# Identification of _Bradyrhizobium elkanii_ Genes Involved in Incompatibility with _Vigna radiata_
## 1 Introduction  
Symbiotic relationships between leguminous plants and soil bacteria, collectively termed rhizobia, are characterized by the formation of root nodules, which are specialized plant organs that accommodate the rhizobia. In the root nodules, rhizobia fix atmospheric dinitrogen into ammonia, which is utilized by the host legumes. This symbiotic association is of critical importance in agricultural production and the nitrogen cycle. The interactions between legumes and rhizobia exhibit high levels of specificity, which is determined by the exchange of signal molecules. Legume roots excrete flavonoid compounds that interact specifically with the transcriptional regulator NodD in rhizobia [1; 2]. The activation of NodD induces the expression of nodulation genes, which are required for the synthesis of lipo-chitooligosaccharides called Nod-factors [1; 2]. The Nod factors function as signals for host receptors, which activate signal pathways leading to rhizobial infection and nodule formation [1; 2]. In addition, rhizobial factors, such as exopolysaccharide, lipopolysaccharides, and secreted proteins, have also been reported to affect nodulation and host specificity [3; 4].  
The rhizobium _Bradyrhizobium elkanii_ forms symbiotic relationships with a number of legumes, including soybean, mung bean (_Vigna radiata_), and groundnuts (_Araclys hnpogaea_). In Brazil, _B. elkanii_ is used as an inoculant for soybean cultivation [5]. The formation of symbiotic root nodules in soybeanis controlled by several loci, which are referred to as the _rj_ or _Rj_ loci [6]. Soybean varieties carrying the dominant _Rj4_ allele, such as the cultivars Hill and BARC2, are incapable of nodulation with _B. elkanii_ USDA61, but are compatible with _Bradyrhizobium diazoefficients_ USDA110 [7]. These studies revealed that _Rj4_ incompatibility is strain-specific and cultivar-specific, and appears to resemble gene-for-gene resistance in plant-pathogen interactions [8]. The recent identification of genes that are encoded by the _Rj2_ and _Rj4_ genes supports this idea. _Rj2_ encodes a member of the Toll-interleukin receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class of proteins [9], while _Rj4_ encodes a thaumatin-like protein [10]. Both of these classes of protein are involved in plant resistance to pathogens.  
Mung bean is an important leguminous crop in tropical and subtropical regions. Growth and seed production by _V. radiata_ are highly dependent on symbiosis with nitrogen-fixing rhizobia [11]. The _V. radiata_ cv. KPS1 cultivar is incompatible with _B. elkanii_ USDA61, resulting in ineffective nodulation [12]. In a previous study [12], we mutated two _B. elkanii_ genes, _rhcC2_ and _rhcJ_, which are well-conserved components of the type III secretion system (T3 SS). The T3 SS is a complex protein transport system that is involved in plant and animal pathogenesis by Gram-negative bacteria, and it is also found in many rhizobia [13; 14; 15; 16; 17]. The _B. elkanii_ T3 SS mutants formed functional nodules on _V. radiata_ cv. KPS1 and _Glycine max_ cv. Hill plants [12]. These results implicated the T3 SS in the control of incompatibility between rhizobia and their plant hosts, however, genetic elements involved in the incompatibility between _B. elkanii_ USDA61 and _V. radiata_ cv. KPS1 have not been fully elucidated. Here, we identified five novel _B. elkanii_ genes that contribute to this incompatibility. Four of these genes also control nodulation restriction in _Rj4_ soybean, suggesting a common mechanism that is underlying both incompatible interactions. The fifth gene encodes a T3 SS-related protein with a host-specific function in KPS1. These findings shed light on the diverse and complicated mechanisms underlying the incompatible symbiosis, and suggest the involvement of a novel T3 SS-related gene in the nodulation restriction of mung bean.