Plant growth-promoting rhizobacteria (PGPR) are free-living bacteria which actively colonize plant roots, exerting beneficial effects on plant development. The PGPR may (i) promote the plant growth either by using their own metabolism (solubilizing phosphates, producing hormones or fixing nitrogen) or directly affecting the plant metabolism (increasing the uptake of water and minerals), enhancing root development, increasing the enzymatic activity of the plant or "helping" other beneficial microorganisms to enhance their action on the plants; (ii) or may promote the plant growth by suppressing plant pathogens. These abilities are of great agriculture importance in terms of improving soil fertility and crop yield, thus reducing the negative impact of chemical fertilizers on the environment. The progress in the last decade in using PGPR in a variety of plants (maize, rice, wheat, soybean and bean) along with their mechanism of action are summarized and discussed here.

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops secretion. TtsI is responsible for the transcriptional activation of nopX, nopA, rhcJ and rhcQ. We confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of some flavonoids to activate these genes were found. The expression of the ttsI and nodA genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod box, but it was restored when a plasmid harbouring the ttsI, rhcC2 and y4xK genes was transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis with Glycine max and Glycyrrhiza uralensis, and detrimental in the case of the tropical legume Erythrina variegata.

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nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.