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ABSTRACT The symbiotic relationship between rhizobia and legumes is critical for sustainable agriculture and has important economic and environmental implications. In this intricate process, rhizobial bacteria colonise plant roots and induce the formation of specialised plant organs, the nodules. Within these structures, rhizobia fix environmental nitrogen into ammonia, significantly reducing the demand for synthetic fertilisers. Multiple bacterial secretion systems (TXSS, Type X Secretion System) are involved in establishing this symbiosis, with T3SS being the most studied. While the Type 6 Secretion System (T6SS) is known as a “nanoweapon” commonly used by diderm (formerly gram‐negative) bacteria for inter‐bacterial competition and potentially manipulating eukaryotic cells, its precise role in legume symbiosis remains unclear. Sinorhizobium fredii USDA257, a fast‐growing rhizobial strain capable of nodulating diverse legume plants, possesses a single T6SS cluster containing genes encoding structural components and potential effectors that could target plant cells and/or act as effector‐immunity pairs. Our research reveals that this T6SS can be induced in nutrient‐limited conditions and, more importantly, is essential for successful nodulation and competitive colonisation of Glycine max cv Pekin. Although the system did not demonstrate effectiveness in eliminating competing bacteria in vitro, its active presence within root nodules suggests a sophisticated role in symbiotic interactions that extends beyond traditional interbacterial competition. The functional type VI secretion system of Sinorhizobium fredii USDA257 is required for successful nodulation with Glycine max cv Pekin.

The establishment of symbiotic interactions between leguminous plants and rhizobia requires complex cellular programming activated by Rhizobium Nod factors (NFs) as well as type III effector (T3E)-mediated symbiotic signaling. However, the mechanisms by which different signals jointly affect symbiosis are still unclear. Here we describe the mechanisms mediating the cross-talk between the broad host range rhizobia Sinorhizobium fredii HH103 T3E Nodulation Outer Protein L (NopL) effector and NF signaling in soybean. NopL physically interacts with the Glycine max Remorin 1a (GmREM1a) and the NFs receptor NFR5 (GmNFR5) and promotes GmNFR5 recruitment by GmREM1a. Furthermore, NopL and NF influence the expression of GmRINRK1 , a receptor-like kinase (LRR-RLK) ortholog of the Lotus RINRK1, that mediates NF signaling. Taken together, our work indicates that S. fredii NopL can interact with the NF signaling cascade components to promote the symbiotic interaction in soybean. This study showed that the type III effector NopL can interact with the Nod factor receptor GmNFR5, and the existence of NopL can promote GmREM1 interacts with GmNFR5, which demonstrates a signaling pathway underlying the symbiosis establishment in soybean.

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.

In the rhizobia-legume symbiotic interaction, bacterial surface polysaccharides, such as exopolysaccharide (EPS), lipopolysaccharide (LPS), K-antigen polysaccharide (KPS) or cyclic glucans (CG), appear to play crucial roles either acting as signals required for the progression of the interaction and/or preventing host defence mechanisms. The symbiotic significance of each of these polysaccharides varies depending on the specific rhizobia-legume couple. In this work we show that the production of exopolysaccharide by Sinorhizobium fredii HH103, but not by other S. fredii strains such as USDA257 or NGR234, is repressed by nod gene inducing flavonoids such as genistein and that this repression is dependent on the presence of a functional NodD1 protein. In agreement with the importance of EPS for bacterial biofilms, this reduced EPS production upon treatment with flavonoids correlates with decreased biofilm formation ability. By using quantitative RT-PCR analysis we show that expression of the exoY2 and exoK genes is repressed in late stationary cultures of S. fredii HH103 upon treatment with genistein. Results presented in this work show that in S. fredii HH103 EPS production is regulated just in the opposite way than other bacterial signals such as Nod factors and type 3 secreted effectors: it is repressed by flavonoids and NodD1 and enhanced by the nod repressor NolR. These results are in agreement with our previous observations showing that lack of EPS production by S. fredii HH103 is not only non-detrimental but even beneficial for symbiosis with soybean.

Background Agrobacterium rhizogenes -mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Results Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A . rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1 , which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. Conclusions We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

Symbiotic nitrogen fixation is the main source of nitrogen for soybean growth. Since the genotypes of rhizobia and soybean germplasms vary, the nitrogen-fixing ability of soybean after inoculation also varies. A few studies have reported that quantitative trait loci (QTLs) control biological nitrogen fixation traits, even soybean which is an important crop. The present study reported that the Sinorhizobium fredii HH103 gene rhcJ belongs to the tts (type III secretion) cluster and that the mutant HH103ΩrhcJ can clearly decrease the number of nodules in American soybeans. However, few QTLs of nodule traits have been identified. This study used a soybean (Glycine max (L.) Merr.) ‘Charleston’ × ‘Dongnong 594’ (C × D, n = 150) recombinant inbred line (RIL). Nodule traits were analysed in the RIL population after inoculation with S. fredii HH103 and the mutant HH103ΩrhcJ. Plants were grown in a greenhouse with a 16-h light cycle at 26 °C and an 8-h dark cycle at 18 °C. Then, 4 weeks after inoculation, plants were harvested for evaluation of nodule traits. Through QTL mapping, 16 QTLs were detected on 8 chromosomes. Quantitative PCR (qRT-PCR) and RNA-seq analysis determined that the genes Glyma.04g060600, Glyma.18g159800 and Glyma.13g252600 might interact with rhcJ.

Background and aims Sinorhizobium fredii  HH103 is a broad host-range rhizobial strain able to induce the formation of nitrogen-fixing nodules in dozens of legumes, including soybean.  S. fredii  HH103 exhibits genistein-induced surface motility. The aim of this work has been to determine whether the  flgJ  gene, which is inducible by genistein and codes for a flagellar protein, is involved in this motility and is relevant for symbiosis with soybean. Methods We have generated two independent mutants in the  flgJ  gene of HH103 and analysed their phenotypes in motility, exopolysaccharide production, biofilm formation, soybean root colonization, symbiosis with soybean, and secretion of effector proteins. We have also further studied the regulation of the expression of  flgJ . Results We show that the expression of  flgJ  is driven by a  tts  box previously not detected, which accounts for its induction by flavonoids and the NodD1 and TtsI transcriptional activators. Inactivation of  flgJ  led to severe impairments in bacterial motility (swimming and genistein-induced surface motility) as well as to a significant reduction in symbiotic performance with soybean when bacteria are not directly inoculated onto the seedling roots. However, the absence of a functional FlgJ protein did not affect the bacterial ability to colonize soybean roots. Conclusion The  flgJ  gene of  S. fredii  HH103 connects the  nod  regulon with the genistein-induced surface motility exhibited by this rhizobial strain.

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.

Leguminous plants can enter into root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. An intriguing but still poorly understood property of the symbiosis is its host specificity, which is controlled at multiple levels involving both rhizobial and host genes. It is widely believed that the host specificity is determined by specific recognition of bacterially derived Nod factors by the cognate host receptor(s). Here we describe the positional cloning of two soybean genes Rj2 and Rfg1 that restrict nodulation with specific strains of Bradyrhizobium japonicum and Sinorhizobium fredii, respectively. We show that Rj2 and Rfg1 are allelic genes encoding a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) proteins. The involvement of host R genes in the control of genotype-specific infection and nodulation reveals a common recognition mechanism underlying symbiotic and pathogenic host-bacteria interactions and suggests the existence of their cognate avirulence genes derived from rhizobia. This study suggests that establishment of a root nodule symbiosis requires the evasion of plant immune responses triggered by rhizobial effectors.

It is well documented that type-III effectors are required by Gram-negative pathogens to directly target different host cellular pathways to promote bacterial infection. However, in the context of legume–rhizobium symbiosis, the role of rhizobial effectors in regulating plant symbiotic pathways remains largely unexplored. Here, we show that NopT, a YopT-type cysteine protease of Sinorhizobium fredii NGR234 directly targets the plant’s symbiotic signaling pathway by associating with two Nod factor receptors (NFR1 and NFR5 of Lotus japonicus ). NopT inhibits cell death triggered by co-expression of NFR1/NFR5 in Nicotiana benthamiana . Full-length NopT physically interacts with NFR1 and NFR5. NopT proteolytically cleaves NFR5 both in vitro and in vivo, but can be inactivated by NFR1 as a result of phosphorylation. NopT plays an essential role in mediating rhizobial infection in L. japonicus . Autocleaved NopT retains the ability to cleave NFR5 but no longer interacts with NFR1. Interestingly, genomes of certain Sinorhizobium species only harbor nopT genes encoding truncated proteins without the autocleavage site. These results reveal an intricate interplay between rhizobia and legumes, in which a rhizobial effector protease targets NFR5 to suppress symbiotic signaling. NFR1 appears to counteract this process by phosphorylating the effector. This discovery highlights the role of a bacterial effector in regulating a signaling pathway in plants and opens up the perspective of developing kinase-interacting proteases to fine-tune cellular signaling processes in general.