Regulatory network of hrp gene expression in Xanthomonas oryzae pv. oryzae

REVIEW FOR THE 100TH ANNIVERSARY

Regulatory network of hrp gene expression in Xanthomonas oryzaepv. oryzae

Seiji Tsuge • Ayako Furutani • Yumi Ikawa

Received: 8 October 2013 / Accepted: 19 December 2013

� The Phytopathological Society of Japan and Springer Japan 2014

Abstract Like other plant-pathogenic bacteria, Xantho-

monas oryzae pv. oryzae, the causal agent of bacterial leaf

blight of rice, has hrp genes that are indispensable for its

virulence. The hrp genes are involved in the construction

of the type III secretion (T3S) apparatus, through which

dozens of virulence-related proteins, called effectors, are

directly secreted into plant cells to suppress and disturb

plant immune systems and/or induce plant susceptibility

genes. The expression of hrp genes is strictly regulated and

induced only in plants and in certain nutrient-poor media.

Two proteins, HrpG and HrpX, are known as key regula-

tors for hrp gene expression. Great efforts by many

researchers have revealed unexpectedly that, besides HrpG

and HrpX, many regulators are involved in this regulation,

some of which also regulate the expression of virulence-

related genes other than hrp. Moreover, it has been found

that HrpG and HrpX regulate not only hrp genes and

effector genes but also genes unrelated to the T3S system.

These findings suggest that the expression of the hrp

gene is orchestrally regulated with other virulence-related

genes by a complicated, sophisticated regulatory network

in X. oryzae pv. oryzae.

Keywords Xanthomonas oryzae pv. oryzae � hrp gene �Type III secretion system � Effector

Introduction

Plants possess immunity systems to protect themselves

from infection by microorganisms (Jones and Dangl 2006;

Schwessinger and Ronald 2012). Plant-pathogenic bacteria,

on the other hand, possess certain systems that overcome

the host immunity to invade plants and grow within. One of

the important systems of many Gram-negative, plant-

pathogenic bacteria is a protein secretion system, called the

type III secretion (T3S) system (Alfano and Collmer 1997;

Buttner and Bonas 2002). The system is conserved in both

animal and plant pathogens, and bacteria introduce viru-

lence-related proteins, so called effectors, directly into host

cell cytoplasm. As bacterial genome sequence data have

accumulated during the past several years, bioinformatic

and functional genomic studies have become possible and

revealed that plant-pathogenic bacteria generally secrete

dozens of effectors, which function to repress or disturb

plant immune systems and/or to increase the susceptibility

of host plants (Buttner and He 2009; Cunnac et al. 2004b;

Furutani et al. 2009; Mukaihara et al. 2010). In some cases,

effectors with an avirulence function are directly or indi-

rectly perceived by cognate plant factors and induce higher

resistance including the hypersensitive response (HR)

(Jones and Dangl 2006). Thus, the T3S system is an

important device in each bacterium both for virulence and

for inducing plant resistance.

The components of the T3S system are encoded by

clustered HR and pathogenicity (hrp) genes (Alfano and

Collmer 1997; Buttner and Bonas 2002). The cluster con-

sists of more than 20 genes on several transcription units,

and, beside the components of the T3S system, each gene

in the cluster encodes a regulator for the expression of

other hrp genes, an effector protein or a chaperone protein

for the translocation of effectors.

S. Tsuge (&) � Y. Ikawa

Laboratory of Plant Pathology, Kyoto Prefectural University,

Kyoto 606-8522, Japan

e-mail: [email protected]

A. Furutani

Gene Research Center, Ibaraki University, Inashiki 300-0393,

Japan

123

J Gen Plant Pathol

DOI 10.1007/s10327-014-0525-3

Xanthomonas oryzae pv. oryzae is the causal agent of

bacterial leaf blight of rice (Oryza sativa L.) (Vauterin

et al. 1995). The pathogen has been reported in all rice-

growing areas of the world (Aldrick et al. 1973; Jones et al.

1989; Lozano 1977; Ou 1985; Vauterin et al. 1995). The

bacterium enters its host through hydathodes or wounds

around the leaf edges, then multiplies in xylem vessels,

producing virulence factors such as extracellular polysac-

charides (EPS) (Watabe et al. 1993) and a general secretion

system through which extracellular hydrolytic enzymes

(e.g., cellulase and xylanase) are secreted (Miyazaki et al.

1976; Ray et al. 2000), eventually resulting in disease

symptoms (Mizukami and Wakimoto 1969; Ou 1985; Ta-

bei 1977). Besides these virulence factors, conserved hrp

genes are essential for virulence of X. oryzae pv. oryzae

(Kamdar et al. 1993; Ochiai et al. 2005; Oku et al. 2004).

The expression of hrp genes in plant-pathogenic bacteria

is highly regulated and is, generally, induced only in plants

and not in nutrient-rich media (Alfano and Collmer 1997;

Schulte and Bonas 1992a). Like other bacterial pathogens,

there are few chemicals to control X. oryzae pv. oryzae in

rice. As a novel control strategy, regulation of hrp gene

expression may be a promising target. Numerous studies

have been revealing that complex regulatory networks with

multiple regulators involved in the infection-specific

expression of hrp genes in plant-pathogenic bacteria. In

this review, we review the regulatory mechanisms of hrp

gene expression in X. oryzae pv. oryzae.

An in vitro hrp gene expression system for X. oryzae pv.

oryzae

As mentioned, the expression of hrp genes in plant-path-

ogenic bacteria is infection-specific and is not induced in

typical nutrient-rich complex media. However, the

expression is induced in certain nutrient-poor and low pH

synthetic media that mimic the apoplastic conditions in

plants. Such hrp-inducing media have been explored for

various plant-pathogenic bacteria and used effectively to

analyze regulatory mechanisms of hrp genes, secretion

mechanisms of effectors and so on (Arlat et al. 1991;

Huang et al. 1991; Marenda et al. 1998; Schulte and Bonas

1992b; Wengelnik et al. 1996a).

XOM2 is a hrp-inducing medium for X. oryzae pv.

oryzae, which contains 0.18 % sugar source, 670 lM

L-methionine, 10 mM sodium L(?)-glutamate monohydrate,

14.7 mM KH2PO4, 40 lM MnSO4, 240 lM Fe(III)-EDTA

and 5 mM MgCl2, adjusted to pH 6.0–6.5 (Furutani et al.

2003; Tsuge et al. 2002). A carbohydrate source (sugar

source) in the medium is likely to be one of the important

factors for hrp gene expression. Among several carbohy-

drates including glucose, sucrose and fructose that we

tested, xylose yielded the best results. Interestingly, Xiao

et al. (2007) showed that hrp genes in another rice pathogen

X. oryzae pv. oryzicola are also induced in xylose-con-

taining medium, while the fructose and sucrose-containing

medium is preferable for hrp expression in the tomato and

pepper pathogen X. campestris pv. vesicatoria (Schulte and

Bonas 1992b; Wengelnik et al. 1996a). Rice plant cell

walls are abundant in xylan, a polymer of xylose (Takeuchi

et al. 1994). Ray et al. (2000) found that X. oryzae pv.

oryzae mutants that are deficient in the general secretory

pathway and unable to secrete xylanase are also avirulent.

Moreover, we found that the X. oryzae pv. oryzae mutant

deficient in phosphoglucose isomerase, which is involved

in xylose utilization, is less virulent and that growth of

the mutant is delayed in plants (Tsuge et al. 2004). It is

likely that xylose is an important sugar for the growth of

X. oryzae pv. oryzae in rice leaves and that the bacterium

acquired the system that enables it to induce hrp gene

expression when xylose is available.

Key regulators of hrp gene expression, HrpG and HrpX

The hrp gene clusters of plant-pathogenic bacteria are divided

into two groups based on their regulatory systems, possession

of similar genes and operon structures (Alfano and Collmer

1997; Gophna et al. 2003). The hrp clusters of Pseudomonas

syringae, Pectobacterium carotovorum and Erwinia spp. are

in group I, and those of Xanthomonas spp. and Ralstonia

solanacearum are in group II. In P. syringae, three key hrp

regulators, HrpR, HrpS and HrpL are involved in the

expression of hrp genes (Hutcheson et al. 2001; Xiao et al.

1994). HrpS and HrpL are also present in E. amylovora (Wei

and Beer 1995). HrpR and HrpS share homology with r54

enhancer binding proteins (Xiao et al. 1994). Activation of

hrpL, encoding an alternative sigma factor, by HrpR and HrpS

leads to activation of all the other hrp genes (Wei and Beer

1995; Xiao et al. 1994). As a negative regulator, Lon protease

is involved in degradation of HrpR (Bretz et al. 2002).

In group II, two regulatory proteins play key roles in

common: HrpG and HrpX in Xanthomonas spp. Unlike

other plant-pathogenic bacteria, key hrp regulator genes,

hrpG and hrpX, are located apart from clustered hrp genes

in xanthomonads. The HrpG protein belongs to the OmpR

family of two-component signal transduction systems

(TCSTS), which are important devices to monitor and

respond to environmental stimuli in bacteria (Laub and

Goulian 2007; Stock et al. 2000; Wengelnik et al. 1996b,

1999), and regulates the expression of another hrp regu-

latory gene, hrpX, along with hrpA (hrcC), which encodes

a component of the T3S apparatus. Although the puta-

tive cognate sensor kinase for the TCSTS response regu-

lator HrpG, named HpaS, was recently identified in

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123

X. campestris pv. campestris (Li et al. 2013), the ortholog

is not found in X. oryzae pv. oryzae. The cognate sensor

kinase and the signal(s) that activates HrpG-containing

TCSTS remain unknown in X. oryzae pv. oryzae.

Another key hrp regulator in xanthomonads, HrpX

(named HrpB in R. solanacearum), belongs to the AraC

regulator family (Kamdar et al. 1993; Wengelnik and Bo-

nas 1996). The protein is indispensable for the expression

of five hrp operons (hrpB, hrpC, hrpD, hrpE and hrpF),

which leads to the construction of the T3S apparatus

essential for bacterial pathogenicity (Oku et al. 2004;

Wengelnik and Bonas 1996). Along with genes for the T3S

apparatus, HrpX regulates many T3S effector genes (As-

tua-Monge et al. 2000; Furutani et al. 2009; Noel et al.

2002). In X. camestris pv. vesicatoria, Koebnik et al.

(2006) reported that HrpX is the most downstream com-

ponent of the hrp regulatory cascade, which activates

the expression of its regulons by directly recognizing the

consensus sequence in each promoter region, called the

plant-inducible promoter box (PIP box; details are below).

Two cis elements in the promoter region of HrpX-

regulated genes

Generally, the promoter region of HrpX-regulated genes

(HrpX regulon) harbors a characteristic sequence for PIP

boxes (consensus: TTCGB-N15-TTCGB; B shows C, G or T).

The consensus sequence functions as the cis-acting regulatory

element controlling the expression of the respective gene

(Fenselau and Bonas 1995; Tsuge et al. 2005; Wengelnik and

Bonas 1996). Additionally, alignments of each promoter

region revealed that another consensus sequence that resem-

bles the -10 binding element of the RNA polymerase r70

factor (YANNRT: Y, C/T; N, A/T/G/C; R, A/G), called -10

box, is conserved 30 to 31 bp downstream of the PIP box

(Tsuge et al. 2005). Also in R. solanacearum, the PIP box

(called the hrpII box) and -10 box-like sequences are con-

served in the promoter region in genes regulated by HrpB, the

homolog of HrpX from xanthomonads (Cunnac et al. 2004a).

The functional similarity between HrpX from Xanthomonas

spp. and HrpB from R. solanacearum is shown by the

observation that the hrpX mutant of X. campestris pv. vesic-

atoria was partially complemented by the introduction of

R. solanacearum hrpB (Wengelnik and Bonas, 1996).

The ‘‘perfect’’ PIP box sequence, TTCGC-N15-TTCGC, is

found in the promoter region of HrpX-regulated hrp gene

operons (hrpB, hrpC, hrpD, hrpE) and hpa1, which is a hrp-

associated protein gene located next to hrp gene operons. We

investigated the flexibility of the two TTCGC sequences and

showed that the introduction of base substitution(s) in the

sequence of the hrpC operon did not always significantly

reduce promoter activity (Tsuge et al. 2005). Several base-

substituted PIP boxes, such as TTCGB-N15-TTCGB,

TRCGB-N15-TTCGB, TTCTB-N15-TTCGB, TTCGB-N15-

VTCGB, TTCGB-N15-TRCGB, TTCGB-N15-TTCHB (B,

C/G/T; H, A/C/T; R, A/G; V, A/C/G), conferred considerable

activity. The result prompted us to search the genome

sequence database of X. oryzae pv. oryzae MAFF311018, a

Japanese strain (Ochiai et al. 2005) for candidates of novel

HrpX-regulated genes preceded by both a perfect or imperfect

PIP box and a -10 box-like sequence in the putative promoter

region, which might include virulence-related genes such as

T3S effector genes. By promoter analyses, we revealed that 11

of the 17 candidates were actually expressed in an HrpX-

dependent manner (Furutani et al. 2006; Tsuge et al. 2005;

Table 1). These HrpX-regulated genes include not only T3S

effector genes but also T3S system-unrelated genes (see

below).

The gene hrpF, one of the HrpX-regulated genes, is

preceded by an ‘‘imperfect’’ PIP box, TTCGC-N8-TTCGC,

in the promoter region. Koebnik et al. (2006) revealed that

this type of imperfect PIP box can also be targeted by

HrpX. Besides hrpF, cysP2, which encodes a putative

cysteine protease, and kgtP, which encodes a-ketoglutarate

transport protein, are preceded by sequences TTCGC-N12-

TTCGC and TTCGA-N21-TTCGC, respectively, and their

expression was also shown to be HrpX dependent in

X. oryzae pv. oryzae (Furutani et al. 2006; Guo et al.

2012a). These findings suggest that the motif sequence of

the PIP box is more flexible than expected and that there

are more HrpX regulons than assumed in the genome of X.

oryzae pv. oryzae.

HrpX-regulated genes other than hrp genes

T3S effector genes

Although HrpX was initially identified as a regulator for

hrp genes, it is now known to regulate the expression of

various genes. T3S effector genes are important members

of the HrpX regulon. T3S effectors of X. oryzae pv. oryzae

can be divided into two groups: the so-called transcrip-

tional activator-like (TAL) effectors and the non-TAL

effectors. TAL effectors harbor a common feature with a

central repeat domain, where units of 34 amino acids are

repeated, nuclear localization signals and an C-terminal

transcriptional activation domain that functions in

eukaryotic (plant) cells, and they activate target plant genes

to increase plant susceptibility (for details see the following

reviews: Boch and Bonas 2010; Doyle et al. 2013). TAL

effectors are conserved in some Xanthomonas spp., and

they form a large group in X. oryzae pv. oryzae and

X. oryzae pv. oryzicola, which harbor up to 26 TAL

effector genes. Interestingly, TAL effector genes are not

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123

preceded by a set of a PIP box and a -10 box-like

sequence, and the expression is not regulated by HrpX.

Unlike TAL effectors, non-TAL effectors generally

have no structural similarity between each other. However,

they have common characteristic features in their N-ter-

minal amino acid compositions, and most non-TAL

effectors in X. oryzae pv. oryzae have at least 3 of fol-

lowing 4 criteria: (1) more than 20 % Ser and Pro residues,

(2) less than 6 % Leu residues in the first 50 aa residues, (3)

either 0 or 1 acidic amino acid residue (Asp or Glu) in the

first 12 residues, and (4) Leu, Ile, Val, or Pro at the third or

fourth residues (Furutani et al. 2009; Table 2). In X. oryzae

pv. oryzae MAFF311018, 20 non-TAL effectors have been

identified or predicted to be present so far, and some of

these are conserved among xanthomonads (Furutani et al.

2009, http://www.xanthomonas.org/t3e.html). Of these 20,

at least 17 effectors are confirmed to be expressed in a

HrpX-dependent manner, although some are not preceded

by a set of the PIP box and -10 box-like sequence

(Furutani et al. 2009; unpublished data, Table 2).

The characteristic N-terminal amino acid compositions

and hrp regulator-dependent expression of X. oryzae

pv. oryzae effectors are consistent with those of other

Table 1 HrpX-regulated gene candidates, which have a PIP box-like

and -10 box-like sequence, found in a search of the genome database

of Xanthomonas oryzae pv. oryzae MAFF311018

Gene

ID

ORF product

(putative)

PIP box (-like)

and -10 box-like

sequencea

Regulated

by HrpXb

0081 Hpa1 (XopA) TTCGC-N15-TTCGC-N31-TACTGT

Yes

0090 HrpB1 TTCGC-N15-TTCGC-N31-TAGCTT

Yes

0091 HrcU TTCGC-N15-TTCGC-N31-CACAAT

Yes

0094 HrcQ TTCGC-N15-TTCGC-N31-TACTTT

Yes

0067 Unknown TTCGC-N15-

TGCGG-N30-

CACCGT

No

0080 Hpa2 TTCGC-N15-

TTCGT-N31-

TATGTT

Yes


TGCGG-N30-

TAAATT

Yes

1551 TolA protein TTCGC-N15-

TTCCC-N31-

CAACGT

No

1669 T3S effector XopK TTCGT-N15-

TTCGT-N30-

CACCAT

Yes


TTCCG-N31-

CAAGAT

No

2732 Unknown TTCTG-N15-

TTCGT-N31-

CACTTT

No

2877 T3S effector XopU TTCGC-N15-

TTCGG-N31-

CAATGT

Yes


TTCGT-N30-

TATGGT

Yes

3557 ABC transporter

substrate binding

protein

TTCGC-N15-

TGCGC-N31-

CATGGT

No

3803 T3S effector XopV TTCGC-N15-

TTCTG-N30-

TACATT

Yes

3824 Anthranilate synthase

component I

TTCGC-N15-

TTCGC-N32-

TACAGT

No


TGCGG-N31-

TAGCAT

Yes

Table 1 continued

Gene

ID

ORF product

(putative)

PIP box (-like)

and -10 box-like

sequencea

Regulated

by HrpXb

4134 T3S effector XopR TTCGG-N15-

TTCGC-N30-

TACGAT

Yes

4208 T3S effector XopQ TTCGT-N15-

TTCAC-N31-

TAACGT

Yes

4259 ISXo1 transposase TTCGC-N15-

TTCAC-N30-

TACCAT

Yes

4367 Phosphatase precursor TTCGC-N15-

TGCGT-N32-

TAATAT

Yes

a Open reading frames preceded by a perfect/imperfect PIP box with

the -10 box-like sequence within 50–500 bp upstream of the putative

start codon were selected as HrpX regulon candidates. Consensus

sequence of a PIP box and -10 box-like sequence is TTCGC-N15-

TTCGC-N30–32-YANNRT (Y, C/T; N, A/T/G/C; R, A/G), and the

imperfect PIP boxes are as follows: TTCGB-N15-TTCGB, TRCGB-

N15-TTCGB, TTCTB-N15-TTCGB, TTCGB-N15-VTCGB, TTCGB-

N15-TRCGB, TTCGB-N15-TTCHB (B, C/G/T; H, A/C/T; R, A/G; V,

A/C/G). Underlines represent base substitutions that differ from the

perfect PIP boxb HrpX-dependent expression of each candidate was examined using

the GUS (b-glucuronidase) reporter (for details, see Furutani et al.

2006)

HrpX-regulated hrp/hpa genes are in bold letters

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http://www.xanthomonas.org/t3e.html

plant-pathogenic bacteria, such as R. solanacearum and P.

syringae strains (Cunnac et al. 2004a; Schechter et al.

2004; Tang et al. 2006).

T3S system-unrelated genes

When X. oryzae pv. oryzae was incubated in the hrp-

inducing medium XOM2, numerous proteins were detected

in the culture supernatant (Furutani et al. 2004). Many of

these proteins disappeared or decreased in amount when

incubated with the HrpX mutant. Interestingly, the secre-

tory protein profile of a mutant lacking the T3S apparatus

was almost similar to that of the wild-type strain except

that a small portion of the proteins was not present. To the

contrary, many of the HrpX-dependent secretory proteins

were either lacking or the quantity was lower than for the

wild type when the mutant deficient in the type II secretion

machinery was cultured in XOM2. By determining the

N-terminal amino acid sequence, we identified one of the

type II secretory proteins as a cysteine protease homolog,

CysP2. Nucleotide sequence analysis revealed that cysP2

has an imperfect PIP box in the promoter region, as

described already, and a deduced signal peptide sequence

at the N-terminus. HrpX-dependent expression of cysP2

Table 2 Type III secretion effectors of Xanthomonas oryzae pv. oryzae MAFF311018

Name (Gene ID) No. of Ser and Proa No. of Leub No. of Asp and Gluc aa PIP box and -10 box-like sequenced

Total Ser Pro 3rd 4th

AvrBs2 (XOO0148) 13 5 8 2 0 I G TTCGC-15-TTCGC-32-TACTGA-27-ATG

XopCe (XOO3221) 11 7 4 4 1 S R TTCGC-15-TACGC-31-CAGAAT-25-ATG

XopF (XOO0103) 13 8 5 3 1 L S TTCGT-15-TTCGC-32-AACAAT-58-ATGh

XopK (XOO1669) 4 1 3 5 0 L N TTCGT-15-TTCGT-30-CACCAT-134-ATG

XopL (XOO1662) 11 3 8 4 1 R V TTCGC-15-TTCGC-31-GATCAT-33-ATG

XopN (XOO0315) 14 8 6 3 0 P A TTCGG-15-TTCTG-31-TTCAAT-201-ATG

XopP (XOO3222) 12 6 6 3 1 R C TTCGT-15-TTCGC-32-TACTAA-294-ATG

XopQ (XOO4208) 13 4 9 3 0 P T TTCGT-15-TTCAC-31-TAACGT-187-ATG

XopR (XOO4134) 10 6 4 2 0 T N TTCGG-15-TTCGC-30-TACGAT-27-ATG

XopT (XOO2210) 12 5 7 5 0 P A TTCAG-15-TTCGC-31-TTCCAT-187-ATG

XopU (XOO2877) 11 3 8 7 1 A L TTCGC-15-TTCGG-31-CAATGT-154-ATG

XopV (XOO3803) 10 7 3 4 0 I S TTCGC-15-TTCTG-30-TACATT-29-ATG

XopW (XOO0037) 12 9 3 2 0 P S Not found

XopXf (XOO4042) 15 8 7 3 2 I Q TTCTG-15-TTCGC-31-GATCAT-53-ATG

XopY (XOO1488) 14 6 8 2 0 P V TTCGC-15-TTCGC-31-CATCCT-27-ATG

XopZ (XOO2402) 17 9 8 3 1 S G TTCTC-15-TTCGC-31-TATTGT-399-ATG

XopAAg (XOO2875) 10 5 5 1 0 I K Not found

XopAB (XOO3150) 13 6 7 3 0 R H Not found

XopAD (XOO4145) 10 4 6 3 0 T H Not found

XopAE (XOO0110) 12 5 7 6 0 N I Not found

Type III secretion effectors of X. oryzae pv. oryzae MAFF311018 are listed according to http://www.xanthomonas.org/t3e.html

T3S-dependent secretion and HrpX-dependent expression were confirmed for effector candidates other than XopC, XopAD and XopAE

(Furutani et al. 2009; unpublished data)a Number of Ser or Pro residues in the N-terminal 50 amino acidsb Number of Leu residues in the N-terminal 50 amino acidsc Number of Asp and Glu residues in the N-terminal 12 amino acidsd Sequences of each motif and number of nucleotide between them are showne Gene xopC probably starts from 141 bp downstream of the start codon indicated in the genome database, because the PIP box and the -10

box-like sequence are found downstream of the start codon indicated in the databasef Gene xopX probably starts from 81 bp downstream of the start codon indicated in the genome database, because the start codon in the database

is located between the PIP box and -10 box-like sequenceg Gene xopAA probably starts from 369 bp upstream of the start codon indicated in the genome database, which is the same with the homolog in

X. campestris pv. vesicatoriah Nucleotide number between -10 box-like sequence and the putative start codon of XOO0104 is shown because XOO0104 and xopF are likely

to be in the same operon

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was confirmed by reverse transcription-PCR analysis. Also

in X. axonopodis pv. citri, HrpX-dependent expression of

type II secretory protein genes are revealed (Guo et al.

2011; Wang et al. 2008; Yamazaki et al. 2008). Thus, the

hrp regulatory protein HrpX is involved in the expression

not only of hrp genes and T3S effector genes but also of

some type II secretory protein genes.

In addition, as shown previously, genes preceded by the

perfect and the imperfect PIP box with the -10 box-like

sequence are scattered in the genome of X. oryzae pv. oryzae,

and at least some portion of them is actually transcriptionally

regulated by HrpX (Table 1) (Furutani et al. 2006; Guo et al.

2012a; Tsuge et al. 2005). Furthermore, microarray analysis

comparing the HrpX mutant and the wild type revealed that

many genes are regulated by HrpX, although some regulation

might be indirect and some genes are not preceded by a set of

two cis-elements (unpublished data).

How the genes without cis-elements are regulated by

HrpX remains unknown. The regulation might be indirect

through unidentified HrpX-regulated transcriptional acti-

vators. Regardless, HrpX is likely to regulate a variety of

genes, though for the most part, they have not been func-

tionally unidentified. Many of the gene products probably

localize in the bacterial cytoplasm and function there

because they possess neither T3S effector-specific N-ter-

minal amino acid compositions nor the type II secretory

protein-specific signal peptide. Also in R. solanacearum,

HrpB regulates dozens of genes including virulence-related

but T3S system-unrelated genes (Mukaihara et al. 2004;

Occhialini et al. 2005). These findings suggest that,

although there have been few reports regarding the

importance of T3S system-unrelated HrpX-regulated genes

in bacterial virulence of X. oryzae pv. oryzae, HrpX reg-

ulates large sets of genes during infection.

Complicated regulatory network for hrp gene

expression

Involvement of novel transcriptional regulators Trh

and two H-NS proteins in the expression of hrpG

In addition to the two key regulators HrpG and HrpX,

several hrp regulatory proteins have been identified

(Fig. 1). Although the phosphorylation of HrpG, a pre-

dicted response regulator of a TCSTS, is one of the most

important key events for the activation of hrp genes, the

cognate sensor histidine kinase, which transmits the phos-

phoryl group to HrpG, has not been identified in X. oryzae

pv. oryzae.

It has been shown that not only the phosphorylation of

HrpG but also the transcriptional activation of hrpG is an

important factor for hrp gene expression. So far, several

proteins that are involved in the regulation of hrpG

expression have been identified. Trh (transcriptional regu-

lator for hrp) functions as an activator of hrpG expression

(Tsuge et al. 2006). In the trh mutant, the expression of

hrpG is lower than that in the wild type; as a result, the

expression of hrpX and the other hrp genes is also lower.

According to a domain analysis, Trh is a member of the

GntR transcriptional regulator family with a conserved

winged helix-turn-helix DNA-binding domain in the

N-terminus and with a ligand-binding domain in the

C-terminus (Hoskisson and Rigali 2009). Trh probably

regulates hrpG indirectly, because GntR proteins generally

function as repressors of gene expression although hrpG is

positively regulated by Trh. Which gene is the direct target

of Trh and which molecule binds to the C-terminal domain

of Trh are still unknown. Interestingly, An et al. (2011)

reported that, in X. campestris pv. campestris, the homolog

of Trh is not involved in hrpG expression; rather, another

GntR family protein, named HpaR1, is negatively

involved. Although the function of the HpaR1 homolog in

X. oryzae pv. oryzae is unclear, at least the involvement of

Trh in hrp gene expression differs between the two Xan-

thomonas species.

The histone-like nucleoid-structuring (H-NS) proteins are

small DNA-binding proteins, which are widely conserved in

Gram-negative bacteria (Dorman 2004; Fang and Rimsky

2008). The protein is an important global regulator, usually

as a repressor of transcription, and regulates a wide range of

Fig. 1 Model of the hrp regulatory network in Xanthomonas oryzae

pv. oryzae. Bold lines indicate the main cascade of hrp gene

expression. Regulatory steps in the model are at the transcriptional

levels except for the phosphorylation of HrpG and hydrolysis of

cyclic di-GMP by RpfC/G. Note that regulators associated with hrp

gene expression are also involved in expression of genes other than

hrp. Grey lines and letters for RpfC/G- and Clp-mediated regulation

of hrp gene expression have been reported for X. campestris pv.

campestris (He et al. 2007; Huang et al. 2009); they have not yet been

reported for X. oryzae pv. oryzae

J Gen Plant Pathol

123

genes including virulence-related genes and environment-

responsive genes. X. oryzae pv. oryzae harbors three h-ns

genes. Feng et al. (2009) revealed that one of the H-NS

proteins, named XrvA, activates hrpG. To the contrary, we

showed that another H-NS protein, XrvB, negatively regu-

lates hrpG expression; that is, in the XrvB mutant, a high

level of the hrpG transcript accumulates followed by acti-

vation of the expression of other hrp genes (Kametani-Ikawa

et al. 2011). A gel retardation assay using XrvB protein

produced by the Escherichia coli protein expression system

indicated that XrvB has DNA-binding activity, but without a

preference for the promoter region of hrpG, suggesting that

an unknown factor(s) mediates the regulation of the hrpG

expression by XrvB. Also, the regulation of XrvA in hrpG

expression may be indirect based on the fact that H-NS

proteins generally function as repressors for target genes.

Microarray analyses using the Trh mutant and XrvB

mutant revealed that both proteins function as global reg-

ulators and are involved in the expression of a variety of

genes, not only hrp-related genes (unpublished data).

Interactions between bacterial cell density sensing

systems and the regulation of hrp gene expression

Lee et al. (2008) reported that a TCSTS, PhoP/PhoQ, is

involved in hrpG expression in X. oryzae pv. oryzae in

response to low Ca2? concentrations. A mutant deficient in

the system has decreased hrpG expression followed by

decreased expression of other hrp genes under the low

Ca2? condition, and also has reduced virulence. Interest-

ingly, the expression of phoP and phoQ is negatively

regulated by another TCSTS, RaxR/RaxH. The system is

required for cell density-dependent production of Ax21, a

quorum-sensing signal molecule of the bacterium (Burd-

man et al. 2004; Lee et al. 2006). The PhoP/PhoQ system is

involved in the expression of genes other than hrp genes in

response to low Ca2? and Mg2? concentrations, such as

corA1, a putative Mg2? transporter, groEL and dnaK,

genes involved in protein folding, cell proliferation/sur-

vival, and self regulation. And the system is required for

resistance to antimicrobial peptides and tolerance to an

acidic environment, which are conditions that X. oryzae pv.

oryzae likely confronts in rice plants (Lee et al. 2008). The

PhoP/PhoQ system plays crucial roles for bacterial survival

and virulence when the bacterium enters into host plants.

In X. campestris pv. campestris, another TCSTS RpfC/

RpfG plays a key role in bacterial virulence (Tang et al.

1991). The system perceives and responds to the cell–cell

signaling molecule DSF (Diffusible Signaling Factor) to

regulate the synthesis of virulence factors such as extra-

cellular enzymes, biofilm structure and motility (Barber

et al. 1997; Dow et al. 2003; Ryan et al. 2007; Slater et al.

2000). The response regulator RpfG contains an HD-GYP

domain and is suggested to function as a di-GMP phos-

phodiesterase, which mediates hydrolysis of the bacterial

second messenger cyclic di-GMP, resulting in the regula-

tion of various cellular functions including expression of

virulence-related genes (Dow et al. 2006; Ryan et al.

2006). Cyclic di-GMP negatively regulates the expression

of a global transcriptional activator protein Clp (catabolite

activator protein or cAMP receptor protein-like protein)

(Ge and He 2008; He et al. 2007; Hsiao et al. 2005, 2009).

Interestingly, Clp is also shown to be involved in hrp gene

expression via two Clp-regulating transcription factors:

FhrR containing a TetR family transcription factor domain

and a zinc uptake regulator Zur belonging to the Fur family

of transcription factors (He et al. 2007; Huang et al. 2009).

In spite of the lack of direct evidence, in X. oryzae pv.

oryzae, the RpfC/RpfG- and Clp-mediated, cell density-

dependent gene expression may also affect the expression

of hrp genes. In support of this idea, the novel TCSTS

PdeK/PdeR, which regulates cyclic-di-GMP turnover, was

found by Yang et al. (2012) to be involved in the expres-

sion of hrpG and hrpX and in the production of EPS in

X. oryzae pv. oryzae.

Relationships between carbohydrate metabolisms

and the regulation of hrp gene expression

As described earlier in this review, carbohydrate metabo-

lism and hrp gene regulation in Xanthomonas species are

likely correlated with each other, because hrp gene

expression in each bacterium is dependent on sugar sour-

ces, such as xylose for X. oryzae pv. oryzae (Tsuge et al.

2002) and X. oryzae pv. oryzicola (Xiao et al. 2007), and

sucrose and fructose for X. campestris pv. vesicatoria

(Schulte and Bonas 1992b; Wengelnik et al. 1996a).

Recently fructose-bisphosphate aldolase (FbaB), which

reversibly converts fructose-1,6-bisphosphate to dihy-

droxyacetone phosphate and glyceraldehydes-3-phosphate

and is essential for glycolysis and gluconeogenesis, has

been shown to be involved not only in carbon metabolism

but also in EPS production, virulence and hrp gene

expression in X. oryzae pv. oryzicola (Guo et al. 2012b).

Compared with the wild-type strain, the FbaB deletion

mutant is less virulent on rice, grows slower and produces

less EPS in a medium with fructose, pyruvate or malate as

the sole sugar source. And the expression of hrpG and hrpX

is repressed by the deletion of FbaB. Interestingly, the

expression of some hrp genes, such as hrcC, hrpE and

hpa3, is higher in the FbaB mutant in contrast to the

expression of hrpG and hrpX. In addition, the expression of

fbaB itself is regulated by HrpG and HrpX. Guo et al.

(2012b) considered that an unknown regulator(s) may

mediate the relationships between FbaB function and hrp

gene expression.

J Gen Plant Pathol

123

HrpG- and HrpX-independent regulation of hrp gene

expression

In addition to FbaB-mediated regulation of hrcC, hrpE and

hpa3 expression in X. oryzae pv. oryzicola (shown above),

HrpG- and HrpX-independent regulation of hrp gene

expression is reported also in X. campestris pv. campestris.

Zhang et al. (2008) reported that the mutant deficient in the

TCSTS ColRXC1049/ColSXC1050 shows low expression of

hrpC and hrpE operon but similar expression of hrpG and

hrpX to the wild type. The TCSTS is also responsible for

bacterial growth both in media and in plants, virulence,

hypersensitive response and stress tolerance. Moreover, in

X. campestris pv. campestris, the involvement of the rsmA

(repressor of secondary metabolism)-like gene, which

presumably encodes an RNA-binding protein playing

important roles as a global post-transcriptional regulator in

various cellular processes, in hrp gene expression has been

shown (Chao et al. 2008). Deletion of the gene reduces the

expression of hrpA to hrpF operons HrpG- and HrpX-

independently. Based on these results shown above, not

only HrpX but also multiple regulators are likely to be

involved in the expression of each hrp operons in xan-

thomonads, probably including X. oryzae pv. oryzae.

Concluding remarks

Since the two key hrp regulators, HrpG and HrpX, were

first identified, intensive research in the last two decades

has revealed the complicated regulatory network with

unexpectedly numerous regulators of hrp gene expression

of X. oryzae pv. oryzae (Fig. 1). The regulators, acting via

the key hrp regulators HrpG and HrpX, also regulate other

virulence-related genes. In R. solanacearum, a global

virulence regulator PhcA, which activates expression of

genes involved in motility, plant cell wall degradation,

and EPS synthesis, negatively regulates the expression of

hrp genes in a quorum sensing-dependent manner (Genin

and Denny 2012; Genin et al. 2005; Yoshimochi et al.

2009). Based on these findings, the T3S system of

R. solanacearum is thought to be important for bacteria to

overcome the plant immunity and to establish colonization

during the early steps of plant infection when the bacterial

population is low, then when the population reaches a

high cell density in the host plant, EPS, cell wall degra-

dation enzymes and other virulence factors are massively

produced with repressing hrp gene expression. Also in

xanthomonads, cell density-related regulation of hrp gene

expression via TCSTSs has been shown as described

above: PhoP/PhoQ and RaxR/RaxH in X. oryzae pv.

oryzae and RpfC/RpfG-mediating Clp in X. campestris pv.

campestris. The cell density-sensing switch may be one of

the main factors for hrp gene expression in X. oryzae pv.

oryzae.

The phosphorylation of HrpG, which is probably a

response regulator of the TCSTS, by the cognate sensor

histidine kinase is another main factor for hrp gene

expression, but the counterpart of HrpG in X. oryzae pv.

oryzae remains unclear. Moreover, the environmental sig-

nals that activate the HrpG-containing TCSTS are com-

pletely unknown. Further study is required to clarify the

initial and important step of hrp gene expression.

HrpX-regulated genes are not limited to hrp and T3S

effector genes. Moreover, some reports show that HrpG

regulates a large number of genes in HrpX- and HrpB-

independent manners in Xanthomonas spp. and R. solan-

acearum, respectively (Guo et al. 2011; Noel et al. 2001,

Valls et al. 2006). Thus, the hrp regulatory cascade is likely

to closely interact with other virulence-related gene regu-

lations, and the complicated and sophisticated network that

orchestrally regulates a variety of genes may enable bac-

teria to overcome plant immune systems to invade and

grow in their host plants.

Acknowledgments We are grateful to Drs. Hirokazu Ochiai

(National Institute of Agrobiological Sciences), Takashi Oku (Pre-

fectural University of Hiroshima), Kazunori Tsuno (Miyazaki Uni-

versity), Yasuhiro Inoue (National Agricultural Research Center),

Kouhei Ohnishi (Kochi University), Yasufumi Hikichi (Kochi Uni-

versity) and many graduate and undergraduate students for their

collaboration. We are supported by Grants-in-Aid for Scientific

Research from the Ministry of Education, Science, Sports and Cul-

ture, Japan.

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Regulatory network of hrp gene expression in Xanthomonas oryzae pv. oryzae

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