ReviewRhizobium type III secretion systems: legume charmers or alarmers?
Introduction
Soil bacteria of the genera Azorhizobium, Bradyrhizobium, Mesorhizobium and Rhizobium (collectively called rhizobia) are prokaryotic symbionts that induce the formation of nodules on leguminous plant roots. Within these newly developed organs, bacteria differentiate into bacteroids that reduce atmospheric nitrogen to ammonia. In exchange for this assimilated nitrogen, the plants supply the bacteria with carbohydrates. Nodule formation requires extensive molecular communication between both partners. Key components for the establishment of symbiosis are plant root-produced flavonoids that, via bacterial activator NodD proteins, induce the transcription of nodulation (nod) genes. Enzymes encoded by these genes are involved in the synthesis of lipochitooligo-saccharides (Nod-factors) that initiate nodule development and bacterial entry 1•., 2•., 3•., 4•..
Although Nod-factors have been shown to be essential for nodule formation, there are many other determinants that influence the extent of the symbiosis. Type III secretion systems (TTSSs) are one such element in rhizobia. First identified in plant and animal pathogens, TTSSs are composed of several proteins that are associated with the inner and outer bacterial membranes. Upon contact with host cells, proteins are secreted via TTSS membrane channels either into the extra-cellular medium or into the eukaryotic cytoplasm where they subvert the functioning of the aggressed cell 5., 6., 7., 8.. The recent discovery of TTSSs in several Rhizobium strains came as a surprise. The purpose of this article is to review knowledge of this machinery, which had been thought to be specific to bacterial pathogens.
Section snippets
Identification of type III secretion genes in Rhizobium strains
A complete rhizobial TTSS was first identified after sequencing the symbiotic plasmid of NGR234 (pNGR234a) [9]. Additonal TTSSs were found by sequencing of the genome of Mesorhizobium loti strain MAF303099 [10•] and the ‘symbiotic island’ (a 410 kilobase region containing nearly all of the known nodulation genes) of Bradyrhizobium japonicum USDA110 [11•]. Two strains of Rhizobium fredii, USDA257 and HH103, also contain TTSS genes. HH103 is capable of nodulating both primitive and advanced
Genetic organisation of type III secretion system genes
In rhizobia, the genes encoding type III secretion machines are clustered within regions of 35–47 kilobases (Fig. 1). Three groups of genes appear to be present in all of the strains for which complete sequence information is available. The three groups include rhcC1–rhcU, y4yQ–y4yS and y4xI–y4xK. At the nucleotide level, the degree of homology with the NGR234 sequence ranges from 98% identical for R. fredii, through approximately 75% for M. loti MAF303099, to 68% for B. japonicum USDA110. The
Characteristics of Rhizobium strains harbouring type III secretion systems
Genomic hybridizations have shown that rhc homologues are also found in B. elkanii USDA76 and B. japonicum CB756 but not in R. meliloti 2011 [16]. The recently completed genomic DNA sequence of R. meliloti 1021 also lacks homologues of genes encoding a TTSS. TTSSs are thus present in some rhizobia but are not ubiquitous (Table 1). No correlation could be found between the genus of rhizobia and the presence of type III secretion machines. For example, two strains of R. fredii contain TTSSs
Regulation of type III secretion system genes
The TTSS genes of pathogenic bacteria are expressed in response to environmental factors that usually correspond to the conditions encountered during infection of a host [8]. In NGR234, transcription of TTSS-related genes requires the presence of flavonoids and two bacterial regulators, NodD1 and y4xI ([16]; C Marie, unpublished data). NodD1 belongs to the LysR family of transcriptional activators. It binds to consensus promoter regions called nod-boxes and, after exposure to flavonoids,
Proteins secreted via the Rhizobium type III secretion systems
NGR234 secretes at least eight proteins in a TTSS-dependent manner, two of which were identified as NolX and y4xL ([16]; C Marie, unpublished data). If the nolXWBTUV locus is not disrupted, R. fredii USDA257 secretes a homologue of NolX and at least four additional proteins ([19]; H Krishnan, personal communication). A mutation in nolT (rhcJ) of HH103 abolishes the secretion of five proteins [13]. In pathogenic bacteria, not all of the secreted proteins exhibit direct anti-host properties, some
What is the function of type III secretion systems during the symbiotic process?
Functionality of TTSSs has been demonstrated in three rhizobia: NGR234, R. fredii HH103 and R. fredii USDA257 (Table 3). A comparison of nodulation efficiency was determined for the wild-type strains against the TTSS mutants. Abolition of protein secretion can affect nodule formation in different ways, ranging from no effect to a reduction or an increase in nodule number 12., 13., 16., 29.. A more drastic phenotype was observed after inoculation of Crotalaria juncea (C Marie, unpublished data)
Conclusions
TTSS genes have been identified in a number of Rhizobium strains. Protein secretion by this apparatus and a role for it in symbiosis have been demonstrated for NGR234 and R. fredii strains. Depending upon the plant studied, the secreted proteins can have diverse effects. How can such varied responses be explained? It is possible that different flavonoids, which might modulate the expression levels of the secreted proteins, are produced by each plant. Alternatively, the secreted proteins could
Acknowledgements
We are grateful to Dr M Göttfert and Dr A Krause (University of Technology, Dresden, Germany) who made data and the map of the TTSS cluster from B. japonicum available to us prior to publication. Dr JE Ruiz-Sainz (University of Seville, Spain) and Dr H Krishnan (University of Missouri, USA) kindly allowed us to review their unpublished results on strains HH103 and USDA257, respectively. We also thank M Bladergroen and Dr HP Spaink (Leiden University, The Netherlands) for the information
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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