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Biotechnology & Applied Microbiology
Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. The final effect of the different effectors can be neutral, beneficial or detrimental for symbiosis, depending on the host plants and the cocktail of effectors secreted. Whereas, in some cases, several T3SS effectors can induce the formation of nodules in the absence of Nod factors, in other cases the recognition of specific effectors by specific plant receptors can block nodulation.
- type 3 secretion system
- T3SS
- effector
- symbiosis
- rhizobium
1. The Symbiotic Type 3 Secretion System
Among all bacterial protein secretion systems, the T3SS is probably the best characterized. The T3SS is a nanomachine present in many Gram-negative bacteria that delivers proteins into the cytosol of eukaryotic host cells to, in most cases, manipulate their functions [61]. Its structure is composed of a set of proteins that spans the inner and outer membranes that serves as a basal complex to polymerize the extracellular component of the system, a long hollow tube of pilins that culminates in a translocon that recognizes host cell membranes where it forms a pore for protein delivery. The whole complex resembles a syringe that injects effector proteins directly from the cytosol of the bacterium to the cytosol of host cell. Although this system has been mainly studied in animal pathogens such as Salmonella, Yersinia, or Escherichia coli [62], its presence in numerous phytopathogens, such as strains of Pseudomonas, Xanthomonas or Ralstonia, has also been explored in depth [63]. Due to the obvious differences among animal and plant host-cell surfaces, T3SS reveals structural differences in the bacteria invading each kind of host, being longer and thinner in the case of plant-interacting bacteria [49]. Analogously, T3SS shows genetic differences among plant-interacting bacteria whether they are beneficial or pathogenic based on gene composition, arrangement, and transcriptional regulation [64].
In rhizobia, genes coding for the T3SS are usually contained on symbiotic plasmids (pSym) or in symbiotic islands in the chromosome. Genes encoding the core machinery are named rhc (Rhizobium conserved) and the secreted proteins (effectors, pilins, and proteins forming the translocon) are known as nodulation outer proteins (Nops) [65]. The rhc loci are quite conserved in most rhizobia and are clustered in regions ranging from 22 to 50 Kb, while genes encoding Nops can also be found dispersed throughout the genome. These Nops sometimes show similarities with T3E secreted by (phyto) pathogens suggesting, in some cases, a similar role in the host cell and even a common ancestor. However, other T3E can be considered Rhizobium-specific. In these cases, it is likely that their function is more associated with specific stages of the symbiotic process [66].
As part of the phylogenetic analysis, four subgroups of rhizobial rhc regions have been identified, where only the Rhc-I (containing members of S. fredii, M. loti, and B. japonicum) has been directly associated with symbiosis [64]. Rhc-II group is restricted to different Sinorhizobium strains (such as NGR234, HH103, and USDA257) but its role is still unclear. The Rhc-III subgroup is characterized by a completely different genetic organization compared to other rhizobia and includes strains of R. etli, R. leguminosarum, and some strains of R. rhizogenes or R. radiobacter. Finally, the β-Rhc gene cluster is represented only by the β-proteobacterium Cupriavidus taiwanensis that harbors a non-canonical genetic organization [64]. Very interestingly, rhizobial T3SS expression is co-regulated with NF production; it is activated by NodD and flavonoids through the induction of the ttsI gene, whose encoded product acts as the positive transcriptional regulator of genes coding for the T3SS apparatus and T3E [67,68]. A recent work showed that T3SS is absolutely required for genistein-induced surface motility in S. fredii HH103, revealing a new and unexpected function of T3SS in rhizobia [69].
2. Rhizobial Type 3 Secretion System Effectors
Although traditionally the assignment of the term “effector” to a putative secreted protein was based on its regulation through NodD and TtsI together with inducer flavonoids, and its translocation to a host cell, the new advances in the combination of multi-omics approaches (transcriptome and quantitative shotgun proteome analysis) with the experimental validation of obtained candidates will provide an ample amount of rhizobial T3E. However, an interesting observation is that while pathogens secrete a plethora of effectors to overcome the different defense responses of target plants (as in the case of P. syringae, which secretes more than 80 effectors), rhizobia have limited their arsenal to a much lower number, especially in Sinorhizobium strains, which has about ten T3E [49,71].
Rhizobial T3E, as well as in pathogens, can be composed by different modules with specific functional domains, and targets a single or diverse subcellular localizations into the plant cell [47,103,112]. Very interestingly, a great number of the studied T3E localize to the plant nucleus, where they can develop their functions. These are the cases of ErnA of Bradyrhizobium sp. ORS3257 [72], NopD of Bradyrhizobium sp. XS1150 and Bel2-5 of B. elkanii USDA61 [81,82,83], and even NopL and NopM of S. fredii NGR234 [93,97]. Some rhizobial T3Es seem to be phosphorylated by plant kinases in vitro, such as NopP, NopL, and NopM of S. fredii NGR234 [92,93,94,95,96,97,100]. More specifically, NopL and NopM are phosphorylated in planta by the mitogen-activated protein kinases (MAPK), salicylic acid-induced protein kinase (SIPK) of Nicotiana tabacum (NtSIPK) [93,96,97]. However, only the physical interactions of NopL and NtSIPK within the plant cell nucleus has been described [93]. Some T3E possess specific domains for the modulation of posttranslational modifications, such as ubiquitinylation or SUMOylation, which can influence diverse plant aspects. Those T3E includes NopM of S. fredii NGR234, and NopD of Bradyrhizobium sp. XS1150 and Bel2-5 of B. elkanii USDA61 from the NopD family of T3E, respectively [66,80,83,96,97]. Finally, some T3E possess proteolytic activity, although the nature of this activity seems to be different. In this regard, NopE1 of B. diazoefficiens USDA110 and NopT of S. fredii NGR234 display autoproteolytic activity, and very interestingly, NopT also cleaves the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and its homologous in soybean, GmPBS1-1, and thus can activate these target proteins [106].
3. The Role of the Rhizobial Type 3 Secretion System in Symbiosis
The first evidence of the existence of a rhizobial T3SS was obtained after the confirmation that S. fredii USDA257 secreted some proteins to the extracellular milieu upon flavonoids induction. This protein secretion determines the incapacity of this strain to nodulate agronomically improved American soybean varieties [113,114]. Since that time, the role of the T3E in symbiosis has been extensively studied in Meso-, Brady-, and Sinorhizobia by different research groups [8,47,49,64] focusing their research mainly on the symbiotic interaction with soybean, and Vigna and Lotus species (Table 2). As previously mentioned, in some cases, the recognition of rhizobial T3E by legume plant protein receptors blocks nodulation. This phenotype resembles the gene-for-gene resistance of the phytopathogen–plant relationship, which in the case of rhizobia–legume interaction is translated to the determination of the host specificity [115]. However, the latest findings indicate that other effectors exert exactly the opposite effect: they are essential for nodulation [72,78,82]. The effect of the different T3E on the symbiotic phenotype will vary from beneficial, to neutral, to detrimental and will always depend on the effector studied and the final balance of the different effects of the different effectors on the symbiotic process.
3.1. Soybean and Wild Soybeans
Glycine max L. Merr. (soybean) cultivation is extended worldwide due to its economical and agronomical importance. In terms of the study of symbiotic signals, soybean is probably the model for the study of T3E function in the rhizobium–legume symbiosis. In fact, all Brady- and Sinorhizobia able to nodulate soybean possess a functional T3SS (but not all rhizobial strains with a T3SS can nodulate soybean). Symbiotic phenotypes vary from nodulation blocking to host-range extension, and even promoting nodulation in the absence of NF.
During soybean domestication, many natural phenotypic changes affecting plant development, flowering time, seed size and protein and oil content, among others, have occurred [157]. In this process of domestication, traits controlling the formation of symbiotic root nodules by several host resistance (R) genes, referred to as Rj/rj genes, have been maintained in agronomically improved soybean cultivars. Four Rj genotypes control nodulation in soybean: (i) Rfg1 soybeans restrict nodulation with some S. fredii strains such as USDA257, USDA205, and USDA193 [143,158]. The Rfg1 gene encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class (TIR-NBS-LRR) of plant resistance proteins; (ii) Rj2 soybeans, carrying an allelic variant of Rfg1, also restrict nodulation with B. japonicum USDA122 [143]; (iii) Rj3 soybeans cannot be nodulated by some B. elkanii strains such as USDA33, BLY3-8, or BLY6-1 [159]; and (iv) strains such as B. japonicum Is-34 or B. elkanii USDA61 cannot nodulate Rj4 soybeans.
The Rj2/Rfg1 protein is the soybean determinant restricting nodulation by some B. japonicum, B. diazoefficiens, and S. fredii strains. Polymorphisms of seven amino acid residues (E452K, I490R, Q731E, E736N, P743S, E756D, and R758S) define three allelic groups of Rj2/Rfg1. Whereas Rj2/rfg1 restricts nodulation with some B. japonicum and B. diazoefficiens strains, rj2/Rfg1 restricts nodulation with strains of S. fredii. On the other hand, rj2/rfg1 allows most B. japonicum, B. diazoefficiens, and S. fredii to form nodules [143,158].
On the other hand, the inability to nodulate Rj4 soybeans is mediated by proteins with a C48 protease domain present in the NopD family of rhizobial effectors [84,127,165]. The catalytic C48-peptidase domain is involved in SUMOylation and de-SUMOylation of host proteins. Small ubiquitin-modifiers (SUMO) are small proteins used by eukaryotic cells to posttranslational modify substrate proteins in a specific manner. These modifications can alter protein stability and activity. While SUMOylation causes the activation/repression of certain transcription factors, de-SUMOylation causes the opposite effect [166]. NopD from B. japonicum is delivered to the nucleus of the host cell and can SUMOylate and de-SUMOylate soybean proteins [83].
Glycine soja (Siebold and Zucc.) is the wild ancestor of the domesticated soybean [168]. Studies carried out by Temprano-Vera et al. [144] indicate that inactivation of the T3SS significantly impacts symbiosis with several G. soja accessions. Thus, accession CH2 from northern China forms nodules with S. fredii NGR234 but not with S. fredii HH103. While T3SS mutants of NGR234 lose their capacity to nodulate this accession, HH103 T3SS mutants gain nodulation. Therefore, one or more T3E secreted by NGR234 can promote CH2 nodulation while the combination of Nops secreted by HH103 prevents it. In the case of accession CH3, also from northern China, HH103 T3SS mutants retain nodulation. However, inactivation of the NGR234 T3SS totally abolishes nodulation. The different effect of the T3SS mutations in the symbiosis with the different G. soja accessions suggests that these wild soybeans could have different Rj/rj genotypes or even new R genes that could have been transferred to cultivated soybeans during the process of domestication.
3.2. Vigna spp.
One of the two most widely consumed types of legumes, together with soybean, belongs to the genus Vigna. Among all the species only a very few have been domesticated so far [169,170]. Vigna unguiculata L. Walp. (cowpea) is one of the most important grain legume crops in the world with a larger zone of occurrence and cultivation, while Vigna radiata L. Wilczek (mung bean) and Vigna mungo var. mungo L. Hepper (black gram) are consumed and produced especially in Asia [170,171].
Rhizobial T3SS induce positive, neutral, and negative effects in symbiosis with Vigna spp. This scenario is well reflected in the Sinorhizobium/Bradyrhizobium–cowpea symbiotic interactions. However, observations turn more interesting with findings from studies about Bradyrhizobium and other Vigna spp. symbiotic relationships. In this sense, this plant could be a very good model to study the role of Bradyrhizobial T3E, because their T3SS are responsible for the symbiotic compatibility/incompatibility phenotype in these legumes and because they possess a wide range of T3E.
Regarding the negative impact on symbiosis, rhizobial T3SS can cause a nodulation-blocking phenotype, as occurs in the symbiosis between B. elkanii USDA61 and V. mungo cv. VM3003 and U-THONG2, V. aconitifolia or V. trinervia [155]. Impressively, the T3SS renders incompatible the symbiotic interaction between various Bradyrhizobia species with several V. radiata cultivars, such as B. vignae ORS3257 with CN72, KPS2, SUT1, SUT4, V4758, and V4785; B. diazoefficiens USDA110 with KPS2, V4718, and V4785; Bradyrhizobium sp. DOA9 with V4718 and V4758; and B. elkanii USDA61 with KPS1 [130,135,156].
3.3. Lotus spp.
Lotus japonicus, together with Medicago truncatula, are the model plants commonly used to study the molecular basis of the symbiotic process due to their small size and the availability of a high variety molecular biology tools. T3E play important and distinct roles in the interaction of Lotus spp. with different rhizobial partners. Regarding the natural partner of Lotus, Mesorhizobium loti, the T3SS (present in MAFF303077 but not in R7A) may play different roles in symbiosis [85,149]: positive with L. corniculatus, L. filicaulis, and L. tenuis INTA PAMPA; neutral with L. japonicus MG-20, and negative with L. halophilus, L. peregrinus, L. subbiflorus, and L. tenuis Esmeralda [85,149]. Regarding S. fredii strains, NGR234 NopL and NopM have a neutral and a positive role with L. japonicus Gifu and MG-20, respectively [92,97], whereas the T3SS have a negative role in the symbiosis of HH103 with L. burttii. Interestingly, S. fredii HH103 cannot nodulate L. japonicus GIFU. However, T3SS mutants gain the capacity to nodulate this legume. When analyzing the effect of the mutation of each T3E secreted by HH103, NopC was revealed to be the one involved in blocking nodulation [148]. With respect to Bradyrhizobium, there are several works regarding the role of T3SS in the symbiosis of Lotus spp. with B. elkanii USDA61 and Bradyhizobium sp. SUTN9-2 [89,147].
3.4. Aeschynomene spp.
Aeschynomene species are tropical plants nodulated by Bradyrhizobial strains. Some interactions were initially considered peculiar cases in Rhizobium–legume symbiosis, since these rhizobia possess a functional T3SS but are incapable of producing NF [23]. Thus, an alternative nodulation process can be developed by an NF-independent pathway in a T3SS-dependent manner. However, growing studies on symbiotic interactions have broken this exception of the rule, due to the occurrence of the symbiotic interaction between B. elkanii USDA61 and G. max cultivar Enrei, as previously mentioned [133]. In general, the T3SS and its T3E exert neutral or positive effects in the Bradyrhizobium-Aeschynomene symbioses studied so far.
This entry is adapted from the peer-reviewed paper 10.3390/ijms231911089
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