NsrA, a Predicted β-Barrel Outer Membrane Protein Involved in Plant Signal Perception and the Control of Secondary Infection in Sinorhizobium meliloti

Anne-Marie Garnerone; Fernando Sorroche; Lan Zou; Céline Mathieu-Demazière; Chang Fu Tian; Catherine Masson-Boivin; Jacques Batut

doi:10.1128/JB.00019-18

DOI: 10.1128/JB.00019-18

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FIG 1
CyaK sensing of signal 1. Ex planta expression of the smc02178-lacZ fusion carried on the pGD2178 plasmid was assessed in the presence of shoot (gray bars) and 14-dpi nodule (black bars) extracts in S. meliloti wild-type (Rm1021), cyaD1, cyaD2, and cyaK genetic backgrounds. Activity was standardized per gram of shoot or nodule extract. ***, P < 0.001. Brackets indicate comparison with the reference sample.
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FIG 2
Need for nsrA for (shoot) signal 1 perception ex planta. (A) Expression of the smc02178-lacZ reporter fusion carried on the pGD2178 plasmid in Rm1021 (light gray bars) and nsrA mutant (dark gray bars) genetic backgrounds. (B) Expression of the smb20495-lacZ reporter fusion carried on the pGD20495 plasmid. *, P < 0.05; **, P < 0.01; °, reference sample. Mean specific activities (i.e., Miller units per gram of fresh weight) are indicated below the graph.
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FIG 3
Plasmid-driven overexpression of nsrA increasing (shoot) signal 1 responses ex planta. Expression of the smc02178-lacZ fusion (pGD2178) was monitored in wild-type S. meliloti Rm1021 (gray bars) or the cyaD1 cyaD2 cyaK triple mutant (black bars). The relevant plasmid genotypes are indicated below the graph. **, P < 0.01; °, reference sample.
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FIG 4
Phenotypes of the nonpolar S. meliloti nsrA mutant in planta. (A) In situ smc02178-lacZ (pGD2178) expression in Medicago sativa nodules (14 dpi) elicited by different S. meliloti strains. The predominant phenotype is shown (see the text and Fig. S6 for details). Scale bars = 100 μm. (B) Nodulation (gray bars) and hyperinfection (green bars) phenotypes of the S. meliloti nsrA mutant on M. sativa at 14 dpi. The pxLGD4 plasmid was introduced in the S. meliloti nsrA mutant to allow eIT visualization. ***, P < 0.001; °, reference sample.
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FIG 5
Working model for signal perception via NsrA, based on the predicted topology of NsrA (see the text for details). In young and mature nodules, signal 1 (S1) interacts with the β-barrel domain of NsrA (yellow). In our model, the periplasmic RIN/FECR (yellow rectangle) and/or TPR (yellow oval) domains of NsrA interact with the inhibitory CHASE2 domain (gold rectangle) of CyaK, releasing the activity of the cytoplasmic AC domain (orange rectangles). In the presence of cAMP, the Clr protein (blue ovals) activates target gene expression and inhibits eIT formation. In young nodules, a hypothetical signal 1′ (S1′) would bind NsrA and allow CyaD1 and/or CyaD2 activity. Together, both signal 1 and signal 1′ and the three ACs control secondary eIT formation. OM, outer membrane; IM, inner membrane.

TABLE 1

Bacterial strains and plasmids used in this study

Strain or plasmid	Description^a	Reference or source
DH5α	E. coli fhuA2 Δ(argF-lacZ)U169 phoA glnV44 ϕ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17	Bethesda Research Laboratories
1021 (Rm1021)	Derivative of S. meliloti strain SU47; Str^r	28
GMI11555	1021 ΔcyaD2::Gm Str^r Gen^r	8
GMI11556	1021 ΔcyaK::Gm Str^r Gen^r	8
GMI11557	1021 cyaD1::pVO155 ΔcyaD2 Str^r Neo^r	8
GMI11558	1021 cyaD1::pVO155 ΔcyaD2 ΔcyaK::Gm Str^r Neo^r Gen^r	8
GMI11561	1021 cyaD1::pVO155 Str^r Neo^r	8
GMI12049	1021 ΔnsrA Str^r	This work
GMI12050	1021(pGMI50331, pGD2178) Str^r Gen^r Tet^r	This work
GMI12051	1021(pGMI50332, pGD2178) Str^r Gen^r Tet^r	This work
GMI12052	1021(pGMI50333, pGD2178) Str^r Gen^r Tet^r	This work
GMI12053	1021 cyaD1::pVO155 ΔcyaD2 ΔcyaK pGMI50331 pGD2178 Str^r Neo^r Gen^r Tet^r	This work
GMI12054	1021 cyaD1::pVO155 ΔcyaD2 ΔcyaK pGMI50332 pGD2178 Str^r Neo^r Gen^r Tet^r	This work
GMI12055	1021 cyaD1::pVO155 ΔcyaD2 ΔcyaK pGMI50333 pGD2178 Str^r Neo^r Gen^r Tet^r	This work
pJQ200-mp19	Suicide vector; Gen^r	25
pGEM-T	Cloning vector; Amp^r	Promega Corp.
pRK600	Helper conjugative plasmid, ColE1 replicon with RK2 transfer region; Chl^r	29
pGD926	pRK290 derivative containing promoterless lacZ gene; Tet^r	30
pXLGD4	hemA-lacZ reporter plasmid; Tet^r	31
pGD2178	pGD926 containing smc02178 promoter region fused to lacZ; Tet^r	8
pGD20495	pGD926 containing smb20495 promoter region fused to lacZ; Tet^r	12
pBBR1MCS-5	Cloning vector; Gen^r	32
pGMI50331	pBBR1MCS-5 derivative expressing nsrA and cyaK; Gen^r	This work
pGMI50332	pBBR1MCS-5 derivative expressing cyaK; Gen^r	This work
pGMI50333	pBBR1MCS-5 derivative expressing nsrA; Gen^r	This work
pGMI50334	pJQ200-mp19 derivative carrying deleted nsrA gene; Gen^r	This work

↵a Str^r, streptomycin resistance; Gen^r, gentamicin resistance; Neo^r, neomycin resistance; Chl^r, chloramphenicol resistance; Tet^r, tetracycline resistance; Amp^r, ampicillin resistance.

Additional Files

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Supplemental material

Supplemental file 1 -
Table S1 (Primers used) and Fig. S1 (Domain organization of NsrA), S2 (PRED-TMBB analysis of NsrA and related proteins), S3 (TMRPres2D representation of NsrA transmembrane region), S4 (RT-PCR monitoring of cyaK and nsrA expression), S5 (nsrA is not needed for cAMP-mediated signaling), and S6 (Nodule expression of smc02178-lacZ fusion)

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