JOURNAL OF BACTERIOLOGY, Feb. 2009, p. 735–746 Vol. 191, No. 30021-9193/09/$08.00�0 doi:10.1128/JB.01404-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Y4lO of Rhizobium sp. Strain NGR234 Is a Symbiotic DeterminantRequired for Symbiosome Differentiation�

Feng-Juan Yang,# Li-Li Cheng,# Ling Zhang, Wei-Jun Dai, Zhe Liu, Nan Yao,Zhi-Ping Xie, and Christian Staehelin*

State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China

Received 7 October 2008/Accepted 25 November 2008

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affectthe nodulation of certain host legumes. The open reading frame y4lO of Rhizobium sp. strain NGR234 encodesa protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family).Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI.Recombinant Y4lO protein expressed in Escherichia coli did not acetylate two representative mitogen-activatedprotein kinase kinases (human MKK6 and MKK1 from Medicago truncatula), indicating that YopJ-likeproteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strainNGR�y4lO) and in NGR�nopL, a mutant that does not produce the T3 effector NopL (strainNGR�nopL�y4lO). When used as inoculants, the symbiotic properties of the mutants differed. Tephrosiavogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguiculata cv. Sui Qing Dou Jiao formed pink effectivenodules with NGR234 and NGR�nopL�y4lO. Nodules induced by NGR�y4lO were first pink but rapidlyturned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of thenodules induced by NGR�y4lO revealed abnormal formation of enlarged infection droplets in ineffectivenodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodulesinduced by NGR234 or NGR�nopL�y4lO. It is concluded that Y4lO is a symbiotic determinant involved in thedifferentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL,suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules.

Legumes establish nodule symbioses with nitrogen-fixingbacteria (called rhizobia). The formation of effective nodules isa complex developmental process which relies on various rhi-zobial signals and determinants from both symbiotic partners.During the infection process, flavonoids exuded by host plantsact as primary signals to induce rhizobial genes required forsymbiosis. The activation of these genes depends on NodDtranscriptional regulators, which bind to conserved DNA se-quences, so-called nod boxes (26, 41). Flavonoids stimulate thesynthesis of rhizobial nodulation factors (Nod factors), whichinduce plant responses, such as root hair deformation, theexpression of early nodulation genes, and the induction ofcortical cell divisions. Rhizobia vary in their ability to enterinto symbiosis with host species and certain strains induceineffective associations, which are detrimental for both part-ners. Symbiotically relevant molecules have been found to bestructurally different from strain to strain. In addition to Nodfactors, the host-specific determinants of rhizobia include exo-polysaccharides (or oligosaccharides derived from them), lipo-polysaccharides, cyclic �-glucans, K antigens, and type 3 (T3)effector proteins secreted by the bacterial T3 secretion system(T3SS) (14, 29, 41).

Pathogenic bacteria use T3SSs to deliver T3 effector pro-teins into host cells where they play a key role in pathogenic

attack, e.g., to overcome defense reactions. On the other hand,many plants developed resistance mechanisms that target T3effectors within the host cell. The direct or indirect recognitionof T3 effectors often results in the induction of resistanceresponses (e.g., a rapid hypersensitive reaction) that block bac-terial multiplication, bacterial spread, and the development ofdisease symptoms (10, 22, 34). Thus, T3 effectors are “two-edged swords” that either act as virulence or as avirulencefactors in plant-pathogen interactions. Similarly, Nops (nodu-lation outer proteins—extracellular components of rhizobialT3SSs and proteins secreted by T3SSs) may play either a pos-itive or negative role in nodule formation (29). Although notproven, it is hypothesized that rhizobial T3 effectors are deliv-ered into legume host cells. Rhizobial T3 effectors are able toinduce striking alterations in plant cells. NopL, a T3 effector ofRhizobium sp. NGR234, interfered with plant defense reac-tions. When expressed in tobacco or Lotus japonicus plants,NopL suppressed the expression of pathogen-related defensegenes (6). NopT, another T3 effector of NGR234, elicited ahypersensitive reaction when expressed in tobacco cells (16).

Rhizobium sp. strain NGR234 possesses a T3SS, which playsa role in the nodulation of certain host plants, such as Tephro-sia vogelii, Phaseolus vulgaris, Pachyrhizus tuberosus, Crotalariajuncea, and Flemingia congesta. In NGR234, the expression ofgenes encoding the T3SS depends on host flavonoids, NodD1,and the transcriptional activator TtsI, which has a nod box inthe promoter region (26, 30, 55). So far, only three bona fideT3 effectors of NGR234 have been characterized, namelyNopL (5, 6, 31), NopP (4, 46), and NopT (16). The openreading frame y4lO on the symbiotic plasmid pNGR234a is

* Corresponding author. Mailing address: State Key Laboratory ofBiocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) Univer-sity, East Campus, Bei San Road, Guangzhou 510006, China. Phone:8620 39332976. Fax: 8620 39332978. E-mail: cst@mail.sysu.edu.cn.

# F.-J.Y. and L.-L.C. contributed equally to this work.� Published ahead of print on 5 December 2008.

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predicted to encode another putative T3 effector of NGR234.The Y4lO sequence displays similarities to proteins belongingto the YopJ effector family (13, 19). Representatives of this T3effector family have been identified and characterized instrains of Yersinia sp. (YopJ/YopP [20, 32]), Salmonella en-terica serovar Typhimurium (AvrA [24]), Vibrio parahaemolyti-cus (VopA [51]), Xanthomonas campestris pv. vesicatoria(AvrRxv [13, 56]; AvrBsT [40]; AvrXv4 [3]; and XopJ [37]),Erwinia amylovora (ORFB [38]), Pseudomonas syringae (ORF5in B728a [1] and AvrPpiG [2]), and Ralstonia solanacearum(PopP1 [28] and PopP2 [17]). These T3 effectors seem to targetspecific signal transduction pathways of the eukaryotic host.YopJ in mammalian host cells inhibits mitogen-activated pro-tein (MAP) kinase and nuclear factor �B (NF-�B) signalingpathways (35) as well as the TLR3-mediated interferon re-sponse (48). In plant-pathogen interactions, resistance mech-anisms (hypersensitive response) have been reported for cer-tain nonhosts that seem to recognize a specific YopJ-likeeffector as avirulence protein (3, 8, 40, 45, 49, 56, 57). InArabidopsis thaliana, ecotype Nd-1 plants carrying the RRS1-Rresistance gene are able to recognize PopP2 (17), and theAvrBsT resistance of ecotype Pi-0 is caused by a mutation in acarboxylesterase, which is predicted to hydrolyze lysophospho-lipids and acylated proteins (15).

For many years, YopJ-like T3 effectors were thought tofunction as cysteine proteases with conserved active site resi-dues (H, D/E, Q, C). Transient expression studies in host cellsprovided certain indications that YopJ and AvrA are deubiq-uitinating enzymes (40, 48, 59, 60), whereas SUMO (smallubiquitin-like modifier) protease activity has been proposedfor AvrXv4 (45). Furthermore, in vitro experiments suggested

that YopJ cleaved the artificial substrate ubiquitin-7-amino-4-methylcoumarin (60) and that AvrA exhibited deubiquitinaseactivity on ubiquitinated I�B� (59). On the other hand, recentbiochemical analysis revealed clear evidence that YopJ andVopA are bacterial acetyltransferases. YopJ uses acetyl-coen-zyme A (acetyl-CoA) to acetylate human MAP kinase kinase 6(MKK6) and the NF-�B pathway kinase IKK�. Acetylationprevented the phosphorylation of these kinases and thus seemsto be a refined strategy to inactivate protein kinases in the hostcell (23, 33, 35, 36, 51).

In this study, we investigated the symbiotic role of y4lO fromRhizobium sp. strain NGR234. Inoculation experiments with amutant strain and ultrastructural analysis of infected nodulecells indicated that y4lO is a symbiotic determinant involved inthe differentiation of symbiosomes.

MATERIALS AND METHODS

Bioinformatic analysis. The promoter region of y4lO was analyzed with theNNPP (Neural Network Promoter Prediction) version 2.2 program (http://www.fruitfly.org/seq_tools/promoter.html). GeneMark version 2.5 (http://opal.biology.gatech.edu/GeneMark/) was used for the identification of alternative startcodons. The molecular weights of predicted proteins were calculated with theCompute pI/Mw tool (http://ca.expasy.org/tools/pi_tool.html). Sequence compar-isons with databases were performed with the BLAST program (http://ncbi.nlm.nih.gov/BLAST/). Y4lO homologues (amino acid sequences) were aligned withthe Clustal W algorithm. The unrooted radial tree (Fig. 1B) was constructed withthe MEGA3.1 program using the neighbor-joining method (27). The amino acidsequence of the short ORF72 upstream of y4lO (Fig. 1C) was analyzed with theMYR prediction server (http://mendel.imp.ac.at/myristate/SUPLpredictor.htm).

Transcriptional and translational fusions. A 870-bp fragment containing theputative promoter region of y4lO (accession number U00090) was insertedupstream of the promoterless gusA gene of vector pRG960 (52), yielding plasmidpRG-y4lOp (transcriptional fusion; Fig. 2A). For the construction of the pRG960

FIG. 1. Analysis of the y4lO sequence of Rhizobium sp. strain NGR234. (A) Genetic map of the y4lO gene and flanking sequences inpNGR234a. The promoter sequence contains a putative tts box. The gene was mutated by the insertion of an � interposon at the indicated position(resulting in strain NGR�y4lO). (B) Unrooted phylogenic tree of predicted Y4lO protein and related T3 effectors (accession numbers: YopJ,ABX88765; YopP, AAK69256, AvrA, AAL21745; AvrRxv, CAJ22102; AvrBsT, AAD39255; XopJ, CAJ23833; AvrXv4, AAG39033; PopP1,CAD14528; PopP2, CAD14570; and VopA, AAT08443). The horizontal bar represents a distance of 0.3 substitution per site. (C) Amino acidsequence alignment of the short ORF upstream of y4lO with the N-terminal sequence of XopJ from X. campestris pv. vesicatoria (CAJ23833).Identical residues are marked with asterisks. Similar residues are marked with single or double dots.

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derivative pRG-891, an 891-bp fragment containing the promoter region of y4lOand 138 bp of the coding region was fused to gusA without the ATG start codon(translational fusion; Fig. 2A). Plasmid pRG-893 with an additional insertion oftwo nucleotides at position 72 of the y4lO coding region, was generated in asimilar way (Fig. 2A).

The plasmids were verified by sequencing and then mobilized into NGR234(50) or NGR�ttsI (53) using a triparental mating procedure with the helperplasmid pRK2013 (18). Where indicated, rhizobia grown in liquid TY (tryptone-yeast extract) medium (7) were induced with 1 �M apigenin and incubated for24 h. Rhizobial cells (equal amounts adjusted to an optical density at 600 nm of0.25) were then transferred to 2-ml test tubes. After centrifugation (13,000 rpmfor 3 min), the pellets were resuspended in 1 ml extraction buffer (50 mMphosphate buffer [pH 7.0], containing 0.1% Triton X-100, 0.1% sarcosyl, 10 mMEDTA, 10 mM 2-mercaptoethanol) and kept on ice for 10 min. �-Glucuronidase(GUS) activity was fluorometrically measured at 37°C with 2 mM 4-methylum-belliferyl-�-D-glucuronide as the substrate.

Expression of proteins in Escherichia coli and Western blots. The DNA ofy4lO (Fig. 1A) from Rhizobium sp. strain NGR234 (accession number U00090)was cloned into pET28b using NdeI and XhoI (yielding plasmid pET-y4lO; Y4lOwith an N-terminal six-His tag). A similar plasmid (pET-y4lO�N) was generatedwith y4lO lacking the first 117 N-terminal nucleotides. Furthermore, the DNA ofy4lO was cloned into the EcoRI and XhoI sites of pGEX-4T-1, an expressionvector for glutathione S transferase (GST) fusion proteins (generating plasmidpGEX-y4lO).

A DNA sequence encoding a MAP kinase kinase of Medicago truncatula,named in this study MtMKK1 (accession number AC144503; position 62783 to61683), was amplified by a PCR-based technique from genomic DNA from M.

truncatula cv. Jemalong A17 with the primers 5�-TTGAAATCATATGAGGCCGATTCAGCTTCC-3� and 5�-TAACTCGAGCAACAAAAATTACATTGACGAAC-3�. The amplification product digested with NdeI and XhoI was thencloned into the pET28b vector, generating plasmid pET-MtMKK1.

Cells of E. coli BL21(DE3) with plasmids verified by sequencing were grownin Luria-Bertani (LB) medium containing 50 �g ml1 kanamycin at 37°C untilcells reached an optical density at 600 nm of 0.6. Protein expression was theninduced with 0.2 mM isopropyl-�-D-thiogalactopyranoside (IPTG), and the cellswere cultured for 12 h at 25°C. The cells were collected by centrifugation andlysed with 2� sodium dodecyl sulfate (SDS) loading buffer, and the proteins wereseparated on SDS-polyacrylamide gels. The proteins were visualized by Coomas-sie brilliant blue R-250 staining. For further characterization of the His-taggedY4lO, the cells were lysed by sonication and the proteins were purified by nickelaffinity chromatography under denaturing conditions according to the manufac-turer’s instructions (Qiagen).

Purified His-tagged Y4lO protein [from BL21(DE3) pET-y4lO�N] was usedto immunize a New Zealand rabbit. For the Western blots, the proteins wereseparated on SDS-polyacrylamide gels and then transferred onto nitrocellulosemembranes by electroblotting. To visualize the Y4lO protein, the membraneswere incubated with the antiserum raised against Y4lO (1:4,000 dilution) andthen with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulinG antiserum (Boster, Wuhan, China). The blots were developed either with3,3�-diamino-benzidine (Boster) or with ECL detection reagents (AmershamBiosciences/GE Healthcare). Secreted proteins from apigenin-induced rhizobialcultures were concentrated according to a published procedure (31) and finallydesalted by dialysis.

FIG. 2. (A) Constructs of transcriptional and translational fusions used in this study. P, PstI; B, BamHI. The black area in pRG-y4lOp is thetranslation initiation site of gusA. Cultures of NGR234 carrying the indicated plasmids were treated with 1 �M apigenin, and GUS activity wasfluorometrically determined 24 h later. (B) The transcriptional activation of y4lO depends on ttsI. Cultures of NGR234 pRG-y4lOp and NGR�ttsIpRG-y4lOp were induced with 1 �M apigenin or left untreated. GUS activity was measured after 24 h. Data represent means (� standard errors)of the results from three independent cultures.

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Acetylation assay. For the acetylation assay, His-tagged Y4lO, His-taggedhuman MKK6 [from E. coli BL21(DE3) carrying pET28a-His-MKK6 (36)], andHis-tagged MtMKK1 were purified by nickel affinity chromatography undernative conditions according to the manufacturer’s instructions (Qiagen). Y4lOwithout a six-His tag was obtained from the digestion of His-tagged Y4lO withthrombin. GST beads from Sigma were used for the purification of GST-Y4lOand GST-YopJ [from E. coli BL21(DE3) carrying pGEX-TEV-YopJ (36)]. Theeluted proteins were concentrated with Microcon centrifugal filter devices (Ami-con; Millipore). Protein quantification was performed on polyacrylamide gelswith Coomassie brilliant blue R-250 and bovine serum albumin as a standard.For the acetylation assays (36), purified enzymes (0.5 to 1 �g) and MAP kinasekinases (4 to 6 �g) were incubated at 30°C for 1 h in the following reactionmixture (20 �l): 50 mM HEPES buffer (pH 7.4), 10% glycerol, 1 mM dithio-threitol, 1 mM phenylmethylsulfonyl fluoride, and 2 �l [14C]-acetyl-CoA (56 �Ci�M1) from Sigma. The proteins were then separated on SDS-polyacrylamidegels and fixed with 50% methanol and 10% glacial acetic acid for 1 h. The gelswere dried and radioactivity was visualized by a Typhoon imaging system(Typhoon 8600 scanner; Amersham Biosciences/GE Healthcare).

Construction of mutant strains. The strains and plasmids used for the con-struction of NGR�y4lO and NGR�nopL�y4lO are listed in Table 1. A 1.5-kbfragment containing y4lO (accession number U00090) was cloned into pBlue-script II SK(�) yielding plasmid pSK-y4lO. A point mutation construct with an

EcoRI site (at position 136 of y4lO [Fig. 1A]) was then generated by PCR-basedsite-directed mutagenesis using pSK-y4lO as a template and DpnI for digestionof the amplification products. A spectinomycin resistance � interposon was thenexcised from pHP45� (42) with EcoRI and ligated into the EcoRI site of themutated pSK-y4lO. Finally, the excised 3.7-kb SacI-XhoI fragment was clonedinto the suicide vector pJQ200SK (44). The construct was mobilized from E. coliDH5� into NGR234 and NGR�nopL (31) by a triparental mating procedurewith the helper plasmid pRK2013 (18). The replacement of the mutated genewas forced by selecting for the resistance of the interposon marker (Spr) and forresistance to sucrose (5% wt/vol). Double-crossover events at homologous siteswere confirmed by Southern blot analysis with rhizobial DNA (12) using the DIGDNA labeling and detection kit as specified by the supplier (Roche).

For the construction of NGR�y4lO constitutively expressing gusA, the mini-Tn5 transposon derivative pFAJ1815 containing gfp-gusAp (58) was introducedinto NGR�y4lO using a biparental mating procedure. The selection of taggedcolonies constitutively expressing gusA was performed on agar plates supple-mented with 0.5 mg ml1 5-bromo-4-chloro-3-indolyl-�-D-glucuronic acid. Aninoculation experiment with P. vulgaris cv. Yudou No. 1 indicated that themTn5gfp-pgusA insertion had no effect on the symbiotic phenotype. For thevisualization of NGR�y4lO expressing gusA, nodules were stained with 5-bromo-4-chloro-3-indolyl-�-D-glucuronic acid as described previously (47).

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Characteristicsa Reference or source

StrainsRhizobium sp.

NGR234 Rhizobium sp. strain NGR234 isolated from Lablab purpureus (Rifr derivative) 50NGR�y4lO NGR234 carrying an � insertion in y4lO (Rifr, Spr) (Fig. 1A) This workNGR�nopL NGR234 carrying an � insertion in nopL (Rifr, Kmr) 31NGR�nopL�y4lO NGR�nopL carrying an � insertion in y4lO (Rifr, Spr) (Fig. 1A) This workNGR�rhcN NGR234 carrying an � insertion in rhcN (Rifr, Spr) 53NGR�ttsI NGR234 carrying an � insertion in ttsI, formerly named NGR�y4xI (Rifr, Spr) 53

E. coliDH5� supE44 �lacU169 ( 80 lacZ�M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Promega Corp.BL21(DE3) F ompT hsdSB (rB

mB) gal dcm (DE3) Novagen (Merck

Chemicals Ltd.)

PlasmidspBluescript II SK(�) High-copy-no. ColE1-based phagemid (Apr) Stratagene (Agilent

Technologies)pSK-y4lO 1.5-kb fragment containing y4lO cloned into pBluescript II SK(�) (Apr) This workpRG960 Broad-host-range vector containing a promoterless gusA gene with start codon

(Spr) (Fig. 2A)52

pRG-y4lOp 870-bp fragment containing the y4lO promoter cloned into pRG960 (Spr) (Fig.2A)

This work

pRG-891 pRG960 derivative with an 891-bp fragment containing the y4lO promoter and138 bp of the coding region fused to gusA lacking the start codon (Spr)(Fig. 2A)

This work

pRG-893 pRG-891 derivative with an insertion of two nucleotides (GC) at position 72 ofthe y4lO coding region (Spr) (Fig. 2A)

This work

pET28b Expression vector for His-tagged proteins (Kmr) Novagen (MerckChemicals Ltd.)

pET-y4lO y4lO cloned into pET28b (N-terminal His-tagged Y4lO) (Kmr) This workpET-y4lO�N y4lO lacking the first 117 N-terminal nucleotides cloned into pET28b (Kmr) This workpET28a-His-MKK6 Human MKK6 (MAPKK6) cloned into pET28a (Kmr) 36pET-MtMKK1 MtMKK1 from M. truncatula (AC144503; position 62783–61683) cloned into

pET28b (Kmr)This work

pGEX-4T-1 Expression vector for GST fusion proteins (Apr) Amersham Biosciences/GE Healthcare

pGEX-y4lO y4lO cloned into pGEX-4T-1 (Apr) This workpGEX-TEV-YopJ YopJ cloned into pGEX-TEV (Apr) 36pJQ200SK Suicide vector used in directed mutagenesis (Ger) 44pRK2013 Tra� helper plasmid for mobilization (Kmr) 18pHP45� Vector containing an � interposon (Apr, Spr) 42pFAJ1815 Mini-Tn5 transposon derivative containing gfp-gusAp (constitutively expressed

gusA) (Kmr)58

a Apr, resistance against ampicillin; Ger, resistance against gentamicin; Kmr, resistance against kanamycin; Rifr, resistance against rifampin; Spr, resistance againstspectinomycin.

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Nodulation tests. Nodulation tests with Crotalaria juncea, Pachyrhizus tu-berosus, Tephrosia vogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguicu-lata cv. Sui Qing Dou Jiao were performed according to previously describedprocedures (47). Briefly, seeds were surface-sterilized and left to germinate onagar plates. The plantlets (1 plant per jar) were then transferred to sterilized300-ml plastic jar units linked with a cotton wick (3:1 [vol/vol] mixture of ver-miculite and expanded clay in the upper vessel; nitrogen-free nutrient solution inthe lower vessel). The plants were inoculated with 109 bacteria (grown in TYmedium, centrifuged, and resuspended in 10 mM MgSO4). The plants weregrown in a plant growth room or air-conditioned greenhouse at 26 � 2°C. If nototherwise indicated, P. vulgaris cv. Yudou No. 1 plants were harvested 4 weekspostinoculation. The other plants were harvested 7 to 8 weeks postinoculation.Biomass (dry weight) data were obtained from lyophilized plant material. Thenitrogen contents of lyophilized and pulverized total plant material were deter-mined with a CHNS analyzer (Elementar Analysensysteme, Hanau, Germany).

Electron and light microscopy. Nodules were excised from roots, sliced man-ually, and immediately fixed in 67 mM sodium phosphate buffer (pH 7.4) con-taining 2.5% (vol/vol) glutaraldehyde and 2% (vol/vol) paraformaldehyde (4°C,overnight). After three washing steps, the samples were postfixed for 90 min in67 mM phosphate buffer (pH 7.4) containing 1% (wt/vol) OsO4. The sampleswere then rinsed in the same buffer (three times for 15 min) and dehydrated withincreasing volumes of ethanol (30%, 50%, 70%, 90%, and 95% for 15 min eachand 100% three times for 30 min each). The samples were then embedded inPoly/Bed 812 resin as specified by the supplier (Polyscience, Warrington, PA).An ultramicrotome equipped with a diamond knife (Leica UC6; Austria) wasused to obtain ultrathin sections (85 nm thick). The sections were stained withuranyl acetate and lead citrate and finally observed in a transmission electronmicroscope. For the light microscopy observations, semithin sections (1 �mthick) from the same samples were stained with 0.6% (wt/vol) toluidine blue.

RESULTS

Sequence analysis of y4lO. The ORF y4lO in the plasmidpNGR234a of Rhizobium sp. strain NGR234 (19) is flanked byseveral ORFs with sequence similarities to insertion sequencehomologs, suggesting transposon-related insertion events (Fig.1A). A putative promoter is predicted by the NNPP version 2.2program with the transcription start sequence CTTGCATATG. The promoter region of y4lO contains a tts box, a puta-tive binding site for the transcriptional activator TtsI (30, 55)(Fig. 1A). Comparisons with sequence databases showed sim-ilarities with YopJ family T3 effectors from bacterial patho-gens. A phylogenetic analysis indicated that the Y4lO se-quence of NGR234 is most closely related to XopJ (27) (Fig.1B). Other rhizobial strains with T3SSs, e.g., Bradyrhizobiumjaponicum USDA110, lack genes homologous to y4lO. Simi-larly to related T3 effectors, four conserved amino acids, pre-dicted to play a role in enzymatic activity, are present in theY4lO sequence (residues H123, E143, Q179, and C185). Theannotated Y4lO protein (29.1 kDa) is encoded by a DNAsequence with the unusual translation start codon TTG. Theprogram GeneMark Version 2.5 (e.g., with the model organismSinorhizobium meliloti plasmid pSymA as a reference) predictsa protein (24.7 kDa) with the N-terminal sequence MSSSL.The annotated Y4lO protein (29.1 kDa) is shorter than thecoding sequences of related T3 effectors, such as XopJ. Se-quence alignment revealed a short ORF of 72 bp (ORF72)upstream of the ORF y4lO (Fig. 1A); the corresponding aminoacid sequence shows similarities with the N-terminal aminoacid residues of XopJ (Fig. 1C).

Transcriptional activation of y4lO. To study the transcrip-tional activation of y4lO, a 870-bp fragment containing theputative promoter region of y4lO was inserted upstream of apromoterless gusA gene of vector pRG960. The resulting tran-scriptional fusion (pRG-y4lOp [Fig. 2A]) was then mobilized

into NGR234 and NGR�ttsI, a strain with a mutated ttsI gene(53). As transcription of ttsI is dependent on NodD1 and fla-vonoids (26), rhizobial cultures were treated with apigenin andGUS activity was measured 24 h later. As shown in Fig. 2B,GUS activity in NGR234 pRG-y4lOp was considerably higherthan in NGR�ttsI pRG-y4lOp, indicating that the transcrip-tional activation of y4lO depended on ttsI. As expected, theGUS activity of the NGR234 derivative was stimulated by thetreatment with apigenin, whereas no elevated values were ob-tained with the NGR�ttsI derivative. These results togetherwith those from a recent study (55) confirm the bioinformaticprediction that y4lO with a tts box in the promoter region isregulated by the transcriptional activator TtsI.

Two translational fusions were constructed to examinewhether the TTG start codon or the ATG codon (at position118 of y4lO) is the translation initiation site of Y4lO. Oneconstruct (pRG-891 [Fig. 2A]) was generated by the in-framefusion of the first 138 nucleotides of y4lO with gusA lacking theATG start codon. The other construct with a frameshift mu-tation was obtained by the insertion of two additional nucleo-tides at position 72 of y4lO (pRG-893 [Fig. 2A]). NGR234derivatives containing these plasmids were grown in the pres-ence of apigenin, and GUS activity was determined 24 h later.As shown in Fig. 2A, GUS activity for NGR234 pRG-891 washigh (comparable to pRG-y4lOp), whereas only backgroundactivity was measured with NGR234 pRG-893. These findingsindicate that the frameshift mutation in pRG-893 abolishedthe formation of a functional GUS protein and that the TTGstart codon is likely the translation initiation site of Y4lO.

Expression of y4lO in E. coli. To characterize the Y4lOprotein, the y4lO sequence was cloned into vector pET28b. E.coli BL21(DE3) containing this plasmid synthesized recombi-

FIG. 3. Expression of Y4lO in E. coli BL21(DE3) and acetylationassay with MAP kinase kinases. (A) Proteins were separated on a SDS-polyacrylamide gel and visualized by staining with Coomassie brilliantblue R-250. Lane 1, BL21(DE3) pET-y4lO; lane 2, BL21(DE3) carryingthe empty vector pET28b; lane 3, purified His-tagged Y4lO protein.(B) Acetylation assay with [14C]-acetyl-CoA and the indicated purifiedproteins. Reaction mixtures were separated on a SDS-polyacrylamide gel,and radioactivity was visualized with a Typhoon imaging system.(C) Western blot analysis with a rabbit serum raised against recombinantY4lO. Lane 1, proteins from E. coli with the empty vector pET28b; lane2, purified His-tagged Y4lO from BL21(DE3) pET-y4lO; lane 3, purifiedHis-tagged Y4lO from BL21(DE3) pET-y4lO�N.

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nant Y4lO protein with an N-terminal six-His tag. Under de-naturing conditions, the purification of the protein by nickelaffinity chromatography resulted in a single band (apparentmolecular weight � 31 kDa) (Fig. 3A). An antiserum raisedagainst the purified His-Y4lO protein [from BL21(DE3) pET-y4lO�N] specifically recognized low amounts of Y4lO proteinon Western blots (detection of �10 ng protein) (Fig. 3C).Similarly, Y4lO expressed as a GST fusion protein (GST-Y4lO) (apparent molecular weight � 54.7 kDa) was obtainedfrom E. coli cells carrying plasmid pGEX-y4lO (data notshown).

His-tagged Y4lO, Y4lO (six-His tag removed by thrombin)and GST-Y4lO proteins purified as native proteins (or affinitybeads with immobilized enzyme) were then used for in vitroacetylation assays with [14C]-acetyl-CoA and two representa-tive His-tagged MAP kinase kinases: human MKK6 (36) andMtMKK1 from the legume Medicago truncatula, which is ho-mologous to SIMKK of Medicago sativa (25). For comparison,the YopJ protein expressed as a GST fusion protein (GST-YopJ) was also purified (36). As shown in Fig. 3B, both MAPkinase kinases were not acetylated by Y4lO, whereas GST-YopJ acetylated MKK6 but not MtMKK1.

Is Y4lO a secreted protein? The T3 effectors of NGR234from bacterial culture supernatants (i.e., NopL, NopP, andNopT) could be detected on immune blots with specific anti-sera. Secreted proteins from mutant derivatives lacking a func-tional T3SS (e.g., strain NGR�rhcN) served as controls todemonstrate T3SS-dependent secretion (4, 16, 31). When sim-ilar experiments were performed with secreted proteins fromrhizobial cultures and the anti-Y4lO antibodies, Y4lO was notdetected on immune blots. In contrast to Y4lO, NopL andNopT secreted by NGR234 were recognized by anti-NopL and

anti-NopT antibodies, indicating that the sample containedT3SS-related nodulation outer proteins. When proteins fromthe total bacteria of NGR234 (pellets) or proteins from nod-ules were probed with the anti-Y4lO antibodies, Y4lO was alsonot detected (not shown), suggesting that Y4lO is produced atlow levels.

Y4lO is a determinant of symbiosis. To explore the effectsof Y4lO during symbiosis with legumes, inoculation tests wereperformed with NGR234 and constructed mutant derivatives,namely NGR�y4lO and the double mutant NGR�nopL�y4lO(mutation of y4lO in NGR�nopL, which is mutated in the T3effector gene nopL [31]). For comparison, the T3SS null mu-tant NGR�rhcN with a nonfunctional T3SS (53) was includedinto the nodulation analysis. C. juncea and P. tuberosus werechosen for inoculation tests, as the effective nodulation ofthese legumes was blocked by nondefined T3SS proteins(31, 53). The symbiotic phenotypes of NGR�y4lO andNGR�nopL�y4lO on these plants were not significantly dif-ferent compared to those of NGR234, however (Table 2).

The nodulation of T. vogelii, another host plant of NGR234,is positively affected by the T3SS (53). A recent mutant analysisshowed that the mutation of nopL did not affect symbiosis withthis host plant. A double mutant with deleted nopL and nopPgenes induced fewer nodules than the parent strain, however(31, 46). Interestingly, T. vogelii challenged with NGR�y4lOhad only ineffective nodules (Fix phenotype) at the time ofharvest. The plants inoculated with NGR234, NGR�nopL�y4lO, or NGR�rhcN formed effective nodules that promotedplant growth (Table 2; Fig. 4A). Ineffective nodules of T. vogeliiinduced by NGR�y4lO were greenish in the infected zone,whereas effective nodules induced by the other strains werepink (Fig. 4B to E). The nitrogen contents of T. vogelii plants

TABLE 2. Nodulation of legumes with NGR234 and the indicated mutant strains

Planta Straina No. ofnodules/plantb

Plant biomass(mg dry weight)b

Phenotype atharvest time

C. juncea NGR234 79 � 11 548 � 200 Fix�/

NGR�y4lO 14 � 2 161 � 22 Fix

NGR�nopL�y4lO 20 � 3 228 � 44 Fix

NGR�rhcN 110 � 6 1,505 � 220 Fix�

P. tuberosus NGR234 0.4 � 0.2 291 � 31 Fix

NGR�y4lO 0.2 � 0.2 286 � 35 Fix

NGR�nopL�y4lO 0.2 � 0.2 341 � 45 Fix

NGR�rhcN 31 � 2 417 � 92 Fix�

T. vogelii NGR234 33 � 4 631 � 51 Fix�

NGR�y4lO 22 � 2 224 � 8 Fix

NGR�nopL�y4lO 33 � 2 547 � 54 Fix�

NGR�rhcN 18 � 2 512 � 45 Fix�

P. vulgaris cv.Yudou No. 1 NGR234 25 � 4 894 � 92 Fix�

NGR�y4lO 28 � 2 771 � 50 Fix

NGR�nopL�y4lO 30 � 4 1,132 � 94 Fix�

NGR�rhcN 31 � 3 962 � 78 Fix�

V. unguiculata cv. Sui QingDou Jiao

NGR234 71 � 12 2,052 � 485 Fix�

NGR�y4lO 134 � 15 555 � 72 Fix

NGR�nopL�y4lO 69 � 8 1,443 � 153 Fix�

NGR�rhcN 75 � 12 1,368 � 148 Fix�

a The plants (one plant per jar) were inoculated with the indicated strains. P. vulgaris cv. Yudou No. 1 plants were harvested 4 weeks postinoculation. The other plantswere harvested 7 to 8 weeks postinoculation.

b Number of nodules and total plant biomass (dry weight) was determined for each plant at the time of harvest. Data indicate means � standard errors (n � 6).

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infected with NGR�y4lO were similar to those of noninfectedcontrol plants. Plants nodulated by the other strains exhibitedconsiderably higher nitrogen contents (Fig. 4L).

Further nodulation assays were performed with P. vulgariscv. Yudou No. 1 (Table 2; Fig. 4F). In this host plant ofNGR234, nodule formation was modulated by the T3 effectorNopT (16). Plants inoculated with NGR234 formed pink ef-fective nodules (Fig. 4G), resulting in well-developed plants.When inoculated with NGR�y4lO, the nodules were pink atfirst but turned greenish later (Fig. 4H). At the time of harvest,the leaves were yellow, plant growth was reduced, and nitrogencontents were low (Fig. 4 M). NGR�rhcN and the doublemutant NGR�nopL�y4lO induced effective nodules (Fig. 4Iand J) that promoted plant growth (Fig. 4F) and increasednitrogen contents in harvested plants (Fig. 4M). These dataindicate that the symbiotic phenotypes of the tested strains onP. vulgaris cv. Yudou No. 1 were similar to those on T. vogelii.

Finally, nodulation studies were performed with the host plantVigna unguiculata cv. Sui Qing Dou Jiao. Previous inoculationtests with NGR�rhcN suggested that the nodulation of V. un-guiculata cv. Red Caloona is independent of a functional T3SSsystem (53). NGR�y4lO and NGR�nopL�y4lO induced pinknodules on V. unguiculata at a frequency similar to that of theparent strain NGR234 when the plants were harvested 4 weekspostinoculation. Differences between NGR�y4lO and NGR234were seen later, however. The leaves of plants inoculated with

NGR�y4lO turned yellow and nodules greenish. At 7 weekspostinoculation, the biomass accumulation was low and numer-ous small ineffective nodules were observed (Table 2).

Mutation in y4lO does not affect early symbiotic stages. Thesymbiotic capacity of NGR�y4lO to induce nodules on P.vulgaris cv. Yudou No. 1 roots was further analyzed in coin-oculation experiments with NGR234. To visualize NGR�y4lObacteria in nodules, a derivative of NGR�y4lO expressing gusAwas constructed. All nodules induced by this strain turned bluewhen stained with 5-bromo-4-chloro-3-indolyl-�-D-glucuronicacid. The coinoculation of NGR234 with NGR�y4lO express-ing gusA resulted in the blue coloration of 50% of the nod-ules (Fig. 4K). A few blue nodules were also seen whenNGR�y4lO expressing gusA was diluted 100-fold in the coin-oculation experiment (not shown). Hence, the mutation iny4lO did not affect the capacity of NGR234 to infect P. vulgariscv. Yudou No. 1. These observations are corroborated by lightmicroscopic analysis. Nodules of P. vulgaris cv. Yudou No. 1induced by NGR234 and NGR�y4lO showed infected cells,which were deeply stained with toluidine blue. Cells infected byNGR234 had a granular matrix, indicating that the cells werecompletely filled with symbiosomes (Fig. 5A). In infected cellsharboring NGR�y4lO, however, the dark blue granular matrixappeared to be aggregated, suggesting differences in the struc-ture of symbiosomes (Fig. 5B).

FIG. 4. Symbiotic phenotype of the mutants NGR�y4lO and NGR�nopL�y4lO. (A) Aerial part of nodulated T. vogelii plants 8 weekspostinoculation. 1, NGR�y4lO; 2, NGR�rhcN; 3, NGR�nopL�y4lO; 4, NGR234. (B) Corresponding ineffective nodule of T. vogelii induced byNGR�y4lO. (C) Effective nodule of T. vogelii induced by NGR�rhcN. (D) Effective nodule of T. vogelii induced by NGR�nopL�y4lO.(E) Effective nodule of T. vogelii induced by NGR234. (F) Aerial part of nodulated P. vulgaris cv. Yudou No. 1 plants 4 weeks postinoculation.1, NGR234; 2, NGR�y4lO; 3, NGR�rhcN; 4, NGR�nopL�y4lO. (G) Corresponding nodule of P. vulgaris cv. Yudou No. 1 induced by NGR234.(H) Ineffective nodule of P. vulgaris cv. Yudou No. 1 induced by NGR�y4lO. (I) Effective nodule of P. vulgaris cv. Yudou No. 1 induced byNGR�rhcN. (J) Effective nodule of P. vulgaris cv. Yudou No. 1 induced by NGR�nopL�y4lO. (K) Nodules of P. vulgaris cv. Yudou No. 1 aftercoinoculation with NGR234 and NGR�y4lO constitutively expressing gusA (ratio, 1:1). Nodules were stained with 5-bromo-4-chloro-3-indolyl-�-D-glucuronic acid. (L) Nitrogen contents of harvested T. vogelii plants infected with the indicated strains and noninfected control plants (blackcolumn). Data indicate means � standard errors (n � 3). (M) Nitrogen contents of harvested P. vulgaris cv. Yudou No. 1 plants infected with theindicated strains and noninfected control plants (black column). Data indicate means � standard errors (n � 3). DW, dry weight. Scale bars �1 mm.

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Ultrastructural analysis of infected cells suggests a role ofY4lO in symbiosome differentiation. To investigate ultrastruc-tural differences in infected nodule cells of P. vulgaris cv. Yu-dou No. 1, nodules induced by NGR234 and NGR�y4lO wereexamined by electron microscopy. In effective nodules 30 dayspostinoculation, cells infected by NGR234 were filled withmany symbiosomes. In most cases, symbiosomes consisted ofone single bacteroid surrounded by a symbiosome (peribacte-roid) membrane. Few bacteroids were also seen in vacuole-likestructures, which can be considered as infection droplets de-rived from infection threads (Fig. 5C). Ineffective nodules in-duced by NGR�y4lO also contained many bacteroids, whichvaried in size and shape. In contrast to NGR234, however,bacteroids of NGR�y4lO were mainly present in enlargedinfection droplets with up to 30 bacteroids. Bacteroids ofNGR�y4lO seem to rapidly divide within the abnormal infec-tion droplets (Fig. 5D).

Under the tested conditions, nodules induced by NGR234were pink, whereas those induced by NGR�y4lO were pink atfirst and later (30 days postinoculation) greenish, indicatingthe degradation of leghemoglobin. To examine symbiosomedifferentiation during nodule development in more detail, nod-ules of P. vulgaris cv. Yudou No. 1 were investigated in atime-course experiment (Fig. 6). In young nodules induced byNGR234 (24 and 26 days postinoculation [Fig. 6A and B]),many bacteroids were present within infection droplets. Somebacteroids already differentiated into symbiosomes harboring asingle bacteroid. Nodules induced by NGR�y4lO (24 and 26days postinoculation) were similar, but most bacteroids wereseen in infection droplets (Fig. 6D and E). In the later stagesof symbiosis, ultrastructural differences between NGR234 andNGR�y4lO nodules were more pronounced (Fig. 6C and F to

L). Bacteroids of NGR234 localized in the periphery of infec-tion droplets were released and rapidly differentiated into sym-biosomes. The infection droplets contained few or finally nobacteroids, resulting in vacuole-like structures (Fig. 6I). Thedifferentiated symbiosomes with bacteroids of NGR234 furtherdivided, thereby increasing the number of symbiosomes withinthe infected cell (Fig. 6G and H). In contrast, most bacteroidsof NGR�y4lO divided within persistent infection droplets(Fig. 6E and J), whereas bacteroids surrounded by individualsymbiosome membranes were only randomly observed. Bacte-roids of NGR�y4lO frequently contained white poly-�-hy-droxybutyrate granules. Bacteroids in infection droplets wereimbedded in a gray matrix, which differed from the halo-likebright material surrounding each bacteroid (Fig. 6J and K).Highly glycosylated nodule extensin is likely an essential com-ponent of the gray matrix material, which is a typical charac-teristic of infection threads and infection droplets (9). At 34days postinoculation, all nodules induced by NGR�y4lO weregreenish, indicating premature senescence. “Tight junctions”formed by adjacent membranes were observed at this stage, atypical feature of senescent nodules (11, 43) (Fig. 6L). Infec-tion droplets in old nodules induced by NGR�y4lO were oc-casionally deteriorated, suggesting that bacteroids had directcontact with the host cytoplasm (not shown).

In addition to P. vulgaris cv. Yudou No. 1, infected nodulecells of T. vogelii were analyzed (Fig. 7A and B). In pinknodules induced by NGR234 (harvested 8 weeks postinocula-tion), most symbiosomes harbored one single bacteroid. Incontrast, persistent infection droplets were frequently ob-served in the ineffective greenish nodules induced byNGR�y4lO. Some of the bacteroids in these atypical struc-tures appear degraded. Hence, mutation in y4lO also nega-tively affected symbiosome differentiation in the interactionwith T. vogelii.

Finally, pictures were taken for nodules from P. vulgaris cv.Yudou No. 1 and T. vogelii plants, which have been inoculatedwith the double mutant NGR�nopL�y4lO. Similarly toNGR234, strain NGR�nopL�y4lO released bacteroids from in-fection droplets, resulting in bacteroids surrounded by symbio-some membranes under the conditions tested (Fig. 7C and D).

DISCUSSION

In many pathogen-host interactions, proteins belonging tothe YopJ T3 effector family play an important role as virulenceor avirulence factors. The y4lO gene of the symbiotic bacte-rium Rhizobium sp. NGR234 shows sequence similarities withT3 effectors belonging to the YopJ effector family. In thisstudy, we provide genetic evidence that the y4lO gene plays animportant role in the symbiosis with various leguminous hostplants. Nodulation tests with a constructed y4lO mutant deriv-ative (strain NGR�y4lO) and ultrastructural analysis of nod-ules suggest that y4lO affects symbiosome differentiation ininfected cells of P. vulgaris cv. Yudou No. 1 and T. vogelii. Inthe interaction with these hosts, Y4lO can be considered as asymbiotic determinant required for the formation of functionalnitrogen-fixing nodules. This is reminiscent to NopL, NopP,and NopT, T3 effectors of NGR234 that play a role in host-specific nodulation (16, 31, 46). As our immunological ap-

FIG. 5. Light and electron microscopic analysis of infected nodulecells from P. vulgaris cv. Yudou No. 1 at 30 days postinoculation.(A) Semithin section of nodule cells infected with NGR234 after tolu-idine blue staining. (B) Nodule cells infected with NGR�y4lO stainedwith toluidine blue. (C) Ultrathin section of an infected cell withNGR234 bacteroids. (D) Ultrathin section of infected cells withNGR�y4lO bacteroids. IC, infected cell; NC, noninfected cell; S, sym-biosome; ID, infection droplet; CW, cell wall. Scale bars in panels Aand B, 10 �m. Scale bars in panels C and D, 2 �m.

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FIG. 6. Morphological differentiation of symbiosomes in nodules from P. vulgaris cv. Yudou No. 1. (A to C) Bacteroids in nodules inducedby NGR234 (at 24, 26, and 28 days postinoculation, respectively). (D to F) Bacteroids in nodules induced by NGR�y4lO (at 24, 26, and 28days postinoculation, respectively). (G to I) Bacteroids in nodules induced by NGR234 (at 30, 32, and 34 days postinoculation, respectively).(J to L) Bacteroids in nodules induced by NGR�y4lO (at 30, 32, and 34 days postinoculation, respectively). S, symbiosome; ID, infectiondroplet; white arrow, symbiosome membrane; black arrow, halo-like matrix surrounding bacteroids; white arrowhead, tight junction formedby adjacent membranes; white asterisk, dividing bacteroids; black asterisk, poly-�-hydroxybutyrate granules. Scale bars � 2 �m.

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proach failed to detect Y4lO protein, it remains an open ques-tion whether Y4lO is a protein secreted by the rhizobial T3SS.

In this study, we tested whether MAP kinase kinases aresubstrates for Y4lO protein. The previously characterizedYopJ protein and human MKK6 served as a positive controlfor the acetylation assay (23, 33, 35, 36). In contrast to YopJ,Y4lO proteins expressed in E. coli lacked acetyltransferaseactivity under the tested in vitro conditions, suggesting thatYopJ-like proteins differ in their substrate specificity. All pro-teins tested did not incorporate radioactivity into MtMKK1, arepresentative plant MAP kinase kinase with the sequenceTMDPCNS in the activation loop (T and S are predictedphosphorylation sites). Thus, further attempts are required to

identify target proteins of Y4lO and to test the possibility thatthe enzyme requires host cell factors for activation.

The symbiotic role of Y4lO extends the function of YopJ-like proteins from pathogens to a symbiotic Rhizobium strain.Genes homologous to y4lO of NGR234 have not been identi-fied in other rhizobial strains, so far. We therefore suggest thaty4lO is an imported gene which derived from a phytopatho-genic bacterium. The possibility of horizontal gene transfer issupported by the genetic organization of the y4lO region in thesymbiotic plasmid pNGR234a. Various ORFs flanking y4lOshow similarities to insertion sequences, suggesting transpo-son-related sequence rearrangements during evolution (Fig.1A). Southern blot analysis indicated a single copy of y4lO inthe genome of NGR234 (not shown). The promoter region ofy4lO contains a predicted tts box, a putative binding site for thetranscriptional activator TtsI (30, 55). Thus, during evolution,the promoter region of y4lO apparently acquired typical fea-tures of a rhizobial promoter with a tts box. Compared to theT3 effector XopJ from X. campestris pv. vesicatoria (Fig. 1B),the predicted Y4lO protein is 46 amino acid residues shorter atthe N terminus. T3 effectors from pathogenic bacteria possessan N-terminal export-signal pattern, which is required forT3SS-dependent secretion (21). Sequence analysis revealed in-deed a short DNA fragment (ORF72) upstream of y4lO withsequence similarities to the N-terminal region of XopJ (Fig. 1C).We therefore suggest that ORF72 is a remnant from an ancestralXopJ-like protein (with the N terminus MGGCISRLS), whichNGR234 acquired from a pathogenic bacterium. The corre-sponding amino acid sequence of ORF72 has characteristicfeatures of an N-myristoylation signal, suggesting that the G2residue of the ancestral T3 effector was myristoylated in eu-karyotic host cells (by the MYR prediction server, probabilityof a false positive prediction for Y4lO, 1.34e4). A recentreport showed that the localization of a XopJ-GFP fusionprotein within plant cells depended on the G2 residue of XopJ,indicating that the myristoylation of XopJ is required for pro-tein targeting to the plasma membrane (49). Our mutant anal-ysis provides evidence for a symbiotic role for Y4lO, whichlacks a glycine residue at the N terminus. Future work isneeded to characterize the spatial distribution of Y4lO withininfected host cells and to elucidate the function of an ORF72-Y4lO fusion protein during symbiosis.

The nodulation tests of this study suggest that Y4lO miti-gated the deleterious effects induced by NopL, which are mostpronounced in P. vulgaris cv. Yudou No. 1 plants. The nodu-lation outer protein NopL is secreted via the rhizobial T3SSand a bona fide T3 effector, whose expression in plant cellscaused the suppression of plant defense reactions in tobaccoand Lotus japonicus (6) as well as disease-like symptoms inother plants (L. Zhang, M. Li, and C. Staehelin, unpublishedresults). Thus, it seems that Y4lO suppressed the deleteriouseffects caused by NopL. In contrast to P. vulgaris cv. Yudou No.1, mutant analysis with NGR�nopL revealed no deleteriouseffects of NopL in the interaction with the host plant T. vogelii(46), and we confirmed these findings under our nodulationtest conditions (data not shown). In combination with NopP,however, NopL seems to be required for the efficient noduleformation of T. vogelii, indicating synergistic symbiosis-pro-moting effects (46). Surprisingly, our nodulation data suggestthat NopL secreted by NGR�y4lO may have deleterious ef-

FIG. 7. Symbiosomes in nodules of T. vogelii and P. vulgaris cv.Yudou No. 1 induced by the double mutant NGR�nopL�y4lO.(A) Symbiosomes in effective T. vogelii nodules induced by NGR234.(B) Infection droplets in ineffective T. vogelii nodules induced byNGR�y4lO. (C) Symbiosomes in effective T. vogelii nodules inducedby NGR�nopL�y4lO. (D) Differentiation into symbiosomes in youngP. vulgaris cv. Yudou No. 1 nodules induced by NGR�nopL�y4lO. S,symbiosome; ID, infection droplet; white arrow, symbiosome mem-brane; black asterisk, poly-�-hydroxybutyrate granules. Scale bars �2 �m.

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fects on T. vogelii. Thus, NopL seems to trigger multiple re-sponses in T. vogelii, and it is tempting to speculate that Y4lOthwarts the cytotoxic effects induced by NopL.

An ultrastructural analysis of the nodules from P. vulgaris cv.Yudou No. 1 and T. vogelii suggests that Y4lO promoted dif-ferentiation into symbiosomes harboring a single bacteroid.Cells infected by NGR�y4lO contained enlarged infectiondroplets. Our electron microscopy analysis of cells infected byNGR234 revealed a rapid differentiation of symbiosomes har-boring a single bacteroid, whereas bacteroids of NGR�y4lOremained entrapped in persistent infection droplets. Thus,Y4lO apparently promoted the endocytotic release of bacte-roids from infection droplets in young nodules. Interestingly,an ineffective pea mutant (sym40) displayed a similar symbioticphenotype with enlarged infection droplets and abnormal en-docytosis, indicating that host genes significantly contribute tosymbiosome differentiation (reference 54 and referencestherein). Factors involved in symbiosome differentiation havebeen poorly characterized (39). Efficient synthesis of mem-branes might play a key role in symbiosome differentiation.Any cellular changes in the host cell affecting membrane syn-thesis could have negative effects on symbiosome differentia-tion. Furthermore, nitrogen starvation and free oxygen couldaffect the formation of abnormal symbiosomes in ineffectivenodules. For example, young nodules of P. vulgaris cv. NegroJamapa infected by nifA or nifH mutants of Rhizobium etlideveloped “multiple-occupancy symbiosomes” (11).

An analysis of the nodules induced by the double mutantNGR�nopL�y4lO revealed a normal differentiation into sym-biosomes, suggesting that NopL negatively affected this pro-cess. It is tempting to speculate that plant defense responsesinduced by NopL action negatively affected symbiosome dif-ferentiation. In other words, Y4lO could function as a suppres-sor of a host defense response.

In conclusion, the findings of this work shed light on thesymbiotic role of Y4lO. In the interaction with P. vulgaris cv.Yudou No. 1 and T. vogelii, Y4lO is crucial for the formationof effective nodules and seems to mitigate the deleterious ef-fects of the T3 effector NopL. Future experiments are requiredto elucidate the molecular function of Y4lO and to investigatewhether Y4lO is a T3 effector delivered into legume host cells.

ACKNOWLEDGMENTS

We express our gratitude to Yi-Hao Ruan and Li-Ming Liang fortheir help with many aspects of this work. Xue-Jiao Chen is acknowl-edged for providing anti-NopL antibodies. We thank Kim Orth (Uni-versity of Texas) for providing pGEX-TEV-YopJ and pET28a-His-MKK6, J. Michiels (K.U. Leuven, Heverlee, Belgium) for pFAJ1815,and William J. Broughton (University of Geneva, Switzerland)for NGR234 and mutant strains (NGR�rhcN, NGR�ttsI, andNGR�nopL). Two anonymous reviewers provided helpful commentson the manuscript.

This work was supported by the National Natural Science Founda-tion of China (grants 30671117 and 30771150) and by the Departmentof Science and Technology of Guangdong Province, China (grant2006B50104004).

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Retraction for Yang et al., Y4lO of Rhizobium sp. Strain NGR234 Is aSymbiotic Determinant Required for Symbiosome Differentiation

Feng-Juan Yang, Li-Li Cheng, Ling Zhang, Wei-Jun Dai, Zhe Liu, Nan Yao, Zhi-Ping Xie, Christian Staehelin

State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou, China

Volume 191, no. 3, p. 735–746, 2009. In the original article describing the symbiotic phenotype of the mutant NGR�y4lO, we concludedthat observed impaired symbiosome differentiation and nitrogen fixation were due to a mutation in the nopJ (formerly y4lO) gene,suggesting a role for nopJ in symbiosome differentiation in host cells of leguminous nodules. In response to a contradictory report byKambara et al. (K. Kambara, S. Ardissone, H. Kobayashi, M. M. Saad, O. Schumpp, W. J. Broughton, and W. J. Deakin, Mol. Microbiol.71:92–106, 2009), we shotgun sequenced the genome of NGR�y4lO and found a 31.9-kb deletion in the symbiotic replicon. Confir-mation of this spontaneous deletion by PCR and phenotypes of two additional nopJ knockout mutants have led us to conclude that theobserved phenotype of NGR�y4lO described in the original paper is due to the deletion of nitrogen fixation (nif) genes rather than toknockout of the nopJ gene. We regret that retraction of this article is necessary and apologize for any inconvenience caused.

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