Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola

Journal Pre-proof Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola Ping-Chuan Zhu, Yi-Ming Li, Xia Yang, Hai-Fan Zou, Xiao-Lin Zhu, Xiang-Na Niu, Ling-Hui Xu, Wei Jiang, Sheng Huang, Ji-Liang Tang, Yong-Qiang He PII:

S0923-2508(19)30122-6

DOI:

https://doi.org/10.1016/j.resmic.2019.10.004

Reference:

RESMIC 3747

To appear in:

Research in Microbiology

Received Date: 23 June 2019 Revised Date:

17 October 2019

Accepted Date: 17 October 2019

Please cite this article as: P.-C. Zhu, Y.-M. Li, X. Yang, H.-F. Zou, X.-L. Zhu, X.-N. Niu, L.-H. Xu, W. Jiang, S. Huang, J.-L. Tang, Y.-Q. He, Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola, Research in Microbiologoy, https:// doi.org/10.1016/j.resmic.2019.10.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

1

Type VI secretion system is not required for virulence on rice

2

but for inter-bacterial competition in Xanthomonas oryzae pv.

3

oryzicola

4 5

Ping-Chuan Zhua, Yi-Ming Lia, Xia Yanga, Hai-Fan Zoua, Xiao-Lin Zhua, Xiang-Na Niua, Ling-Hui Xua, Wei Jianga, Sheng Huanga, Ji-Liang Tanga, *, Yong-Qiang Hea, b, *

6 7 8

a

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004, China b

9 10

National Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, 100 Daxue Road, Nanning Guangxi, 530004, China

11 12

*Corresponding author: ([email protected])

13

Abstract:

Yong-Qiang

He

([email protected])

or

Ji-Liang

Tang

14

The type VI secretion system (T6SS), a multifunctional protein secretion device,

15

plays very important roles in bacterial killing and/or virulence to eukaryotic cells.

16

Although T6SS genes have been found in many Xanthomonas species, the biological

17

function of T6SSs has not been elucidated in most xanthomonads. In this study, we

18

identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly

19

sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which

20

causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays

21

demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01

22

on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role

23

in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is

24

actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several

25

candidate T6SS effectors were predicted by bioinformatics analysis that might play a

26

role in the antibacterial activity of Xoc. This is the first report to investigate the type

27

VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play

28

an important role in inter-bacterial competition, allowing this plant pathogen to gain 1

29

niche advantage by killing other bacteria.

30

Keywords: Xanthomonas oryzea pv. oryzicola; type VI secretion system (T6SS);

31

bacterial competition; Hcp secretion; T6SS effectors

32 33

1. Introduction

34

The type VI secretion system (T6SS), widely distributed in the Gram-negative

35

Proteobacteria, is a contact-dependent protein secreting device that directly

36

translocates effector proteins to target other bacteria or eukaryotic cells, playing

37

multiple roles ranging from inter-bacterial relationships and biofilm formation, to

38

cytotoxicity and persistent survival [1-3]. Thirteen T6SS core proteins (TssA-TssM)

39

and a PAAR protein assemble into a bacteriophage tail-like structure, a dynamic

40

protein injection machine containing two distinct interacting subassemblies. Of the

41

T6SS core proteins, hemolysin-coregulated protein (Hcp), an important component of

42

T6SS tube and the chaperone for T6SS effectors, is considered as the hallmark of

43

T6SS. TssB and TssC form a contractile sheath, enclosing the Hcp hexamer ring tube.

44

At the top of the Hcp tube is a puncturing device consisting of trimeric VgrG spikes,

45

coated with PAAR protein [4]. The cytoplasmic portion of T6SS may dock with a

46

membrane complex (TssLMJ) by interacting with a phage-like structure [5]. The ClpV

47

ATPase binds and disintegrates the contracted sheath, thus resetting the system for

48

reassembly of an extended sheath that is ready to trigger again. T6SSs are usually

49

very modular and can accommodate different combinations of VgrG/PAAR proteins

50

to form tips. Effector proteins, associated noncovalently with the Hcp, VgrG or PAAR

51

subunits (cargo effectors) or occurring as additional domains on these proteins

52

(specialized effectors), are thus introduced and delivered through the formation of

53

tubular Hcp proteins into the target cells they kill. These T6SS effectors (T6SEs) are

54

all toxic to bacteria; to prevent self-intoxication, bacteria also encode T6SS effector

55

immunity proteins (T6SIs) that make them immune to T6SE toxins [6].

56

It has been estimated that about 25% of Proteobacteria encode at least one

57

T6SS system, including bacterial pathogens of humans, animals and plants, as well as 2

58

symbiotic, antagonistic and environmental bacteria [2, 7]. The functions of T6SSs

59

have been characterized in many bacteria, associated with virulence, host immunity

60

suppression, antibacterial and anti-eukaryotic activities [8]. Different bacterial species

61

and strains use their T6SS(s) for specific roles according to the niche and strategy of

62

the organism. In general, T6SS coding genes are clustered in the genomes, with the

63

obvious features of horizontal gene transfer [9]. Genomic and phylogenetic analysis

64

showed that the existing T6SS clusters could be classified into three types, type i, ii

65

and iii [10]. Type i system in turn contains six subtypes (i1, i2, i3, i4a, i4b and i5),

66

while the type ii class is uniquely populated by the Francisella pathogenicity

67

island-encoded system. The type iii class was identified in Bacteroidetes. In

68

plant-associated bacteria, T6SS clusters were phylogenetically grouped into five main

69

clades, group 1–5, based on the core component protein TssB [11], which is almost

70

equal to the corresponding relationship with type i system [10]. In many cases, more

71

than one T6SS gene clusters were found in a single bacterial genome, and they often

72

have different biological functions [3]. In some bacteria, one T6SS is used for

73

multiple roles, e.g., antibacterial and anti-eukaryotic, whereas in other cases different

74

T6SSs may play distinct roles, or only promote inter-bacterial competition [12]. Like

75

other bacterial secretion systems, the expression and activation of T6SS is closely

76

related to the niche conditions and the competitors to which bacteria are confronted,

77

to maintain optimal performance in suppressing opponents and energy consumption

78

[13]. It was found that the T6SS systems respond to various environmental conditions,

79

e.g. pH, temperature, ion concentration and cell density, and the cellular extracts of

80

their hosts or opponents. The expression of T6SS genes might be controlled by certain

81

regulators in some bacterial species [14]. Recently, a variety of T6SS-dependent

82

effectors and cognate immunity proteins have been described, including superfamilies

83

of antibacterial and anti-eukaryotic effectors [2], which further expanded our

84

understanding of the biological functions of T6SS system.

85

Although the functions of T6SSs are widely characterized in many animal

86

pathogens, symbionts and probiotics, their roles still remain unclear in many

87

phytobacterial pathogens [3, 11]. Xanthomonas is a genus of Proteobacteria, most of

88

which are important plant pathogens. It has been calculated that about 80% of 3

89

Xanthomonas species or strains with whole genome sequenced possess one to three

90

T6SS clusters, significantly higher than 25%, the ratio of Gram-negative bacteria

91

containing T6SS clusters [11]. However, with the exception of Xanthomonas citri [15],

92

there are no other reports about the functions of T6SS genes in Xanthomonas genus.

93

Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice bacterial leaf

94

streak (BLS), one of the most destructive diseases of rice (Oryza sativa L.), the

95

important staple food crop. Xoc can invade host leaves via stomata and wounds, and

96

colonizes intercellular spaces in the mesophyll, causing water-soaked interveinal

97

lesions that develop into translucent streaks. A variety of virulence factors or systems,

98

such as adhesins, extracellular polysaccharides (EPS), type II and III secretion

99

systems, quorum sensing (QS) system, have been identified successively [16, 17].

100

Recently, we identified two phylogenetically distinct T6SS gene clusters, T6SS1 and

101

T6SS2, in a newly sequenced Chinese strain GX01 of Xoc. To elucidate their

102

biological functions, we have constructed the single mutants of each T6SS clusters

103

and two hcp genes, and double mutants of two clusters and two hcp genes,

104

respectively. In this study, we describe the functional characterization of T6SS

105

systems in the important bacterial phytopathogen.

106

2. Materials and Methods

107

2.1. Bacterial strains, plasmids and growth conditions

108

Microbial strains, plasmids, and primers used in this study are listed in Table 1.

109

Xanthomonas oryzae pv. oryzicola strains were grown at 28

with shaking at 200

110

rpm in nutrient-rich medium NB (3 g/L beef extract, 1 g/L yeast extract, 5 g/L

111

polypeptone, and 10 g/L sucrose, pH7.0) or on nutrient agar (NA), and minimal

112

medium XOM3 (1.8 g/L D-xylose, 670 µM D, L-methionine, 10 µM sodium

113

L-glutamate, 240 µM NaFe2+-EDTA, 5 µM MgCl2, 14.7 mM KH2PO4, 40 µM MnSO4,

114

pH7.0). Ochrobactrum oryzae strains were grown at 30

115

the medium (3 g/L beef extract, 5 g/L polypeptone, 5 g/L NaCl, pH7.0). E. coli DH5α

116

strains were grown at 37

117

extract, 10 g/L polypeptone, 10 g/L NaCl, pH7.0). Yeast was cultured in liquid PYG

118

medium (20 g/L polypeptone, 10 g/L yeast extract, 20 g/L glucose). Antibiotics were

with shaking at 200 rpm in

with shaking at 200 rpm in this medium (5 g/L yeast

4

119

used at the following final concentrations: Tetracycline (Tc), 15 µg/ml, and

120

kanamycin (Km), 50 µg/ml, for E. coli; rifampicin (Rif), 50 µg/ml, Tc, 5 µg/ml, and

121

Km, 50 µg/ml, for Xoc strains.

122

2.2. Construction of Mutant Strains

123

To construct the deletion mutant of T6SS1 (XOCgx_2341 to XOCgx_2348)

124

(Figure 1), the region from 554 bp upstream to 666 bp downstream was amplified by

125

the

126

DMT6SS1-RF(X)/DMT6SS1-RR(H) (Supplementary Table S1), and fused into the

127

suicide plasmid pK18mobsacB to create the recombinant plasmid pKDT6SS1. The

128

plasmid pKDT6SS1 was transferred into the Xoc GX01 by triparental conjugation,

129

followed by the selection of colonies on NA plates which have no sucrose containing

130

Rif and Kan. The ∆T6SS1 deletion mutant was obtained by further selection on NA

131

supplemented with Rif and 10% sucrose. The mutants were then checked for Kan

132

sensitivity and were further confirmed by PCR. The confirmed mutant was named

133

∆T6SS1. ∆T6SS2 (XOCgx_3583 to XOCgx_3589) mutants were constructed using

134

the

135

DMT6SS2-RR(H) (Supplementary Table S1), in accordance with above method for

136

constructing ∆T6SS1. By introducing the recombinant plasmid pKDT6SS1 into

137

∆T6SS2, the double mutant ∆T6SS1∆T6SS2 was generated and confirmed by PCR.

primer

primer

pairs

pairs

DMT6SS1-LF(E)/DMT6SS1-LR(X)

DMT6SS2-LF(E)/DMT6SS2-LR(X)

and

and

DMT6SS2-RF(X)/

138

The deletion mutants ∆hcp1 (XOCgx_2343) and ∆hcp2 (XOCgx_3588) were

139

constructed using the above-mentioned method with ∆T6SS1 or ∆T6SS2, and the

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generation of double mutant ∆hcp1∆hcp2 (XOCgx_2343 and XOCgx_3588) also

141

followed the method for creating double mutant ∆T6SS1∆T6SS2. The mutants ∆hcp1,

142

∆hcp2 and ∆hcp1∆hcp2 were confirmed by PCR using primers listed in

143

Supplementary Table S1.

144

For complementation of the hcp2 mutant, a 716-bp DNA fragment containing the

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hcp2 coding region and extending from 200 bp upstream of the ORF was amplified

146

using the primer set C3588-F(H)/C3588-R(X) (Supplementary Table S1), and the

147

amplified DNA fragment was cloned into the plasmid pXUK (Table 1) to generate the 5

148

recombinant plasmid pXUKhcp2 (Table 1). The recombinant plasmid was transferred

149

into the deletion mutant of ∆hcp2 by triparental conjugation, resulting in the

150

complemented strain C∆hcp2, further confirmed by PCR (Table 1).

151

2.3. Phenotypic Characterization of the T6SS Mutant Strains

152

In order to assess the T6SS mutational effects in Xoc GX01 mutants, different

153

phenotypic assessments were determined and compared to the wild-type strain. The

154

growth of the strains was assayed both in rich and defined media. For the assessment

155

of EPS production, 2 µl of the cell suspension (OD600 = 0.1) of each strain was

156

pipetted onto NA with 2% sucrose. The plates were kept for 72 h at 28

157

measure the activity of extracellular protease, 2 µl of bacterial culture (OD600 = 1.0)

158

was spotted on NA plates containing 1% (m/v) skim milk powder. After incubation at

159

28

160

hydrolytic zones around bacterial colony [17]. The biofilm formation assay was

161

performed as follows. 100 µl of the cell suspension (OD600 =1.0) of each strain was

162

pipetted into 10 ml LB in universal glass bottles. After 4 days incubation at 28

163

steadily, the liquid medium in each bottle was removed gently. The bottles were rinsed

164

with water and stained with 10 ml of 1‰ crystal violet for 5 min, rinsed with water

165

until all unbound dye was removed. The dye was solubilized with acetic acid solution,

166

and the absorbance of each sample was determined at 630 nm using a

167

spectrophotometer. All of the assays were performed at least in triplicates.

168

2.4. Plant Assays

169

[17]. To

for 36 h, protease activity was measured and compared according to the

Virulence tests were carried out in a glasshouse at about 28

. In brief, Xoc

170

strains were cultivated in NB broth at 28

with appropriate antibiotics. Cell pellets

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were suspended in water, adjusted to OD600 = 0.5, and infiltrated into leaves of

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2-month-old rice, Oryza sativa L. ssp. japonica cv. Nipponbare, using needleless

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syringe. Disease lesion length was measured 14 days post-inoculation, at least twenty

174

leaves were inoculated for each Xoc strain.

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Hypersensitive response (HR) assays were performed by infiltration of bacterial

176

suspension into intact tobacco (Nicotiana benthamiana) leaves using a needleless 6

177

syringe. The bacterial suspensions of Xoc strains cultured in NB medium to the

178

logarithmic growth phase were diluted in 10 mM MgCl2 at an approximate OD600 of

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0.5 and spot-infiltrated into intact tobacco. The water soaking spots formed on the

180

leaves inoculated, i.e., the symptoms of HR elicited by Xoc strains, were observed at

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48 and 72 h after infiltration [17]. All trials were repeated at least three times.

182

2.5. Intercellular competition experiments.

183

In order to clearly show the intercellular competitions between Xoc strains and

184

their potential rivals, two broad host range plasmids carrying green fluorescence

185

protein gene (gfp) and mCherry gene, i.e., pVLacGreen and pKLacRed (Table 1),

186

were introduced into Xoc strains or their competitors by electroporation to construct

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fluorescence labelled bacterial strains (Table 1). Inter-bacterial competition assay. RFP-labelled Xoc strains (GX01-RFP,

188 189

∆hcp1-RFP,

∆hcp2-RFP,

∆hcp1∆hcp2-RFP,

∆T6SS1-RFP,

∆T6SS2-RFP,

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∆T6SS1∆T6SS2-RFP) and E. coli DH5α-GFP (Table 1) were cultivated to OD600 =

191

1.0, centrifuged, washed and resuspended to OD600 = 0.5. These Xoc cells were then

192

mixed with E. coli diluted at a 10:1 of Xac: E. coli ratio with 10 mM MgCl2. 4 µl

193

mixtures were spotted on NB plates and allowed to grow together as a co-culture at

194

30

195

SZX16 equipped with filters for GFP and RFP. The results of competition

196

experiments were quantified by determining the CFUs of Xoc and E. coli by dilutions

197

on selective medium plates, as the Xoc-RFP were resistant to rifampicin and

198

tetracycline but DH5α-GFP resistant to kanamycin. Competition experiments of Xoc

199

cells against Ochrobactrum oryzae MTCC 4195T (Table1) at 50:1 ratio with 10 mM

200

MgCl2 were performed as above and analyzed after 40 h of incubation. Finally, the

201

CFUs of MTCC 4195T-GFP were calculated by dilutions on kanamycin LA medium

202

plates [18].

for 40 hours. Fluorescence microscopy was carried out using an Olympus

203

Yeast competition assay. Xoc GX01and its mutant derivatives were cultivated in

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NB medium, and the NMY51 yeast was cultured in liquid PYG medium. Xoc and

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yeast cells were suspended in 10 mM MgCl2 and diluted to an OD600 of 0.5. After 40 7

206

hours of incubation at 30

, all of the cells in a well were suspended in 10 mM MgCl2

207

and the yeast cells were counted with Bürker cell counting chamber under microscope

208

using an Olympus SZX16. For each sample, six 0.04-mm squares with a 0.1-mm

209

depth were counted at least three repeats [22].

210

2.6. Quantitative RT-PCR analysis

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RNA was extracted from cells which in log-phase growth with the PureLinkTM

212

RNA Mini Kit (12183018A, Thermo Fisher, USA). We added 500 ng RNA sample to

213

produce cDNA with the reverse transcription-based reaction (00567293, RevertAid

214

First Strand cDNA Synthesis Kit, Thermo Fisher). Quantitative PCR (qPCR) was

215

performed using Fast SYBR Green Chemistry (Q411-02, Vazyme, China) according

216

to the manufacturer's instructions on an qTOWER2.0 Real-Time PCR System

217

(Analytik Jena AG, Germany). A three-step RT-PCR program (95

218

then 41 cycles of 95

219

specific primers (from BBI Life Sciences Corporation, China; listed in Supplementary

220

Table S1). Gene expression was analyzed according to 2-∆∆Ct methods, and 16S rRNA

221

was used as reference control [23].

222

2.7. Semi-quantitative RT-PCR

for 15 s and 60

for 5 min, and

for 60 s) was used for amplification with

223

RNA was reverse transcribed by the reverse transcription-based reaction

224

(Thermo Fisher), according to the manufacturer’s instruction. The cDNA was diluted

225

and used for semi-quantitative RT-PCR with specific primers (Supplementary Table

226

S1). Relative quantification of gene expression was performed using gapA gene as the

227

control.

228

2.8. Detection of Xoc T6SS secretion by Western blot

229

To assess the T6SS secretion of Xoc strains, a recombinant plasmid, named

230

pJXGhcp2 (Table 1), was constructed by cloning the hcp2-encoding ORF

231

(XOCgx_3588) without its stop codon into the vector pJXG [21]. In pJXGhcp2, the

232

3-end of the hcp2 ORF was fused to the 3× Flag-tag coding sequence to ensure the

233

expression of an Hcp2 protein with a 3× Flag-tag at its C-terminus. The recombinant

234

plasmid pJXGhcp2 was introduced into Xoc strains by triparental conjugation. The 8

235

recombinant strains obtained were grown to 0.8 at OD600, and harvested by

236

centrifugation at 4000 g for 30 min. The proteins in the cells were dissolved in lysis

237

buffer (8 M urea, 2 mM EDTA, 10 mM DTT, 1% cocktail proteinase inhibitor) on ice

238

with 30 min after washing twice with PBS, and ultrasonicating for 1 min, collecting

239

supernatants after centrifugation with 13000 rpm for 10 min. The proteins secreted in

240

the supernatant fraction were filtered with 0.45 µm filter paper, precipitated with

241

chilled 10% trichloroacetic acid (TCA)/ acetone at -20

242

acetone and resuspended in dissolution solution (8 M urea, 0.1 M triethylammonium

243

bicarbonate). For western blot, proteins were separated in 12% SDS-PAGE gel and

244

transferred to a polyvinylidene difluoride membrane (Roche) by electrophoresis. Then

245

covered with 5% skimmed milk at room temperature for 2 h and incubated with

246

primary antibodies (Pierce) at room temperature for 4 h. Finally, the membrane was

247

rinsed with TBST buffer (150 mM NaCl, 50 mM Tris, 0.05% Tween 20, pH7.6) three

248

times and incubated with a secondary antibody (Pierce) at room temperature for 2 h.

249

Signals were detected using Chemiluminescence immunoassay (Thermofisher) and

250

collected by gel imaging system.

251

2.9. Bioinformatic and Statistical Analysis

overnight, and washed with

252

T6SS Gene IDs of Xoc GX01 used in this study are from the new annotation

253

version of Xoc GX01 Genome (Table S2). Potential protein-coding sequences were

254

analyzed manually using BLAST suite of programs, including BLASTN, BLASTP,

255

BLASTX. The putative T6SS gene clusters were identified and aligned by using the

256

web-based tool VR profile [24]. MEGA6.0 was used for phylogenetic tree analysis,

257

GraphPad prism software version 5 was used for all the statistical analyses. The

258

prediction of T6SEs and T6SIs was conducted by using a combined method and

259

homology search (http://db-mml.sjtu.edu.cn /SecReT6/) [25]. Signal peptide was

260

detected by the SignalP program (http://www.cbs.dtu.dk/services/ SignalP/) [26].

261

Primers were designed using Vector NTI software. Circular maps of the chromosome

262

and plasmid were generated with CGView Server. T-test was used to determine the

263

statistical significance of differences between the least three biological replicates in an

264

experiment. ANOVA with p ≤ 0.05 was considered. 9

265 266

3. Results

267

3.1. Xoc GX01 encodes two phylogenetically distinct Type VI secretion systems

268

Genome sequence analysis revealed two T6SS clusters encoding T6SS apparatus

269

components on the chromosome of Xanthomonas oryzea pv. oryzicola GX01, named

270

T6SS1 and T6SS2, respectively. Table 2 lists the T6SS structural elements encoded in

271

both clusters. T6SS1 is 30.5 Kb containing 12 core T6SS genes, including hcp1 and

272

T6SS2 is 50.1 Kb containing 13 core genes, including hcp2. It was found that the

273

gene coding for the TssJ protein, which is part of the membrane complex [5], is

274

present only in T6SS2 cluster (Fig. 1, Table 2). Genetic organizations of the two T6SS

275

clusters of Xoc GX01 are not identical, and T6SS2 was interrupted by clusters of

276

conserved hypothetical genes and transposase genes, whereas homologs of tssB, tssC,

277

tssD (hcp), tssE, tssF, tssG and tssH are organized together in sytenic pattern between

278

T6SS clusters (Fig. 1C). Phylogenetic analysis showed that the T6SS1 has a long

279

evolutionary distance with T6SS2 (Fig. S1). The identities of the corresponding

280

proteins encoded within two clusters were not high, ranging from 13.1 to 50.8%

281

(Table 2). These observations indicate that the two clusters may have arisen from the

282

different origin, and not from a recent direct duplication.

283

Moreover, 17 T6SS genomic islands (GIs), were annotated on the chromosome

284

of Xoc GX01 and several vgrG orphan genes were found scattered on the

285

chromosome (Fig. 1, Table S2). Three genes (XOCgx_0030p, XOCgx_0031p and

286

XOCgx_0032p) on the indigenous plasmid of Xoc GX01 might encode proteins for

287

both T4SS and T6SS [27]. Obviously, several transposase genes were linked or

288

inserted in both T6SS clusters and all of the T6SS GIs, strongly suggesting that T6SS

289

genes in Xoc GX01 might have originated from later horizontal transfer events (Fig.

290

1).

291

3.2. Both T6SS1 and T6SS2 of GX01 are not required for virulence on rice

292

To elucidate the biological function of T6SS systems in Xoc GX01, we

293

constructed single mutants of each T6SS cluster and each hcp gene, and the double

294

mutants of two clusters and two hcp genes, using pK18mobsacB vector. The T6SS

295

mutants constructed from Xoc GX01 were confirmed by PCR, and named ∆T6SS1, 10

296

∆T6SS2, ∆hcp1 and ∆hcp2, representing the deletion mutants of each T6SS cluster or

297

each hcp gene, respectively. ∆T6SS1∆T6SS2 and ∆hcp1∆hcp2 refer to double

298

mutants of two clusters and two hcp genes, respectively (Table 1). No growth

299

difference was observed between the wild type and mutant strains in both rich and

300

minimal media (Fig. S3).

301

To analyze whether the T6SS system of Xoc GX01 plays a role in the adaptation

302

to adverse conditions, we have conducted a series of plate assays, including

303

morphologic, auxotrophic assays and tolerance tests [17]. There was no significant

304

difference between the wild type and any mutant strain of T6SS in growth,

305

phenotypes and tolerance to high osmotic pressure, heavy metal stress, and

306

antioxidant reactions and sodium dodecyl sulfate (SDS) lysis (data not shown).

307

To determine whether the T6SS system is involved in the pathogenicity of Xoc

308

GX01, we have carried out the assays of the production of major virulence factors of

309

Xoc strains, including extracellular protease ability, EPS production, motility and

310

biofilm formation, and the plant tests, including virulence test on rice host

311

(Nipponbare) and HR induction on tobacco (Nicotiana benthamiana). The results

312

showed that none of the virulence factors tested were influenced by mutations in any

313

T6SS cluster or hcp gene (Fig. 2A, B). Notably, after the mutants were inoculated into

314

rice leaves, they behaved as the wild type strain GX01, produced almost identical

315

disease symptoms during the experimental period (Fig. 2C). These results suggest that

316

T6SS system is not required for full virulence of Xoc GX01 on rice.

317

3.3. T6SS2 but not T6SS1 plays an important role in bacterial competition

318

To verify whether the T6SS confers a competitive advantage to Xoc in

319

intercellular competitions, a series of co-culture confrontation trials between Xoc

320

strains and other bacteria have been conducted in this study. When Xoc cells carrying

321

a plasmid for the expression of red fluorescent protein (RFP) , the RFP-labeled strains,

322

and E. coli DH5α cells expressing green fluorescent protein (GFP), the GFP-labeled

323

strains, were mixed and allowed to grow together after 40 hours of co-culture on agar

324

plates, we observed a significant increase in both the number and the size of the green

325

E. coli colonies zones in the green background of the ∆T6SS2, ∆T6SS1∆T6SS2,

326

∆hcp2 and ∆hcp1∆hcp2 strains when compared with that of the Xoc WT strain (Fig. 11

327

3A a, c, d, f, g), but there was no significant difference in fluorescence between

328

∆T6SS1, ∆hcp1 and the Xoc WT strain (Fig. 3A. b, e). Xoc competitiveness is

329

recovered when the Hcp2 protein is expressed in the ∆hcp2 mutant (Fig. 3A. h).

330

Quantitatively, after 40 h of co-culture, the Xoc/E. coli ratio was four orders of

331

magnitude lower in the treatments of ∆T6SS2, ∆T6SS1∆T6SS2, ∆hcp2 and

332

∆hcp1∆hcp2 strains than those of the wild type, ∆T6SS1, ∆hcp1 and C∆hcp2 strain

333

with pXUKhcp2 (Fig. 3B).

334

Xoc competitiveness against Ochrobactrum oryzae, the endophytic bacterium

335

isolated from deep-water rice [18], was also observed to be T6SS dependent in the

336

experiments (Fig. 3C) after 40 h of co-culture. We initially compared two groups,

337

∆T6SS2 and GX01, and the ∆T6SS2 mutant showed a significantly reduced capacity

338

for bacterial competition. We then compared ∆T6SS1, ∆T6SS1∆T6SS2, ∆hcp1,

339

∆hcp2 and ∆hcp1∆hcp2 strains (Fig. 3C). In the same way, we observed a significant

340

increase in the number of O. oryzae in the presence of ∆T6SS1∆T6SS2, ∆hcp2 and

341

∆hcp1∆hcp2 which showed a marked decline in competitive capacity, and the defect

342

was restored by pXUK3888. The results demonstrated that T6SS2 in Xoc GX01 was

343

predominantly involved in competition with E. coli DH5α and O. oryzae, the rice

344

endophytic bacterium.

345

3.4. Genes in T6SS1 of Xoc GX01 were inactive under various conditions

346

In our previous RNA-seq results of Xoc GX01 both in NB medium and in planta

347

[28], we found that most of the genes in T6SS1 cluster showed very low expression

348

level. In order to verify the expression status of T6SS genes in Xoc GX01, four genes

349

in each cluster encoding T6SS core components were selected as targets and gapA as

350

the control. The semi-quantitative PCR tests showed that there were detectable bands

351

of the four marker genes in T6SS2, but no detectable bands of the marker genes in

352

T6SS1, when the Xoc strains grew both in nutrient medium and minimal medium (Fig.

353

4), suggesting that T6SS1 was inactive when Xoc cultured in nutrient-rich and defined

354

condition. In addition, although we tried to use different temperature and pH

355

conditions, genes in T6SS1 remained inactive (Fig. S6, Fig. S7).

356

3.5. Hcp2 protein secretion is dependent on T6SS2 in Xoc GX01

357

Hcp release is dependent on the T6SS and is a reliable marker for assessing 12

358

functionality of the system [1]. To confirm the secreting function of T6SS in Xoc

359

GX01, we engineered Xoc strains producing a 3× Flag-tagged version of Hcp2 for

360

tracing Hcp protein. Hcp2 was readily detected in the supernatant of wild type

361

cultures but not in the T6SS2 mutant (Fig. 5), demonstrating that Hcp2 protein

362

secretion is dependent on T6SS2 in Xoc GX01, thus further establishing that the

363

T6SS2 is a functional secretion machine in the bacterium. We assume that the

364

antibacterial activity of GX01 might result from the secretion of T6SS effectors.

365

3.6. Bioinformatics analysis predicted several Xoc GX01 genes encoding candidate

366

T6SS effectors

367

To predict the candidate T6SS effectors in Xoc GX01, a genome-wide searching

368

was conducted based on combined methods and sequence homology searching using

369

BLASTP search (http://db-mml.sjtu.edu.cn /SecReT6/) [25], with identity ≥ 35% and

370

E-value < 10-10. The amino acid sequences of the collected proteins were checked

371

manually. Proteins with length less than 100 residues or containing a signal peptide

372

checked by the SignalP program were removed. To avoid inaccuracy of the data, we

373

also examined the length deviation between all hit query sequences and identified

374

protein sequences [29]. A total of 23 T6SE candidates and 9 T6SI candidates were

375

predicted in Xoc GX01 and listed in Table S3, most of which are Hcp, VgrG and

376

Rhs-repeat proteins. Some effectors are not only encoded in the T6SS main cluster but

377

also found in the T6SS GIs located outside the T6SS main clusters. Several vgrGs are

378

scattered on the T6SS gene cluster or orphan vgrG islands.

379

It has been proposed that effectors bind tightly to the VgrG structural element

380

[30]. Some genes linked with vgrG were also co-regulated with some T6SS

381

phylogenetic clusters, suggesting that these genes are also related to T6SS.

382

Interestingly, XOCgx_3596, associated with an orphan vgrG XOCgx_3595, encodes a

383

protein containing no signal peptide and contains a glycosidase domain, making it a

384

good candidate T6SE, which might be secreted through the T6SS and play a role in

385

degrading bacterial cell wall peptidoglycan. It is noteworthy that XOCgx_3597 was

386

located near the predicated effector XOCgx_3596 and they might encode

387

toxin/immunity pairs.

388 13

389

4. Discussion

390

Type VI secretion system (T6SS) is a versatile bacterial weapon which endows

391

its owner with advantages for survival in a variety of competitions, manifesting as

392

bacterial killing or/and eukaryotic toxicity [3]. A survey of available Xanthomonas

393

genomes in GenBank indicated that the majority of the Xanthomonas species encode

394

at least one T6SS system, and T6SS-related genes account for almost 1.20% of the

395

total genome of a given Xanthomonas strain. However, T6SS was only reported to be

396

required for mediating resistance of X. citri to the predation of a eukaryote

397

Dictyostelium discoideum so far [15]. In this study, we demonstrated that T6SS play

398

an anti-bacterial role in Xoc strain GX01. Considering the relatively high frequency of

399

T6SS genes in Xanthomonas, it is reasonable to anticipate that the biological

400

functions of T6SSs in this genus could be much more complicated.

401

Phylogenetic analysis indicated that T6SS clusters in Xanthomonads are grouped

402

into two clades, e.g., clade 3 and clade 4b in the T6SS classification of

403

plant-associated bacteria based on tssB nucleotide sequence [11]. T6SS1 and T6SS2

404

of Xoc GX01 belong to clade 3 and clade 4b, respectively. Genome annotation

405

predicted that 17 genomic islands in Xoc GX01 might to be related to T6SS system.

406

These observations suggest that the occurrences of T6SS systems in Xoc GX01 might

407

have arisen from horizontal gene transfer and not as the result of duplication, which is

408

in conformity with the inferences by Bernal et al. (2018). It was found that the clade 3

409

T6SS system (T6SS1 in Xoc GX01) is commonly present in Xanthomonas strains

410

which have T6SS genes and the clade 4b is only present in X. oryzae [11]. Our results

411

support the previous studies showing that T6SSs from different clades might have

412

diverse functions. B. thailandensis E264 contains six T6SS clusters [12], only one

413

cluster involved in host manipulation (T6SS-5). P. putida KT2440 is equipped with

414

three T6SSs [31], but only K1-T6SS is a potent antibacterial device. In our work, we

415

found that only T6SS2 has the anti-bacterial activity.

416

It is worth mentioning that clade 3 T6SS system, the homolog of T6SS1 of Xoc

417

GX01, is required for mediating resistance of X. citri to the predation of Dictyostelium

418

discoideum [15]. However, although we tested the mutant strains of T6SS1 or hcp1 in

419

various conditions, we found no significant difference with the wild type strain in 14

420

phenotypic traits, tolerance to adverse conditions and virulence on host plants. Genes

421

in T6SS1 cluster always remained inactive both in transcriptional tests and proteomic

422

assays, under various experimental conditions [28]. We performed semi-quantitative

423

PCR to confirm expression at the transcript level, and while hcp2 mRNA was clearly

424

visible, hcp1 mRNA could not be detected, suggesting that no hcp1 transcript was

425

present in Xoc GX01. Previously, it was reported that T6SS in many other bacteria

426

could be induced under certain conditions, such as temperature, pH, presence of chitin

427

or antibiotics, and their expression was controlled by certain regulators or quorum

428

sensing systems [32, 33]. In this study, we did not find suitable conditions for

429

induction of T6SS1. Thus, hcp1 may be a silent gene or it is expressed under

430

as-yet-uncharacterized conditions. Moreover, the expression of T6SS2, the functional

431

T6SS in Xoc GX01, was found to be slightly affected by these conditions.

432

T6SSs were initially reported as virulence factors and proven to be involved in

433

the pathogenesis in several pathogens [1]. Among phytopathogenic bacteria,

434

Pectobacterium atrosepticum, a pectolytic bacterium that produces soft rot in plants,

435

is one of the first plant pathogens whose T6SS activity was linked with virulence [34].

436

In R. solanacearum, a destructive plant pathogen of solanaceae plants having a wide

437

range of plant hosts, the virulence of a tssB mutant is reduced when compared with

438

the wild-type strain [35]. This mutation has a considerable effect on antibacterial

439

activity and biofilm formation, which may indirectly affect virulence. In order to

440

confirm the functions of T6SS with Xoc GX01, the T6SS strains were inoculated into

441

rice leaves to verify whether T6SS of Xoc GX01 plays a role in plant virulence. The

442

results revealed that T6SS plays no role in Xoc GX01 virulence. This is consistent

443

with the result in P. syringae pv. tomato DC3000, a plant-pathogenic bacterium found

444

in a wide variety of agricultural environments, in which T6SS does not significantly

445

contribute to virulence in the plant host [22]. Moreover, we found that there were no

446

significant differences in expression of major pathogenicity genes between T6SS

447

mutants and wild type strain (Fig. S6) or in hcp genes between pathogenicity gene

448

mutants and wild type strain (Fig. S7). Studies showed that T6SS has been also

449

related with biofilm formation in many other plant pathogenic bacteria such as

450

Acidovorax citrulli [36] and the non-pathogenic P. fluorescens MFE01 strain [37]. A 15

451

possible link between biofilm formation and T6SS has also been found in animal

452

pathogens such as P. aeruginosa [38]. However, in our study, mutations in T6SS

453

genes had no effect on biofilm formation, EPS production and extracellular protease

454

activity of the pathogen.

455

Growing evidence revealed that the primary function of the T6SS is as a device

456

for inter-bacterial competition [3]. In this study, we found that Xoc has a significant

457

competitive effect not only on the model organism E. coli, but also on a rice

458

endophytic bacterium Ochrobactrum oryzae [18]. Xoc mainly invades rice through the

459

stomata and wounds and colonizes intercellular space in the rice leaf mesophyll [16].

460

Our preliminary investigations showed that the rice leaf surface and interior inhabit

461

the complex multi-microbial groups, which maintain a fierce competitive environment

462

in epiphytic and endophytic growth of Xoc. We demonstrated that T6SS systems

463

might play important role in bacterial killing, but the competitive advantage of Xoc

464

with T6SS over the plant microbiota during infecting plant hosts need to be verified

465

further. Besides, the T6SSs promotes bacterial competition with eukaryotic microbes,

466

i.e., X. citri with Dictyostelium discoideum [15], P. syringae pv. tomato DC3000 with

467

yeast [22]. In our work, although we tested various conditions, we did not find any

468

evidence that T6SS of Xoc GX01 plays a role in competition with yeast.

469

Once the biological functions of T6SS are revealed, it is an important work to

470

discover and identify T6SS effectors (T6SEs) in certain bacteria, which is an

471

important basis to reveal the functional mechanism of T6SSs. T6SEs have been

472

identified by a variety of approaches, including bioinformatics analysis, genetic

473

analysis of T6SS-associated genes, proteomics-based method, and mutant library

474

screening [39]. Since T6SS effectors are all toxic to bacteria, and they are all located

475

near genes that code for proteins that give bacteria immunity to toxins that prevent

476

them from self-intoxication, suggesting that effectors and their cognate immunity

477

proteins are encoded from their adjacent genes [6]. Based on their enzymatic activity,

478

the known effectors can be divided into amidases (Tae), glycosyl hydrolases (Tge),

479

lipolytic enzymes (Tle) and nucleases (Tde) [40]. In this study, we predicted 21

480

candidates of the T6SE in Xoc GX01 based on a homology search [25], most of which

481

are Hcp, VgrG and Rhs-repeat proteins. Effectors are not only encoded in the T6SS 16

482

main clusters but also found in the other T6SS GIs, some of which were found near

483

vgrG [6]. Genes encoding toxin/immunity pairs were also found in this study,

484

suggesting that they are good candidates for T6SS effectors in our prediction.

485

In this study, we have demonstrated that a T6SS system plays an important role

486

in bacterial competition, allowing this plant pathogen to survive under conditions in

487

which it has to compete with other microorganisms for resources. This is the first

488

study to investigate the type VI secretion system in Xanthomonas oryzae. Our results

489

will serve useful reference and guidance of T6SS studies in phytopathogenic bacteria

490

in future.

491

Conflict of interest

492 493

The authors declare that they have no any conflict of interests. Acknowledgements

494

We thank Mr. Andrew Read (Plant Pathology and Plant-Microbe Biology Section,

495

School of Integrative Plant Science, Cornell University Ithaca, NY, USA) for

496

providing the broad host range plasmids, pVLacGreen. The work is supported by the

497

National Natural Science Foundation of China (31270139 and 31660505), the

498

National Key R&D Program of China (2018YFD02003), the Ba Gui Scholar Program

499

of Guangxi Zhuang Autonomous Region of China (2014A002), and the Natural

500

Science Foundation of Guangxi (2017GXNSFAA198310).

501

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502

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[40] Li M, Le Trong I, Carl MA, Larson ET, Chou S, De Leon JA, et al. Structural basis for type

604

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605

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606 607

Legend

608

Fig. 1. The locations of annotated T6SS genes and T6SS genomic islands on the

609

genomic map of Xoc GX01. (A) Circular map of the chromosome of Xoc GX01.

610

The internal circle shows the scale in Kb, with 0 representing the origin of

611

replication; the first ring from the interior denotes GC content; second circle denotes

612

GC skew (+) were green, skew (-) were purple; third circle and fourth circle indicate

613

forward genes and reverse genes, respectively; the outer circle shows the distribution

614

of the T6SS islands (red), T6SS1 and T6SS2 cluster (green) in the Xoc GX01

615

chromosome. (B) Circular map of plasmid pXOCgx01 in Xoc GX01. The circles from

616

inside to outside denote the same details as the circular map of the chromosome of

617

Xoc GX01; the outer circle shows predicted T6SS genes (yellow) in the GX01

618

plasmid. (C) Genomic organization of the GX01 T6SS1 and T6SS2 clusters. Different

619

colors represent different genes; red indicates hcp, blue indicates vgrG, and blackish

620

green stands for transposase gene; the gray indicates non-T6SS genes, like unknown

621

or hypothetal protein genes.

622

Fig. 2. Phenotypic characterization and virulence of the T6SS Mutant Strains. (A) The

623

EPS assays for T6SS mutants of Xoc GX01 strain. Two microliters of each bacterial

624

cell suspension (OD600 = 0.1) was pipetted onto NA with 2% sucrose in a Petri dish.

625

The plates were kept for 72 h at 28

626

of Xoc GX01 strains. 100 µL of each bacterial cell suspension (OD600 = 1.0) was

627

pipetted into 10 ml LB, and incubated 4 days at room temperature. (C) Virulence

628

assay of Xoc GX01 strains. The bacterial suspensions concentration of each Xoc strain

629

was adjusted to OD600 = 0.5 by bacteria free water. Bacterial suspensions were

630

infiltrated into 2-month-old rice leaves (Nipponbare), using needleless syringe on the

. (B) Biofilm formation assays for T6SS mutants

21

631

back of the main vein side, virulence length was measured 14 days after inoculation.

632

Fig. 3. Xoc cells competition with bacteria. (A) Xoc cells carrying a plasmid for the

633

expression of RFP (red) and E. coli DH5α cells expressing GFP (green) were mixed at

634

a 10:1 ratio, spotted on agar plates and allowed to grow together as a co-culture 40

635

hours. The images show fluorescence emitted from the resultant colonies. Xoc strains

636

used were: a wild-type (WT); b, ∆T6SS1; c, ∆T6SS2; d, ∆T6SS1∆T6SS2; e, ∆hcp1; f,

637

∆ hcp2; g, ∆hcp1∆ hcp2; h, C∆hcp2. (B) Ratio of the number of viable Xoc and E.

638

coli in experiments similar to those show in a-h growth after 0 and 40 h of co-culture.

639

Xoc WT, ∆T6SS1, C∆T6SS2, ∆T6SS1∆T6SS2, ∆hcp1, ∆ hcp2, ∆hcp1∆ hcp2 and

640

C∆hcp2 were used. The error bars represent the standard deviation of three

641

experiments. (C) Competition assays of Xoc strains against Ochrobactrum oryzae.

642

Xoc and Ochrobactrum oryzae cells expressing GFP (green) were mixed at a 50:1

643

ratio, spotted on agar plates and allowed to grow together as a co-culture 40 hours.

644

The graphs show the number of fluorescent colonies produced. Significance was

645

tested by Student’s T-test (*indicates significance at P < 0.05, **indicates significance

646

at P < 0.01). The error bars represent the standard deviation of three experiments.

647

Fig. 4. The expression of T6SS of Xoc GX01. Semi-quantitative PCR was used to

648

detect the expression of T6SS, with gapA as the reference gene, and samples were

649

cultured with NB medium. tssB, tssC, hcp and tssH genes code for contractile sheath,

650

tube sheath, landmark effector and ATPase respectively. (A) Samples were cultured in

651

NB medium. (B) Samples were cultured in XOM3 medium.

652

Fig. 5. Production and secretion of Hcp2 in the Xoc GX01 wild type and the ∆T6SS2

653

mutant strains. The Flag-tagged Hcp2 protein was detected by western blot analysis

654

using an anti-Flag antibody. Detection of the β-subunit of the RNA polymerase

655

(β-RNAP) was used as the control. The position of the molecular size marker (in kDa)

656

is indicated.

657 658

Table 1 Bacterial strains and plasmids used in this study.

659

Table 2 Lists the T6SS structural elements of both clusters and compares the amino 22

660

acid sequences of the components of Xoc GX01.

661 662

Supplementary Materials

663

Fig. S1. Phylogenetic distribution of T6SS clusters in typical stains. Maximum

664

likelihood tree with 1000 bootstrap replicates were built with Mega 6 for the core

665

component protein Hcp. A grey circle indicated T6SSs involved in interbacterial

666

competition whereas a grey pentagram represented systems involved in virulence.

667

And a grey triangle indicated the hcp of Xoc GX01, which highlighted them in red.

668

Fig. S2. PCR identification of mutants ∆hcp1, ∆hcp2 and ∆hcp1∆hcp2 constructed

669

from Xoc GX01. (A) Principle for identifying the four mutants by using primers

670

T1/T2, T3/T4, T5/T6 and T7/T8; (B) PCR identification of mutant strains. The

671

genomes used in the PCR were indicated above the lane. Primers T1/T2 (lane1, 3, 9,

672

13), T5/T6 (lane 5, 7, 11, 15), T3/T4 (lane 2, 4, 10, 14), T7/T8 (lane6, 8, 12, 16) were

673

used for identification.

674

Fig. S3. The growth of the wild type and T6SS mutant strains in NB medium（A）and

675

XOM3 medium (B).

676

Fig. S4. The expression of f hcp1 and hcp2 genes genes in Xoc Wild strain under

677

different pH.

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Fig. S5. The expression of f hcp1 and hcp2 genes genes in Xoc Wild strain under

679

different temperature.

680

Fig. S6. The expression of major pathogenic genes in Xoc strain.

681

Fig. S7. The expression of hcp1 and hcp2 genes in Xoc strain

682

Table S1 PCR Primers used for T6SS core genes.

683

Table S2 Predicted T6SS genes and T6SS islands in Xoc GX01.

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Table S3 T6SE candidates predicted in Xoc GX01 based on a homology search.

685

23

Table 1 Bacterial strains and plasmids used in this work. Strains or plasmids

Relevant characteristics

Source

Strains Xoc GX01

Xoc Chinese strain GX01, Rifr

[17]

∆hcp1

Deletion mutant of XOCgx_2343 (hcp1) gene in Xoc GX01, Rifr

This study

∆hcp2

Deletion mutant of XOCgx_3588 (hcp2) gene in Xoc GX01, Rifr

This study

∆hcp1∆ hcp2 ∆T6SS1

r

Double deletion mutant of hcp1 and hcp2 in Xoc GX01, Rif

This study

r

This study

r

Deletion mutant of cluster T6SS1 in Xoc GX01, Rif

∆T6SS2

Deletion mutant of cluster T6SS2 in Xoc GX01, Rif

This study

∆T6SS1∆T6SS2

Double deletion mutant of T6SS1and T6SS2 in Xoc GX01, Rifr

This study

GX01-RPF

GX01 carrying pKLacRed, Rifr, TCr

This study

∆hcp1-RPF ∆hcp2-RPF ∆hcp1∆hcp2--RPF ∆T6SS1-RPF ∆T6SS2-RPF

r

r

r

r

∆hcp1 carrying pKLacRed, Rif , TC ∆hcp2 carrying pKLacRed, Rif , TC

This study This study r

∆hcp1∆hcp2 carrying pKLacRed, Rif , TC r

r

r

r

∆T6SS1 carrying pKLacRed, Rif , TC ∆T6SS2 carrying pKLacRed, Rif , TC

r

This study This study This study

r

r

∆T6SS1∆T6SS2-RPF

∆T6SS1∆T6SS2 carrying pKLacRed, Rif , TC

This study

C∆hcp2

Complement strain, ∆hcp2 carrying pXUKhcp2, Rifr, Kanr r

This study

Deletion mutant of hrpG gene in Xoc GX01, Rif

Lab collection

∆rpfC

r

Deletion mutant of rpfC gene in Xoc GX01, Rif

Lab collection

∆rpfG

Deletion mutant of rpfG gene in Xoc GX01, Rifr

Lab collection

E. coli DH5α

Used for molecular cloning and competitive test

Lab collection

∆hrpG

r

DH5α-GFP

DH5α carrying pVLacGreen, Kan

This study

MTCC 4195T

Ochrobactrum oryzae wild type strain, used for competitive test r

MTCC 4195T-GFP

MTCC 4195T carrying pVLacGreen, Kan

NMY51

Yeast reporter strain, used for competitive test

[18] This study Lab collection

plasmids Suicide vector to recombination, Kanr

pKLacRed

A derivative of broad host range plasmid pKEB31 carrying mCherry gene under LacZ promoter, TCr

Lab collection

pVLacGreen

A derivative of broad host range plasmid carrying a gfp under LacZ promoter, Kanr

[20]

pRK2013

A help plasmid used in triparental conjugation, Kanr

Lab collection

pXUK

A broad host range plasmid with LacZ promoter, Kanr

Lab collection

pXUKhcp2

create

pXUK carrying hcp2 gene, Kan

mutant

r

by

double

crossover

[19]

pK18mobsacB

This study

pLAFRJ

A derivative of broad host range vector pLAFR3 containing the Lab collection multiple cloning sites of pUC19, Tcr

pJXG

pLAFRJ containing 3×Flag, Tcr

pJXGhcp2 a b

[21] r

pJXG containing ORF exclusive termination codon of hcp2, Tc

This work

Kanr, Rifr, and Tcr = Kanamycin-, Rifampicin-, and Tetracycline-resistant, respectively. ∆T6SS1, deletion from XOCgx_2341 to XOCgx_2348 genes; ∆T6SS2, deletion from XOCgx_3583 to XOCgx_3589

genes.

Table 2 Lists of the T6SS structural genes in both clusters and comparison of the amino acid sequences of the components of both T6SS systems in Xoc GX01a Unified T6SS nomenclature TssA TssB TssC TssD TssE TssF TssG TssH TssI TssJ TssK TssL TssM a

b

T6SS structural genes in T6SS1

T6SS structural genes inT6SS2

Identitiesb

Function

XOCgx_2363 XOCgx_2341 XOCgx_2342 XOCgx_2343 (hcp1) XOCgx_2345 XOCgx_2346 XOCgx_2347 XOCgx_2349 XOCgx_2353 XOCgx_2340 N* XOCgx_2356 XOCgx_2357 XOCgx_2358

XOCgx_3623 XOCgx_3590 XOCgx_3589 XOCgx_3588 (hcp2) XOCgx_3586 XOCgx_3585 XOCgx_3584 XOCgx_3583 XOCgx_3595 XOCgx_3581 XOCgx_3592 XOCgx_3593 XOCgx_3594 XOCgx_3623

26.60% 40.60% 49.00% 29.70% 27.70% 34.00% 33.90% 50.80% 18.80% 49.80% N/A 31.30% 27.00% 22.00%

Baseplate contractile sheath contractile sheath Hcp Tube Baseplate Baseplate Baseplate ATPase VgrG spike Membrane complex Baseplate Membrane complex Membrane complex

T6SS Gene IDs of Xoc GX01 used in this study are from the new annotation version of Xoc GX01 Genome (Table S2). The identities are the results of comparison of the amino acid sequences of the components of both T6SS systems in Xoc GX01.

* N indicate no presence of the gene.