要約のみTohoku University佐藤修正課

Arashida Haruka

Tohoku University博士（生命科学）博士学位論文 (Thesis(doctor))要約のみthesi

Arashida, Haruka

Tohoku University Repository (TOUR) / 東北大学機関リポジトリ

Elucidation of cause and natural feature ofcheating rhizobia, and host defense mechanismagainst them著者 Arashida Haruka学位授与機関 Tohoku University学位授与番号 11301URL http://hdl.handle.net/10097/00137188            博⼠論⽂ (要約) Elucidation of cause and natural feature of cheating rhizobia,  and host defense mechanism against them.  (Cheating 根粒菌の生成機構と生態、及びそれに対する宿主マメ科植物の防御機構の解明)         令和 4 年度 東北大学大学院生命科学研究科 生態発生適応科学専攻 共生ゲノミクス分野 嵐田 遥    【Introduction】         Rhizobia can establish mutualistic relationship with legumes resulting in symbiotic N2-fixation (SNF). In contrast, commensal relationship could be established by “cheating rhizobia”, which possess nodulation ability, but lack N2-fixing ability. Cheating rhizobia can exploit carbon source from the host plants, and thus their widespread persistence can destabilize the mutualism. However, the reality of cheating rhizobia including of the generative mechanism or symbiotic performance in nature has been unknown, since their isolation is difficult due to the presence of the host sanction system (Fig. 1) (Kiers et al., 2003). The generation of cheating rhizobia would be mainly caused by defect in symbiosis islands (or symbiotic plasmids), which is the distinct packages of symbiotic related genes within rhizobial genome (Batut et al., 2004; Ormeño-Orrillo & Martínez-Romero, 2019; Poole et al., 2018). On the symbiosis islands, many of insertion sequences (ISs) were inserted, which could read the structural change in symbiosis islands (Kaneko et al., 2011). As a first step to approaching the origin of cheating rhizobia, I demonstrated the structural variation within the symbiosis islands of Bradyrhizobium strains depending on ISs at laboratory scales (Results and discussions 1). Moreover, our group isolated pink4 mutant (pink4) of Lotus japonicus with a defect in host sanction system. Taking the advantage of pink4, natural cheaters (Mesorhizobium sp. S24, W4, S35, and Aminobacter sp. S33) were isolated from soil microbiota in Kashimadai field (Miyagi, Osaki city). Following that, I performed the phenotypic and genomic analyses of the natural cheaters to reveal characteristics of cheating rhizobia in nature (Results and discussions 2). In parallel, pink4 mutant defective in the host sanction system also allowed me the opportunity to approach the molecular mechanism of the host sanction system, which has also remained an open question. Therefore, I analyzed the host reaction mediated by PINK4 against cheating rhizobia to understand the host sanction mechanism (Results and discussions 3). Following that, I tried to identify the causal gene of pink4 mutant, and discussed the transcriptional regulation and phylogenetical features of the isolated LjPINK4 (Results and discussions 4). Through these comprehensive researches, I tried to provide a new concept about mutualistic and parasitic lifestyles of host leguminous plants and rhizobia.  【Results and discussions】 1) Insertion sequence-mediated deletion and duplication on rhizobial symbiosis islands Effector-triggered immunity of soybean carrying the Rj2 allele is triggered by NopP (a type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (symA) in Bradyrhizobium diazoefficiens USDA122 (Sugawara et al., 2018b). However, this immunity was occasionally overcome by natural mutation, resulting in the formation of sporadic nodules. The rhizobial isolates showed deletions of T3SS (rhc) and N2 fixation (nif) genes on symA, by homologous recombination between ISs. To demonstrate the structural variations on symA during free-living growth, I cultured the USDA122 strain with a marker gene sacB inserted into the rhc gene cluster. As a result, most of the sucrose resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants described above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that Fig. 1 Process of endosymbiosis with rhizobia, and host sanction system against cheating rhizobia.  Infected cellMature nodule(Red)Immature nodule(White)Infection threadWT rhizobia(N2-fixation +)  Cheater (N2-fixation -)  Host sanction systemIS-mediated deletions occurred during free-living growth and the host plants selected the mutants. I also found not only IS-mediated deletions but also IS-mediated duplications during the growth in free-living stage. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resulting pools of rhizobial populations. These results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating structural rearrangements in the symbiosis islands.   2) Phenotypic and genomic variation of cheating rhizobia in nature   I found that IS-mediated deletions containing nif gene cluster were remarkably detected during the growth in free-living stage, which results in the creation of cheating rhizobia. However, it was under the laboratory condition. In the situation, I got interested in the reality of cheating rhizobia in natural environments. Then, I altered my research target to the cheating rhizobia isolated from natural field using pink4 mutant. To investigate phenotypic variation of these field isolated cheating rhizobia, I evaluated the nodulation and N2-fixing abilities on wild type, L. japonicus MG-20 (MG-20). S24 and W4 held no N2-fixing ability, and formed white nodules. S35 and S33 held low level N2-fixing ability in the order of S35 to S33, indicating the presence of variation in symbiotic phenotypes among natural cheaters. Genome sequencing of these field isolated cheating rhizobia revealed that the causes of the defect in SNF were not IS-mediated deletions as is the case of Bradyrhizodium strains, but point mutations in coding or possibly promoter regions related with regulation of SNF. In addition, the sequences of the symbiosis island were conserved between S24/W4, and between S35/S33, indicating that the horizontal transfers of the symbiosis island in Kashimadai field including inter genus level. Based on the comparison between S24/W4 and S35/S33, the symbiosis islands of these rhizobia were composed of highly conserved small regions of essential symbiotic genes (nod, nif, and fix) separated by non-conserved regions with totally different sequences. On these non-conserved regions, numerous ISs were accumulated. These results suggest that IS-mediated rearrangements would frequently occur in Mesorhizobium, resulting in concentration of essential symbiosis genes. These genomic features of Mesorhizobium strains possibly contributed to the features of mutations on the symbiotic genes in the natural cheater strains, not by IS-mediated duplications but by point mutations.  3) Host sanction system against cheating rhizobia  By comparing the symbiotic phenotypes between pink4 and MG-20 inoculated with these natural cheaters and artificial cheating rhizobium ΔnifH, it was confirmed that PINK4 provided multiple levels of sanctions depending on the Fig. 2 Section of nodule cells in L. japonicus MG-20 and pink4 under inoculations with ΔnifH, S35 and wild-type rhizobia, M. japonicum MAFF303099 visualized by transmission electron microscopy. Scale bars = 0.5 μm.  ΔnifHN2-fixation : -S35N2-fixation : lowMAFF303099 N2-fixation : +Rhizobia Host plantsMG-20 Sanction : +pink4 Sanction : -nitrogen-fixing ability of cheaters. Cytological analysis with an electron microscope demonstrated that ΔnifH strains in the MG-20 nodule were lysed by PINK4 function (Fig. 2), and the lytic reaction could be divided into two steps, 1) multiple cheating rhizobia were incorporated by large vesicles, and 2) large vesicles were digested by fusion with the vacuole. These steps resemble endocytosis pathway for lytic digestion. The similar sanction was observed under inoculation with S35, but the large vesicles did not completely fuse with the vacuole, and thus the lytic reaction was milder than against ΔnifH. The transcriptomic analysis revealed that expression of the genes encoding membrane trafficking / tethering with vacuole (e.g., VPS protein SKD1, SNARE protein VTI13), were significantly upregulated depending on PINK4 under inoculation with ΔnifH, while the upregulation levels of these genes were lower in inoculation with S35, supporting the results of the cytological observations. Furthermore, genes related to oxidation-reduction process (e.g., cytochrome p450 family protein, peroxidases) were also significantly upregulated depending on PINK4, especially in inoculation with ΔnifH, suggesting that respiratory burst like reaction could be occurred in the process of digestion, as is the case of phagocytosis. These results suggested that L. japonicus would provide the sanction mediated by PINK4 against cheaters by vesicle transport and vacuolar degradation in the similar way with phagocytosis, and the sanction level could be controlled by the process of fusion with vacuole. 4) Identification of LjPINK4, the key factor of host sanction system against cheating rhizobia By using 23 segregants with pink4 phenotype from the F2 population of cross between wild-type Gifu and pink4, bulked resequencing analysis was carried out to identify the causal gene of pink4. By searching the SNP site with homozygous alternative in all segregants, a single non-synonymous SNP with C to T base change on the coding region of a gene on chr4 was identified as a candidate. Taking advantage of the availability of a large-scale transposon insertion library with insertion site information, three independent lines with insertion on the candidate gene could be selected. As all of three tag-lines formed pink nodules against inoculation with ΔnifH, the candidate gene was confirmed as LjPINK4 gene. LjPINK4 encodes a protein with 642 amino acids length, which is annotated as “unknown protein” with no known motif sequence. The phylogenetic analyses revealed that orthologs of PINK4 were conserved not only in legumes but also in angiosperms. Based on the transcriptome data on the nodules inoculated with cheaters, highest level of expression of LjPINK4 was observed in ΔnifH inoculation, followed by S35 inoculation. In addition, high level expression of LjPINK4 was observed in uninoculated root, suggesting that PINK4 system functioned in general defense system, and L. japonicus applied it to the sanction system against infection of the cheating rhizobia.  【General discussion】        In this study, I explored comprehensive researches on legumes and rhizobia to understand their parasitic and mutualistic lifestyles, and provided new concepts about their realistic behaviors in ecosystems.  Regarding the parasitic behavior of rhizobia, I elucidated that nitrogen-fixation genes on the symbiosis island of Bradyrhizobium strains were deleted via IS elements during free-living growth, which could be the first example to demonstrate the possible generative mechanism of cheating rhizobia under the laboratory condition. In the phenotypic analysis of L. japonicus under the inoculation with cheating rhizobia isolated from natural environments, these strains demonstrated variety in nodule phenotypes and nitrogen fixation abilities with L. japonicus MG-20, and these nodulations were suppressed by the function of PINK4 depending on the nitrogen ability of the rhizobim. These results suggest that L. japonicus possess PINK4 meidated sanction system as the countermeasure against cheating rhizobia that occasionally occurred and showed various symbiotic performances.  In terms of molecular mechanism of the host sanction system, cytological and transcriptomic dataset suggested that L. japonicus activated the sanction system by vesicle transport and vacuolar degradation in the similar way with phagocytosis, and the sanction level was controlled in the process of the fusion with vacuole, depending on the nitrogen-fixing ability in the symbiosome. Regarding phagocytosis in plants, it has been reported that plants conserve the components that are involved in the principal process of phagocytosis (Yutin et al., 2009), and phagocytosis between protoplasts was observed in the 1970s (Ueda et al., 1978), suggesting that plants possess the basal machinery for phagocytosis. In the process of endosymbiotic interaction between legumes and rhizobia, significant overlaps, especially in the vesicle trafficking system, with phagocytosis have been reported (Peleg-Grossman et al., 2007), implying that the process of endosymbiosis of rhizobia itself could be considered as originating from the process of phagocytosis. Considering that LjPINK4 was conserved among angiosperms and constantly expressed in uninoculated roots, PINK4 would be generally associated with phagocytosis, and symbiosomes would originally bound to enter into the lysis process in the presence of PINK4. Under the inoculation with cheating rhizobia, pH level in the symbiosome should become lower along with acidification by proton-pumping V-ATPase, which would be a signal to proceed to the lysis process triggered by PINK4 function, as is the case of phagosome maturation (Levin et al., 2016, Uribe-Querol and Rosales 2020). Under the inoculation with honest rhizobia, on the other hand, pH level in the symbiosome could be increased by the production of ammonia by nitrogen fixation, and the elevated pH level could be a signal to prevent to enter the lytic process. Based on the hypothesis, SNF in legumes would establish as a result of prevention of phagocytic lysis mediated by PINK4, presumably.  To demonstrate the function and universality of PINK4 among angiosperms, the analyses of the gene expression/localization in the plant-tissue or nodule cell and the functional complementation test by the PINK4 orthologs in other legumes or non-legumes would be worth carrying out, in the future.   

Cheating根粒菌の生成機構と生態、及びそれに対する宿主マメ科植物の防御機構の解明

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Cheating根粒菌の生成機構と生態、及びそれに対する宿主マメ科植物の防御機構の解明

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