK

The interaction was further confirmed by coimmu-noprecipitation (Co-IP). SIE3 was tagged with GFP,whereas SymRK was expressed as a Myc-tagged pro-tein in N. benthamiana leaves. Coexpression of the tworecombinant proteins was achieved by infiltration of amixture of two A. tumefaciens strains (1:1) that con-tained pSIE3-GFP and pSymRK-myc, respectively. Ascontrols, a mixture of A. tumefaciens strains (1:1) con-taining pSymRK-myc and pBA-myc, respectively, orcontaining pSIE3-GFP and pBA-myc, respectively, wasused for infiltration. Three days after infiltration, theexpression of the recombinant proteins in leaves wasconfirmed by immunoblot analysis using anti-GFP andanti-Myc antibodies (Supplemental Fig. S4). Leaf cellextracts were precipitated with anti-GFP followed bydetection using anti-Myc antibody. As shown in Fig-ure 3C, when Myc-SymRK was expressed alone, anti-GFP antibody could not pull down SymRK (Fig. 3C).If SIE3-GFP was expressed alone, no protein bandcorresponding to Myc-SymRK could be pulled downby anti-GFP antibody (Fig. 3C). These results indicate

Figure 1. A novel RING finger E3 ubiquitin ligase from L. japonicus. A, SIE3 comprises three conserved domains, the CTLH(green), CRA (blue), and RING finger (purple). B, Phylogenetic tree of SIE3 and its homologs from Populus trichocarpa (Pt), M.truncatula (Mt), rice (Os), maize (Zm), Vitis Vinifera (Vv), Ricinus communis (Rc), and Arabidopsis lyrata (Al). C, The RINGdomain of SIE3-related ligases. Numbers indicate the positions of the eight residues (Cys, Ser, or His) involved in Zn2+ coor-dination. Positions 2, 5, and 6 in some proteins are substituted with Leu, Ser, or Thr (red), which may disable Zn2+ coordination(blue circle).

108 Plant Physiol. Vol. 160, 2012

Yuan et al.

Dow



ber 2021




that the SIE3-SymRK interaction takes place in plantcells.

Self-Ubiquitination of SIE3

The RING domain of SIE3 belongs to the degen-erated C3HC4-type variant (Fig. 1C). It is known that acanonical Cys and His pattern in a RING domain isnot critical for the activity of E3 ubiquitin ligases. Wefirst tested if SIE3 had an enzyme activity for self-ubiquitination, a hallmark property of E3 ubiquitinligases. His-SIE3 was expressed in Escherichia coli andpurified using nickel beads. Self-ubiquitination of His-SIE3 was assayed in the presence of wheat (Triticumaestivum) E1 activating enzyme, human (Homo sapiens)

Figure 2. Interaction of SIE3 with SymRK in yeast cells. A, Functionaldomains and deletion constructs of SIE3 and SymRK. SIE3-DCTLHrepresents the shortest coding region of clones isolated from the libraryscreen. SIE3-DRING lacks the RING domain. SymRK contains a signalpeptide (SP), a Malectin-like motif, a tetrapeptide Gly-Asp-Pro-Cysmotif, three Leu-reach repeats (LRR), a transmembrane domain (TM),and PK. Extracellular domains without the signal peptide. B, Interac-tion between SIE3 and SymRK in yeast cells. Proteins were fused withthe Gal4 DNA binding domain (BD) in pGBKT7 or with its activationdomain (AD) in pGADT7. Yeast cells harboring the constructs weremaintained on SD/-Trp-Leu medium (SD-2) and selected for protein-protein interaction on SD/-Trp-Leu-His-Ade/X-gal (SD-4). The interac-tion between murine tumor-suppressor p53 and simian virus 40 largeT-antigen (pGBKT7-53 and pGADT7-SV40) was used as a positivecontrol, whereas the combination between human lamin C (Lam) andSV40 (pGBKT7-Lam and pGADT7-SV40) served as a negative control.C, Specificity of SIE3-SymRK interaction. The PK domain of NFR1,NFR5, and LHK1 and the extracellular region of LHK1 were used totest for interaction with SIE3. Yeast cells harboring the constructs weremaintained on SD/-Trp-Leu medium (SD-2) and selected for protein-protein interaction on SD/-Trp-Leu-His-Ade (SD-4).

Figure 3. Interaction of SIE3 with SymRK in planta. A, BiFC assay. N.benthamiana leaves were cotransfected with A. tumefaciens carryingconstructs for expression of full-length SIE3 and SymRK that weretagged with SCN and SCC, respectively. The interaction between SIE3and SymRK reconstituted the CFP and allowed fluorescence detectionby a confocal microscope. Coexpression of Arabidopsis CIPK24 andCBL served as a positive control. The split CFP fragments were used innegative controls. Bars = 50 mm (Top) row, 25 mm (Bottom). B, Im-munoblot analysis of proteins expressed in N. benthamiana leaves.Anti-HA and anti-FLAG antibodies detected the HA and FLAG tagspresent in the SCC and SCN domains, respectively. Antitubulin anti-body was used for protein loading references. C, Co-IP of SIE3 withSymRK. N. benthamiana leaves were infiltrated with A. tumefacienscells harboring pSIE3-GFP and pMyc-SymRK (Lane 1), pSIE3-GFP andpBA-myc (Lane 2), and pMyc-SymRK and pBA-myc (Lane 3). Leafextracts were incubated first with anti-GFP antibody and then withimmobilized Protein G-beads. Proteins bound to the beads wereseparated on SDS-PAGE and immunoblotted with anti-Myc antibody.The positions of IgG H chain, Myc-SymRK, polyubiquitinated Myc-SymRK, and protein mass markers are indicated. Asterisk (*) demarks anonspecific band.



Dow



ber 2021

E2 (UBCh5b) conjugating enzyme, and Arabidopsisubiquitin. SIE3 was converted to a mixture of high-Mrubiquitinated protein products (Fig. 4A, lane 3), suggest-ing that SIE3 is capable of undergoing self-ubiquitination.

Ubiquitination of SymRK by SIE3 Ligase

Myc-SymRK and SIE3-GFP were coexpressed in N.benthamiana leaves. Anti-Myc antibody was used forCo-IP, and either anti-Ub (for ubiquitin) or anti-Mycwas used for immunoblotting. When Myc-SymRK wasexpressed alone, Myc-SymRK and its ubiquitinatedproducts that were likely to be generated by endoge-nous E3 enzymes were detected (Fig. 4B, lane 2). WhenSIE3 was coexpressed with SymRK, Myc-SymRK andits polyubiquitinated products were detected (Fig. 4B,lane 1). The intensity of SymRK-related bands weremuch stronger in the leaf cells coexpressing SymRKand SIE3 (Fig. 4B, lane 1) than those expressing SymRKalone (Fig. 4B, lane 2). These results showed that SIE3could promote in vivo ubiquitination of SymRK. Usinganti-Ub antibody, we were able to confirm that thehigh molecular mass smear ladders (Fig. 4B, lane 1)and the degraded Myc-SymRK (Fig. 4B lane 2) bandswere indeed ubiquitinated (Fig. 4B, right).

SIE3 Gene Expression

Real-time quantitative reverse transcription (qRT)-PCR results showed that the SIE3 mRNA was present

in all tested tissues of L. japonicus plants (Fig. 5A). M.loti-inoculated roots with developing nodules werefound to have the highest expression level of SIE3mRNA. The lowest expression was found in maturenodules. During the nodulation period, the expressionof SIE3 was elevated 8 d after rhizobial infection andcontinued to stay at high levels 10 d post inoculation(Fig. 5B). These observations were consistent with theproposed role of SIE3 in rhizobial entry and noduleorganogenesis (see below).

Subcellular Localization

GFP-SIE3 was expressed in transgenic onion (Alliumcepa) epidermal cells by infiltration with Agrobacteriumrhizogenes. Subcellular localization of GFP-SIE3 wasobserved using a confocal microscope. GFP aloneserved as a control. As expected, GFP was found inboth the cytoplasm and nuclei (Fig. 5C, top). The greenfluorescence of GFP-SIE3 was concentrated to the nu-clei and plasma membrane, and could also be found inthe cytoplasm and extracellular space in plasmolyzedcells induced by 4% (w/v) NaCl (Fig. 5C, bottom).When expressed in transgenic hairy roots of L. japoni-cus and transgenic N. benthamiana leaves, a strongfluorescent signal was found in the nuclei and plasmamembrane, and a weak fluorescent signal was alsoobserved in the cytoplasm (Fig. 5, D and E). Com-paring the subcellular localization data (Fig. 5, C–E)with the BiFC results of interaction with SymRK(Fig. 3A; Supplemental Fig. S3), we conclude that the

Figure 4. Ubiquitination of SymRK by SIE3. A, Self-ubiquitination of SIE3 in vitro. His-SIE3 was incubated with wheat E1,human E2 (UBCh5b), and Arabidopsis ubiquitin. Reaction products were immunoblotted using anti-Ub antibody. The positionsof Ub, monoubiquitinated E2 (Ub-E2) and polyubiquitinated SIE3 (PUb-SIE3) are indicated. Protein mass markers are shown inkD. B, Ubiquitination of SymRK by SIE3 in N. benthamiana leaf cells. Leaf extracts expressing pSIE3-GFP and pMyc-SymRK(Lane 1), pMyc-SymRK and pBA-myc (Lane 2), and pMyc-GFP and pBA-myc (Lane 3) were immunoprecipitated with anti-Mycantibody. The immunocomplex was absorbed to Protein G beads and resolved on SDS-PAGE. Anti-Myc and anti-Ub antibodieswere used to detect Myc-tagged proteins and ubiquitinated products. Protein mass markers are shown in kD. The positions ofIgG H chain, Myc-SymRK, ubiquitinated SymRK (Ub-SymRK), and degraded SymRK are indicated.


Yuan et al.

Dow



ber 2021


SIE3-SymRK interaction occurs at both sides of theplasma membrane, although SIE3 may have additionalfunctions in the nuclei.

Promotion of Nodulation by SIE3 Overexpression

SIE3 was expressed under the control of the maize (Zeamays) ubiquitin promoter (SIE3OX) in transgenic hairyroots of L. japonicus (Kumagai and Kouchi, 2003; Chenet al., 2012). The phenotypes of nodulation were scored 4weeks after inoculation with M. loti MAFF303099, whichexpresses b-galactosidase (lacZ) as a constitutive markerfor the presence of rhizobial cells (Tansengco et al., 2003).Real-time qRT-PCR analysis indicated that the expressionlevel of the SIE3 transgene in SIE3OX hairy roots was 10-fold of that in the control (Fig. 6B). Significantly morenodules were produced in SIE3OX hairy roots than in thecontrol in two large-scale experiments (SupplementalTable S1; Fig. 6A). Nodule numbers per root system wereincreased from 7.6 in the control to 13.3 in SIE3OX hairy

roots in experiment I, and from 4.0 in the control to 10.2in SIE3OX hairy roots in experiment II (SupplementalTable S1; Supplemental Fig. S5A). These results suggestthat SIE3 plays an important role as a positive regulatorof nodule initiation in L. japonicus.

Suppression of Rhizobial Infection by SIE3 RNAi

An SIE3-specific RNAi construct was prepared totarget a 210-bp fragment containing the 59-untranslatedregion and a short sequence of the coding region.Control hairy roots were generated using cloning vectorpCAMBIA1301-35S-int-T7. Transgenic hairy roots wereinoculated with M. loti strain MAFF303099 for nodula-tion. Real-time qRT-PCR analysis showed that the SIE3transcript was reduced to approximately 50% in RNAihairy roots as compared with that in the control. Nod-ulation phenotypes were scored 4 weeks after rhizobialinoculation. As shown in Figure 7A and SupplementalTable S2, nodule numbers in SIE3 RNAi hairy roots

Figure 5. Gene expression and subcellular localization of SIE3. A, Expression of the SIE3 gene in different tissues of L. japo-nicus. Total RNAwas isolated from uninoculated roots (UR), M. loti-inoculated roots (IR), stems (S), leaves (L), and nodules (N).The expression levels of the SIE3mRNA transcripts were determined by real-time PCR. Relative expression levels of SIE3mRNAin different tissues were calculated with reference to that in the uninoculated roots. B, Expression of the SIE3 gene duringM. lotiinfection and nodule development in L. japonicus. Roots were inoculated with M. loti and harvested at different time intervalspost inoculation. Total RNAwas isolated and the expression of the SIE3 gene was measured by real-time PCR. The values of SIE3expression were normalized using polyubiquitin as an internal control. Roots treated with water served as the uninoculationcontrol. Relative expression levels were calculated with reference to the value of the control roots harvested 5 h after mocktreatment with water. C, Subcellular localization of SIE3 protein in transgenic onion cells by infiltration with A. rhizogenesLBA1334 cells carrying pSIE3-GFP (bottom). GFP alone served as a control (top). The onion cells were treated with 4% (w/v)NaCl to induce plasmolysis before microscope observation. Bars, 30 mm. D and E, Subcellular localization of SIE3 protein intransgenic hairy roots of L. japonicus (D) and N. benthamiana leaves (E) using A. rhizogenes LBA1334 cells. N, nucleus. Bars =20 mm.



Dow



ber 2021








were significantly lower than those in the control in allthree large-scale experiments. Analysis of the distribu-tion of nodule numbers per plant revealed that 40% to70% of SIE3 RNAi hairy roots developed only one orno nodule. In contrast, 90% of the control hairy rootsproduced more than six nodules (Supplemental Fig.S5B). These data suggest that SIE3 plays a positiveregulator role in nodulation in L. japonicus, which isconsistent with the conclusion drawn from the analysisof SIE3OX hairy roots.

To investigate if nodulin gene expression was af-fected by SIE3 RNAi, we examined the expressionlevels of NIN and ENOD40-2, two early nodulin genesimplicated in the processes of rhizobial entrance,nodule initiation, and subsequent organogenesis(Schauser et al., 1999; Kumagai et al., 2006), as well asthe expression of Lb (for leghemoglobin), a typical nod-ulin gene required for nitrogen fixation (Ott et al.,2005). Real-time PCR analysis showed that the ex-pression levels of the two early nodulin genes (NINand ENOD40-2), but not Lb, were reduced drasticallyin the SIE3 RNAi hairy roots, as compared with thosein the control hairy roots (Fig. 7C; Supplemental Fig.S6). Thus, the reduction in nodulation in SIE3 RNAihairy roots could be a result of disruption in the pro-cesses of nodule development.

L. japonicus SymRK is known to play a role in ITinitiation (Stracke et al., 2002). We scored IT numbers inSIE3 RNAi hairy roots 8 d after rhizobial inoculation.ITs were visible as blue threads under a microscopeafter staining with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside acid (X-gal) that reacted with thelacZ-labeled M. loti cells (Tansengco et al., 2003;Kumagai et al., 2006). ITs were divided into four groupsaccording to the cellular locations of their growing tips:1) those emerging from curled root hairs, 2) those

growing from elongated root hairs to the root epider-mis, 3) those penetrating through the root cortex, and4) those reaching to the nodule primordia, as shownin Supplemental Figure S7. The numbers of ITs in allcategories were reduced drastically in SIE3 RNAi hairyroots as comparedwith the control (Fig. 7D; SupplementalTable S3). These results demonstrate a role of SIE3 in theearly events of rhizobial infection. Taken together, wehave identified an E3 ligase of SymRK that acts as a keyregulator of nodule development.

DISCUSSION

SymRK is believed to function in both NF perceptionand downstream signal transduction, and is requiredfor the earliest detectable root hair responses (Endreet al., 2002; Stracke et al., 2002). However, little isknown about how SymRK transduces the NF signal todownstream components. In this report, we describe anovel E3 ubiquitin ligase, SIE3, which mediated ubiq-uitination of SymRK. Our results demonstrate a piv-otal role of SIE3 in promotion of rhizobial infectionand nodule initiation in L. japonicus.

A Novel RING Finger E3 Ubiquitin Ligase

SIE3 is related to and distinct from human RMD5and yeast GID2. It is not closely related to any previ-ously characterized RING finger E3 ligases, such as thecullin Ring-based ligases (CRLS) from higher plants(Petroski and Deshaies, 2005; Deshaies and Joazeiro,2009). It defines a novel class of RING domain E3 li-gases, and will be referred to hereafter as the SIE3-related ligases. First, this family of ligases contains acharacteristic arrangement of three conserved domains,

Figure 6. Effect of SIE3 over-expression on nodulation. A–E,Nodulation phenotypes of trans-genic hairy roots 4 weeks afterinoculation with M. loti. Hairyroots expressing vector pU1301served as a control (A). Fourindependent transgenic plants(SIE3OX-1 to -4) were chosen forphotography (B–E). F, Analysisof the transcript levels of SIE3 inthe control and SIE3OX hairyroots (SIE3OX-1 to -4). Total RNAisolated from individual rootsystem was used for real-timePCR. Relative expression levelsof SIE3 transcripts in SIE3OX

hairy roots were calculated withreference to that of the controlhairy roots. Bars = 5 mm. [Seeonline article for color versionof this figure.]


Yuan et al.

Dow



ber 2021








the CTLH, CRA, and a degenerate RING finger. Al-though the function of the CTLH motif remains un-known, the CRA and RING domains have beenimplicated in protein-protein interactions. Yeast GID2E3 ligase does not contain the CRA domain. Second,SIE3-related ligases have a characteristic pattern ofsubstitutions in the RING domain, where Cys residuesat positions 2 and 6 are replaced with Ser or Thr (S/T),resulting in the coordination of only one zinc ion. Thistype of RING belongs to the RING-S/T variant and

consists of C-x2-S/L-x14-C-x-H-x2-C-x2-S/T-x13-C-x2-C,where x could be any amino acid. In contrast, the ca-nonical C3HC4 RING (C-x2-C-x9-39-C-x1-3-H-x2-3-C-x2-C-x4-48-C-x2-C) is capable of coordinating with two zincions (Lorick et al., 1999; Fang and Weissman, 2004;Deshaies and Joazeiro, 2009). Human RMD5 has aneven more degenerated RING finger, which may notbind zinc at all and may be involved only in protein-protein interactions (Santt et al., 2008). Third, SIE3-related ligases are conserved in plants (Fig. 1B;

Figure 7. Effect of SIE3 RNAi on rhizobial infection and nodulation. A, Transgenic hairy roots expressing pSIE3-RNAi or vector pCAMBIA1301-35S-int-T7 (Control). Nodulation phenotypes were examined 4 weeks after inoculation with M. loti MAFF303099. The SIE3 RNAi hairy roots weredivided into 4 groups: RNAi-1 (no nodule per plant), RNAi-2 (1 nodule per plant), RNAi-3 (3 to 7 nodules per plant), and RNAi-4 (.7 nodules perplant). Bars = 5 mm. B, Real-time qRT-PCR analysis of SIE3 expression in SIE3 RNAi hairy roots. Total RNAwas isolated from each of the 4 groups ofSIE3 RNAi hairy roots. Relative SIE3 expression levels were calculated with reference to the SIE3 transcript level in control hairy roots. C, Real-timeqRT-PCR analysis of the expression levels of NIN and ENOD40-2 transcripts in groups 1 and 2 of SIE3 RNAi transgenic hairy roots. D, SIE3 RNAihairy roots were stained with X-gal 8 d postinoculation withM. loti and the presence of ITs were scored under a microscope. A root segment of 2-cmlong was randomly chosen to represent each root for analysis of IT phenotypes. Forty-two SIE3 RNAi hairy roots segments and 46 control hairy rootssegments were scored. Four types of infection threads were recorded according to the cellular locations of IT tips, i.e. infection threads in the roothair, root epidermis, root cortex, and nodule primordium. [See online article for color version of this figure.]



Dow



ber 2021

Supplemental Fig. S1). Arabidopsis contains threeSIE3-related ligase genes (At2g22690, At3g55070, andAt4g37880), none of which has been characterized sofar. Fourth, SIE3-related proteins exist as a reverseparallel homodimer (Supplemental Fig. S2A). The di-merization of SIE3 could be detected when expressedin yeast cells (Supplemental Fig. S2B). Therefore, SIE3-related proteins represent a new class of RING-domainE3 ligases from plants.

Ubiquitination of SymRK by SIE3

In the presence of E1, E2, and ubiquitin, SIE3exhibited ligase activity for self-ubiquitination in vitro(Fig. 4A). When expressed in plant cells, the level ofubiquitination of SymRK was increased by coex-pression of SIE3 (Fig. 4B). There are two main modesof action by which an E3 could exert an effect at thecellular level through interaction with an RLK. First,an E3 could mediate the degradation of an RLKthrough ubiquitination. Second, the RLK could mod-ulate, via phosphorylation, the enzyme activity of theE3, which in turn regulates the stability of downstreamcomponent(s). Although plant E3 ligases have previ-ously been shown to interact with RLKs (Gu et al.,1998; Kim et al., 2003; Wang et al., 2006; Samuel et al.,2008; Mbengue et al., 2010), these E3 ligases belongto the Ankyrin motif-containing proteins and the plantU-box (PUB) enzymes, which differ in protein struc-ture from SIE3-related ligases, and their direct role inRLK ubiquitination has not been shown. In rice (Oryzasativa), the kinase domain of XA21 binds and phos-phorylates the E3 ligase XB3 (Wang et al., 2006). InArabidopsis, the kinase domain of the S-locus receptorkinase binds and phosphorylates the E3 ligase ARC1(Samuel et al., 2008). Similarly in M. truncatula, thekinase domain of Nod factor receptor LYK3 phos-phorylates the E3 ligase PUB1 (Mbengue et al., 2010).Because there has not been evidence for direct ubiq-uitination of RLKs by these E3 ligases, the possiblemode of action for these E3 ligases is the second one;The activity of these E3 ligases is modulated by RLKs,and they then regulate downstream components. ForSIE3, both modes of action could apply. Our obser-vation clearly shows that SIE3 could mediate ubiq-uitination of SymRK. However, whether the SIE3activity is modulated by SymRK and whether SIE3regulates the stability of downstream components re-mains to be investigated.

A Positive Regulator of Nodulation

SymRK is the checkpoint of bacterial entry and es-sential for nodulation (Stracke et al., 2002; Radutoiuet al., 2003; Yoshida and Parniske, 2005). SIE3 couldexert its biological function through ubiquitination ofSymRK, and it is possible that ubiquitinated SymRKmay allow sustained signal transduction to downstream

host responses. In SIE3OX hairy roots, the elevated SIE3level promoted nodule formation (Supplemental TableS1, Fig. 6). Consistent with this finding, fewer ITs andnodules were developed in hairy roots expressing SIE3RNAi (Supplemental Table S2; Fig. 7, A, B, and D).These data support the notion that both SIE3 andSymRK serve as positive regulators of NF signaling,rhizobial infection, and nodulation. Ubiquitination is aposttranslational modification specific to eukaryotesand has been recognized as a major means in regu-lation of protein stability. Recently, more and morenonproteolytic functions of ubiquitination have beendiscovered. It can regulate the transcription activity oftranscription factors (Herrera and Triezenberg, 2004),and modulate the enzyme activity and protein sub-cellular localization (Chen and Sun, 2009; Komander,2009; Yang et al., 2010). We propose that SIE3 mayexert its positive effect on symbiotic signaling viaubiquitination of SymRK. There are at least two pos-sible mechanisms. First, SIE3 may promote SymRKsignaling through activating its protein kinase activity.Second, ubiquitination of SymRK by SIE3 may helpmaintain the protein conformation in the membrane,which could be useful for its biological function.However, the exact mechanism by which SIE3 regu-lates SymRK remains to be further investigated. SIE3represents a new class of RING-domain E3 ligasesfrom higher plants. This work has opened a new av-enue for future research on ubiquitination and receptorkinases in plants.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of wild-type Lotus japonicus ‘MG-20’ were surface-sterilized andgerminated on agar plates. Seedlings grown in sterilized conditions were usedfor generation of transgenic hairy roots via Agrobacterium rhizogenes. Wild-typeNicotiana benthamiana plants were grown in a growth chamber at 22°C and70% relative humidity under a 16-h-light/8-h-dark photoperiod for about 4weeks before infiltration with Agrobacterium spp. cells. After infiltration, plantswere kept under the same growth conditions.

Yeast Two-Hybrid Screen

Screening of interaction clones was performed as described previously (Zhuet al., 2008). A total of 107 transformants were screened for protein-proteininteraction on synthetic defined medium SD/-Leu-Trp-His-Ade. Plasmidsfrom positive clones were verified for interaction by retransformation intoyeast (Saccharomyces cerevisiae) AH109 cells containing pGBKT7-SymRK-EC.Positive colonies were also tested for expression of the lacZ reporter (Chenet al., 2012).

Plasmid Construction for Protein-Protein Interactionsin Yeast

Full-length cDNA of SIE3 and SIE3DRING cDNAs were amplified by PCRand inserted into NdeI/EcoRΙ sites of pGBKT7 and pGADT7; The SymRK-PKand SymRK-EC cDNAs were amplified by PCR and inserted into NdeI/EcoRΙsites of pGBKT7 and pGADT7; NFR5-PK cDNAs were amplified by PCR andinserted into EcoRI/XhoI of pGADT7 and EcoRI/salI of pGBKT7; NFR1-PKcDNAs were amplified by PCR and inserted into EcoRI/SalI of pGBKT7;LHK1-PK cDNAs were amplified by PCR and inserted into EcoRI/SalI of


Yuan et al.

Dow



ber 2021







pGBKT7; and LHK1-EC cDNAs were amplified by PCR and inserted intoBamHI/SalI of pGBKT7. Sequences of primers are provided in SupplementalTable S4.

Real-Time PCR

RNA samples were treated with DNase I (Promega, Tokyo, Japan) andreverse-transcribed using SYBR PrimeScript RT-PCR kit II (Takara) with oligo(dT) as primer. The cDNA from RT of about 300 ng of RNA was used astemplate for real-time PCR using a Mini Opticon real-time PCR system(LightCycler 480). Primer sets used for real-time PCR were listed in Table 4.Cycling conditions were as follows: preheating at 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 12 s, followed by 72°C for 5 s. Poly-ubiquitin (AW720576) transcript was used as an internal control. The identityof amplified PCR products was verified by DNA sequencing. Gene expressiondata were normalized on the basis that the expression levels of ubiquitin wereassumed to be the same in all RNA samples.

BiFC Experiments

Full-length cDNA of SymRK, SymRK-EC, and SymRK-PK were cloned intothe SpeΙ/SalΙ site of pSCYCE-R vector to obtain SymRK:SCC, SymRK-EC:SCC,and SymRK-PK:SCC fusions (Waadt et al., 2008). Full-length cDNA of SIE3was cloned into the BamHI/XhoΙ site of pSCYNE vector to obtain SIE3:SCNfusion. Plasmids were introduced into Agrobacterium tumefaciens strain GV3101cells by electroporation. A. tumefaciens cells containing plasmids were pelletedand resuspended in the infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl2,150 mM acetosyringone). A. tumefaciens strains containing different plasmidswere mixed to a final optical density at 600 nm of 0.5 for each A. tumefaciensstrain. The mixture of A. tumefaciens strains was incubated on bench for 2 to4 h at room temperature and used for infiltration into the top leaves of 4- to 6-week-old N. benthamiana plants using a 2-ml syringe. Cyan fluorescence wasassayed 2 to 5 d after infiltration with A. tumefaciens using a Zeiss LSM510laser scanning microscope with a CFP filter set (excitation wavelength at405 nm, emission wavelength at 477 nm). Cyan fluorescence of leaf cellsexpressing the two proteins (SIE3:SCN and SymRK-EC:SCC or SymRK-PK:SCC) were observed using a confocal microscope (Olympus BX61WI).

Generation of Transgenic Hairy Roots

Wild-type L. japonicus ‘MG-20’ was used to produce transgenic hairy rootsusing an A. rhizogenes-mediated procedure as described previously (Kumagaiand Kouchi, 2003; Chen et al., 2012; Ke et al., 2012). Briefly, seedlings germi-nated on sterile agar-plates were cut at the base of the hypocotyls and placedin suspension of A. rhizogenes LBA1334 cells containing plasmids in a petridish for 30 min. The seedlings with cotyledons were transferred onto agarplates of Murashige and Skoog (MS; Sigma) medium containing 1.5% (w/v)Suc and cocultivated for 5 d in a growth chamber. The plants were transferredonto agar plates containing 250 mg/ml of cefotaxime and grown for an ad-ditional 10 d until hairy roots (a few cm long) developed from the base ofhypocotyls. A short root tip (2- to 3-mm long) was removed from each hairyroot and tested for GUS activity in staining solution (100 mM sodium phos-phate buffer, pH 7.0, 0.1% (v/v) Triton X-100, 0.1% (w/v) N-laurylsarcosine,10 mM Na2EDTA, 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6, and 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-b-glucuronic acid) overnight at 37°C in the dark. Each hairyroot was properly labeled. Hairy roots with GUS-negative tips were removedfrom the hypocotyl. Hairy roots with GUS-positive tips were preserved andallowed to continue to grow. Each seedling was allowed to have 1 to 2transgenic hairy roots. Plants with transgenic hairy roots were transferred topots filled with vermiculite and sand (1:1) with one-half-strength Broughton &Dilworth (B&D) medium (Broughton and Dilworth, 1971) and grown in achamber in a 16-/8-h day/night cycle at 22°C. After a week of adaptation,plants were inoculated with Mesorhizobium loti strain MAFF303099 and grownin the same medium without ammonium nitrate.

Subcellular Localization of SIE3

The full-length coding sequence of SIE3 was cloned into the NcoI/SpeI siteof pCAMBIA1302 vector, generating pSIE3-GFP. A. rhizogenes strain LBA1334cells carrying pSIE-GFP was used to induce hairy roots of L. japonicus plants.GFP signals in transgenic hairy roots were observed using confocal micro-scope Zeiss LSM510. GFP-SIE3 was also expressed in transgenic N.

benthamiana leaves using A. rhizogenes strain LBA1334 cells carrying pSIE3-GFP. Green fluorescent signal was observed using confocal microscope Olym-pus BX61WI.

A. rhizogenes strain LBA1334 cells carrying pSIE3-GFP were also used forexpression of GFP-tagged SIE3 in onion (Allium cepa) epidermal cells via A.rhizogenes infiltration. A 50-ml overnight culture of A. rhizogenes was collectedby centrifugation and resuspended in 20 ml of 0.53 MS medium supple-mented with 150 mM acetosyringone. After 3 to 4 h growth at 28°C, surfactantSilwet L-77 was added at a final concentration of 0.01% (v/v). Onion bulbslices (1.0 cm2) were surface-sterilized in 75% (v/v) ethanol for 1 min andwashed with 13 MS liquid medium, followed by treatment with 2% (w/v)sodium hypochlorite for 2 min. After four times of washing with 13MS liquidmedium, the onion slices were treated with 4 M NaCl for 20 min, and thenincubated with the A. rhizogenes cell culture in 0.01% (v/v) Silwet L-77 for 90min. After removal of the surface liquid, onion slices were placed onto MSmedium-agar plates and grown at 26°C for 36 to 48 h in the dark. GFP signalswere observed using confocal microscope Zeiss LSM510.

Overexpression of SIE3

The full-length coding sequence of SIE3 was cloned into the KpnI/BamHIsite of pU13011, generating pMUb:SIE3. A. rhizogenes strain LBA1334 cellscarrying pMUb:SIE3 were used to induce hairy root formation in L. japonicus.Nodulation phenotypes of transgenic hairy roots were scored 4 weeks forinoculation with M. loti. Transgenic hairy roots expressing the empty vector(pU1301) were used as a control.

SIE3-Specific RNAi

A 210-bp fragment of the 59-region of SIE3 cDNA containing a short se-quence of the coding region was amplified by PCR and cloned into pCAM-BIA1301-35S-int-T7, generating pSIE3-RNAi in which the sense and antisenseSIE3 RNA sequences would be linked in tandem separated by the Arabidopsis(Arabidopsis thaliana) Actin-11 intron. This SIE3-specific intron-hairpin RNAwas expressed under the control of the cauliflower mosaic virus 35S promoter.A. rhizogenes LBA1334 cells harboring pSIE3-RNAi was used to induce for-mation of transgenic hairy roots in L. japonicus. After inoculation with M. lotiMAFF303099, plants with transgenic hairy roots were grown for 4 weeks andnodulation phenotypes were scored.

Protein Extraction and Immunoblot Analysis

Proteins were extracted from N. benthamiana leaves expressing recombinantproteins using two protein extraction buffers as described in Liu et al., 2010.The denaturing buffer contained 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 4 M

urea, and 1 mM phenylmethylsulfonyl fluoride or protease inhibitor cocktailCompleteMini tablets (Roche). The native extraction buffer 1 contained 50 mM

Tris-MES, pH 8.0, 0.5 M Suc, 1 mM MgCl2, 10 mM EDTA, 5 mM dithiothreitol,and protease inhibitor cocktail CompleteMini tablets (Roche). The extractswere used for immunoblot analysis and immunoprecipitation. Proteins preparedusing the denaturing buffer was subjected to western-blot analysis using theMillipore Chemiluminescent HRP Substrate kit (Millipore). The sources anddilutions of antibodies used in these experiments were as follows: anti-HA an-tibody (Santa Cruz, 1:500), anti-Myc antibody (Santa Cruz, 1:500), anti-GFPantibody (Clontech, 1:1,000), antiubiquitin monoclonal antibody (obtained fromthe Xie laboratory, Chinese Academy of Sciences, 1:5,000), and goat antimousehorseradish peroxidase-conjugated antibody (Proteintech, 1:2,500).

Immunoprecipitation

SymRK-Myc protein and the protein coexpressed SymRK-myc and SIE3-GFP extracted using the denative extraction buffer and SIE3-GFP proteinextracted using the native extraction buffer were used for immunoprecipi-tation. Corresponding antibodies were added to the extract (10 mg/mlproteins). MG132 was also added at a final concentration of 50 mM to pre-vent protein degradation. The mixtures were kept at 4°C with gentleshaking for 3 h to overnight. Protein G agarose beads (Millipore) was addedto the mixture at 20 mg/ml in order to immobilize the immunocomplex.After gentle shaking at 4°C for 3 h, the agarose beads were recovered bycentrifugation at 4,000 rpm for 1 min and washed three times with coldphosphate-buffered saline.



Dow



ber 2021



Ubiquitination Assay

For in vitro E3 ubiquitin ligase activity assays, purified His-tagged SIE3 wasincubated with wheat (Triticum aestivum) E1 (GenBank ID GI:136632), human(Homo sapiens) E2 (UBCh5b), and Arabidopsis ubiquitin (UBQ14) as describedelsewhere (Liu et al., 2010). Reactions were carried out with agitation for 1.5 hat 30°C. Proteins were resolved on SDS-PAGE, followed by immunoblotanalysis using antiubiquitin monoclonal antibody.

To test if SymRK could serve as a substrate for ubiquitination by SIE3, weexpressed Myc-tagged SymRK and GFP-tagged SIE3 in leaves of N. ben-thamiana plants via infiltration with A. tumefaciens strain EHA105. We per-formed ubiquitination assays using SymRK as a substrate in vivo systems. Forin vivo ubiquitination assays, we transfected plants with pMyc-SymRK alone,or a combination of pMyc-SymRK and pSIE3-GFP. We used pMyc-GFP toreplace pMyc-SymRK in the control. Protein extracts from transfected N.benthamiana leaves were immunoprecipitated with anti-Myc antibody. West-ern-blot analysis was carried out using anti-Myc antibody and antiubiquitinmonoclonal antibody.

Rhizobial Infection Assay

For rhizobial infection assay, transgenic hairy roots were inoculated withM.loti strain MAFF303099, which harbored the lacZ gene as a constitutive marker(Tansengco et al., 2003), and was grown in pots containing sand and ver-miculite at a 1:1 volume ratio. Seven days after rhizobial inoculation, trans-genic hairy roots were stained for lacZ activity (Tansengco et al., 2003) andobserved using an Olympus BX51 microscope under bright-field illumination.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Amino acid sequence alignment of SIE3-likeubiquitin ligases from plants.

Supplemental Figure S2. Protein structure and dimerization of L. japonicusSIE3.

Supplemental Figure S3. BiFC assay of interaction of SIE3 with the extra-cellular or intracellular portion of SymRK.

Supplemental Figure S4. Time course of protein expression in N. benthami-ana leaves.

Supplemental Figure S5. Nodulation phenotypes of transgenic hairy rootswith altered SIE3 transcript levels.

Supplemental Figure S6. Real-time qRT-PCR analysis of gene expressionin SIE3 RNAi transgenic hairy roots.

Supplemental Figure S7. Classification of infection threads in transgenichairy roots.

Supplemental Table S1. Nodulation phenotypes of SIE3OX hairy roots.

Supplemental Table S2.Nodulation phenotypes of SIE3 RNAi hairy roots.

Supplemental Table S3. Phenotypes of rhizobial infection in SIE3 RNAihairy roots.

Supplemental Table S4. Primers used in this study.

ACKNOWLEDGMENTS

S.Y., Z.Z., and Z.H. designed this work and wrote the manuscript. S.Y.performed most of the experiments, and H.Z., H.G., W.F., L.L., T.C., D.K.,H.K., and Q.X. contributed substantially to the completion of this work.

Received April 22, 2011; accepted July 18, 2012; published July 20, 2012.

LITERATURE CITED

Banba M, Gutjahr C, Miyao A, Hirochika H, Paszkowski U, Kouchi H,Imaizumi-Anraku H (2008) Divergence of evolutionary ways among com-mon sym genes: CASTOR and CCaMK show functional conservation

between two symbiosis systems and constitute the root of a common sig-naling pathway. Plant Cell Physiol 49: 1659–1671

Broughton WJ, Dilworth MJ (1971) Control of leghaemoglobin synthesis insnake beans. Biochem J 125: 1075–1080

Capoen W, Goormachtig S, De Rycke R, Schroeyers K, Holsters M (2005)SrSymRK, a plant receptor essential for symbiosome formation. ProcNatl Acad Sci USA 102: 10369–10374

Catoira R, Galera C, de Billy F, Penmetsa RV, Journet EP, Maillet F,Rosenberg C, Cook D, Gough C, Dénarié J (2000) Four genes of Med-icago truncatula controlling components of a nod factor transductionpathway. Plant Cell 12: 1647–1666

Chen T, Zhu H, Ke D, Cai K, Wang C, Gou H, Hong Z, Zhang Z (2012) AMAP kinase kinase interacts with SymRK and regulates nodule orga-nogenesis in Lotus japonicus. Plant Cell 24: 823–838

Chen ZJ, Sun LJ (2009) Nonproteolytic functions of ubiquitin in cell sig-naling. Mol Cell 33: 275–286

D’Haeze W, Holsters M (2002) Nod factor structures, responses, and per-ception during initiation of nodule development. Glycobiology 12: 79R–105R

Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. AnnuRev Biochem 78: 399–434

Ehrhardt DW, Wais R, Long SR (1996) Calcium spiking in plant root hairsresponding to Rhizobium nodulation signals. Cell 85: 673–681

Endre G, Kereszt A, Kevei Z, Mihacea S, Kaló P, Kiss GB (2002) A re-ceptor kinase gene regulating symbiotic nodule development. Nature417: 962–966

Fang S, Weissman AM (2004) A field guide to ubiquitylation. Cell Mol LifeSci 61: 1546–1561

Felle HH, Kondorosi E, Kondorosi A, Schultze M (1999) Elevation of thecytosolic free [Ca2+] is indispensable for the transduction of the Nodfactor signal in alfalfa. Plant Physiol 121: 273–280

Gu T, Mazzurco M, Sulaman W, Matias DD, Goring DR (1998) Binding ofan arm repeat protein to the kinase domain of the S-locus receptor ki-nase. Proc Natl Acad Sci USA 95: 382–387

Herrera FJ, Triezenberg SJ (2004) Molecular biology: what ubiquitin candispatch do for transcription. Curr Biol 14: 622–624

Ke D, Fang Q, Chen C, Zhu H, Chen T, Chang X, Yuan S, Kang H, Ma L,Hong Z, et al (2012) The small GTPase ROP6 interacts with NFR5 andis involved in nodule formation in Lotus japonicus. Plant Physiol 159:131–143

Kevei Z, Lougnon G, Mergaert P, Horvath GV, Kereszt A, Jayaraman D,Zaman N, Marcel F, Regulski K, Kiss GB, et al (2007) 3-Hydroxy-3-methylglutaryl coenzyme A reductase1 interacts with NORK and iscrucial for nodulation in Medicago truncatula. Plant Cell 19: 3974–3989

Kim M, Cho HS, Kim DM, Lee JH, Pai HS (2003) CHRK1, a chitinase-related receptor-like kinase, interacts with NtPUB4, an armadillo repeatprotein, in tobacco. Biochim Biophys Acta 1651: 50–59

Kistner C, Parniske M (2002) Evolution of signal transduction in intra-cellular symbiosis. Trends Plant Sci 7: 511–518

Komander D (2009) The emerging complexity of protein ubiquitination.Biochem Soc Trans 37: 937–953

Kosuta S, Held M, Hossain MS, Morieri G, Macgillivary A, Johansen C,Antolín-Llovera M, Parniske M, Oldroyd GE, Downie AJ, et al (2011)Lotus japonicus symRK-14 uncouples the cortical and epidermal symbi-otic program. Plant J 67: 929–940

Kumagai H, Kinoshita E, Ridge RW, Kouchi H (2006) RNAi knock-downof ENOD40s leads to significant suppression of nodule formation inLotus japonicus. Plant Cell Physiol 47: 1102–1111

Kumagai H, Kouchi H (2003) Gene silencing by expression of hairpin RNAin Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16:663–668

Lefebvre B, Timmers T, Mbengue M, Moreau S, Hervé C, Tóth K,Bittencourt-Silvestre J, Klaus D, Deslandes L, Godiard L, et al (2010) Aremorin protein interacts with symbiotic receptors and regulates bac-terial infection. Proc Natl Acad Sci USA 107: 2343–2348

Liu L, Zhang Y, Tang S, Zhao Q, Zhang Z, Zhang H, Dong L, Guo H, XieQ (2010) An efficient system to detect protein ubiquitination by agro-infiltration in Nicotiana benthamiana. Plant J 61: 893–903

Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM(1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci USA 96: 11364–11369

Madsen EB, Madsen LH, Radutoiu S, Olbryt M, Rakwalska M,Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, et al (2003) A


Yuan et al.

Dow



ber 2021












receptor kinase gene of the LysM type is involved in legume perceptionof rhizobial signals. Nature 425: 637–640

Madsen LH, Tirichine L, Jurkiewicz A, Sullivan JT, Heckmann AB, BekAS, Ronson CW, James EK, Stougaard J (2010) The molecular networkgoverning nodule organogenesis and infection in the model legumeLotus japonicus. Nat Commun 1: 10

Mbengue M, Camut S, de Carvalho-Niebel F, Deslandes L, Froidure S,Klaus-Heisen D, Moreau S, Rivas S, Timmers T, Hervé C, et al (2010)The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with theLYK3 symbiotic receptor and negatively regulates infection and nodu-lation. Plant Cell 22: 3474–3488

Mitra RM, Gleason CA, Edwards A, Hadfield J, Downie JA, Oldroyd GE,Long SR (2004) A Ca2+/calmodulin-dependent protein kinase requiredfor symbiotic nodule development: gene identification by transcript-based cloning. Proc Natl Acad Sci USA 101: 4701–4705

Miwa H, Sun J, Oldroyd GE, Downie JA (2006) Analysis of calciumspiking using a cameleon calcium sensor reveals that nodulation geneexpression is regulated by calcium spike number and the developmentalstatus of the cell. Plant J 48: 883–894

Oldroyd GE, Murray JD, Poole PS, Downie JA (2011) The rules ofengagement in the legume-rhizobial symbiosis. Annu Rev Genet 45:119–144

Ott T, van Dongen JT, Günther C, Krusell L, Desbrosses G, Vigeolas H,Bock V, Czechowski T, Geigenberger P, Udvardi MK (2005) Symbioticleghemoglobins are crucial for nitrogen fixation in legume root nodulesbut not for general plant growth and development. Curr Biol 15: 531–535

Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RINGubiquitin ligases. Nat Rev Mol Cell Biol 6: 9–20

Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M,Sato S, Nakamura Y, Tabata S, Sandal N, et al (2003) Plant recognitionof symbiotic bacteria requires two LysM receptor-like kinases. Nature425: 585–592

Radutoiu S, Madsen LH, Madsen EB, Jurkiewicz A, Fukai E, QuistgaardEMH, Albrektsen AS, James EK, Thirup S, Stougaard J (2007) LysMdomains mediate lipochitin-oligosaccharide recognition and Nfr genesextend the symbiotic host range. EMBO J 26: 3923–3935

Samuel MA, Mudgil Y, Salt JN, Delmas F, Ramachandran S, Chilelli A,Goring DR (2008) Interactions between the S-domain receptor kinasesand AtPUB-ARM E3 ubiquitin ligases suggest a conserved signalingpathway in Arabidopsis. Plant Physiol 147: 2084–2095

Santt O, Pfirrmann T, Braun B, Juretschke J, Kimmig P, Scheel H,Hofmann K, Thumm M, Wolf DH (2008) The yeast GID complex, a

novel ubiquitin ligase (E3) involved in the regulation of carbohydratemetabolism. Mol Biol Cell 19: 3323–3333

Schauser L, Roussis A, Stiller J, Stougaard J (1999) A plant regulatorcontrolling development of symbiotic root nodules. Nature 402: 191–195

Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T, Tabata S,Sandal N, Stougaard J, Szczyglowski K, et al (2002) A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature 417:959–962

Tansengco ML, Hayashi M, Kawaguchi M, Imaizumi-Anraku H,Murooka Y (2003) crinkle, a novel symbiotic mutant that affects the in-fection thread growth and alters the root hair, trichome, and seed de-velopment in Lotus japonicus. Plant Physiol 131: 1054–1063

Timmers AC, Auriac MC, Truchet G (1999) Refined analysis of earlysymbiotic steps of the Rhizobium-Medicago interaction in relationshipwith microtubular cytoskeleton rearrangements. Development 126:3617–3628

Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008)Multicolor bimolecular fluorescence complementation reveals simulta-neous formation of alternative CBL/CIPK complexes in planta. Plant J56: 505–516

Wais RJ, Galera C, Oldroyd G, Catoira R, Penmetsa RV, Cook D, GoughC, Denarié J, Long SR (2000) Genetic analysis of calcium spiking re-sponses in nodulation mutants of Medicago truncatula. Proc Natl AcadSci USA 97: 13407–13412

Wais RJ, Keating DH, Long SR (2002) Structure-function analysis of nodfactor-induced root hair calcium spiking in Rhizobium-legume symbi-osis. Plant Physiol 129: 211–224

Wang YS, Pi LY, Chen X, Chakrabarty PK, Jiang J, De Leon AL, Liu GZ, LiL, Benny U, Oard J, et al (2006) Rice XA21 binding protein 3 is aubiquitin ligase required for full Xa21-mediated disease resistance. PlantCell 18: 3635–3646

Yang WL, Zhang X, Lin HK (2010) Emerging role of Lys-63 ubiquitinationin protein kinase and phosphatase activation and cancer development.Oncogene 29: 4493–4503

Yoshida S, Parniske M (2005) Regulation of plant symbiosis receptor ki-nase through serine and threonine phosphorylation. J Biol Chem 280:9203–9209

Zhu H, Chen T, Zhu M, Fang Q, Kang H, Hong Z, Zhang Z (2008) A novelARID DNA-binding protein interacts with SymRK and is expressedduring early nodule development in Lotus japonicus. Plant Physiol 148:337–347



Dow



ber 2021

a ubiquitin ligase of symbiosis receptor kinase involved in nodule

Documents