Table 1.
Bacterial strains and plasmids.
Table 2.
DNA oligonucleotide primers used in this study.
Fig 1.
Organization of the HH103 nopC locus.
(A) Position of the annotated open reading frames (ORFs) nopC, nopA, y4yQ, rhcV, and y4yS. (B) Neighbor joining phylogenies of the NopC tree of several rhizobial strains. Bootstrap values ≥ 60 are indicated for each node. The cluster analysis to group the strains by NopC sequence similarity was done using the program CLUSTAL W in the MEGA5 software package with the algorithm neighbor-joining method. Tree robustness was assessed by bootstrap resampling (1000 replicates each).
Fig 2.
The expression of the S. fredii HH103 nopC gene is regulated by flavonoids, NodD1 and TtsI.
(A) qRT-PCR analysis of the expression of nopC in the parental strain HH103 RifR and the mutant strains HH103 RifR nodD1::lacZ-GmR and HH103 RifR ttsI::Ω in the absence (-) or presence (+) of the inducer flavonoid genistein (3.7 μM). Final expression was calculated relative to the expression of the HH103 RifR strain in the absence of flavonoids. Expression data shown are the mean (± standard deviation of the mean) for two biological replicates performed at least in triplicates. Each expression value was individually compared with the HH103 RifR strain in the absence of flavonoids using the Mann-Whitney non-parametrical test. Asterisks indicate that numbers are significantly different at the level α = 5% (p< 0.05). (B) Immunodetection of NopC in extracellular proteins extracts of the parental strain HH103 RifR and the ttsI and nodD1 mutants in the presence or absence of genistein (3.7 μM). Molecular masses (kDa) of the marker are shown on the left. Samples were separated by 15% SDS-PAGE.
Fig 3.
The in frame mutation of the nopC gene did not block secretion of the rest of the S. fredii HH103 Nops.
Silver-stained gel of secreted extracellular proteins of HH103 RifR, the HH103 RifR ΔnopC mutant and the nopC mutant complemented with plasmid pMUS986 in the absence (-) or presence (+) of genistein (3.7 μM). Proteins whose secretion depends on genistein and a functional T3SS are indicated with an asterisk and indicated on the right. Molecular masses (kDa) of the marker are shown on the left. Samples were separated by 15% SDS-PAGE.
Fig 4.
Immunodetection of several S. fredii HH103 Nops.
Immunodetection of NopA, NopB, NopC, NopP, and NopX in extracellular proteins extracts of the parental strain HH103 RifR, the HH103 RifR ΔnopC mutant and the nopC mutant complemented with plasmid pMUS986 in the absence (-) or presence (+) of genistein (3.7 μM). Molecular masses (kDa) of the marker are shown on the left. Samples were separated by 15% SDS-PAGE.
Fig 5.
The S. fredii HH103 NopC is translocated into Glycine max cv. Williams 82 root cells.
(A) In vivo monitoring of the activation of the tts box upstream nopC in vermiculite assays. Bioluminescence was measured in soybean plants inoculated with the HH103 RifR strain carrying plasmid pMUS1207 (plasmid pMP92 containing the tts box fused to luxCDABE). Bioluminiscence is shown by colored areas and indicated with arrows. (B) cAMP levels measured in soybean nodules harvested 18 d.p.i. from plants inoculated with several strains carrying the nopC-cya fusion. Data shown are the mean (± standard deviation of the mean) for two biological replicates. Each cAMP value was individually compared to that obtained in plants inoculated with the HH103 RifR strain using the Mann-Whitney non-parametrical test. Asterisks (*) indicate that numbers are significantly different at the level α = 5% (p< 0.05).
Table 3.
Plant responses to inoculation of Glycine max cv. Williams 82 with different Sinorhizobium fredii HH103 strains.
Table 4.
Plant responses to inoculation of Vigna unguiculata with different Sinorhizobium fredii HH103 strains.