# Identification of _Bradyrhizobium elkanii_ Genes Involved in Incompatibility with _Vigna radiata_
## 4 Discussion  
In fields, the application of high nitrogen-fixing rhizobia to their target legume crops often fails to increase the crop yields. One of the major causes of this phenomenon is that more competitive indigenous rhizobial strains, with low nitrogen fixation activity, occupy most of the nodules. This problem is known as the competition problem, and it is very important in the cultivation of legumes. Besides the competitiveness of different rhizobial strains, compatibility between leguminous plants and rhizobia also affect nodule occupancy. In soybean, _Rj_ genes control nodulation: plants carrying specific _Rj_ genes cannot form nodules when inoculated with specific rhizobial strains [6]. This compatibility/incompatibility might be reminiscent of host-pathogen interactions. Consistent with this observation, the _Rj2_ and _Rj4_ genes encode TIR-NBS-LRR and thaumatin-like proteins, respectively [10], and these proteins are involved in plant resistance to pathogens. Similarly, host-controlled nodulation occurs in mung beans, and the compatibility is dependent on both the host cultivar and the rhizobial strain. In a previous report, we showed that two genes that encode components of the T3 SS, _rhcC2_ and _rhcJ_, are necessary for the incompatibility between _V. radiata_ cv. KPS1 and _B. elkanii_ USDA61 [12]. The T3 SS is involved in plant and animal pathogenesis, and many rhizobial strains possess a functional T3 SS [13; 14; 15; 16; 17]. The secreted proteins, called nodulation outer proteins (Nops), affect symbiosis positively or negatively, depending on the host plant [23; 30; 31]. The expression of the rhizobial T3 SS is controlled by flavonoids that are derived from the host legume. Flavonoids perceived by the NodD protein activate the expression of _ttsI_, a transcriptional activator of the type III secretion gene cluster (_ttsI_). TtsI subsequently activates the _ttsI_ gene cluster, and proteins are secreted via the T3 SS. A conserved motif, called the _tts_ box, is located in the promoter region of genes encoding structural components and secreted effector proteins [23; 32; 33]. In the present study, we explored additional genetic determinants of _B. elkanii_, which are involved in the nodulation incompatibility between _V. radiata_ KPS1 and _B. elkanii_ USDA61. We performed Tn5 transposon mutagenesis and identified five Tn5 insertion mutants of _B. elkanii_ USDA61, which were able to form functional nodules on _V. radiata_ KPS1 and enhance the growth of the plants. The BE5 mutant contains a Tn5 insertion in the _innA_ gene, which encodes cytosine deaminase. Cytosine deaminase deaminase cytosine to form uracil and ammonia [34]. Although the mechanism for the involvement of cytosine deaminase in nodulation restriction remains unclear, the BE5 mutant efficiently nodulated and promoted the growth of both KPS1 and _Rj4_ plants. The disruption of _innA_ might cause the inactivation of the T3 SS, which abolishes the secretion of effector proteins that are responsible for nodulation restriction in both _Rj4_ soybean and _V. radiata_ KPS1. The BE53 mutant carries transposon in the _innB_ gene, which encodes a hypothetical protein with an unknown function. The _innB_ gene is preceded by the consensus _tts_ box, and the _innB_ homologue in _M. loti_, _mlr6327_, occurs in the cluster of genes encoding the T3 SS [28]. Intriguingly, the _innB_-deficient mutant formed nodules on _V. radiata_ KPS1 plants, but not on _Rj4_ soybean. This suggests that _innB_ encodes a protein with a host-specific function in KPS1. One possibility is that _innB_ encodes an effector protein that is recognized specifically in _V. radiata_ KPS1. It is likely that the rhizobial effector proteins that are recognized by KPS1 and _Rj4_ soybean differ. The InnB might be recognized specifically by a resistance (R) protein in KPS1, causing nodulation restriction in KPS1, but not in _Rj4_ soybean. The BE85 mutant contains a Tn5 insertion in _innC_, encoding TerB family tellurite resistance protein that is conserved among the _Bradyrhizobiaceae_.