# Type IV Secretion System Is Not Involved in Infection Process in Citrus

Authors: Tiago Rinaldi Jacob; Marcelo Luiz de Laia; Leandro Marcio Moreira; Janaína Fernandes Gonçalves; Flavia Maria de Souza Carvalho; Maria Inês Tiraboschi Ferro; Jesus Aparecido Ferro
Journal: International Journal of Microbiology
 (2014)

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## Abstract

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA
substrates across the cell envelope and into target cells.Xanthomonas citri subsp. citri contains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS in Xcc, we analyzed, in vitro and in planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.


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## Body

## 1. Introduction

Xanthomonas citri subsp. citri (Xcc) is a Gram-negative plant pathogenic bacteria that causes severe disease in many economically important citrus plants [1]. The citrus canker induced by Xcc is a destructive disease characterized by canker lesions on leaves, stems, and fruits; furthermore the pathogen induces defoliation, which results in reduced yield and premature fruit drop [2]. Control is difficult in areas where the disease is already established, and plant eradication is the only effective way to control and prevent the disease spread. Recurrent and severe attacks of the pathogen are responsible for serious economic losses in citrus groves around the world [2].The focus of the present study is aimed at understanding the role of bacterial secretory systems, specifically the type IV secretory system (T4SS). Seven secretory systems are known and described in prokaryotic organisms, and each one is related to a physiological process [3–5]. Of these, the best studied is the type III secretory system (T3SS), which enables bacterial pathogens to deliver effector proteins into eukaryotic cells [6]. Some bacterial pathogens, including species from Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia, and Yersinia, depend on the T3SS to induce damage to the host. In contrast, other organisms including Agrobacterium tumefaciens, Helicobacter pylori, and Legionella pneumophila depend on the type IV secretory system (T4SS) for virulence induction [7].The T3SS contains at least twenty distinct proteins, which are subdivided into three parts. The basal body of the system [8–11] is associated with an ATPase and likely facilitates the entry of substrates into the secretion apparatus [12]. This basal body is also associated with a protein needle (the second part of the T3SS), which binds the bacterium to host cells and acts as a conduit for effectors secretion. The third part of the T3SS is composed of three proteins that are exported through the bacterial needle and form a pore on the surface of the host cell, which facilitates the export of toxins into the target cytoplasm [13].On the other hand, the T4SS is a multiprotein complex that consists of a protein channel (encoded byvirB and virD) through which proteins or protein-DNA complexes can be translocated between bacteria (cell-to-cell communication) and into host cells [14]. Translocation is driven by a number of cytoplasmic ATPases that potentially energize large conformational changes in the translocation complex [15]. There are three functional types of T4SS. The first type, found in many Gram-positive and Gram-negative bacteria and some Archaea, functions in conjugation and in the transfer of T-DNA into plant cells by A. tumefaciens. A second type of T4SS mediates DNA uptake in the transformation process (found in H. pylori). A third type of T4SS is used to transfer toxic effector proteins or protein complexes into the cytoplasm of host cells (found in Bordetella pertussis, Legionella pneumophila, Pseudomonas aeruginosa, and Xcc) [15].Substrates transported by the T4SS modulate various cellular processes including apoptosis, vesicular traffic, and ubiquitination; furthermore, the number of type IV effector proteins continues to increase [16–19]. However, most of these substrates have not been functionally characterized, and their role in bacterial pathogenesis remains unknown [20].In many bacteria, these two secretory systems types are well characterized; however, inXanthomonas spp. only the T3SS has been intensively studied [1], in contrast to the limited number of studies involving the T4SS [21, 22]. In Xcc the T4SS deserves more attention, especially since the genome contains two copies of this system (chromosomal and plasmid-borne) [23]. The plasmid copy is homologous to others Xanthomonads that contain the T4SS on extrachromosomal DNA. However, only Xcc and Xanthomonas campestris contain the chromosomal copy of the T4SS.To understand the conditions that stimulate expression of T4SS genes inXcc we used qPCR to analyze the in vitro and in planta expression of 18 ORFs from the T4SS (both chromosomal and plasmid copies) and 7 hypothetical ORFs flanking the T4SS. To validate the qPCR data, we compared expression of 29 ORFs from the T3SS, which are known to be expressed in planta.Our results showed that the T4SS is not induced during the infection process inXcc, but may be very important in cell-to-cell communication.


## 2. Materials and Methods

### 2.1. Microbiological Procedures

Xcc strain 306 was previously sequenced by da Silva et al. [23]. Xcc was maintained in sterile tap water and grown on nutrient agar (NA) medium containing 3 g beef extract, 5 g peptone, and 15 g agar in 1 L of distilled water at 28°C. After 24 h, three single colonies were transferred to new NA plates and incubated for 12 h at 28°C. These Xcc solid cultures were used as pre-inoculum for in planta and in vitro studies. For in planta studies, pre-inoculum cultures were aseptically transferred to sterile flasks containing 50 mL of nutrient broth (NB) and the bacterial concentration was adjusted to 108 CFU/mL (OD600 = 0.3). This bacterial suspension was taken up in a 1 mL needleless syringe and used to infiltrate orange leaves (Citrus sinensis cv. pera) grown in 20 L capacity pots. A total of nine plants were inoculated (20 leaves per plant) and maintained for 72 h in a growth room at 28°C, with a 12 h photoperiod and light intensity ~2,000 lux. Three plants were used for each incubation period. After multiplication, inoculated leaves were collected, sliced into thin strips with a razor blade, and placed in a beakers with sterile distilled water in an ice bath with gentle agitation. Samples from each plant were placed in separate beakers for bacterial exudation. After 5 min, leaf debris was removed by filtration through gauze, and bacterial cells were recovered by centrifugation at 5,000 ×g for 5 min at 4°C. Total RNA was extracted immediately, as described below. For in vitro studies, 1 mL of each Xcc suspension at 108 CFU/mL were aseptically transferred to three Erlenmeyer flasks and incubated for 12 h at 28°C with shaking (200 rpm). After incubation, bacterial cells were harvested by centrifugation at 5,000 ×g for 5 min. Total RNA was extracted as described below. Thus, we obtained three independent biological replicates for Xccgrowth in vitro and in planta. All cultivation media were obtained from Difco Chemical Co., Detroit, USA. For confirmatory RT-qPCR, the same procedures were followed except the plant incubation period was only 72 h, due to RNA quality and concentration sufficient to yield reliable results. The time point 72 hours was the shortest period to allow obtaining satisfactory bacterial mass to the proposed analyzes involving gene expression. The bacterial cells concentration in short time (12 or 24 hours) don't allows this study. For this reason, some works are using culture media that mimic vegetable conditions to investigate the infectious process under these time scales [24, 25].


### 2.2. Extraction of RNA fromXcc

Each inoculated flask represented one independent biological replicate. RNA was extracted using an Illustra-RNAspin Mini RNA Isolation Kit (Amersham Biosciences) following the manufacturer’s instructions. To ensure that samples did not contain DNA, PCR was performed using RNA samples treated with DNase I as a template. PCR conditions consisted of an initial denaturing step of 94°C for 3 min, followed by 35 cycles of a denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 2 min. A final elongation step of 72°C for 4 min was performed, and then samples were maintained at 4°C until needed. The amplification reaction was conducted in a total volume of 25μL containing 200 ng RNA in 2.5 μL, PCR buffer (2.5 μL, Invitrogen), 1.5 mM MgCl2, 0.2 mM dNTP, 300 nM 16S rRNA primer [26], and 1 U Taq DNA polymerase (Invitrogen). The products were electrophoresed in a 1% agarose gel with TAE buffer, stained with ethidium bromide, and visualized using a UV transilluminator. No products were observed (data not shown). To verify the quality of extracted RNA, the samples were analyzed by electrophoresis in a 1% agarose gel using TAE buffer followed by ethidium bromide staining. The A260/280 ratio of RNA samples was measured, and RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA samples were stored at −80°C until needed.


### 2.3. Gene Expression Using RT-qPCR

First strand cDNA synthesis and all RT-qPCR reactions were done using the SuperScript III First-Strand Synthesis SuperMix for RT-qPCR (Invitrogen) as recommended by the manufacturer’s specifications except for the amount of cDNA in each reaction, which was 20 ng. All PCR was performed with SYBR Green on a 7500 Real-Time PCR instrument (Applied Biosystems) using three biological replicates and three technical replicates (one for each biological replicate). PCR was for 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. To determine PCR efficiency, standard curves were generated using cDNA samples at five dilutions and measured in triplicate.rpoB, atpD, and gyrB were used as reference genes in all experiments [26]. To perform relative expression analysis, we used the 2
-
Δ
Δ CT method [27]. Primer features are presented in Table 1.Table 1
General features of the T3SS and T4SS primer sequences.
XAC ID
Gene
Gene ID
Product (NCBI Annotation)
Primers (Forward)
Primers (Reverse)
Amplicon (bp)
System
XAC0293
hrpB
1154364
ATP-dependent RNA helicase
GCCGTATCCCGTTGACCTT
ACCGCCCCTTCATCTCTTTT
81
T3SS
XAC0393
hpaF
1154464
HpaF protein
GGCGAGCGTTTTGACAA
CCGGAGATCCTGGCAGTTT
57
T3SS
XAC0394
hrpF
1154465
HrpF protein
GCCGCGACGCAGTTG
CGGTGATCGAGTTGTTCAA
62
T3SS
XAC0396
hpaB
1154467
HpaB protein
GTCCGTGTACGCGCAAGAC
TGACGCCGGCATCATTG
53
T3SS
XAC0397
hrpE
1154468
HrpE protein
ACGAGGCCCAGAAGTCCAT
CCACGTTGAAGTCCAGATCATTT
66
T3SS
XAC0398
hrpD6
1154469
HrpD6 protein
ACCGATACGGTCACCCAAGA
GATCCGCCGCCTTGGT
55
T3SS
XAC0399
hrpD5
1154470
HrpD5 protein
GCCCTGAAGTGCGTTCGTTA
TCACGGACGGGTCTTGCT
54
T3SS
XAC0400
hpaA
1154471
HpaA protein
CGCCACCCCAACAGAAAA
ACGGCATCAGCTCCATCAA
59
T3SS
XAC0401
hrcS
1154472
HrcS protein
CGACGATCTAGTGCGATTTACCT
CGACCACCGGCAAGGA
68
T3SS
XAC0402
hrcR
1154473
Protein T3SS
ACACGGGAACGCGAAAAA
CCTTGGGCCAGATCTGTTGT
58
T3SS
XAC0403
hrcQ
1154474
HrcQ protein
GCAGCTGGAAGTGGACCAA
GGCTGCAAACCCGACAAC
57
T3SS
XAC0404
hpaP
1154475
HpaP protein
TGTCGATGGCACGAAATACC
CCCAACCGCGCCATT
58
T3SS
XAC0405
hrcV
1154476
HrcV protein
GCCGCAAGTTGGTCGAGTT
CTCTGCCATATCGCGATTCC
58
T3SS
XAC0406
hrcU
1154477
HrcU protein T3SS
AGCCGCGGTGCTCTTTG
TAGTCGCCGGCCGATTT
51
T3SS
XAC0407
hrpB1
1154478
HrpB1 protein
CTGCGGCCAGAGCTGAAG
ACCGCGCTTGATGGAAATC
60
T3SS
XAC0408
hrpB2
1154479
HrpB2 protein
CAGCGCAGCAGATCAAGTTG
CGCCGACGCTGACATTG
69
T3SS
XAC0409
hrcJ
1154480
HrcJ protein
GAGCGGCACGCCAATC
TGTCGAGATCAAGCCATCCTT
54
T3SS
XAC0410
hrpB4
1154481
HrpB4 protein
GGACAACACGCGGATCGA
GTCCGTCCAGCGCTGAA
52
T3SS
XAC0411
hrpB5
1154482
Protein  T3SS HrpB
CGCACGCCTGGGATATG
AGGCCGGATTCGTTCCA
63
T3SS
XAC0412
hrcN
1154483
T3SS ATPase protein
TCGATGGGCACCTGATTCTC
CGTCGATTGCCGGGTACT
63
T3SS
XAC0413
hrpB7
1154484
HrpB7 protein
CCGCGCTGATGGACAAG
GCTGCAGGATCGTGTCGATA
60
T3SS
XAC0414
hrcT
1154485
HrcT protein
ATCCAGCAATCCGACAGCAT
CACCGGTGCGGACAGTTT
54
T3SS
XAC0415
hrcC
1154486
HrcC protein
CAGATGCGGCTGGATGTG
CCGTCCACGGTGTTGGAT
59
T3SS
XAC0416
hpa1
1154487
Hpa1 protein
GCAGCAGGCCGGTCAGT
TTCATCAGCATCTGGGTGTATTG
57
T3SS
XAC0459
phaE
1154530
monovalent cation/H + antiporter subunit E
CCATCCCGGCTTCATCTG
GCGATGCCGTGAATGTTG
54
T3SS
XAC0618
hrpM
1154689
Glucosyltransferase MdoH
CAAGGGCCTGCACTGGAT
GCATCAACAGACCCCACATC
86
T3SS
XAC1265
hrpG
1155336
HrpG protein
CGGCAAGCCGATCGTATT
GAAAAGAGCAGCCAGGCAAT
54
T3SS
XAC1266
hrpXct
1155337
HrpX protein
GCCTACAGCTACATGATCACCAAT
TGCGGCCACTTCGTTGA
63
T3SS
XAC1520
hrcA
1155591
Heat-inducible transcriptional repressor
AGTTCGCGTTTCGGCATATC
CGGTTCTGCACCTCGTTGT
91
T3SS
XAC1994
hrpX
1156064
HrpX-like protein
CTGGTCTGATGCGAGCTTTCT
GCGTACTCGTCCACGATCAA
90
T3SS
XAC2047
phaE
1156118
PHA synthase subunit
AGCAGGGTGCATCGAAGAAG
TTTTCGGAGCCGCCTTTT
63
T3SS
XAC2606
—
1156677
Hypothetical protein
CCCTCAGTGTGGGCCAGAT
AGGAGGCGGTTGAGGGATA
58
H_T4SS_C
XAC2611
—
1156682
Hypothetical protein
GGGAGCCCGCAATGTG
GGAGGCGTGGGAATATCATCT
59
H_T4SS_C
XAC2612
virB6
1156683
VirB6 protein
TGCAGCAGGGAGGATTAGGA
CGGCGGTGCGGTGAT
56
T4SS_C
XAC2613
—
1156684
Hypothetical protein
CGAAGGGAGATGGAAGCAGTT
GCTCAACAGATCGGCTGGAA
59
H_T4SS_C
XAC2614
virB4
1156685
VirB4 protein
GACGTCGCTTGGTGGAGAAA
TTCCTCGGCGCGAATCT
53
T4SS_C
XAC2615
virB3
1156686
VirB3 protein
GCCCCGCCATGTTTTTG
CGCCCGCACCAATGAA
54
T4SS_C
XAC2616
virB2
1156687
VirB2 protein
TCGCCGATGCCAAAATG
CAGCAACGAAATGAATAGCAATG
62
T4SS_C
XAC2617
virB1
1156688
VirB1 protein
CGTATCGAGTCGTCGCGTAAT
CACCAGGCGTCCACCAA
57
T4SS_C
XAC2618
virB11
1156689
VirB11 protein
CGCTGGTCAACCACATTCC
CCTGGCGTCCTCAATCGT
56
T4SS_C
XAC2619
virB10
1156690
VirB10 protein
CCATTGCAGCTCTCTTGATTCTC
GAATCCTCCCCGCTTTGC
61
T4SS_C
XAC2620
virB9
1156691
VirB9 protein
GCTGAGCCCGAACGAAAAA
GCTCCCATCCACCAGTGAA
59
T4SS_C
XAC2621
virB8
1156692
VirB8 protein
TCGTCATGGCAGATGCGTAT
CCGAAGTTGGGCTCAAGCT
61
T4SS_C
XAC2622
—
1156693
Hypothetical protein
GGCGGATTCCAATATGCAGTT
TGGCCCGATCAAGGTGTAG
59
H_T4SS_C
XAC2623
virD4
1156694
VirD4 protein
TCGCTGGAATCCATTGACCTA
TCAAATCCGAAACGCGAAAT
59
T4SS_C
XAC2922
hrpW
1156993
HrpW protein
GGCGGACACACCGACATC
TGCCTTTCAGGGTGGAGTCTT
58
T3SS
XAC3122
hrpA
1157193
ATP-dependent RNA helicase
TGCGCGCTCTGAATTCG
GGACTGGGTAAGATCTTCATGCA
76
T3SS
XACb0035
—
1158522
Hypothetical protein
CGAGACGAGAACGGCCATT
TGTCACGCAGCAATTCGAA
55
H_T4SS_P
XACb0036
virB1
1158523
VirB1 protein
CGTTACAACCTCGCCAAATATG
CTGCCCGCCTTGAGATTG
74
T4SS_P
XACb0037
virB11
1158524
VirB11 protein
CCCGGAAGCGGTTGAGT
CGTGAGCGACCCCTTGTG
58
T4SS_P
XACb0038
virB10
1158525
VirB10 protein
TCTACGTGAACAAAGACCTCGATT
CGCTTGGCTCCGTCGAT
64
T4SS_P
XACb0039
virB9
1158526
VirB9 protein
GCTCACGCGGCGAAGTT
GCACTTGCTTGACACGATCATC
58
T4SS_P
XACb0040
virB8
1158527
VirB8 protein
CTCGAACAAGCTCAAACAGGAA
AACAGCAGCAACCCGATGA
60
T4SS_P
XACb0041
virB6
1158528
VirB6 protein
GCGCAGCTCGTCATCGT
CGCGGACACCCAACATG
55
T4SS_P
XACb0042
—
1158529
Hypothetical protein
CCGGGAACCCGCTTTTA
GGAAGCCACTGCCGAAACT
52
H_T4SS_P
XACb0043
—
1158530
Hypothetical protein
TTCGCGGCCCTCATCTC
AGGGCGACTTGTTGAACTTGTT
58
H_T4SS_P
XACb0044
virB5
1158531
VirB5 protein
CAGGCCGCCTACGAGAAG
GGGATTGGCGAACAGGTCTT
57
T4SS_P
XACb0045
virB4
1158532
VirB4 protein
GGAACAGGCAAAAGACGTCAAC
CCCGACAAGGTTTGAACGA
57
T4SS_P
XACb0046
virB3
1158533
VirB3 protein
TCTTGCGATGAGCTTTTATTTCC
CCCAAAGTGATCGCGTGATT
59
T4SS_P
XACb0047
virB2
1158534
VirB2 protein
CGATCTGGCGGCTAACGT
ATGCGAGCTGCGGAAGAA
56
T4SS_P
XACb0048
—
1158535
Hypothetical protein
GGCGGATGACGTTGAAGCT
CGCGGTAACGACCTCATAAAC
56
H_T4SS_P
XACb0049
—
1158536
Hypothetical protein
ACCACGGATCCTGGCAAAT
AGCAGCCCCCGTGAACA
51
H_T4SS_P
This table provides primers sequences, ORF's identification ID, gene's name, and respectiveXanthomonas citri subsp. citri secretory systems. T4SS: type IV secretion system; T3SS: type III secretion system; C: chromosomal copy; P: plasmid copy; H: hypothetical genes associated with the T4SS.


## 2.1. Microbiological Procedures

Xcc strain 306 was previously sequenced by da Silva et al. [23]. Xcc was maintained in sterile tap water and grown on nutrient agar (NA) medium containing 3 g beef extract, 5 g peptone, and 15 g agar in 1 L of distilled water at 28°C. After 24 h, three single colonies were transferred to new NA plates and incubated for 12 h at 28°C. These Xcc solid cultures were used as pre-inoculum for in planta and in vitro studies. For in planta studies, pre-inoculum cultures were aseptically transferred to sterile flasks containing 50 mL of nutrient broth (NB) and the bacterial concentration was adjusted to 108 CFU/mL (OD600 = 0.3). This bacterial suspension was taken up in a 1 mL needleless syringe and used to infiltrate orange leaves (Citrus sinensis cv. pera) grown in 20 L capacity pots. A total of nine plants were inoculated (20 leaves per plant) and maintained for 72 h in a growth room at 28°C, with a 12 h photoperiod and light intensity ~2,000 lux. Three plants were used for each incubation period. After multiplication, inoculated leaves were collected, sliced into thin strips with a razor blade, and placed in a beakers with sterile distilled water in an ice bath with gentle agitation. Samples from each plant were placed in separate beakers for bacterial exudation. After 5 min, leaf debris was removed by filtration through gauze, and bacterial cells were recovered by centrifugation at 5,000 ×g for 5 min at 4°C. Total RNA was extracted immediately, as described below. For in vitro studies, 1 mL of each Xcc suspension at 108 CFU/mL were aseptically transferred to three Erlenmeyer flasks and incubated for 12 h at 28°C with shaking (200 rpm). After incubation, bacterial cells were harvested by centrifugation at 5,000 ×g for 5 min. Total RNA was extracted as described below. Thus, we obtained three independent biological replicates for Xccgrowth in vitro and in planta. All cultivation media were obtained from Difco Chemical Co., Detroit, USA. For confirmatory RT-qPCR, the same procedures were followed except the plant incubation period was only 72 h, due to RNA quality and concentration sufficient to yield reliable results. The time point 72 hours was the shortest period to allow obtaining satisfactory bacterial mass to the proposed analyzes involving gene expression. The bacterial cells concentration in short time (12 or 24 hours) don't allows this study. For this reason, some works are using culture media that mimic vegetable conditions to investigate the infectious process under these time scales [24, 25].


## 2.2. Extraction of RNA fromXcc

Each inoculated flask represented one independent biological replicate. RNA was extracted using an Illustra-RNAspin Mini RNA Isolation Kit (Amersham Biosciences) following the manufacturer’s instructions. To ensure that samples did not contain DNA, PCR was performed using RNA samples treated with DNase I as a template. PCR conditions consisted of an initial denaturing step of 94°C for 3 min, followed by 35 cycles of a denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 2 min. A final elongation step of 72°C for 4 min was performed, and then samples were maintained at 4°C until needed. The amplification reaction was conducted in a total volume of 25μL containing 200 ng RNA in 2.5 μL, PCR buffer (2.5 μL, Invitrogen), 1.5 mM MgCl2, 0.2 mM dNTP, 300 nM 16S rRNA primer [26], and 1 U Taq DNA polymerase (Invitrogen). The products were electrophoresed in a 1% agarose gel with TAE buffer, stained with ethidium bromide, and visualized using a UV transilluminator. No products were observed (data not shown). To verify the quality of extracted RNA, the samples were analyzed by electrophoresis in a 1% agarose gel using TAE buffer followed by ethidium bromide staining. The A260/280 ratio of RNA samples was measured, and RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA samples were stored at −80°C until needed.


## 2.3. Gene Expression Using RT-qPCR

First strand cDNA synthesis and all RT-qPCR reactions were done using the SuperScript III First-Strand Synthesis SuperMix for RT-qPCR (Invitrogen) as recommended by the manufacturer’s specifications except for the amount of cDNA in each reaction, which was 20 ng. All PCR was performed with SYBR Green on a 7500 Real-Time PCR instrument (Applied Biosystems) using three biological replicates and three technical replicates (one for each biological replicate). PCR was for 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. To determine PCR efficiency, standard curves were generated using cDNA samples at five dilutions and measured in triplicate.rpoB, atpD, and gyrB were used as reference genes in all experiments [26]. To perform relative expression analysis, we used the 2
-
Δ
Δ CT method [27]. Primer features are presented in Table 1.Table 1
General features of the T3SS and T4SS primer sequences.
XAC ID
Gene
Gene ID
Product (NCBI Annotation)
Primers (Forward)
Primers (Reverse)
Amplicon (bp)
System
XAC0293
hrpB
1154364
ATP-dependent RNA helicase
GCCGTATCCCGTTGACCTT
ACCGCCCCTTCATCTCTTTT
81
T3SS
XAC0393
hpaF
1154464
HpaF protein
GGCGAGCGTTTTGACAA
CCGGAGATCCTGGCAGTTT
57
T3SS
XAC0394
hrpF
1154465
HrpF protein
GCCGCGACGCAGTTG
CGGTGATCGAGTTGTTCAA
62
T3SS
XAC0396
hpaB
1154467
HpaB protein
GTCCGTGTACGCGCAAGAC
TGACGCCGGCATCATTG
53
T3SS
XAC0397
hrpE
1154468
HrpE protein
ACGAGGCCCAGAAGTCCAT
CCACGTTGAAGTCCAGATCATTT
66
T3SS
XAC0398
hrpD6
1154469
HrpD6 protein
ACCGATACGGTCACCCAAGA
GATCCGCCGCCTTGGT
55
T3SS
XAC0399
hrpD5
1154470
HrpD5 protein
GCCCTGAAGTGCGTTCGTTA
TCACGGACGGGTCTTGCT
54
T3SS
XAC0400
hpaA
1154471
HpaA protein
CGCCACCCCAACAGAAAA
ACGGCATCAGCTCCATCAA
59
T3SS
XAC0401
hrcS
1154472
HrcS protein
CGACGATCTAGTGCGATTTACCT
CGACCACCGGCAAGGA
68
T3SS
XAC0402
hrcR
1154473
Protein T3SS
ACACGGGAACGCGAAAAA
CCTTGGGCCAGATCTGTTGT
58
T3SS
XAC0403
hrcQ
1154474
HrcQ protein
GCAGCTGGAAGTGGACCAA
GGCTGCAAACCCGACAAC
57
T3SS
XAC0404
hpaP
1154475
HpaP protein
TGTCGATGGCACGAAATACC
CCCAACCGCGCCATT
58
T3SS
XAC0405
hrcV
1154476
HrcV protein
GCCGCAAGTTGGTCGAGTT
CTCTGCCATATCGCGATTCC
58
T3SS
XAC0406
hrcU
1154477
HrcU protein T3SS
AGCCGCGGTGCTCTTTG
TAGTCGCCGGCCGATTT
51
T3SS
XAC0407
hrpB1
1154478
HrpB1 protein
CTGCGGCCAGAGCTGAAG
ACCGCGCTTGATGGAAATC
60
T3SS
XAC0408
hrpB2
1154479
HrpB2 protein
CAGCGCAGCAGATCAAGTTG
CGCCGACGCTGACATTG
69
T3SS
XAC0409
hrcJ
1154480
HrcJ protein
GAGCGGCACGCCAATC
TGTCGAGATCAAGCCATCCTT
54
T3SS
XAC0410
hrpB4
1154481
HrpB4 protein
GGACAACACGCGGATCGA
GTCCGTCCAGCGCTGAA
52
T3SS
XAC0411
hrpB5
1154482
Protein  T3SS HrpB
CGCACGCCTGGGATATG
AGGCCGGATTCGTTCCA
63
T3SS
XAC0412
hrcN
1154483
T3SS ATPase protein
TCGATGGGCACCTGATTCTC
CGTCGATTGCCGGGTACT
63
T3SS
XAC0413
hrpB7
1154484
HrpB7 protein
CCGCGCTGATGGACAAG
GCTGCAGGATCGTGTCGATA
60
T3SS
XAC0414
hrcT
1154485
HrcT protein
ATCCAGCAATCCGACAGCAT
CACCGGTGCGGACAGTTT
54
T3SS
XAC0415
hrcC
1154486
HrcC protein
CAGATGCGGCTGGATGTG
CCGTCCACGGTGTTGGAT
59
T3SS
XAC0416
hpa1
1154487
Hpa1 protein
GCAGCAGGCCGGTCAGT
TTCATCAGCATCTGGGTGTATTG
57
T3SS
XAC0459
phaE
1154530
monovalent cation/H + antiporter subunit E
CCATCCCGGCTTCATCTG
GCGATGCCGTGAATGTTG
54
T3SS
XAC0618
hrpM
1154689
Glucosyltransferase MdoH
CAAGGGCCTGCACTGGAT
GCATCAACAGACCCCACATC
86
T3SS
XAC1265
hrpG
1155336
HrpG protein
CGGCAAGCCGATCGTATT
GAAAAGAGCAGCCAGGCAAT
54
T3SS
XAC1266
hrpXct
1155337
HrpX protein
GCCTACAGCTACATGATCACCAAT
TGCGGCCACTTCGTTGA
63
T3SS
XAC1520
hrcA
1155591
Heat-inducible transcriptional repressor
AGTTCGCGTTTCGGCATATC
CGGTTCTGCACCTCGTTGT
91
T3SS
XAC1994
hrpX
1156064
HrpX-like protein
CTGGTCTGATGCGAGCTTTCT
GCGTACTCGTCCACGATCAA
90
T3SS
XAC2047
phaE
1156118
PHA synthase subunit
AGCAGGGTGCATCGAAGAAG
TTTTCGGAGCCGCCTTTT
63
T3SS
XAC2606
—
1156677
Hypothetical protein
CCCTCAGTGTGGGCCAGAT
AGGAGGCGGTTGAGGGATA
58
H_T4SS_C
XAC2611
—
1156682
Hypothetical protein
GGGAGCCCGCAATGTG
GGAGGCGTGGGAATATCATCT
59
H_T4SS_C
XAC2612
virB6
1156683
VirB6 protein
TGCAGCAGGGAGGATTAGGA
CGGCGGTGCGGTGAT
56
T4SS_C
XAC2613
—
1156684
Hypothetical protein
CGAAGGGAGATGGAAGCAGTT
GCTCAACAGATCGGCTGGAA
59
H_T4SS_C
XAC2614
virB4
1156685
VirB4 protein
GACGTCGCTTGGTGGAGAAA
TTCCTCGGCGCGAATCT
53
T4SS_C
XAC2615
virB3
1156686
VirB3 protein
GCCCCGCCATGTTTTTG
CGCCCGCACCAATGAA
54
T4SS_C
XAC2616
virB2
1156687
VirB2 protein
TCGCCGATGCCAAAATG
CAGCAACGAAATGAATAGCAATG
62
T4SS_C
XAC2617
virB1
1156688
VirB1 protein
CGTATCGAGTCGTCGCGTAAT
CACCAGGCGTCCACCAA
57
T4SS_C
XAC2618
virB11
1156689
VirB11 protein
CGCTGGTCAACCACATTCC
CCTGGCGTCCTCAATCGT
56
T4SS_C
XAC2619
virB10
1156690
VirB10 protein
CCATTGCAGCTCTCTTGATTCTC
GAATCCTCCCCGCTTTGC
61
T4SS_C
XAC2620
virB9
1156691
VirB9 protein
GCTGAGCCCGAACGAAAAA
GCTCCCATCCACCAGTGAA
59
T4SS_C
XAC2621
virB8
1156692
VirB8 protein
TCGTCATGGCAGATGCGTAT
CCGAAGTTGGGCTCAAGCT
61
T4SS_C
XAC2622
—
1156693
Hypothetical protein
GGCGGATTCCAATATGCAGTT
TGGCCCGATCAAGGTGTAG
59
H_T4SS_C
XAC2623
virD4
1156694
VirD4 protein
TCGCTGGAATCCATTGACCTA
TCAAATCCGAAACGCGAAAT
59
T4SS_C
XAC2922
hrpW
1156993
HrpW protein
GGCGGACACACCGACATC
TGCCTTTCAGGGTGGAGTCTT
58
T3SS
XAC3122
hrpA
1157193
ATP-dependent RNA helicase
TGCGCGCTCTGAATTCG
GGACTGGGTAAGATCTTCATGCA
76
T3SS
XACb0035
—
1158522
Hypothetical protein
CGAGACGAGAACGGCCATT
TGTCACGCAGCAATTCGAA
55
H_T4SS_P
XACb0036
virB1
1158523
VirB1 protein
CGTTACAACCTCGCCAAATATG
CTGCCCGCCTTGAGATTG
74
T4SS_P
XACb0037
virB11
1158524
VirB11 protein
CCCGGAAGCGGTTGAGT
CGTGAGCGACCCCTTGTG
58
T4SS_P
XACb0038
virB10
1158525
VirB10 protein
TCTACGTGAACAAAGACCTCGATT
CGCTTGGCTCCGTCGAT
64
T4SS_P
XACb0039
virB9
1158526
VirB9 protein
GCTCACGCGGCGAAGTT
GCACTTGCTTGACACGATCATC
58
T4SS_P
XACb0040
virB8
1158527
VirB8 protein
CTCGAACAAGCTCAAACAGGAA
AACAGCAGCAACCCGATGA
60
T4SS_P
XACb0041
virB6
1158528
VirB6 protein
GCGCAGCTCGTCATCGT
CGCGGACACCCAACATG
55
T4SS_P
XACb0042
—
1158529
Hypothetical protein
CCGGGAACCCGCTTTTA
GGAAGCCACTGCCGAAACT
52
H_T4SS_P
XACb0043
—
1158530
Hypothetical protein
TTCGCGGCCCTCATCTC
AGGGCGACTTGTTGAACTTGTT
58
H_T4SS_P
XACb0044
virB5
1158531
VirB5 protein
CAGGCCGCCTACGAGAAG
GGGATTGGCGAACAGGTCTT
57
T4SS_P
XACb0045
virB4
1158532
VirB4 protein
GGAACAGGCAAAAGACGTCAAC
CCCGACAAGGTTTGAACGA
57
T4SS_P
XACb0046
virB3
1158533
VirB3 protein
TCTTGCGATGAGCTTTTATTTCC
CCCAAAGTGATCGCGTGATT
59
T4SS_P
XACb0047
virB2
1158534
VirB2 protein
CGATCTGGCGGCTAACGT
ATGCGAGCTGCGGAAGAA
56
T4SS_P
XACb0048
—
1158535
Hypothetical protein
GGCGGATGACGTTGAAGCT
CGCGGTAACGACCTCATAAAC
56
H_T4SS_P
XACb0049
—
1158536
Hypothetical protein
ACCACGGATCCTGGCAAAT
AGCAGCCCCCGTGAACA
51
H_T4SS_P
This table provides primers sequences, ORF's identification ID, gene's name, and respectiveXanthomonas citri subsp. citri secretory systems. T4SS: type IV secretion system; T3SS: type III secretion system; C: chromosomal copy; P: plasmid copy; H: hypothetical genes associated with the T4SS.


## 3. Results and Discussion

### 3.1. qPCR Results for the T3SS

The results obtained fromXcc growing in planta or in vitro are presented in Figure 1. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation (Figure 1(b)). hpa1, hpaB, hrpD6, hrpE, and hrpF showed the highest rates of expression, whereas phaE, hrpG, hrpX, and hrcA showed the lowest levels of expression. Interestingly, hrpB4and hrpXct, which were shown to be necessary for Xcc pathogenesis [28], were not among the most upregulated genes in this study.T3SS genes inXanthomonas citri subsp. citri and their expression levels. Schematic representation of T3SS genes in Xanthomonas citri subsp. citri and their expression levels. (a) shows functional maps of T3SS genes on the chromosome. The expression levels of genes in the T3SS (b) are indicated. Solid green bars indicate in vivo expression with respect to their expression in vitro (black bars). Error bars indicate standard deviation of the replicates.
(a)
(b)


### 3.2. qPCR Results for the T4SS

The results for the expression levels of T4SS genesin planta and in vitro are shown in Figure 2. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation (Figure 2(c)). We also analyzed the expression of hypothetical genes located near virB in chromosomal DNA (XAC2606, XAC2611, XAC2613, and XAC2622) and in plasmid pXAC64 (XACb0035, XACb0042, XACb0043, XACb0048, and XACb0049) (Figure 2(d) and Table 1). ORFs representing hypothetical genes in both the chromosome and plasmid were down-regulated when Xcc was cultivated in Citrus sinensisfor 72 h. An exception was XAC2611, where expression was similar when Xcc was cultivated in culture medium or in planta (Figure 2(d)).T4SS genes inXanthomonas citri subsp. citri and their expression levels. Schematic representation of T4SS genes in Xanthomonas citri subsp. citri and their expression levels. (a) and (b) show functional maps of T4SS genes on the chromosome (a) and plasmid pXAC64 (b), respectively. The expression levels of genes in the T4SS (c) and ORFs localized near the virB genes (d) are indicated. Solid green bars indicate in vivo expression with respect to their expression in vitro (black bars). Error bars indicate standard deviation of the replicates.
(a)
(b)
(c)
(d)Our results clearly show different gene expression profiles for the T3SS and T4SS inXcc during Citrus infection. The T4SS was previously described in A. tumefaciens, where it mediates the transfer of DNA and protein substrates to plants and other organisms via a cell contact-dependent mechanism [14]. In Xcc, the genome sequence revealed the presence of two virB operons, one on the chromosome and a second copy in the 64 kb plasmid pXAC64 [23]. The chromosomal genes virB1, virB3, virB4, virB8, virB9, and virB11 and the plasmid-encoded genes virB1, virB2, virB4, virB6, virB9, and virB11 were all down-regulated in planta (Figure 2(c)). This was somewhat surprising because the T4SS is essential for a successful infection in many Gram-negative pathogenic bacteria [29]. The fact that ORFs representing hypothetical genes in both the chromosome and plasmid near the virB genes were also down-regulated under the same condition (Figure 2(d)) indicates that they could be related to the T4SS system in Xcc. Recent studies in Brucella suis [30] and in A. tumefaciens [31] showed that alterations in VirB8 result in protein dimerization, a process that modifies the T4SS structure and affects bacterial virulence. In Streptococcus suis [32], the knockout of the virD4-89K and virB4-89K of the T4SS eliminated the lethality of a highly virulent strain and impaired its ability to trigger host immune responses in mice. Recent studies continue to demonstrate the importance of the T4SS and its components in virulence; thus the lack of induction of Xcc T4SS genes in the present study is intriguing. However, in Xanthomonas campestris pv. campestris T4SS-deletion mutant displayed the same virulence as wild type and authors conclude that T4SS is not involved in pathogenicity in that Xanthomonas [33].Wang et al. [34] presented evidence that in planta transfer of a 37 kb plasmid (pXcB) from Xanthomonas aurantifolii to Xanthomonas citri can occur via T4SS. Thus, at least the T4SS copy present in Xcc plasmid can play a hole in horizontal gene transfer.All but one of the twenty-nine genes from the T3SS ofXcc were up-regulated in planta. Laia et al. [28] showed that Xcc mutants containing mutations in hrpB4 or hrpXct failed to cause disease and growth in citrus leaves was lower than the wild-type Xcc strain 306. These two genes, which are not among the most up-regulated genes in the present study, are part of the hrp (hypersensitive reaction and pathogenicity) system and comprise part of the T3SS [35]. In the related pathogen, Xanthomonas campestris pv. vesicatoria (Xcv), a hrpB4 mutant was unable to cause disease in susceptible pepper plants or the hypersensitive reaction in pepper plants carrying the respective compatible R gene [36]. Previously, hrpXv was shown to be necessary for transcriptional activation of five hrp genes (loci hrpB to hrpF) [37], and hrpB4 was required for complete functionality of the T3SS in Xcv [36]. Thus it is apparent that a gene does not have to be strongly up-regulated during infection to play an important role in virulence. Multiple genes, including hpaA, hpaE, hpaF, hpaP, hrpB, hrpB1, hrpB2, hrpB7, hrpD5, hrpM, hrpW, hrcC, hrcQ, hrcS, hrcT, and hrcV, were expressed at levels similar to hrpXct and hrpB4 and are good candidates for further studies into their roles in citrus canker disease.Among the most up-regulated genes,hrpD6 warrants further attention. A hrpD6 mutant of Xanthomonas oryzae pv. oryzae (Xoo) failed to trigger a hypersensitive response in tobacco and was nonpathogenic in rice because the mutation in hrpD6 impacts the secretion of T3SS effectors, such as Hpa1, which is translocated through the T3SS [38]. Our qPCR data showed that hpa1 was the most up-regulated gene during Citrus sinensis infection by Xcc, followed by hrpD6 (Figure 1(b)). Conversely, hrpA, which encodes an important component of the T3SS pilus [39], was down-regulated in our study. Previously, hrpA mutants of Pseudomonas syringae pv. tomato DC3000 showed reduced accumulation of structural components of the hrp pilus [39]. Haapalainen et al. [40] showed that soluble plant cell signals induce the expression of hrp/hrc genes and specifically upregulate hrpA. Interestingly, they found that HrpA does not accumulate to high levels intracellularly, suggesting that some kind of feedback regulation takes place between secretion and production of HrpA, perhaps through degradation of intracellular HrpA. Our work shows that this may also be applicable to hrpA gene in Xcc: its mRNA may be degraded faster, just as happen with HrpA in Pseudomonas syringae.


## 3.1. qPCR Results for the T3SS

The results obtained fromXcc growing in planta or in vitro are presented in Figure 1. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation (Figure 1(b)). hpa1, hpaB, hrpD6, hrpE, and hrpF showed the highest rates of expression, whereas phaE, hrpG, hrpX, and hrcA showed the lowest levels of expression. Interestingly, hrpB4and hrpXct, which were shown to be necessary for Xcc pathogenesis [28], were not among the most upregulated genes in this study.T3SS genes inXanthomonas citri subsp. citri and their expression levels. Schematic representation of T3SS genes in Xanthomonas citri subsp. citri and their expression levels. (a) shows functional maps of T3SS genes on the chromosome. The expression levels of genes in the T3SS (b) are indicated. Solid green bars indicate in vivo expression with respect to their expression in vitro (black bars). Error bars indicate standard deviation of the replicates.
(a)
(b)


## 3.2. qPCR Results for the T4SS

The results for the expression levels of T4SS genesin planta and in vitro are shown in Figure 2. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation (Figure 2(c)). We also analyzed the expression of hypothetical genes located near virB in chromosomal DNA (XAC2606, XAC2611, XAC2613, and XAC2622) and in plasmid pXAC64 (XACb0035, XACb0042, XACb0043, XACb0048, and XACb0049) (Figure 2(d) and Table 1). ORFs representing hypothetical genes in both the chromosome and plasmid were down-regulated when Xcc was cultivated in Citrus sinensisfor 72 h. An exception was XAC2611, where expression was similar when Xcc was cultivated in culture medium or in planta (Figure 2(d)).T4SS genes inXanthomonas citri subsp. citri and their expression levels. Schematic representation of T4SS genes in Xanthomonas citri subsp. citri and their expression levels. (a) and (b) show functional maps of T4SS genes on the chromosome (a) and plasmid pXAC64 (b), respectively. The expression levels of genes in the T4SS (c) and ORFs localized near the virB genes (d) are indicated. Solid green bars indicate in vivo expression with respect to their expression in vitro (black bars). Error bars indicate standard deviation of the replicates.
(a)
(b)
(c)
(d)Our results clearly show different gene expression profiles for the T3SS and T4SS inXcc during Citrus infection. The T4SS was previously described in A. tumefaciens, where it mediates the transfer of DNA and protein substrates to plants and other organisms via a cell contact-dependent mechanism [14]. In Xcc, the genome sequence revealed the presence of two virB operons, one on the chromosome and a second copy in the 64 kb plasmid pXAC64 [23]. The chromosomal genes virB1, virB3, virB4, virB8, virB9, and virB11 and the plasmid-encoded genes virB1, virB2, virB4, virB6, virB9, and virB11 were all down-regulated in planta (Figure 2(c)). This was somewhat surprising because the T4SS is essential for a successful infection in many Gram-negative pathogenic bacteria [29]. The fact that ORFs representing hypothetical genes in both the chromosome and plasmid near the virB genes were also down-regulated under the same condition (Figure 2(d)) indicates that they could be related to the T4SS system in Xcc. Recent studies in Brucella suis [30] and in A. tumefaciens [31] showed that alterations in VirB8 result in protein dimerization, a process that modifies the T4SS structure and affects bacterial virulence. In Streptococcus suis [32], the knockout of the virD4-89K and virB4-89K of the T4SS eliminated the lethality of a highly virulent strain and impaired its ability to trigger host immune responses in mice. Recent studies continue to demonstrate the importance of the T4SS and its components in virulence; thus the lack of induction of Xcc T4SS genes in the present study is intriguing. However, in Xanthomonas campestris pv. campestris T4SS-deletion mutant displayed the same virulence as wild type and authors conclude that T4SS is not involved in pathogenicity in that Xanthomonas [33].Wang et al. [34] presented evidence that in planta transfer of a 37 kb plasmid (pXcB) from Xanthomonas aurantifolii to Xanthomonas citri can occur via T4SS. Thus, at least the T4SS copy present in Xcc plasmid can play a hole in horizontal gene transfer.All but one of the twenty-nine genes from the T3SS ofXcc were up-regulated in planta. Laia et al. [28] showed that Xcc mutants containing mutations in hrpB4 or hrpXct failed to cause disease and growth in citrus leaves was lower than the wild-type Xcc strain 306. These two genes, which are not among the most up-regulated genes in the present study, are part of the hrp (hypersensitive reaction and pathogenicity) system and comprise part of the T3SS [35]. In the related pathogen, Xanthomonas campestris pv. vesicatoria (Xcv), a hrpB4 mutant was unable to cause disease in susceptible pepper plants or the hypersensitive reaction in pepper plants carrying the respective compatible R gene [36]. Previously, hrpXv was shown to be necessary for transcriptional activation of five hrp genes (loci hrpB to hrpF) [37], and hrpB4 was required for complete functionality of the T3SS in Xcv [36]. Thus it is apparent that a gene does not have to be strongly up-regulated during infection to play an important role in virulence. Multiple genes, including hpaA, hpaE, hpaF, hpaP, hrpB, hrpB1, hrpB2, hrpB7, hrpD5, hrpM, hrpW, hrcC, hrcQ, hrcS, hrcT, and hrcV, were expressed at levels similar to hrpXct and hrpB4 and are good candidates for further studies into their roles in citrus canker disease.Among the most up-regulated genes,hrpD6 warrants further attention. A hrpD6 mutant of Xanthomonas oryzae pv. oryzae (Xoo) failed to trigger a hypersensitive response in tobacco and was nonpathogenic in rice because the mutation in hrpD6 impacts the secretion of T3SS effectors, such as Hpa1, which is translocated through the T3SS [38]. Our qPCR data showed that hpa1 was the most up-regulated gene during Citrus sinensis infection by Xcc, followed by hrpD6 (Figure 1(b)). Conversely, hrpA, which encodes an important component of the T3SS pilus [39], was down-regulated in our study. Previously, hrpA mutants of Pseudomonas syringae pv. tomato DC3000 showed reduced accumulation of structural components of the hrp pilus [39]. Haapalainen et al. [40] showed that soluble plant cell signals induce the expression of hrp/hrc genes and specifically upregulate hrpA. Interestingly, they found that HrpA does not accumulate to high levels intracellularly, suggesting that some kind of feedback regulation takes place between secretion and production of HrpA, perhaps through degradation of intracellular HrpA. Our work shows that this may also be applicable to hrpA gene in Xcc: its mRNA may be degraded faster, just as happen with HrpA in Pseudomonas syringae.


## 4. Conclusions

Our finds confirm that the T3SS genes are induced in the successful infection ofCitrus sinensis by Xcc, and therefore are excellent targets to help control or decrease the severity of citrus canker, whereas T4SS seems not have any relation with virulence. On the other hand, our results show that T4SS is not induced under the same infection conditions, which could indicate that it is not necessary for infection but only for cell-to-cell communication.



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*Source: 763575-2014-02-23.xml*