Plant Pathol. J. 37(6) : 566-579 (2021)
https://doi.org/10.5423/PPJ.OA.06.2021.0087
pISSN 1598-2254 eISSN 2093-9280
Research Article Open Access Fast Track

The Plant Pathology Journal©The Korean Society of Plant Pathology

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in
Inducing Hypersensitive Response on Eggplant (Solanum melongena cv.
Senryo-nigou)
Nur Laili1,2, Takafumi Mukaihara3, Hidenori Matsui1, Mikihiro Yamamoto1, Yoshiteru Noutoshi1,
Kazuhiro Toyoda1, and Yuki Ichinose 1*
1
Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama
700-8530, Japan
2
Research Center for Biology, Research Organization for Life Sciences, National Research and Innovation Agency(BRIN), Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor, West Java 16911, Indonesia
3
Research Institute for Biological Sciences, Okayama (RIBS), 7549-1 Yoshikawa, Kibichuo-cho, Okayama 716-1241, Japan(Received on June 1, 2021; Revised on October 8, 2021; Accepted on October 12, 2021)
Ralstonia syzygii subsp. indonesiensis (Rsi, former
name: Ralstonia solanacearum phylotype IV) PW1001,
a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum
melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance inmany organisms. We found that trehalose is required
for eliciting HR on eggplant by plant pathogen Rsi
PW1001. In R. solanacearum, it is known that the OtsA/OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate(T6P) synthase and T6P phosphatase, respectively. We
generated otsA and otsB mutant strains and found thatthese mutant strains reduced the bacterial trehalose
concentration and HR induction on eggplant leaves
compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Re*Corresponding author.
Phone) +81-86-251-8308, FAX) +81-86-251-8388
E-mail) yuki@okayama-u.ac.jp
ORCID
Yuki Ichinose
https://orcid.org/0000-0002-7767-7912
Handling Editor : Cecile Segonzac
This is an Open Access article distributed under the terms of theCreative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0) which permits unrestrictednoncommercial use, distribution, and reproduction in any medium,provided the original work is properly cited.
Articles can be freely viewed online at www.ppjonline.org.

quirement of trehalose in HR induction is not common
in R. solanacearum species complex because mutationof otsA in Ralstonia pseudosolanacearum (former name:
Ralstonia solanacearum phylotype I) RS1002 did notaffect HR on the leaves of its non-host tobacco and wild
eggplant Solanum torvum. Further, we also found thateach otsA and otsB mutant had reduced ability to growin a medium containing NaCl and sucrose, indicatingthat trehalose also has an important role in osmotic
stress tolerance.
Keywords : eggplant, hypersensitive response, Ralstonia
solanacearum species complex, trehalose

Ralstonia solanacearum species complex (RSSC) is wellknown as one of the most destructive bacterial plant pathogens due to its aggressiveness, extensive host range, broad
geographical distribution, and long persistence under diverse environmental conditions (Genin, 2010; Meng, 2013;
Prior et al., 2016). RSSC causes bacterial wilt disease inover 200 plant species worldwide and affects a wide rangeof economically important crops including tomato, potato,
eggplant, chili, tobacco, banana, groundnut, and ginger (Duet al., 2017; Meng, 2013). R. solanacearum was ranked
2nd in a list of the top 10 most scientifically and economically important plant-pathogenic bacteria (Mansfield etal., 2012). RSSC has been classified into four phylotypes,
depending on its geographical distribution and host plants.
In 2014, Safni et al. proposed a new classification of RSSC
and further taxonomic revision of the diverse group ofTrehalose Required for HR by Ralstonia syzygii

strains in phylotype IV. Phylotype I and III are newly classified as R. pseudosolanacearum, phylotype II as R. solanacearum, and phylotype IV as R. syzygii. Then, R. syzygii
was divided into three subspecies: R. syzygii subsp. syzygii,
R. syzygii subsp. celebesensis, and R. syzygii subsp. indonesiensis (Rsi).
RSSC enters host plants through natural openings orwounds in the root system or at sites of secondary root formation, colonizes the cortex and vascular parenchyma, thenenters the xylem vessels and rapidly spreads up into thestems and leaves (Du et al., 2017; Meng, 2013). This bacterium forms biofilms in the xylem, produces large amountsof extracellular polymeric substances (EPS), and proliferates rapidly (Minh Tran et al., 2016; Mori et al., 2016).
The rapid proliferation of bacteria and abundant EPS in
the vascular system will block xylem vessels and disrupt
water flow, causing wilt disease symptoms and eventually the death of infected plants (Genin and Deny, 2012).
The internal symptoms are progressive discoloration of thevascular tissue in the early stages of infection, the diseasedevelops in the pith and cortex, and then complete necrosis
occurs. The external symptoms of infected plants are wilting, stunting, and yellowing of the foliage with the diseaseprogression until the plant completely collapses (Àlvarez etal., 2010).
Rsi causes disease in a number of solanaceous plants andclove plants in Indonesia and other Asian countries (Safni
et al., 2014). In Japan, Rsi strains have been isolated only
from Solanum tuberosum (Horita et al., 2010), and showedvarying levels of pathogenicity for tomato, groundnut, andtobacco. However, none of the strains tested were pathogenic for S. melongena (Suga et al., 2013). Rsi PW1001 is
an RSSC phylotype IV biovar 2N that was isolated frompotato plants in Japan and causes wilt disease on potato.
However, the response of non-host plants to this pathogen
has not been investigated so far. In this study, we foundthat inoculation of Rsi PW1001 into its non-host eggplant
S. melongena cv. Senryo-nigou induced hypersensitive response (HR).
HR is a plant defense response to inoculation by pathogens, which is often accompanied by a rapid localized andprogrammed cell death; it restricts bacterial multiplication
at the inoculation site and is associated with disease resistance (Balint-Kurti, 2019; Nakano and Mukaihara, 2019).
Disease resistance (R) proteins have been evolved in plants
to detect pathogen effectors inside plant cells during pathogen inoculation. The recognition of pathogen effectors by
R proteins induces a strong defense response, called effector-triggered immunity (ETI), which includes ion effluxes,
production of reactive oxygen species, phytohormone accu-

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mulation, and transcriptional activation of defense-related
genes (Cui et al., 2015; Feys and Parker, 2000). Inductionof the ETI response results in an HR, which is followed byelicitation by a pathogen or metabolite, electrolyte leakage
due to membrane damage, the cessation of cyclosis, andcellular collapse (Balint-Kurti, 2019).
To induce HR, most bacterial pathogens require a typeIII secretion system (T3SS) to directly translocate someeffectors into the plant cytoplasm. R. solanacearum injects approximately 70 effector proteins into host cells via
the Hrp T3SS during infection (Mukaihara et al., 2010;
Peeters et al., 2013). Nowadays, several effector proteinsof R. solanacearum have been shown to act as avirulence
determinants in certain plants. RipAA (formerly AvrA)
triggers HR cell death and induces bacterial wilt resistancein Nicotiana tabacum (Carney and Denny, 1990). RipP1(formerly PopP1), an acetyl-transferase effector of the
YopJ family, restricts bacterial growth in Petunia St40 (Lavie et al., 2002). RipP2 (formerly PopP2) induces bacterial
wilt resistance in Arabidopsis thaliana Nd-1 (Deslandes
et al., 2003). RipP1 and RipAA from the R. solanacearumstrain GMI1000 have been shown to act as avirulence determinants in N. tabacum and N. benthamiana (Poueymiro
et al., 2009). RipAX2 (formerly Rip36) induces HR in thewild eggplant S. torvum (Nahar et al., 2014) and restricts
the growth of R. solanacearum in eggplant AG91-25 (Morelet al., 2018). RipBN is recognized in tomato and induces
resistance (Mazo-Molina et al., 2019). RipB is recognized
as an Avr effector in N. benthamiana, N. tabacum, and N.
otophora (Nakano and Mukaihara, 2019), and RipX (formerly PopA) and RipI are also reported as Avr effector in
N. benthamiana (Sun et al., 2020; Zhuo et al., 2020). Furthermore, RipTPS which has trehalose-6-phosphate (T6P)
synthase (TPS) has been reported to elicit HR-like responseon tobacco (Poueymiro et al., 2014).
In this study, we investigated the involvement of trehalose in the interaction of bacterial pathogen and non-hostplant. Trehalose is a non-reducing disaccharide consistingof two glucose molecules linked by α-1,1-glucosidic bond(Fernandez et al., 2010). Trehalose is synthesized in a twostep reaction from UDP-glucose (UDPG) and 6-phosphoglucose by TPS and trehalose-6-phosphate phosphatase(TPP) (Miao et al., 2016). Bacteria have several trehalose
synthesis pathways, including trehalose synthase (TreS),
TreY/TreZ, and OtsA/OtsB pathways. The TreS pathway
synthesizes trehalose from maltose using TreS, the TreY/
TreZ pathway synthesizes trehalose from maltooligosaccharides using maltooligosyl-trehalose synthase (TreY)
and maltooligosyl-trehalose trehalohydrase (TreZ). In the
OtsA/OtsB pathway, trehalose is synthesized from glucose-568

Laili et al.

6-phosphate and UDPG, which is catalyzed by TPS (OtsA),
then dephosphorylated by TPP (OtsB). MacIntyre et al.
(2020) revealed that the OtsA/OtsB pathway is the predominant pathway for trehalose synthesis in R. solanacearum
GMI1000. Rsi PW1001 also has an otsB/otsA gene cluster
as an operon for trehalose synthesis in the OtsA/OtsB pathway.
Trehalose is a common metabolite that has been shown
to be involved in conferring tolerance to a variety of environmental stresses, such as dehydration, heat, oxidative andosmotic stresses, and freezing (Hayner et al., 2017). Trehalose is also well-known player in biological signaling,development, and metabolic regulation (Lunn et al., 2014).
Trehalose biosynthesis in some bacterial pathogens is also
necessary for fitness and virulence, and has been an antibiotic target (Tournu et al., 2013). Despite comprehensive
evidence of the biological benefits of trehalose in manyorganisms, relatively few reports about how trehalose andrelated metabolites function during plant-microbe interactions. Freeman et al. (2010) reported that mutating the treS
and treY/Z pathways in Pseudomonas syringae pv. tomato
DC3000 decreased bacterial phyllosphere fitness in tomato
leaves. In P. aeruginosa, trehalose is a key virulence factor
to cause full disease symptoms on A. thaliana. Mutation oftreS and treY/Z, genes for the synthesis pathway in P. aeruginosa, decreased the acquisition of nitrogen-containing
nutrients and xyloglucan components of the plant cellwall, thereby inhibiting the replication of P. aeruginosa inintercellular spaces of leaves (Djonović et al., 2013). Thisevidence suggests that trehalose metabolism is requiredfor bacterial movement into intercellular spaces and nitrogen uptake. Mutation of otsA/B in the trehalose synthesis
pathway in Xanthomonas citri reduced virulence on citrus(Piazza et al., 2015). Furthermore, MacIntyre et al. (2020)
generated ∆otsA and ∆treY/treS/otsA in R. solanacearum
GMI1000, and found that the level of disease index andbacterial propagation of these mutants on tomato werereduced, indicating that trehalose synthesis contributes tofitness and virulence.
However, the study of the role of trehalose synthesis for
HR induction in plant-pathogen interaction has not beenreported yet. Therefore, we mutated otsA and otsB in Rsi
PW1001 and investigated trehalose concentration, effect on
HR induction, and osmotic tolerance. The role of trehalose
synthesis in the interaction of bacterial pathogen and plants
will be discussed.

Materials and Methods
Plant material and culture conditions. Eggplant (S. mel-

ongena cv. Senryo-nigou), wild eggplant S. torvum, andtobacco plants (N. tabacum cv. Xanthi) were grown in a
growth chamber under the following conditions: a 16/8 h(light/dark) photoperiod at 28°C and 80% relative humidity, and 7-week-old plants were used for inoculation experiments.
Bacterial strains, plasmids, media, and culture condition. The bacterial strains and plasmids used in thisstudy are listed in Tables 1 and 2. The strain Rsi PW1001
was spontaneously resistant to nalidixic acid (Nal) of
MAFF327034 (Horita et al., 2010; Suga et al., 2013). Rsi
PW1001 and R. pseudosolanacearum RS1002 strains wereroutinely cultured in BG medium (1% bacto peptone, 0.1%
casamino acid, 0.1% yeast extract, and 0.5% glucose) at
27°C, and supplemented with antibiotics when needed (3050 µg/ml of nalidixic acid, 30 µg/ml of kanamycin, 18 µg/
ml of tetracycline, or 30 µg/ml of gentamicin). Escherichia
coli strains were routinely cultured in Luria-Bertani medium at 37°C and supplemented with appropriate antibiotics(100 µg/ml of ampicillin, 50 µg/ml of kanamycin, 30 µg/
ml of tetracycline, or 50 µg/ml of gentamicin).
Construction of hrpB, hrcV, hpaB, and ripTPS mutants
in Rsi PW1001. To generate hrpB and hrcV mutants, partsof hrpB and hrcV fragments (0.8 kb) were amplified by
PCR with primer pairs of P3747 and P3748 for hrpB, andP3749 and P3750 for hrcV. All primer sequences used inthis study are listed in Supplementary Table 1. KOD-FX
Neo DNA Polymerase (Toyobo Inc., Osaka, Japan) wasused for all PCR for plasmid construction. Resulting PCR
products were inserted into the plasmid pARO191 using
an In-Fusion HD cloning kit (Takara, Shiga, Japan). Bothresulting plasmids were transformed into E. coli S17-1 andintegrated into wild-type (WT) of Rsi PW1001 by conjugation.
To generate ∆hpaB and ∆ripTPS of Rsi PW1001, thehpaB and ripTPS coding region with their surrounding region was amplified with primer pairs of hpaB-F and hpaBR for hpaB, and ripTPS-F and ripTPS-R for ripTPS, andinserted into a pGEM-T Easy vector (Promega, Madison,
WI, USA). To delete the hpaB and ripTPS open reading
frame, inverse PCR was performed with appropriate primer
pairs, hpaB-d1, hpaB-d2, ripTPS-d1, and ripTPS-d2, respectively. Then, the PCR product was digested with XbaIand DpnI, and self-ligated with a Ligation-convenience kit(Nippon Gene, Tokyo, Japan). The ∆hpaB and ∆ripTPS
DNA construct was subcloned into pK18mobsacB at an
EcoRI site (Schäfer et al., 1994) to obtain pK18-∆hpaB
and pK18-∆ripTPS, and transformed into E. coli S17-1.

Trehalose Required for HR by Ralstonia syzygii

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Table 1. Bacterial strains used in this study
Bacterial strain
Ralstonia syzygii subsp.
indonesiensis
PW1001
PW1002
PW1003
PW1004
PW1005PW1006
PW1007
PW1008PW1078PW1079PW1080
PW1081PW1082
PW1083
PW1085
PW1086
PW1087
PW1088R. pseudosolanacearum
RS1002
RS1211RS1273
RS1662
RS1736Escherichia coli
DH5α
S17-1
S17-1 λpir

Relevant characteristics

Reference or source

Spontaneously resistant strain to nalidixic acid of MAFF327034
PW1001 hrpB::pARO (Nalr and Kmr)
PW1001 hrcV::pARO (Nalr and Kmr)
PW1001 ∆hpaB (Nalr)
PW1001 otsA::pARO (Nalr and Kmr)
PW1001 otsB::pARO (Nalr and Kmr)
PW1001 otsA::pARO chr::miniTn10 otsBA (Nalr, Kmr and Tetr)
PW1001 otsB::pARO chr::miniTn10 otsBA (Nalr, Kmr and Tetr)
PW1001 ∆ripTPS (Nalr)
PW1001 possessing ripX::lacZ (Nalr and Gmr)
PW1005 possessing ripX::lacZ (Nalr and Gmr)
PW1006 possessing ripX::lacZ (Nalr and Gmr)
PW1007 possessing ripX::lacZ (Nalr and Gmr)
PW1008 possessing ripX::lacZ (Nalr and Gmr)
PW1005 ripX::’cya (expressing RipX-’Cya, Nalr and Gmr)
PW1006 ripX::’cya (expressing RipX-’Cya, Nalr and Gmr)
PW1007 ripX::’cya (expressing RipX-’Cya, Nalr and Gmr)
PW1008 ripX::’cya (expressing RipX-’Cya, Nalr and Gmr)

This study
This study
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RS1000 (Nalr)
RS1002 ∆hpaB (Nalr)
RS1002 ∆hrpY (Nalr)
RS1002 ∆ripAX2 (Nalr)
RS1002 otsA::pARO (Nalr and Kmr)

Mukaihara et al. (2004)
Mukaihara et al. (2004)
Mukaihara et al. (2004)
Nahar et al. (2014)
This study

F–, λ–, ø80dLacZΔM15, Δ(lacZYA-argF)U169, recA1, endA1,
hsdR17(rK–mK+), supE44, thi-1, gyrA, relA1
Thi pro hsdR-hsdM + recA [chr::RP4-2-Tc::Mu-Km::Tn7]
λpir lysogeny of S17-1

Nippon Gene
Schäfer et al. (1994)
Simon et al. (1983)

Gm, gentamicin; Km, kanamycin; Nal, nalidixic acid; Tet, tetracycline; r, resistance.

The ∆hpaB (PW1004) and ∆ripTPS (PW1078) deletionmutants were generated by conjugation and homologous
recombination with Rsi PW1001 WT. To confirm the precise deletion of target genes, colony PCR of the conjugants
was carried out using the respective primer pair.
Construction of otsA and otsB mutants and complemented strains in Rsi PW1001 and R. pseudosolanacearum RS1002. For construction of the otsA mutant
in Rsi PW1001, a part of the otsA gene fragment (678 bp)
was amplified by a primer pair of otsA-F_IF and otsA-R_
IF and was inserted into the plasmid pARO191 using an
In-Fusion HD cloning kit (Takara). A part of the otsB fragment (600 bp) of Rsi PW1001 was amplified with a primer

pair, otsB-F_NEB and otsB-R_NEB, and inserted into the
EcoRI site of pARO191 using NEBuilder Assembly (NewEngland Biolabs, Tokyo, Japan). Both resulting plasmids
were transformed into E. coli S17-1 and integrated into WTof Rsi PW1001. The homology of the DNA sequence ofotsA between Rsi PW1001 and RS1002 was 93% identity.
Therefore, to introduce mutation of otsA in RS1002, we integrated the plasmid pARO-otsA of Rsi PW1001 into WT
strain RS1002, and generated otsA mutant RS1736. The
mutants PW1005 (otsA::pARO), PW1006 (otsB::pARO),
and RS1736 (otsA::pARO) were selected after conjugation
on BG agar plates with 30 µg/ml of kanamycin and 30 µg/
ml of Nal, and the mutation of the surviving colonies was
confirmed by PCR using primer otsA-1 and M13-R for570

Laili et al.

Table 2. Plasmids used in this study
Plasmid

Relevant characteristics

Reference or sourcerpGEM-T EasypGEM-otsBA

Cloning vector for PCR product, Amp
pGEM-T Easy carrying 3006 bp PCR product with otsBA with their native promoter from PW1001, Ampr

Promega
This study

pGEM-hpaB

pGEM-T Easy carrying 1911 bp PCR product with hpaB with its native
promoter from PW1001, Ampr

This study

pGEM-ripTPS

pGEM-T Easy carrying 2958 bp PCR product with ripTPS with its native
promoter from PW1001, Ampr

This study

pGEM-ripX-1

pGEM-T Easy carrying 449 bp PCR product with ripX with its native
promoter from PW1001, Ampr

This study

pGEM-ripX-2

pGEM-T Easy carrying 1324 bp PCR product with ripX with its native
promoter from PW1001, Ampr
Small mobilizable vector, sucrose-sensitive (sacB), Kmr
pK18mobsacB with upstream and downstream regions of hpaB, Kmr
pK18mobsacB with upstream and downstream regions of ripTPS, Kmr
The mobilizable narrow-host-range vector derived from pUC19, Kmr
pARO191 containing 0.8 kb hrpB-DNA fragment from PW1001, Kmr
pARO191 containing 0.8 kb hrcV-DNA fragment from PW1001, Kmr
pARO191 containing 678 bp otsA-DNA fragment from PW1001, Kmr
pARO191 containing 600 bp otsB-DNA fragment from PW1001, Kmr
Cya reporter plasmid, KmrpARO-HA-’cya containing 1324 bp of ripX fragment and its promoter
region at XbaI and XhoI sites, Kmr

This study

pBSL199

Mini-Tn10 transposon vector derived from pBSL177, Ampr, Tetr

pBSL199-otsBA
pHRP315

pBSL199 connecting with pGEM-otsBA at XbaI site, Ampr, Tetr
Cohort vector for directed cloning of promoter fragment, Ampr

Alexeyev and Shokolenko(1995)
This study
Parales and Harwood(1993)

pHRP315-ripX

pHRP315 containing 449 bp ripX-DNA fragment and its promoter region
at KpnI and NotI sites, Ampr

This study

pHRP309

Broad-host-range lacZ transcriptional fusion vector based on the IncQ
plasmid RSF1010, Gmr

Parales and Harwood(1993)

pHRP309-ripX

pHRP309 possessing XbaI-EcoRI fragment of pHRP315-ripX, ripX-lacZtranscriptional fusion vector, Gmr
Vector containing Gentamicin-cassette, Gmr, Ampr
pARO-ripX::’cya with Gm-cassette from pMS255 at EcoRI site, Kmr,
GmrThis study

pK18mobsacB
pK18mobsacB-∆hpaB
pK18mobsacB-∆ripTPS
pARO191
pARO191-hrpB
pARO191-hrcV
pARO191-otsA
pARO191-otsB
pARO-HA-’cyapARO-ripX::’cyapMS255pARO-ripX::’cya-Gm

Schäfer et al. (1994)
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Parke (1990)
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Murata et al. (2006)
This study

Becker et al. (1995)
This study

Amp, ampicillin; Gm, Gentamicin; Km, kanamycin; Tet, tetracycline; r, resistanceotsA mutant, and otsB-1 and M13-R for otsB mutant.
For complementation of PW1005 and PW1006 in Rsi,
the entire otsBA operon with its native promoter was amplified with primer pair (otsB-F and otsA-R), cloned into
pGEM-T Easy and ligated with plasmid pBSL199 (Alexeyev and Shokolenko, 1995) at XbaI site. The resultant
plasmid was transformed into E. coli S17-1 and introduced
into PW1005 and PW1006 by conjugation. The comple-

mented strains PW1007 and PW1008 were selected on BG
plates with nalidixic acid and tetracycline.
Construction of ripX::lacZ promoter plasmid and generation of Rsi PW1001 and its derivative strains possessing ripX­::lacZ. For construction of the ripX::lacZ, apart of the ripX gene fragment and its promoter region (449bp) was amplified by a primer pair of ripX-F and ripX-R

Trehalose Required for HR by Ralstonia syzygii

and was inserted into the pGEM-T Easy vector (Promega)to generate pGEM-ripX-1. Then the ripX promoter fragment was excised by KpnI and NotI digestions, and was inserted into cohort vector pHRP315 (Parales and Harwood,1993) at the same sites. A XbaI-EcoRI fragment containing
the ripX from pHRP315-ripX was introduced into a broad
host range lacZ transcriptional fusion vector, pHRP309(Parales and Harwood, 1993) to obtain pHRP309-ripX.
pHRP309-ripX was introduced into Rsi PW1001 WT and
its derivative strains by conjugation and b-galactosidase assay was carried out.
HR induction by bacterial inoculation. To examine HR
induction on non-host plant S. melongena cv. Senryo-nigou
by Rsi PW1001 and on S. torvum and N. tabacum cv. Xanthi by RS1002, the infiltration was assayed as described by
Mukaihara et al. (2004) with some modifications. The bacterial strains were cultured overnight in BG broth medium
at 27°C, and the cells were harvested by centrifugation.
The cell pellets were washed with 10 mM MgSO4, thenadjusted the OD600 of 0.3 (ca. 3 × 108 cfu/ml). The bacterial suspensions were infiltrated into 7-week-old eggplant,
tobacco, and wild eggplant leaves with a 1 ml needlelesssyringe. Infiltrated plants were incubated at 28°C, 80%
humidity with 16/8 h photoperiod (light/dark) for 3 days,and HR was observed every 24 h. To investigate the effectof exogenous trehalose in HR induction, trehalose solution
was added to bacterial suspensions at concentrations 0.25
mM or 2.5 mM, and infiltrated into 7-week-old eggplantleaves. HR development was observed for 3 days.
Measurement of ion leakage. The severity of cell deathin HR induction was quantified by the degree of electrolyte
leakage from eggplant leaves. Leaf disks (8 mm in diameter) were collected from individual plants and immersed
in 1 ml of water for 2 h at room temperature with gentle
shaking, as previously described (Nakano et al., 2021). The
ion conductivity of the water was measured using a conductivity meter (LAQUAtwin, Horiba, Kyoto, Japan).
Quantitative analysis of trehalose concentration. For
measurement of the trehalose concentration in Rsi PW1001
and its derivatives, the bacterial strains were grown overnight in 5 ml of minimal medium (50 mM KH2PO4, 7.6
mM (NH4)2SO4, 1.7 mM MgCl2, 1.7 mM NaCl at pH 7.0)
at 27°C. Bacterial cells were harvested by centrifugation at
9,800 ×g for 10 min, the cell pellet was resuspended in 1
ml of distilled deionized water, and the cells were disrupted
twice using a sonicator (Branson Sonifier, Danbury, CT,
USA) at 40% duty cycle for 10 s with a 30 s interval in an

571

ice bath. After sonication, disrupted cells were heated at
95°C for 10 min to inactivate trehalose-degrading enzymes,
and the cell debris was removed by centrifugation at 9,800
×g for 10 min (Li et al., 2012; MacIntyre et al., 2020).
The concentration of trehalose was determined by an
enzymatic trehalose assay using a Trehalose Assay Kit(Megazyme, Wicklow, Ireland) in a microtube following
the manual procedure. Briefly, 400 µl of distilled water,
40 µl of trehalose sample, 40 µl of solution 1 (buffer), 20µl of solution 2 (NADP+, ATP), and 4 µl of suspension 3(hexokinase/glucose-6-phosphate dehydrogenase) wereadded to the microtubes. Solutions were mixed and incubated for 5 min, then the absorbance values of a half volume of the solutions were read at 340 nm (A1) using UVVis Spectrophotometer UV mini 1240 (Shimadzu, Kyoto,
Japan). Subsequently, 2 ml of suspension 4 (trehalase) was
added into the remaining volume of the solutions in the microtube, mixed, incubated for 8 min at room temperature,and the absorbance values were measured at 340 nm (A2).
The amount of NADPH formed in this reaction is stoichiometric with the amount of D-glucose and thus with twice
the amount of trehalose. The pre-existing glucose concentrations for both blank and sample were determined in acontrol reaction without trehalase (A1) and subtracted fromthe total glucose (A2). The trehalose concentration was calculated using Megazyme Mega-Calc per l of samples.
β-Galactosidase activity assay to monitor ripX gene
expression. The b-galactosidase activity was measured as
described by Miller (1992) with some modifications. The
bacterial strains possessing pHRP309-ripX were cultured
in the BG and minimal medium pH 7.0 at 27°C for 16 h,then bacterial densities were adjusted to OD595 of 0.2 in
BG and minimal medium, and further incubated for 1 h. bgalactosidase activities were determined in 10 min reaction
time and shown in Miller units.
Construction of ripX-’cya fusion plasmid and adenylate cyclase assay to investigate RipX translocation. To
investigate effector translocation into plant cells Cya translocation assay system (Murata et al., 2006) was used, andripX was selected as one of representative T3SS genes. For
construction of the ripX::’cya fusion gene, a 1,324 bp DNA
fragment of the promoter and entire ripX open reading
frame without stop codon was amplified by a primer pairof ripX-F2 and ripX-R2 and cloned in pGEM-T Easy vector (Promega). The resultant plasmid, pGEM-ripX-2, wasdigested with XbaI and XhoI and then inserted into pAROHA-’cya (Murata et al., 2006) to obtain pARO-ripX::’cya.
Because both complemented strains are resistant to Nal,

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Km and Tet, we used Gm as a selection marker. A Gmcassette was excised from pMS255 vector (Becker etal., 1995) by EcoRI digestion, and inserted into pAROripX::’cya at EcoRI site to obtain pARO-ripX::’cya-Gm.
This reporter plasmid was introduced into otsA and otsB
mutant strains and otsA and otsB complemented strains.
The resulting strains expressing ripX::’cya were used for inplanta adenylate cyclase assay.
The in planta adenylate cyclase assay was performed as
described by Murata et al. (2006) with some modifications.
Briefly, bacterial strains expressing the ripX::’cya fusion
gene were grown overnight in BG medium at 27oC. The
bacterial cells were centrifuged, washed twice with distilled water, then resuspended at OD600 of 0.3. The bacterial
suspension was infiltrated into 7-week-old eggplant leaves,then incubated at 28°C, 80% humidity with 16/8 h photoperiod (light/dark). After 15 h incubation, three-leaf disks (8mm in diameter) from each sample were collected, frozen
with liquid nitrogen, then ground with mortar and pestle.
The leaf homogenates were boiled in 200 µl of 0.05 M sodium acetate buffer containing 0.02% bovine serum albumin for 5 min. Cell debris was removed by centrifugation
twice for 10 min each, and the supernatant was collected
in new microtubes and stored at -80°C until assayed forcAMP. The cAMP was measured using a Cyclic AMP EIA
kit (Cayman Chemical Company, Ann Arbor, MI, USA).
Examination of role of trehalose synthesis in osmotic
stress tolerance. Overnight cultures of Rsi PW1001 andderivatives were centrifugated, rinsed twice using BG fresh
medium, and resuspended to OD600 of 0.05 (1 × 107 cfu/ml)
in BG broth supplemented with or without 0.15 M NaClor 10% sucrose in culture tubes. Tubes were incubated in a
Bio-Shaker BR-21FP (Taitec, Kumagaya, Japan) at 27°C.
The bacterial growth was measured using a CO7500 colorimeter (Biochrom, Holliston, MA, USA) at OD600 every
12 h for 72 h. Each experiment included two replicates pertreatment from two independent experiments.
Data analysis. Statistical analyses were carried out using
GraphPad Prism version 9 package (GraphPad SoftwareInc., San Diego, CA, USA). All results are expressed asmeans with standard deviation (SD). The data were analyzed by one-way/two-way ANOVA, and significant differences between means were calculated by Tukey’s multiple comparison test at P < 0.05 considered significant.

Results
Important role of trehalose synthesis in HR induc-tion of S. melongena cv. Senryo-nigou. Rsi PW1001 is
avirulent pathogen on eggplant. When we infiltrated it on
eggplant at low cell density condition (OD600 = 0.0003),
bacteria did not propagate and symptom did not appear
even at 6 days post inoculation (dpi) (Supplementary Fig.
1). Although inoculation of WT strain PW1001 induced
HR on eggplant Senryou-nigou, inoculation of hrpB andhrcV mutants abolished HR induction (Fig. 1A), indicating the importance of T3SS in HR induction. Furthermore,the ∆hpaB mutant also abolished HR induction, indicating
T3SS-dependent effector transfer was required for HR. To
investigate the role of trehalose biosynthesis in HR induction by Rsi PW1001 on eggplant S. melongena cv. Senryonigou, we generated otsA and otsB gene-disrupted mutants
and their complemented strains and infiltrated these bacteria into the eggplant leaves. WT strain PW1001 caused
HR at 2-3 dpi, whereas both mutants of otsA (PW1005)
and otsB (PW1006) abolished HR induction to the level of
hrp mutants (Fig. 1). The introduction of intact otsBA into
the otsA and otsB mutants completely restored the development of HR. HR cell death on infiltrated leaves was accompanied by electrolyte leakage. We measured the degreeof ion leakage from the leaf disks at 2 and 3 dpi. As expected, the otsA and otsB mutants had significantly reduced
ion leakage compared to the WT (Fig. 1B), suggesting thattrehalose biosynthesis might be required for HR induction
on eggplant by Rsi PW1001.
We investigated the potential involvement of RipTPS
in Rsi PW1001-induced HR on eggplant. However, infiltration of ∆ripTPS still induced HR-like WT on eggplantat 2-3 dpi (Fig. 1C), and the degree of ion leakage wasalso similar to the WT (Fig. 1D). This result suggests that
RipTPS is not involved in Rsi PW1001-induced HR in
eggplant, although both OtsA and RipTPS encode TPS in
the trehalose biosynthesis pathway.
Importance of intracellular trehalose in HR induction.
To investigate the effect of exogenous trehalose on HR
induction in eggplant, we added trehalose at two concentrations (0.25 mM and 2.5 mM) to the bacterial cultures.
We observed that infiltration with only trehalose, both at
0.25 and 2.5 mM, did not induce HR. Although WT strain
PW1001 induced HR, otsA and otsB mutants did not induce
it, like the WT strain (Fig. 2A). The addition of exogenous
trehalose to the WT strain did not affect HR induction, andaddition to the otsA and otsB mutants did not restore HR.
To confirm that exogenous trehalose did not affect the HRlevel, we measured ion leakage from the infiltrated leaves.
The otsA and otsB mutants had significantly reduced ion
leakage, even with trehalose as compared to the WT (Fig.

Trehalose Required for HR by Ralstonia syzygii

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Fig. 1. Hypersensitive response (HR) on eggplant Solanum melongena cv. Senryo-nigou. (A, C) HR in leaves inoculated with the bacterial suspension (OD600 of 0.3) of Ralstonia syzygii subsp. indonesiensis PW1001 (WT), PW1002 (hrpB), PW1003 (hrcV), PW1004(∆hpaB), PW1005 (otsA), PW1006 (otsB), the complemented strains PW1007 (otsA TnotsBA+) and PW1008 (otsB TnotsBA+), PW1078(∆ripTPS), and 10 mM MgSO4 (Mock). Photographs show representative results from three independent experiments with similar results
at 2 and 3 dpi. (B, D) The degree of ion leakage from strain-infiltrated eggplant leaves was measured at 3 dpi using a conductivity meter.
Values are means ± SD of two replicates from two independent experiments. Different letters indicate significant differences among thenine inoculations (B) and six inoculations (D) as defined by Tukey’s multiple comparison test (P < 0.05).

2B). Namely, exogenous trehalose did not affect the ionleakage in infiltrated eggplant leaves.
Reduced trehalose concentration in otsA and otsB mutants of Rsi PW1001. To confirm that otsA and otsB mutants reduced the ability to synthesize trehalose, we used a
Trehalose Assay Kit to quantify the trehalose concentrationin the bacterial cells (Fig. 3). Both mutant strains had significantly reduced trehalose concentrations compared to
WT. The trehalose concentration of WT strain PW1001,
otsA mutant, and otsB mutant was 73.8, 13.3, and 6.5 mg/lof each trehalose sample, respectively. The complemented

strains of otsA and otsB completely recovered their ability
to synthesize trehalose; moreover, the trehalose concentration was higher than in the WT strain. The trehalose
concentrations of complemented strains of otsA and otsB
were 121.0 and 147.0 mg/l of each trehalose sample. In thisstudy, we found that Rsi PW1001 does not export trehalose
extracellularly because there was almost no trehalose in the
spent media (Supplementary Fig. 2).
Effects of trehalose synthesis on the expression of effector gene ripX and translocation of RipX. To investigate whether trehalose biosynthesis affects T3SS gene

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Fig. 2. The effect of exogenous trehalose in Ralstonia syzygii subsp. indonesiensis (Rsi)-induced hypersensitive response of eggplant
Solanum melongena cv. Senryo-nigou. (A) Leaves were infiltrated with the bacterial suspension (OD600 of 0.3) of Rsi PW1001 (WT),PW1005 (otsA), or PW1006 (otsB) and without exogenous trehalose or with trehalose at 0.25 mM (T1) and 2.5 mM (T2) final concentrations. Photographs were taken at 3 dpi from three independent experiments with similar results; representative results are shown. (B)The degree of ion leakage from leaves was measured at 3 dpi using a conductivity meter. Values are means ± SD of two replicates from
two independent experiments. Different letters indicate significant differences among the four inoculations as defined by Tukey’s multiple comparison test (P < 0.05).

Fig. 3. Trehalose concentration of trehalose samples preparedfrom bacterial cells that were cultured in minimal medium for 16
h. The trehalose concentration was measured using a Trehalose
Assay Kit. Values are means ± SD of four replicates. Different
letters indicate significant differences among the five strains as
defined by Tukey’s multiple comparison test (P < 0.05).

expression, the effector gene ripX expression was investigated as a representative T3SS gene. The expression of
ripX is known to be under the control of HrpB (Sun etal., 2020). We measured β-galactosidase activity of thebacterial strains possessing ripX::lacZ in BG and minimal
medium. As shown in Fig. 4, the β-galactosidase activity
in BG medium was lower than in minimal medium, suggesting that ripX gene expression was induced in minimal
medium. However, there was no significant difference inthe β-galatosidase activities between WT, otsA, and otsB

Fig. 4. Gene expression of ripX. The ripX gene expression in Ralstonia syzygii subsp. indonesiensis (Rsi) PW1079 (WT), PW1080(otsA), PW1081 (otsB), the complemented strains PW1082 (otsA
TnotsBA+) and PW1083 (otsB TnotsBA+). The ripX promoter was
fused to the lacZ gene, and the effect of trehalose biosynthesis on
b-galactosidase activity in Rsi PW1079 and its derivative strains
was examined. The b-galactosidase activities are indicated asMiller units. Values are means ± SD of four replicates from two
independent experiments. Different letters indicate significant
differences among the five strains as defined by Tukey’s multiple
comparison test (P < 0.05).

mutants, and their complemented strains. In addition, an effector translocation study using ripX::’cya construct in otsAand otsB mutants, and their complemented strains was conducted. The cAMP was low without ripX::’cya, but significant amount of cAMP was detected with ripX::’cya in all
strains. However, there is no significant difference among
the strains (Supplementary Fig. 3). These results indicate

Trehalose Required for HR by Ralstonia syzygii

575

Fig. 5. Trehalose biosynthesis is required for Ralstonia syzygii subsp. indonesiensis PW1001 osmotic stress tolerance, either in NaClor sucrose. (A) The bacterial growth in each strain in BG medium with/without 0.15 M NaCl at 36 h and 60 h incubation time. (B) The
bacterial growth in each strain in BG medium with/without 10% sucrose at 36 h and 60 h incubation time. Values are means ± SD of two
replicates from two independent experiments. Different letters indicate significant differences among different conditions of five strains
as defined by Tukey’s multiple comparison test (P < 0.05).

that expression of ripX and probably other T3SS genes and
RipX translocation to eggplant is likely independent on trehalose biosynthesis of Rsi.
Importance of trehalose synthesis for osmotic stress tolerance. To determine if trehalose biosynthesis contributes

to tolerance of osmotic stress in Rsi PW1001, we measured
the bacterial growth of WT, otsA and otsB mutants, andtheir complemented strains in BG medium supplemented
with/without 0.15 M NaCl or 10% sucrose at 27°C for 36 h
and 60 h. There was no significant difference in the bacterial growth between WT, otsA and otsB mutants, and theirFig. 6. Hypersensitive response (HR) on wild eggplant Solanum torvum (A, B) and tobacco Nicotiana
tabacum cv. Xanthi (C, D) by infiltration of Ralstonia
pseudosolanacearum RS1002 and its derivatives. (A)HR on wild eggplant leaf inoculated with RS1002 (WT),
RS1211 (hpaB), RS1273 (∆hrpY), RS1662 (∆ripAX2),
RS1736 (otsA), and 10 mM MgSO4 (mock) at 2 dpi.
(B) The degree of ion leakage from wild eggplantleaves was measured at 2 dpi. (C) HR on tobacco leaf
inoculated with R. pseudosolanacearum RS1002 (WT),
RS1211 (∆hpaB), RS1273 (∆hrpY), RS1736 (otsA),
and 10 mM MgSO4 (mock) at 3 dpi. (D) The degree ofion leakage from tobacco leaves was measured at 3 dpi.
Values are means ± SD of two replicates from three
independent experiments with similar results and representative results are shown. Different letters indicate
significant differences among the six (B) and five (D)inoculations as defined by Tukey’s multiple comparison test (P < 0.05).

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Laili et al.

complemented strains in BG medium without NaCl (Fig.
5A) nor sucrose (Fig. 5B). However, both mutant strains
had lower growth than WT under osmotic stress conditions, both with NaCl (Fig. 5A) or sucrose (Fig. 5B), andcomplemented strains partially with NaCl and almost completely with sucrose restored their growth. These results
indicate that Rsi PW1001 requires trehalose biosynthesis
for full osmotic stress tolerance.
Role of trehalose synthesis in HR induction in RS1002.
In order to investigate whether trehalose synthesis is required for HR induction in other strains of RSSC, we generated an otsA mutant in R. pseudosolanacearum RS1002
and confirmed that otsA mutant strain, RS1736 also significantly reduced the trehalose concentration compared to WT
RS1002 (Supplementary Fig. 4). However, we observedthat the RS1736, otsA mutant of RS1002 induced HR-like
WT at 2 dpi, both on wild eggplant S. torvum and tobacco N.
tabacum (Fig. 6). This result indicates that the involvementof trehalose synthesis for HR induction is not common in
RSSC.

Discussion
Requirement of trehalose of Rsi PW1001 in HR induction on eggplant. Trehalose is a non-reducing disaccharide
found in many organisms, including bacteria, fungi, insects, and plants, and plays a wide range of functional andstructural roles. Currently, there are five known trehalosebiosynthetic routes, and the OtsA/OtsB pathway is themost widespread pathway in prokaryotes and eukaryotes(Paul et al., 2008). This pathway produces the intermediate
T6P, which is an important key signal molecule in plantpathogenic bacteria interactions and affects many metabolic pathways, developmental processes, and stress responses(Lunn et al., 2014). Several studies also revealed the roleof T6P in regulation of carbon assimilation and the sugar
status in plants (Paul et al., 2008; Zhang et al., 2009).
Poueymiro et al. (2014) reported that R. solanacearum
encodes RipTPS, a type III-secreted effector that is a functional TPS, in addition to OtsA. RipTPS is translocated intoplant cells by T3SS, and elicits HR in N. tabacum. Thisfinding proposed speculation that the pathogen directly
induces T6P synthesis during the course of infection tosubvert plant cell metabolism through the endogenous T6P
concentration in the plant rather than to stimulate trehalosebiosynthesis. However, the involvement of RipTPS incompatible interactions was not revealed yet. In line withthis function of RipTPS, we found that ∆ripTPS mutant Rsi
PW1078 did not abolish HR and ion leakage on eggplantcompared to WT (Fig. 1C and D), indicating that RipTPS
is not avirulence factor in eggplant.
In this study, we provided evidence that trehalose biosynthesis in Rsi PW1001 is required for HR induction on
eggplant because mutation of otsA and otsB significantly
reduced HR level (Fig. 1). However, T3SS is indispensable for HR induction because the hrpB, hrcV, and ∆hpaB
mutants abolished HR. The hrpB, hrcV, and hpaB genes
are required for the expression T3SS genes, biogenesisof Hrp pili and the translocation of the T3Es into the hostcells (Asolkar and Ramesh, 2018; Lonjon et al., 2016;
Mukaihara et al., 2004). Thus, a T3SS-dependent effector
may induce HR. However, trehalose biosynthesis does notappear to affect T3SS gene the expression or T3Es translocation. The ripX is a representative effector gene in RSSC
and RipX induces HR on tobacco plants (Sun et al., 2020).
We observed that the β-galactosidase activities of all bacterial strains possessing ripX::lacZ were not significantly
different in a minimal medium, suggesting that trehalosebiosynthesis did not affect ripX expression. This finding
suggests that trehalose biosynthesis might have no effectfor hrpB-dependent effector gene expression. There might
be a novel effector present in Rsi strains to induce HR on
eggplant, whose expression is hrpB-independent and requires trehalose biosynthesis in Rsi. However, this effector
still requires Hrp-apparatus for its secretion and translocation.
Importance of intracellular trehalose in HR induction.
The addition of exogenous trehalose to the bacterial culture
did not affect the HR level or ion leakage in the infiltrated
eggplant leaves. Infiltration of trehalose alone also did not
induce HR and ion leakage (Fig. 2). In contrast, it is reported that application of exogenous trehalose induced resistance to the biotrophic fungus Blumeria graminis, whichcauses powdery mildew in wheat (Tayeh et al., 2014) andtriggers plant defense responses in citrus leaves (Piazza etal., 2015).
Based on the results, we conclude that only intracellulartrehalose was involved in HR induction of eggplant by
Rsi PW1001. This conclusion was further supported bymeasurement of the trehalose concentration in the bacterial cells. We observed low intracellular trehalose concentrations in the otsA and otsB mutants. This finding is inagreement with a previous study revealing the intracellular
localization of trehalose in R. solanacearum GMI1000(MacIntyre et al., 2020). In that study, they measured thetrehalose concentration by liquid chromatography‒tandem
mass spectrometry analysis, and trehalose was not detectedin spent cell media, suggesting that R. solanacearum does

Trehalose Required for HR by Ralstonia syzygii

not export trehalose extracellularly. Thus, trehalose biosynthesis-defective mutants in R. solanacerum GMI1000
showed reduced in planta multiplication in host tomato
plant and disease incidence (MacIntyre et al., 2020). Unlike this observation, although trehalose is required forvirulence toward Arabidopsis leaves, extracellular trehalose was important in P. aeruginosa PA14 (Djonović etal., 2013). The P. aeruginosa PA14 ∆treYZ∆treS mutant
reduced in planta growth, while application of exogenous
trehalose restored bacterial growth in a dose-dependent
manner (Djonović et al., 2013). The role of trehalose inplant-pathogen interactions appears to depend on the typeof pathogen.
Importance of trehalose biosynthesis for osmotic stress
tolerance. Trehalose accumulation in intracellular spaces
functions as a compatible solute to contend with osmotic
stress to an equilibrium at which water no longer moves
out of the cell into high osmolarity environments (Argüelles,
2000; Csonka, 1989). In line with this function, we foundthat the otsA and otsB mutants showed lower growth than
WT in BG medium supplemented with 0.15 M NaCl or
10% sucrose. This evidence suggests that trehalose has important role as an osmoprotectant. Compatible solutes liketrehalose could help this soilborne pathogen survive in an
osmotically stressful environment (van Elsas et al., 2001).
Rsi may experience hyperosmolarity in plant xylem vessels during infection. In this study, although we attempted
to generate deletion mutants of otsA and otsB using thesucrose-sensitive suicide vector pK18mobSacB (Schäfer etal., 1994), we obtained only WT strains after 10% sucrose
selection. This result suggests that high concentration of
sucrose could be an osmotic stress factor and affect themultiplication of trehalose-deficient strains.
Different role of trehalose biosynthesis in HR inductionof RSSC. In contrast to the effect of the HR phenotype ofthe otsA mutant Rsi PW1005 on eggplant S. melongena
cv. Senryo-nigou, RS1736, a mutant of otsA in RS1002
strain induced HR-like the WT on its non-host plants, wild
eggplant and tobacco plant (Fig. 6). Because otsA mutant
of RS1002 still induced HR on wild eggplant and tobacco,
trehalose biosynthesis is not required for HR induction,and defect of trehalose biosynthesis probably did not interfere T3SS gene expression. On the other hand, trehalosedefective mutants in Rsi reduced HR on eggplant, suggesting that the gene expression of particular effector that could
induce HR might be affected by trehalose biosynthesis.

577

Conflicts of Interest
No potential conflict of interest relevant to this article wasreported.

Acknowledgments
This work was supported in part by Joint Research Project
by Okayama Prefecture.

Electronic Supplementary Materials
Supplementary material is available at The Plant Pathology
Journal website (http://www.ppjonline.org/).

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