National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

Assistant Professor:
KAWADE, Kensuke
Specially Appointed Assistant Professor:
OKUMA, Nao
Technical Staff:
FUKADA-TANAKA, Sachiko
Postdoctoral Fellow:
TSUGANE, Mika
YANO, Koji
HASHIMOTO, KayoJSPS Postdoctoral Fellow: FUKUDOME, Mitsutaka
Visiting Scientist:
NAKAGAWA, Tomomi
SOKENDAI Graduate Student: GOTO, Takashi
Visiting Graduate Student: NAKAGAWA, Soya
Technical Assistant:
TSUKAO, Chihaya
ODA, Akiko
Admin Support Staff:KOSUGI, Eikorhizobia with their photosynthetic products. To accomplish this biotic interaction, leguminous plants develop
nodules on their roots. However, more than 80% of land
plant families have symbiotic relationships with arbuscular mycorrhizal (AM) fungi. Despite marked differences
between the fungal and bacterial symbioses, common
genes are required for both interactions. Using a model
legume, Lotus japonicus, we are trying to elucidate themolecular mechanisms of both symbiotic systems.

I. Root nodule symbiosis

Legumes develop de novo organs known as root nodules
to accommodate symbiotic bacteria called rhizobia.
Nodule formation involves two distinct processes: rhizobial infection in epidermis and nodule development
accompanied by cell division in cortex (Figure 1).

Figure 1. Processes for nodule formation.

Rhizobium–legume symbiosis is one of the most successful mutually beneficial interactions on Earth. In thistype of symbiosis, soil bacteria called rhizobia supplies
the host legumes with ammonia produced through bacterial nitrogen fixation. In return, host plants provide theVisual overview of this lab’s work.

50The cortical cell division provides an indispensable
scaffold for rhizobia progression from epidermis to cortex(Figure 1; pink line), leading to successful nodule formation. Cortical cell division occurs just below the site of
rhizobial infection in epidermis. Therefore, there appears
to be a spatiotemporal coordination across epidermis
and cortex in this symbiotic organogenesis. Epidermal
expression of genes required for early symbiotic genes
is sufficient for nodule development in cortex, suggesting that some kinds of signals generated in epidermis
trigger cortical cell division (Figure 1; blue dotted arrow).
However, little is known about the mechanism that coordinates these two events.
In this study, we conducted a time-course transcriptome
analysis using a L. japonicus non-nodulation mutant“daphne”, which has uncoupled symbiotic events inepidermis and cortex, in that it promotes excessive rhizobial infection in epidermis but does not produce nodule
primordia in cortex. Among genes that showed different
expression patterns in daphne and wild type, we foundFigure 2. Auxin methylation in nodule symbiosis. IAMT1 converts IAA
into MeIAA (left). Relative MeIAA amount of wild type (WT) anddaphne at 0 (non-inoculation) or 2 d after inoculation (DAI) (right).

IAA CARBOXYL METHYLTRANSFERASE 1 (IAMT1),
which encodes the enzyme that specifically converts
auxin (indole-3-acetic acid; IAA) into its methyl ester(MeIAA). A significant MeIAA increase after rhizobial
infection was detected by using daphne roots, in which
excess rhizobia are infected (Figure 2).
In Arabidopsis, IAMT1 reportedly serves development
and differential growth of shoot. On the other hand, wefound that IAMT1 is duplicated in the legume lineage,and one of the duplicates (named IAMT1a) was expressedby rhizobial infection in root of L. japonicus. LjIAMT1aknockdown inhibited nodule formation (Figure 3).
LjIAMT1a overexpression promoted MeIAA accumulation and nodule formation. In contrast, overexpressionof LjMES17, a MeIAA esterase, decreased MeIAA levels
and nodule number. These showed that auxin methylation
is essential for nodule formation.

II. Arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is the oldest
beneficial mutualistic relationship between the majorityof terrestrial plants and AM fungi. AM fungal spores
germinate and elongate their hyphae toward the rootof host plants then enter the root cells and form finely
branched structures, arbuscules, for exchanging nutrientswith plants (Figure 5). AM fungi deliver mineral nutrients especially phosphorus in the soil to host plants. In
return, they obtain organic carbon sources such as sugars
which are produced through photosynthesis. AM fungiare obligate symbionts depending on their host plants foressential nutrients, thus they are incapable of propagationby themselves.

Figure 3. LjIAMT1a-knockdown
inhibited nodule formation. Hairy
roots harboring empty vector (forcontrol; left) or LjIAMT1a-RNAivector (for knock-down; right).
Nodules are indicated by arrowheads. (Scale bars, 1 cm.)

Notably, application of MeIAA significantly induced
expression of a symbiotic gene, NIN, without rhizobia(Figure 4). NIN is a key regulator for cortical cell
division; therefore, auxin methylation could act on theprocess of cortical cell division. Whereas IAMT1a wasexpressed mainly in root epidermis, it would be interesting to see how MeIAA affects cortex. The result alsosuggests that auxin methylation is not simply due toalteration of auxin homeostasis, contrary to what manystudies assumed, MeIAA, as well as other auxin secondary metabolites, is also an inactive molecule of IAA.
Understanding the function of auxin methylation andMeIAA in nodule development should open a new avenuefor auxin metabolisms in the plant biology.

Figure 4. Exogenous MeIAA significantly induced NIN expression
without rhizobia. Relative expression levels of NIN after treatment
with DMSO as mock (white bar),IAA (gray bar), or MeIAA (bluebar) for 24 h.

Figure 5. AM fungus R. clarus HR1 growing with carrot hairy roots.
(A) Expanded extraradical hyphae and formed symbiotically generated
spores between the roots. Bar, 500 µm. (B) Fungal structures stained bygreen fluorescent (Wheat germ agglutinin Oregon green 488) in the root.
Arrowheads, arbuscules inside root cortical cells. Bar, 100 µm.

In the history of AM fungal culture research, it wasshown that an AM fungus Rhizophagus irregularis could
complete its life cycle in co-culture with mycorrhizahelper bacterium Paenibacillus validus in the absence of
host plants. Over the last several years, it was reported
that AM fungi receive not only sugars but also fatty acids.
Subsequently, from these experiments, it was demonstrated that some fatty acids such as palmitoleate andmyristate induce R. irregularis spore formation under an
asymbiotic (completely host-free) culture media.
We found that a fungus isolated from Aichi prefecture, Rhizophagus clarus HR1, whose genome has beensequenced, shows better growth on the asymbiotic culture
medium, thus is more suitable for this culture method. Afatty acid, myristate promotes R. clarus hyphal growth,
but the addition of only myristate could not ensure induction of asymbiotically-generated spores (AS) formation.
Furthermore, the number of AS in this culture was muchsmaller than those of symbiotically-generated spores (SS)which are produced in the co-culture system with hairy roots.
Some phytohormones have been reported that they
have important roles as signals during AM symbiosis.
Especially, strigolactone is well known as a major signal
which induces hyphal elongation and branching and stimulates their mitochondrial activity in the pre-symbiotic
stage. By supplementing synthetic strigolactone, GR24with myristic acid, the time of germination and AS formation in R. clarus was accelerated. Although not all R.
clarus spores formed AS in the medium supplemented51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

only with myristic acid, the addition of GR24 resulted ina quite high efficiency of AS formation to nearly 100 %.
Besides strigolactone, it was reported that there was anincrease of jasmonate levels in AM roots and the upregulation of genes involved in jasmonic acid biosynthesisin the cells containing arbuscules. Application of jasmonates in the medium with myristic acid and GR24 led R.
clarus to produce a higher number of AS with larger sizes(Figure 6).

Publication List:〔Original papers〕

T., Soyano, T., Liu, M., Mori, T., and Kawaguchi, M. (2022).
• Goto,Auxin methylation by IAMT1, duplicated in the legume lineage,••••

Figure 6. Produced spores on asymbiotic culture medium, supplemented
with only myristate (A), myristate and GR24 (B), myristate, GR24 andmethyl jasmonate (C). Arrowheads, parent spores. Bars, 500 µm.

Although the number of AS produced in the medium
with myristate, GR24 and jasmonate has been close
to that of SS, the size of AS is still smaller than SS.
However, AS can be subcultured with the same efficiency
as the first asymbiotic culture. Moreover, AS was capableof infecting hairy roots and plants. The growth of Welsh
onions was significantly promoted by inoculation withAS as well as with SS (Figure 7). Our findings would lead
us to more extensive studies of AM symbiosis and to theapplication of AS as an inoculum for basic research andfor sustainable agriculture.

••promotes root nodule development in Lotus japonicus. Proc. Natl.
Acad. Sci. U.S.A. 119, e2116549119. DOI: 10.1073/pnas.2116549119
Hayashi-Tsugane, M., and Kawaguchi, M. (2022). Lotus japonicus
HAR1 regulates root morphology locally and systemically under amoderate nitrate condition in the absence of rhizobia. Planta 255, 95.
DOI: 10.1007/s00425-022-03873-8
Misawa, F., Ito, M., Nosaki, S., Nishida, H., Watanabe, M., Suzuki,T., Miura, K., Kawaguchi, M., and Suzaki, T. (2022). Nitrate transport
via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression ofroot nodulation in Lotus japonicus. Plant Cell 34, 1844–1862. DOI:
10.1093/plcell/koac046
Nishida, H., Nosaki, S., Suzuki, T., Ito, M., Miyakawa, T., Nomoto,M., Tada, Y., Miura, K., Tanokura, M., Kawaguchi, M., and Suzaki,T. (2021). Different DNA-binding specificities of NLP and NIN
transcription factors underlie nitrate-induced control of root nodulation.
Plant Cell 33, 2340–2359. DOI: 10.1093/plcell/koab103
Tanaka, S., Hashimoto, K., Kobayashi, Y., Yano, K., Maeda, T.,Kameoka, H., Ezawa, T., Saito, K., Akiyama, K., and Kawaguchi, M.
(2022). Asymbiotic mass production of the arbuscular mycorrhizal
fungus Rhizophagus clarus. Commun. Biol. 5, 43. DOI: 10.1038/s42003-021-02967-5
Fukudome, M., Shimokawa, Y., Hashimoto, S., Maesako, Y., UchiFukudome, N., Niihara, K., Osuki, K.I, and Uchiumi, T. (2021).
Nitric oxide detoxification by Mesorhizobium loti affects root nodule
symbiosis with Lotus japonicus. Microbes Environ. 36, ME21038.
DOI: 10.1264/jsme2.ME21038
Kobayashi, Y., Katsuren, M., Hojo, M., Wada, S., Terashima,y., Kawaguchi, M., Tokuda, G., Kinjo, K., and Shigenobu, S. (2022).
Taxonomic revision of Termitomyces species found in Ryukyu
Archipelago, Japan, based on phylogenetic analyses with three loci.
Mycoscience 63, 33-38. DOI: 10.47371/mycosci.2021.11.001〔Review articles〕

N., and Kawaguchi, M. (2021). Systemic optimization of• Okuma,legume nodulation: A shoot-derived regulator, miR2111. Front. Plant
Sci. 12, 682486. DOI: 10.3389/fpls.2021.682486

T., Liu, M., Kawaguchi, M., and Hayashi, M. (2021).
• Soyano,
Leguminous nodule symbiosis involves recruitment of factors

contributing to lateral root development. Curr. Opin. Plant Biol. 59,
102000. DOI: 10.1016/j.pbi.2020.102000

Figure 7. Inoculation of AM fungal spores to Welsh onions. Numbers
following AS or SS are the number of spores for inoculation. (A) An
appearance of plants inoculated with AM spores (upper). Bar, 5 cm.
Ink-stained fungal structures in the roots (bottom). Bars, 50 µm. (B) Dry
weight of Welsh onion shoots. Different letters indicate significant differences among treatments in each trial using Wilcoxon rank-sum test withBonferroni correction, p < 0.05.

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