Elucidation of cause and natural feature of cheating rhizobia, and host defense mechanism against them

概要

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】
Mature nodule

Rhizobia can establish mutualistic relationship
Immature nodule

with legumes resulting in symbiotic N2-fixation (SNF). In
contrast, commensal relationship could be established by

Infection thread

(Red)
Infected cell

(White)

“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

WT rhizobia
(N2-fixation +)
Cheater
(N2-fixation -)

Host sanction
system

Fig. 1 Process of endosymbiosis with rhizobia, and host
sanction system against cheating rhizobia.

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

IS-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

Rhizobia

Mesorhizobium, resulting in concentration of

ΔnifH
N2-fixation : -

essential symbiosis genes. These genomic

S35
N2-fixation : low

MAFF303099
N2-fixation : +

features of Mesorhizobium strains possibly
symbiotic genes in the natural cheater strains, not
by IS-mediated duplications but by point
mutations.

3) Host sanction system against cheating
rhizobia

Host plants

contributed to the features of mutations on the

MG-20
Sanction : +

pink4
Sanction : -

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.

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. ...

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