# Identification of _Bradyrhizobium elkanii_ Genes Involved in Incompatibility with _Vigna radiata_
## 3 Results
### B. elkanii Genes Responsible for the Symbiotic Incompatibility  
To identify the genes that were disrupted by the Tn5 insertions, Y-linker PCR was performed using genomic DNA from the Tn5 mutants. All of the mutant genomes contain the Tn5 fragment, and the PCR fragment sizes differ among strains, indicating that they are independent mutations. The genome of _B. elkanii_ USDA61 has been completely sequenced [25], and the Tn5 flanking regions all contain sequences that are found in the genome (Table 2). The location map and genome context of the identified genes indicated that these genes are located outside T3 SS gene cluster (Figures S1 and S2). BE5 contains a Tn5 insertion in a 1212 bp open reading frame (ORF), designated as \(innA\), that encodes a cytosine deaminase, which is similar to those in rhizobia and other Gram-negative bacteria. BE85 contains a Tn5 insertion in a 504 bp ORF (designated as \(innC\)) that encodes a tellurite resistance protein (TerB). The gene products are similar with those in other rhizobia and pathogenic bacteria. BE103 has an insertion in a 1089 bp ORF (\(innD\)) that encodes an ABC-transporter substrate-binding protein, whose homologues were well conserved among rhizobia as well as species of _Afipia_, _Rhodopseudomonas_, _Azospirillum_, and _Agrobacteriumum_. One of its homologues in _B. diazoefficients_ USDA110, blr3743, has been reported as a secreted protein [28]. BE168 has an insertion in a 2292 bp ORF (\(innE\)) encoding GTP pyrophosphokinase (Table 2). The \(innE\) product has 88% and 60% identity with the products of \(relA\) genes in rhizobia and \(spT\) genes in other bacteria including _Rhodopseudomonas palustris_, _Xanthomonas campestris_, _Pseudomonas fluorescens_, and _Salmonella enterica_.  
BE53 mutant carries a Tn5 insertion in the 2280 bp ORF (\(innB\)), which encodes a hypothetical protein that is conserved among rhizobia as well as _Xanthomonas_ and _Rhodopseudomonas_. Notably, a \(tts\) box motif (5'-TATGGGACCTAGCTTTCGAAAAGCTAGCAG-3') is present at 96 bp upstream of the \(innB\) ORF. The \(tts\) box motifs are often located in the promoter regions of genes encoding structural components of the T3 SS and secreted proteins. Furthermore, a homologue of \(innB\) in _Mesorrizobium_\(loti\), mlr6327 (GenBank accession No. BAB52639.1), is located in the cluster of genes that are encoding the T3 SS [29].  
In order to confirm that the observed phenotypes of the mutants were caused by transposon insertion and not by a secondary mutation independent of the transposon, we reintroduced the mutation into the wild-type background. The \(innB\) gene was mutated via single-crossover recombination in wildtype USDA61 and the newly constructed mutant, designated BE53S, was used to inoculate _V. radiata_ cv. KPS1 plants, and its symbiotic phenotypes were examined. As a result, the BE53S formed numerous mature nodules similar to the transposon mutant (BE53) (Figure 2A-C), confirming that transposon insertion in BE53 was responsible for the altered nodulation phenotype.  
Figure 4: Symbiotic properties of _G. max_ cv. BARC2 (_Rj4 Rj4_) inoculated with _B. elkanii_ USDA61, Tn5 mutants and type III secretion system-deficient mutant BErhcJ. (**A**) Nodule number, (**B**) nodule weight and (**C**) plant weight measured at 30 days post inoculation. Open bars and closed bars in (**A**) show the numbers of small nodules (<2 mm) and large nodules (\(\geq\)2 mm), respectively. Nodulation tests were performed at least two times, the values are means of six soybean plants, and the error bars indicate standard deviations. Statistical analysis ([PERSON]’s method) was performed comparing the total nodule numbers induced by USDA61, BErhcJ and Tn5 mutants. Means followed by the same letters are not significantly different at 5% level of significance by [PERSON]’s test. N.D.: Not determined.