# Identification of _Bradyrhizobium elkanii_ Genes Involved in Incompatibility with _Vigna radiata_
## 4 Discussion
It has been reported that the Tellurium resistance proteins of _Frankia_ spp. are induced by seed phenolic compounds that are produced by their actinorhizal host plants [35]. Considering that the disruption of _innC_ induced nodulation on both KPS1 and _Rj4_ plants, the gene might be involved in the T3 SS. However, the function of these proteins in rhizobial symbiosis remains unknown. The BE103 mutant carries a Tn5 insertion in the gene _innD_, which encodes a substrate-binding protein in the ABC transporter family. These substrate-binding proteins are located in the periplasm, and are involved in the active transport of substrates into the cytoplasm [36; 37; 38]. Although the function of the _innD_ protein remains unclear, its homologue in _B. diazoefficients_ USDA110 (_blr3743_, BAC49008.1)is secreted [28], and is upregulated in bacteroids in nodules [39; 40]. Clearly, functional analyses are needed to determine the contribution of these transporters to the compatibility/incompatibility between rhizobia and their hosts. Since the inactivation of the \(imD\) gene by the Tn5 insertion canceled the nodulation restriction in both KPS1 and _Rj4_ plants, the \(imD\) product might be involved in the T3 SS. Further molecular experiments will be performed to explore this possibility. The BE168 mutant contains a Tn5 insertion in \(imE\), which encodes a GTP pyrophosphokinase (ppGpp synthetase) that is conserved among various rhizobia and pathogenic bacteria. \(imE\) shares similarity with the _relA_ genes of _B. diazoefficens_ USDA110 (BAC50330.1) and other bacteria. It has been proposed that ppGpp synthetase binds to, and alters the ability of, RNA polymerase to initiate and elongate transcription. ppGpp synthetase activates genes that function in the biosynthesis of amino acids and transcription factors that are responsible for stress responses [41; 42]. In addition, ppGpp synthetase plays important roles in the virulence and the persistence of phytopathogens within host plants [43]. In _Rhizobium etli_, the _relA_ gene regulates gene expression in bacteroid cells [44]. A _relA_ mutant of _Sinorhizobium melioti_ shows a non-modulation phenotype on alfalfa [45]. These findings suggest that the mutation in \(imE\) affects cellular ppGpp synthetase levels and leads to changes in cell functions that affect the strain specific incompatibility between _B. elkanii_ USDA61 and mung bean and soybean. It should be noted that, except for the \(imB\) gene, which was confirmed by the newly constructed mutant BE53S, the other genes also need to be confirmed by mutational and/or complementation analysis to unequivocally associate the gene with the phenotype. In addition to the genes and gene products discussed above, rhizobial factors have been reported to be involved in incompatible symbioses, including the type IV secretion system [46], extracellular polysaccharides [47], and peptidase that cleaves host-derived signaling peptides [48]. T3 SSs in other rhizobia involved in incompatible symbioses have been reported for the following interactions: _Rhizobium_ sp. NGR234 with _Crotalaria junca_[49], _M. loti_ MAFF30309 with _Lotus halophilus_[28], _Sinorhizobium fredii_ USDA257 with _G. max_ cv. McCall [15], _B. elkanii_ USDA61 with _Rj4_ soybean [8], _Bradyrhizobium japonicum_ USDA122 with _Rj2_ soybean [50], and _B. diazoefficens_ USDA110 with _V. radiata_ KPS2 [51]. The type III-secreted proteins \(\mathrm{NopE1}\) and \(\mathrm{NopE2}\) of _B. diazoefficens_ USDA110 were reported as negative effectors of compatibility with _V. radiata_ KPS2 [51]. In our preliminary results, _B. diazoefficens_ USDA110 formed nodules on _V. radiata_ KPS1 and promoted plant growth efficiently (data not shown), indicating that \(\mathrm{NopE1}\) and \(\mathrm{NopE2}\) do not control incompatibility with KPS1, and that _B. elkanii_ might possess other effectors that are recognized by KPS1.