Bacterial inducible expression of plant cell wall-binding protein yeso through conflict between glycine max and saprophytic bacillus subtilis

ABSTRACT Saprophytic bacteria and plants compete for limited nutrient sources. _Bacillus subtilis_ grows well on steamed soybeans _Glycine max_ to produce the fermented food, _natto_. Here

we focus on bacterial responses in conflict between _B. subtilis_ and _G. max_. _B. subtilis_ cells maintained high growth rates specifically on non-germinating, dead soybean seeds. On the

other hand, viable soybean seeds with germinating capability attenuated the initial growth of _B. subtilis_. Thus, _B. subtilis_ cells may trigger saprophytic growth in response to the

physiological status of _G. max_. Scanning electron microscope observation indicated that _B. subtilis_ cells on steamed soybeans undergo morphological changes to form apertures,

demonstrating cell remodeling during saprophytic growth. Further, transcriptomic analysis of _B. subtilis_ revealed upregulation of the gene cluster, _yesOPQR_, in colonies growing on

steamed soybeans. Recombinant YesO protein, a putative, solute-binding protein for the ATP-binding cassette transporter system, exhibited an affinity for pectin-derived oligosaccharide from

plant cell wall. The crystal structure of YesO, in complex with the pectin oligosaccharide, was determined at 1.58 Å resolution. This study expands our knowledge of defensive and offensive

strategies in interspecies competition, which may be promising targets for crop protection and fermented food production. SIMILAR CONTENT BEING VIEWED BY OTHERS THE _BRADYRHIZOBIUM

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EFFICACY IN _BACILLUS VELEZENSIS_ 118 Article Open access 13 February 2025 INTRODUCTION Plant seeds, each consisting of an embryo, storage tissues, and protective outer coat, remain dormant,

waiting for favorable environmental conditions for germination. Stored nutrient sources, such as carbohydrates, lipids, and proteins, are available to the germinating embryo, and also to

seed microbiota, a community of bacteria and fungi surrounding the seeds1,2. Plants have evolved sophisticated mechanisms to prevent phytopathogen colonization and infection. However, once

ungerminated seeds undergo senescence, decreased antimicrobial activity of dead seeds and, thus, increased accessibility to nutrients may permit microbial growth using seed decomposition

products, i.e., saprophytic growth3,4,5. The molecular basis for recognition of seed physiological status is required to understand the competition between microorganisms and plants.

_Bacillus subtilis_ is a Gram-positive, spore-forming, saprophytic bacterium ubiquitously isolated from soil, water, air, decaying plant materials, and fermented foods6,7,8,9. During

production of _natto_, a traditional fermented soybean food in Japan, soybean seeds are soaked and steamed before inoculation with _B. subtilis_. This process, which kills seeds, is regarded

as essential for enhancing _B. subtilis_ growth. Therefore, _B. subtilis_ cells growing on soybean seeds may be a useful model to study cell responses and molecular recognition at

saprophytic growth onset. _B. subtilis_ is widely used in emerging research fields as a Gram-positive model organism10,11; however, its physiological responses and behaviors on soybean seeds

remain unclear. Saprophytic growth requires nutrient uptake by decomposer organisms from decaying plant material. Bacterial ATP-binding cassette (ABC)-family transporters translocate a

variety of substrates into cells, using energy from ATP hydrolysis12,13,14. A typical ABC transporter consists of three components: integral membrane proteins that form pores,

nucleotide-binding proteins (NBPs) that bind and hydrolyze ATP, and solute-binding proteins (SBPs) that recognize and deliver target compounds into cells. SBPs are critical elements for

substrate specificity and high affinity of ABC transporter systems15,16,17,18. For instance, a conformational change of SBP in pathogenic bacteria is critical for selective recognition and

import of mammalian host glycosaminoglycans19,20. Pathogenic and symbiotic microorganisms secrete numerous enzymes to penetrate and degrade plant cell walls to provide

nutrition21,22,23,24,25. Thus, cell wall decomposition products are potential targets for saprophytic bacteria, which may use them to monitor plant physiological status. Primary plant cell

walls consist of a cellulose-hemicellulose framework embedded in an inter-fibrillar matrix of pectins26,27. Complex and diverse pectic polysaccharides include homogalacturonan (HG),

rhamnogalacturonan type I (RG-I), and rhamnogalacturonan type II (RG-II). HG is a linear polymer of α-1,4-linked galacturonic acid. RG-I is composed of a backbone, based on

disaccharide-repeating units of rhamnose and galacturonic acid, with side chains of galactans, arabinans, and arabinogalactans. RG-II contains HG as a backbone, with complex side chains

composed of a variety of sugars. Although studies on HG degradation have progressed28,29,30, bacterial decomposition of RG-I and RG-II has been reported in a limited number of papers so

far31,32,33. This report is the first on the competition between saprophytic _B. subtilis_ and soybean _Glycine max_ on the surfaces of soybean seeds. This article further focuses on the

physiological and transcriptomic traits of _B. subtilis_, and provides structural insight into SBP-dependent recognition of RG-I-derived trisaccharide during the saprophytic growth. RESULTS

AND DISCUSSION GROWTH OF _B. SUBTILIS_ ON LIVE AND DEAD SOYBEANS _Natto_, a traditional Japanese fermented food, is manufactured from steamed soybean seeds7,9. Saprophytic _B. subtilis_

cells grow on dead soybeans, using decomposition products as nutrients. Steaming may cause not only loss of viability but also denaturation of proteins; it may alter other soybean components

as well. Therefore, what exactly triggers saprophytic growth is an open question. Heat-related impacts to soybean components were reduced by adopting physiological senescence, induced at

different temperatures, by prolonged seed storage. Six-month storage at 4 °C maintained viability, but six-month storage at an ambient temperature severely limited seed germination

(Supplementary Fig. S1A). Thus, seeds stored under the former and the latter conditions were used as live and dead soybeans, respectively. _Bacillus subtilis_ cells were found to grow

vigorously on non-germinating dead soybean seeds, but not on live seeds (Supplementary Video S1, Fig. 1A). Thus, seed germination and bacterial growth inhibition may be closely linked. We

first discovered this phenomenon using _B. subtilis natto_-producing strain NBRC 1644934. Quantitative analysis of colony-forming units (CFUs) indicated that _B. subtilis_ initially

proliferated on both live and dead soybeans. However, growth attenuated specifically on live soybeans when bacterial cell numbers reached 106 CFUs per seed (Fig. 1B). _B. subtilis_

laboratory standard strain 16835 exhibited a similar growth profile on live and dead seeds (Fig. 1C). Thus, live soybeans can severely limit _B. subtilis_ growth on their surfaces. Invading

bacteria and germinating plant embryos may compete for nutrients in soybean seeds. _B. subtilis_ cells gain access to seed nutrients when the seeds lose germination ability. _B. subtilis_

growth on soybean seeds is a promising model for bacterial-plant interactions in seed microbiota. MORPHOLOGICAL TRAITS OF _B. SUBTILIS_ DURING SAPROPHYTIC GROWTH To understand the strategy

of _B. subtilis_ for saprophytic growth on dead soybeans during _natto_ production, morphological and transcriptional responses were examined. Scanning electron microscope (SEM) analysis

revealed cell morphological changes of _B. subtilis_ NBRC 16449 during saprophytic growth on steamed seeds (Fig. 2A–C). The early logarithmic growth phase, 5 h after inoculation, was

characterized by elongated, rod-shaped cells, 6–10 μm long. As the growth progressed, most cells displayed short-rod forms, approximately 2 μm long, with distinctive aperture-like structures

(Fig. 2C). _B. subtilis_ NBRC 16449 (Supplementary Fig. S2) and 168 (Fig. 2D–F) strains exhibited apertures on soybean seed-mimicking solid medium (Supplementary Fig. S1B). No apertures

were observed in liquid medium (Fig. 2G–I), suggesting that a solid substrate is needed for this culture-specific response. Similar aperture-like structures are previously reported in

_Clostridium sporogenes_ during formation of endospores36. _B. subtilis_ cell membranes are subject to engulfment during spore development under nutrient-depleted conditions37,38, and

saprophytically growing _B. subtilis_ may undergo similar morphological remodeling. How solid-state culture stimulates the starvation-triggered sporulation pathway is of great interest from

a signal transduction viewpoint. TRANSCRIPTOMIC PROFILE OF _B. SUBTILIS_ DURING SAPROPHYTIC GROWTH Comprehensive understanding of saprophytism-specific recognition and responses was sought

using the transcriptomic profiles of _B. subtilis_ NBRC 16449 cells from steamed soybeans and from soybean seed-mimicking solid medium, using RNA-Seq analysis. Reads were mapped to the

genomic sequence of _B. subtilis_ subsp. _natto_ BEST195 strain39,40. Expression of 93 and 107 genes among a total of 4271 analyzed genes was over tenfold upregulated and downregulated,

respectively, in _B. subtilis_ grown on steamed soybeans. Upregulated transcripts included several ABC transporter components and their neighboring genes (Fig. 3A). The gene cluster

_yesOPQR_ was highly expressed, as previously reported in _B. subtilis_ 168 cells grown on RG-I, a component of plant cell wall pectin32. Based on sequence homology, _yesO_ and _yesPQ_ genes

encode a putative SBP and transmembrane permeases for the ABC-family sugar transport system, respectively. Deleting _yesOPQ_ has a negative impact on utilization of RG-I as a carbon

source41. YesR is a cytosolic hydrolase that acts on unsaturated rhamnogalacturonan disaccharide derived from RG-I31. Another upregulated gene, _yurJ_, may encode a functional NBP for YesOPQ

and other ABC transporters41,42. Thus, _B. subtilis_ may recognize and use plant cell wall-derived RG-I as a carbon source by inducing _yesOPQR_ and _yurJ_ genes during saprophytic growth

(Fig. 3B). AFFINITY BETWEEN YESO AND RG-I OLIGOSACCHARIDES YesO is a putative SBP involved in RG-I assimilation, but its binding substrate is unknown. Recombinant YesO protein and the

decomposition product of RG-I backbones (oligo-RG-I) were prepared to address this knowledge gap. Comparisons between YesO of _B. subtilis_ laboratory strain 168 and its orthologs from

_Bacillus sonorensis_, _Bacillus licheniformis_ DSM 13, _Paenibacillus_ sp. Aloe-11, and _Paenibacillus macerans_ revealed low conservation of the N-terminus (Supplementary Fig. S3). Thus,

N-terminus-deleted YesO, of approximately 45 kDa, was overexpressed in _Escherichia coli_, and purified to homogeneity (Fig. 4A,B). To obtain oligo-RG-I, RG-I backbones were treated with

exolyase YesX (Fig. 4C). Thin-layer chromatography (TLC) analysis of reaction products showed that released oligosaccharides consisted mainly of unsaturated rhamnogalacturonan disaccharide,

as reported previously32. The interaction between YesO and oligo-RG-I was analyzed by measuring YesO fluorescent intensity in the presence or absence of oligo-RG-I (Fig. 4D). Adding

oligo-RG-I decreased the fluorescent intensity from tryptophan residues of YesO by 3–7%. Based on the plot, the dissociation constant (_K_d) was 1.6 μM. No significant decrease of

fluorescent intensity was observed when other disaccharides, such as sucrose, maltose, cellobiose, chitobiose, and digalacturonic acid, were used as a ligand (Supplementary Fig. S4). YesO

appears to recognize oligo-RG-I as a substrate selectively. BINDING MODE OF YESO TO OLIGO-RG-I Oligo-RG-I recognition by YesO was analyzed with X-ray crystallography. The crystal structures

of YesO and its complex form with oligo-RG-I (YesO/oligo-RG-I) were determined at 1.97 Å and 1.58 Å resolution, respectively. Data collection and refinement statistics are summarized in

Supplementary Table S1. Ligand-free YesO structure (Fig. 4E) was similar to a previously determined structure (PDB ID 4R6K). YesO is an α/β protein with 15 α helices (residues 23–39, 52–65,

76–86, 92–96, 111–113, 135–140, 152–166, 177–187, 205–219, 253–262, 312–318, 328–336, 340–354, 368–383, and 381–405) and 8 β sheets (residues 15–20, 45–55, 71–73, 122–126, 130–133, 248–252,

267–270, and 288–290). Note that amino acid residue numbers of our YesO are 20 fewer than those of the full-length YesO to adjust to PDB ID 4R6K. The substrate-binding cleft is formed

between two globular N- and C-domains (Fig. 4F), as typically observed among SBPs of ABC transport systems15,16,17,18. YesO is categorized as a Class II SBP, based on a β2–β1–β3–β8–β4

topology of the β sheet core in the N-domain. N- and C-domains are connected through two short hinges (residues Val126-Leu129 and Leu281-Lys284), assigning YesO to Cluster D SBPs. Comparison

of ligand-free YesO with YesO/oligo-RG-I revealed a 26.2°-closed conformation upon binding of a ligand molecule (Fig. 4G), suggesting a Venus-flytrap conformational change43. Although our

prepared oligo-RG-I was mostly disaccharide (Fig. 4C), X-ray crystallography of YesO/oligo-RG-I indicated an interaction between YesO and the trisaccharide, ΔGalUA-Rha-Gal, composed of

unsaturated galacturonic acid and rhamnose from the main chain and galactose from the side chain (Fig. 4H). This result reproduced another independent YesO/oligo-RG-I crystal structure. The

molecular docking simulation suggested that rhamnogalacturonan tetrasaccharide is too large to fit in the ligand-binding cleft (Supplementary Fig. S5). Thus, YesO may selectively recognize

ΔGalUA-Rha-Gal trisaccharide as a ligand. ΔGalUA-Rha-Gal binds to the YesO cleft via hydrogen bonds and C–C contacts (Table 1). There were 19 hydrogen bonds (ΔGalUA, 10; Rha, 2; Gal, 7) and

44 C–C contacts (ΔGalUA, 22; Rha, 17; Gal, 5). ΔGalUA-interacting residues, Arg25, Asn127, Asn254, and Ser286, are highly conserved among the known YesO orthologs, highlighting the

importance of ΔGalUA in the YesO ligand. RECOGNITION OF ΔGALUA-RHA-GAL DURING SAPROPHYTIC GROWTH In the present study, _B. subtilis yesOPQR_ was identified as an upregulated gene cluster

during saprophytic growth on dead soybean surfaces (Fig. 3B). YesO was found to act as an SBP for uptake of RG-I-derived ΔGalUA-Rha-Gal trisaccharide. YesPQ are putative transmembrane

permeases, and YesR catalyzes intracellular degradation of unsaturated rhamnogalacturonan disaccharide31. Expression of the _yurJ_ gene, encoding a putative NBP for YesOPQ40, was also

induced on dead soybeans. Thus, YesOPQR/YurJ-dependent assimilation of ΔGalUA-Rha-Gal is likely to play a major role in saprophytic growth. Assuming that plant cell wall degradation is

closely associated with wounding, pathogen attack, or cell death, induction of the _yesOPQR_ and _yurJ_ genes might be one of the initial responses for saprophytic growth on dead plant

surfaces. Our hypothesis is that ΔGalUA-Rha-Gal is used as a nutrient or signaling molecule, or both. ΔGalUA-Rha-Gal is degraded into assimilable monosaccharides by an unidentified

intracellular β-1,4-galactosidase and YesR. Since the soybean surface is a low-nutrient environment, pectin-derived oligosaccharides may be the carbon source that supports saprophytic

growth. Further, ΔGalUA-Rha-Gal may also serve as a signaling molecule to trigger saprophytism. Some bacterial two-component regulatory systems adopt SBPs for signal recognition18. Our

recent study demonstrated that SPH1118, an SBP in _Sphingomonas_ sp. strain A1, is responsible not only for pectin assimilation but also for chemotaxis toward pectin44. Assuming such

alternative functions of YesO, recognizing ΔGalUA-Rha-Gal may trigger signal transduction for proliferation or adaptation under saprophytic conditions. Based on the YesO/oligo-RG-I

three-dimensional structure, YesO traps ΔGalUA-Rha-Gal trisaccharide, although most of the oligo-RG-I used in this study was disaccharide. How is ΔGalUA-Rha-Gal generated via pectin

degradation? _B. subtilis_ cells secrete a series of enzymes for RG-I degradation, such as YesWX lyases to cleave α-1,4-glycosidic linkages between rhamnose and galacturonic acid residues in

the RG-I backbone32, GanAB galactosidases to degrade galactan branches45, and AbfA and Xsa arabinofuranosidases to catalyze hydrolysis of terminal arabinofuranoside bonds of arabinogalactan

or arabinan46. YesWX-dependent degradation of RG-I backbones, with a single galactose residue at each branch point, may release ΔGalUA-Rha-Gal trisaccharide (see red areas in Fig. 3B). In

conclusion, this report is the first to describe the competition between saprophytic _B. subtilis_ and soybean _G. max_ on soybean seeds surfaces. Live soybeans prevent bacterial invasion,

while _B. subtilis_ YesO recognizes pectin-derived ΔGalUA-Rha-Gal trisaccharide to establish saprophytic growth on dead soybean seeds. This study will help elucidate the diversity of

bacterial-plant interactions, particularly for seed microbiota. MATERIALS AND METHODS MATERIALS AND MICROORGANISMS Soybean seeds of Suzumaru (_G. max_) were purchased from Mamehei (Japan).

RG-I (from potatoes) was purchased from Megazyme (Ireland). _B. subtilis_ laboratory standard strain 168 was obtained from the National Bio Resource Project (NBRP), Japan. _B. subtilis_

strain NBRC 16449 was obtained from the NITE Biological Research Center (NBRC), Japan. _E. coli_ strain BL21-Gold(DE3) (Agilent, USA) was used for recombinant protein overexpression. _E.

coli_ strain DH5α was used for cloning and plasmid maintenance. Bacterial cells were routinely grown in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.2) at 37 °C.

GROWTH TEST Soybean seeds were stored at 4 °C and at room temperature for 6 months to prepare live and dead soybeans, respectively. The seeds were washed and soaked in pure water at room

temperature for 3.5 h. The seed surfaces were sterilized by 20-min ultraviolet exposure. This exposure did not produce severe effects on living soybean seeds’ germination capability

(Supplementary Fig. S1A). Soybean seed-mimicking solid LB medium blocks (solidified by 3% agar, 300 μl each; Supplementary Fig. S1B) were prepared aseptically in microtubes. Thirty seeds or

blocks were used for each growth test. _Bacillus subtilis_ was precultured at 37 °C in liquid LB medium overnight, washed, and resuspended in 0.85% sodium chloride. Approximately 104 cells

per seed or solid medium block were inoculated and incubated at 37 °C. To assay CFUs, _B. subtilis_ cells on the sampled seeds or blocks were collected in 0.85% sodium chloride, spread on LB

agar plates, and incubated at 37 °C overnight. SEM ANALYSIS _Bacillus subtilis_ cells were grown on steamed soybean seeds, on soybean mimicking solid LB medium blocks, and in liquid LB

medium at 37 °C. Steamed soybeans were prepared by washing and soaking live seeds with pure water at room temperature for 3.5 h, followed by autoclaving at 121 °C for 20 min. For protein

fixation, samples (soybean seeds or collected cells) were mixed with 4% formaldehyde and incubated for 1 h. Subsequently, for lipid fixation, samples were washed with 10 mM potassium

phosphate buffer or pure water three times and immersed in 1% osmium oxide for 1–2 h. Fixed samples were washed with 10 mM potassium phosphate buffer or pure water three times, dehydrated

sequentially with 50%, 70%, 90%, and 100% ethanol, and immersed in _t_-butyl alcohol at 4 °C. Freeze-dried samples were coated with a platinum-palladium layer under argon gas and observed

with a field emission (FE)-SEM SU8230 (Hitachi, Japan) using a 1.5 kV electron beam. RNA-SEQ ANALYSIS _Bacillus subtilis_ NBRC 16449 cells were grown on steamed soybean seeds or

soybean-mimicking solid LB medium blocks to the logarithmic phase. Cells on the seeds or blocks were collected in 0.85% sodium chloride, treated with RNAprotect bacteria reagent (Qiagen,

Netherlands), and frozen immediately in liquid nitrogen. Total RNA was prepared using the hot phenol method47 by Nihon Gene Research Laboratories, Inc. (Japan). RNA-Seq analysis was

performed by Macrogen Japan Corp. (Japan). In brief, paired-end sequencing of the constructed library was performed using HiSeq 2500 (Illumina, USA). Raw data were filtered using Trimmomatic

v0.3248 to remove adapter sequences and bases with a Phred score below 30. Trimmed reads were aligned against the _B. subtilis_ subsp. _natto_ BEST195 reference genome (DDBJ accession no.

DRA000001)39,40, using Bowtie as the alignment algorithm49. Differential expression analysis was conducted using HTSeq50. RNA-Seq data, generated from this study, were deposited in the NCBI

Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE109523. EXPRESSION AND PURIFICATION OF RECOMBINANT YESO PROTEIN To construct an expression system for

YesO that lacks the weakly conserved N-terminus (Supplementary Fig. S3), the _yesO_ gene of _B. subtilis_ strain 168 was amplified by genomic PCR, using primers

5′-GGCATATGACACTCAGAATCGCGTGGTGGGGC-3′ and 5′GGCTCGAGTCAATTATTCCTCTCTAATATCTCATT-3′ (with underlined restriction sites), and cloned into the NdeI-XhoI site of pET21b(+) (Novagen, USA). The

resultant pET21b(+)-_yesO_ plasmid was introduced into _E. coli_ BL21-Gold(DE3) cells. _Escherichia coli_ cells harboring the plasmid were cultured in LB liquid medium with 100 μg ml−1

ampicillin at 30 °C. When optical density at 600 nm (OD600) reached 0.3, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.1 mM. After further

incubation at 16 °C for 2 days, cells were washed, suspended in 20 mM Tris–HCl (pH 7.5), and disrupted with an ultrasonicator 201 M (Kubota, Japan). The supernatant obtained by

centrifugation (20,000 × _g_, 4 °C, 20 min) was used for YesO purification. Cell extracts were applied to an anion exchange chromatography system with a TOYOPEARL DEAE-650M column (Tosoh,

Japan). Proteins were eluted using a linear gradient of sodium chloride (0–250 mM) in 20 mM Tris–HCl (pH 7.5). After separation with 12.5% SDS-PAGE, fractions containing the target protein

were combined and purified by gel filtration chromatography using HiLoad 16/60 Superdex 200 pg resin (GE Healthcare, USA) equilibrated with 200 mM sodium chloride in 20 mM Tris–HCl (pH 7.5).

After separation with 12.5% SDS-PAGE, fractions containing the target protein were combined. PREPARATION OF OLIGO-RG-I Substrate RG-I backbones were prepared from commercially available

RG-I, as previously reported32. A reaction mixture, consisting of 1% RG-I backbone, 50 mM Tris–HCl (pH 7.5), 20 mM manganese(II) chloride, and lysates of YesX-expressing _E. coli_ cells32,

was incubated at 30 °C for 3 h. The mixture was boiled for 5 min and centrifuged (7200 × _g_, room temperature, 5 min). The supernatant was passed through a Centriprep centrifugal filter (3 

kDa NMWL; Merck Millipore, USA) to obtain decomposition products. Resultant oligo-RG-I was detected by TLC analysis, as reported previously32. FLUORESCENCE SPECTRUM ANALYSIS Fluorescence of

protein, with a peak around 340 nm, is partially quenched by adding a binding ligand51. Fluorescent intensities of YesO, with increasing concentrations of oligo-RG-I, were measured by

spectrophotometer FP-6500 (JASCO, Japan). Measurement parameters during reactions were: excitation band width, 1 nm; emission band width, 10 nm; response, 2 s; sensitivity, high; excitation

wavelength, 280 nm; start to end emission wavelength, 300–500 nm; data pitch, 1 nm; and scan speed, 100 nm min−1. The reaction mixture contained 0.14 μM YesO, 20 mM Tris–HCl (pH 7.5), and

0–40 μM ligands. Decreases in fluorescent intensity by adding ligands were plotted, and the dissociation constant (_K_d) was determined based on nonlinear regression with one site-specific

binding model51. X-RAY CRYSTALLOGRAPHY Ligand-free YesO and YesO/oligo-RG-I were crystallized by sitting drop vapor diffusion, using the JBScreen crystallization kit (Jena Bioscience,

Germany). In 96-well sitting drop plates, 1 μl of 15 mg ml−1 YesO (without or with 1 mM oligo-RG-I) and 1 μl of reservoir solution were mixed. The mixture was kept at 20 °C until crystals

grew sufficiently. Ligand-free YesO was crystallized in the drop, consisting of 100 mM malonate-imidazole-boric acid (MIB) buffer (pH 6.0), 25% polyethyleneglycol (PEG)-1500, and 1 mM

digalacturonate. YesO/oligo-RG-I was crystallized in the drop, consisting of 100 mM Tris–HCl (pH 8.5), 20% PEG-1000, and 25% glycerol. Each single crystal was picked up using a nylon loop

from the drop, soaked in a reservoir solution containing 20% ethylene glycol, and instantly frozen using cold nitrogen gas. Synchroton radiation X-ray irradiated the crystals at 1.00 Å

wavelength, and X-ray diffraction data were collected using a MAR225HE CCD detector (Rayonix, USA) at the BL-26B1 and BL-38B1 beamlines in SPring-8 (Japan). Diffraction data were processed

using the HKL-2000 program52. The crystal structures of YesO and YesO/oligo-RG-I were determined through molecular replacement with the Molrep program53 in the CCP4Interface package, using

the ligand-free YesO structure (PDB ID 4R6K) as a reference model. Structure refinement was conducted using the phenix.refine54 and REFMAC553 programs. At each refinement cycle, the model

was adjusted manually with the WinCoot program55. Images of protein structures were prepared using the PyMOL molecular graphics system (Schrödinger, USA). The coordinates and structure

factors (accession codes 5Z6C for ligand-free YesO and 5Z6B for YesO/oligo-RG-I) have been deposited in the PDB. CODE AVAILABILITY _Accession codes_ RNA-Seq data were deposited in the NCBI

GEO and are accessible through GEO series accession number GSE109523. Protein structures of YesO and YesO/oligo-RG-I were deposited in the PDB under accession codes 5Z6C and 5Z6B,

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(2004). Article  PubMed  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS Diffraction data were collected at the BL26B1 and BL38B1 stations of SPring-8 (Hyogo, Japan) with the

approval of JASRI (proposal nos. 2016A2574, 2016B2574, 2017B2547, and 2017B2592). This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the

Promotion of Science (to W.H.), and Research Grant (to W.H.) from Fuji Foundation for Protein Research. The authors would like to thank Enago (https://www.enago.com) for the English language

review. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture,

Kyoto University, Uji, Kyoto, 611-0011, Japan Haruka Sugiura, Ayumi Nagase, Sayoko Oiki, Daisuke Watanabe & Wataru Hashimoto * Laboratory of Applied Structural Biology, Division of

Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan Bunzo Mikami Authors * Haruka Sugiura View author publications You can also search for

this author inPubMed Google Scholar * Ayumi Nagase View author publications You can also search for this author inPubMed Google Scholar * Sayoko Oiki View author publications You can also

search for this author inPubMed Google Scholar * Bunzo Mikami View author publications You can also search for this author inPubMed Google Scholar * Daisuke Watanabe View author publications

You can also search for this author inPubMed Google Scholar * Wataru Hashimoto View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS W.H.

designed the study; H.S., A.N., S.O., B.M., and W.H. performed the experiments; H.S., A.N., S.O., B.M., D.W., and W.H. analyzed the data; H.S., D.W., and W.H. wrote the manuscript.

CORRESPONDING AUTHOR Correspondence to Wataru Hashimoto. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE

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