Type III Secretion System of Bradyrhizobium sp. SUTN9-2 Obstructs Symbiosis with Lotus spp.

The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

Rhizobia induce the growth of symbiotic nitrogen-fixing organs, called nodules, on the roots of leguminous plants. Rhizobial nodulation factors (NFs) are key molecules for symbiosis (Lerouge et al., 1990). NFs are lipochitooligosaccharides with a chitin oligomer backbone, the length and modifications of which are specific to rhizobial species (Ardourel et al., 1994;Haeze and Holsters, 2002). After recognizing a compatible NF, the host legume activates nodulation signaling (Radutoiu et al., 2003;Radutoiu et al., 2007).
In addition to NFs, the rhizobial type III secretion system (T3SS) is an important factor for initiating symbiosis. Bacterial T3SS proteins, known as "nano syringes" or "injectisomes", deliver effector proteins (type III effector proteins, T3Es) into target cells (Ryan and Stebbins, 2016). The T3SS of plant pathogenic bacteria, such as Pseudomonas syringae, suppress plant immunity and contribute to the virulence of the pathogen (Jakobek et al., 1993). On the other hand, rhizobial T3SS may either promote or inhibit the establishment of symbiosis, depending on the host legume (Miwa and Okazaki, 2017). Rhizobial T3SS facilitate nodulation to promote symbiosis (Okazaki et al., 2013), whereas T3SS trigger plant immune responses that suppress nodulation to inhibit symbiosis (Sugawara et al., 2018;Kusakabe et al., 2020).
Bradyrhizobium sp. SUTN9-2 was originally isolated from Aeschynomene americana nodules (Noisangiam et al., 2012). SUTN9-2 has a wide host range and establishes sym-biosis with legume species in several genera (Noisangiam et al., 2012;Hashimoto et al., 2019). To investigate the role of the T3SS of SUTN9-2 in symbiosis, a T3SS inactivation (ΔT3SS) mutant of SUTN9-2, which cannot deliver T3Es, was constructed by disrupting the rhcJ gene, which encodes a T3SS component (Piromyou et al., 2015). Inactivation of the T3SS did not affect symbiosis of SUTN9-2 with the original host A. americana, which belongs to the Dalbergioids legume clade (Piromyou et al., 2015). However, in symbiosis with Vigna radiata and Macroptilium atropurpureum (these plants belong to the Phaseolids legume clade), SUTN9-2ΔT3SS mutants induced a greater number of pink nodules and more effectively promoted plant growth than wild-type SUTN9-2 (Piromyou et al., 2015). Thus, the T3SS of SUTN9-2 has a negative effect on symbiosis with V. radiata and M. atropurpureum. However, it currently remains unclear whether the T3SS of SUTN9-2 affects symbiosis with other host plants. In the present study, we focused on the symbiotic phenotypes of SUTN9-2 and its ΔT3SS mutant with a model legume of Lotus japonicus ecotypes B-129 Gifu and MG-20 Miyakojima as well as Lotus burttii B-303, which all belong to the Galegoids clade. We found that the T3SS of SUTN9-2 obstructed symbiosis with these three Lotus spp.
The seeds of Lotus spp. were surface-sterilized in concentrated sulfuric acid for 10 min followed by 0.2% sodium hypochlorite and 0.1% Tween 20 for 40 min, and then washed with sterilized water. After surface sterilization, the seeds were transferred onto 0.8% agar plates and germinated Article ME20041 at 28°C. Two-day-old seedlings were transferred to the top of a test tube containing vermiculite with buffered nodulation medium (Ehrhardt et al., 1992) and grown at 28°C with a 12/12-h light/dark cycle. After 1 week, each seedling was inoculated with 1 mL of a rhizobial suspension adjusted to an OD 600 =1.0 with sterilized distilled water. Plant fresh weights, nodule numbers, and acetylene reduction activity (ARA; a marker of nitrogenase activity) were measured at 5 or 8 weeks post-inoculation (wpi) according to Hashimoto et al. (2019).
When grown with L. japonicus Miyakojima, Bradyrhizobium sp. SUTN9-2 induced only white nodules with no ARA (Fig. 1B and E), and host plant growth was not promoted ( Fig. 1A and C). However, SUTN9-2ΔT3SS induced pink nodules with ARA ( Fig. 1B and E) and promoted host plant growth ( Fig. 1A and C). The number of white nodules induced by SUTN9-2ΔT3SS was significantly lower than that induced by SUTN9-2 (Fig. 1D).
A previous study reported that the T3SS of Bradyrhizobium sp. SUTN9-2 negatively affected symbiosis with V. radiata and M. atropurpureum, but not symbiosis with the original host A. americana (Piromyou et al., 2015). In the present study, we also found that the T3SS of SUTN9-2 obstructed symbiosis with Lotus spp. The inoculation with wild-type SUTN9-2 induced only white nodules, whereas that with SUTN9-2ΔT3SS induced pink nodules and promoted the growth of L. japonicus Miyakojima. In symbiosis with L. japonicus Gifu and L. burttii, SUTN9-2 induced pink nodules; however, the number of nodules and  degree of plant growth promotion were lower than those with SUTN9-2ΔT3SS. These results suggest that the T3E(s) of SUTN9-2 interfere with symbiosis with Lotus spp. Kusakabe et al. (2020) examined symbiosis between Bradyrhizobium elkanii USDA61 and Lotus spp. A phylogenetic analysis among Bradyrhizobium strains showed that SUTN9-2 belonged to the same clade as B. yuanmingense isolated from Lespedeza cuneata (Zhu et al., 2002) and B. liaoningense isolated from soybean (Xu et al., 1995), but to a different clade than B. elkanii strains (Fig. S1). However, the present results were consistent with the findings reported by Kusakabe et al. (2020) (Fig. 4), suggesting that the T3SS of Bradyrhizobium obstructed symbiosis with Lotus spp.. NopM (Nodulation outer protein M) of USDA61 is a T3E that suppresses pink nodule formation on L. japonicus Miyakojima (Kusakabe et al., 2020) (Fig. 4A). M. loti, a symbiont of Lotus spp., does not possess the nopM gene on its genome (Kusakabe et al., 2020). Based on comparisons with genome sequence data available in the MicroScope database (https://mage.genoscope.cns.fr/microscope/home/ index.php) (Vallenet et al., 2020), SUTN9-2 possesses a putative nopM gene (the accession number in MicroScope is shown in Table. S1 as SUTN92_v1_640013), the product of which shows approximately 75% amino acid sequence identity with NopM (accession number in DDBJ, LC471585) of USDA61. The putative NopM of SUTN9-2 contained the same leucine-rich repeat (LRR) and ubiquitin ligase domain as NopM of USDA61 (Fig. S2). The nopM gene of SUTN9-2 may inhibit the formation of pink nodules on this plant; however, the T3E(s) of SUTN9-2 responsible have yet to be identified.
The nopM of USDA61 also suppressed the formation of pink nodules on L. burttii (Kusakabe et al., 2020) (Fig. 4C). SUTN9-2 induced pink nodules on L. burttii despite the presence of a putative nopM ( Fig. 3B and D), similar to the nopM disruption mutant of USDA61 on L. burttii (Kusakabe et al., 2020) (Fig. 4C). Interestingly, the putative NopM protein of SUTN9-2 had a smaller number of LRR than that of the NopM protein in USDA61 (Fig. S2). LRR in proteins are generally involved in interactions with other molecules. These results suggest that the NopM proteins of these two strains either have different affinities for their targets or have different targets. The difference in the LRRnumber of NopM may be related to the different phenotypes in their symbiosis with L. burttii.
A recent study by Kusakabe et al. (2020) suggested that not only NopM, but also other T3E(s) of USDA61 interfered with symbiosis with L. burttii (Fig. 4C). However, the T3E(s) responsible have yet to be identified. Similar to USDA61, the inactivation of T3SS in SUTN9-2 showed a better symbiotic phenotype than that of the wild-type strain (Fig. 4C). This result suggests that T3E(s) common to SUTN9-2 and USDA61, but not to M. loti, interfere with symbiosis with L. burttii.
SUTN9-2 induced pink nodules on L. japonicus Gifu, in contrast to USDA61 (Fig. 2B, D, and 4B). The NopF pro- tein (accession number in DDBJ, LC471586) of USDA61 has been identified as a T3E that inhibits rhizobial infection and nodulation on L. japonicus Gifu (Kusakabe et al., 2020). Based on comparisons with genome sequence data available in the MicroScope database (Vallenet et al., 2020), SUTN9-2 does not possess a gene encoding NopF. The absence of NopF in SUTN9-2 may explain why SUTN9-2 exhibited a better nodulation ability than USDA61 on L. japonicus Gifu (Fig. 4B). However, the ability of the USDA61 nopF disruption mutant (ΔnopF in Fig. 4) to induce the formation of pink nodules was lower than that of wild-type SUTN9-2 (Fig. 4B). This result suggests that, in addition to NopF, USDA61 may possess specific T3E(s) that interfere with symbiosis with L. japonicus Gifu. The ΔT3SS mutants derived from both SUTN9-2 and USDA61 more effectively promoted the growth of Lotus spp. than their respective wild-type strains, but not as well as the original microsymbiont M. loti (Fig. 1, 2, and 3; Kusakabe et al., 2020). This result suggests that not only T3SS, but also unknown rhizobial factor(s) of SUTN9-2 and USDA61 obstruct symbiosis with Lotus spp. In addition, the functions and target molecules of these T3Es in Lotus spp. cells remain unknown. Comparisons of the sequences of these putative T3Es among SUTN9-2, USDA61, and M. loti may provide a more detailed understanding of the functions of T3E proteins in Lotus spp. cells. Further functional experiments will reveal the functions of T3E proteins in Lotus spp. cells. Lotus spp. used in the present study are useful lines for further investigations to identify the target of T3E in host plant cells. Thus, the present results will contribute to clarifying the mechanisms by which rhizobial T3Es inhibit Bradyrhizobium-Lotus symbiosis.