The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp. citri

Journal of Integrative Agriculture 2015, 14(7): 1338–1347 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp. citri Guo Jing, SonG Xue, Zou Li-fang, Zou Hua-song, CHen Gong-you School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R.China

Abstract Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hypersensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri. Keywords: Xanthomonas citri subsp. citri, carbamoyl-phosphate synthase, pathogenicity, citrus canker, hrp gene

1. Introduction The Gram-negative bacterium Xanthomonas citri subsp. citri is the causal agent of canker disease, one of the most serious diseases of citrus (Vauterin et al. 1995). X. citri subsp. citri has been proposed to use several mechanisms for citrus canker development. Epiphytic survival and biofilm formation are involved in the early step of the

Received 21 July, 2014 Accepted 30 September, 2014 Correspondence ZOU Hua-song, Tel: +86-21-34205873, E-mail: [email protected]; CHEN Gong-you, Tel: +86-2134205873, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60965-5

pathogenicity process (Rigano et al. 2007). A large set of genes have been identified to be associated with biofilm formation via genomic and proteomic methods (Li and Wang et al. 2011; Malamud et al. 2013; Zimaro et al. 2013). The monofunctional catalase KatE and the rpf cell-to-cell quorum-sensing system also contribute to the development of canker symptoms (Siciliano et al. 2006; Tondo et al. 2010). X. citri subsp. citri possesses a complete hrp gene cluster that encodes the type III secretion system (T3SS), which is required for canker development in susceptible citrus plants and the hypersensitive response (HR) in non-host plants (Alegria et al. 2004; Dunger et al. 2005). The hrp clusters in X. citri subsp. citri are similar to those of other Xanthomonas strains in that it contains the front hpa2 and hpa1 genes as well as six conserved transcription units from hrpA to hrpF, which are all regulated by two genes, hrpG and hrpX (Guo et al. 2011). This system has been predicted to secrete the effector proteins (Dunge et al. 2012; Sgro et al. 2012),

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and PthA, a well-characterized member of the avrBs3 gene family, targets the host susceptible gene Lateral organ boundaries 1 (AI-Saadi et al. 2007; Hu et al. 2014). Carbamoyl-phosphate synthase (CPS) is an enzyme composed of a small and a large subunits. The small subunit (CPSI) is coded by the carA gene, and the large subunit (CPSII) is coded by the carB gene. CPS catalyzes the synthesis of carbamoyl-phosphate from biocarbonate, ATP and glutamine (Stapleton et al. 1996) or ammonia (Jones and Spector 1960). This action represents the first committed step in pyrimidine and arginine biosynthesis in prokaryotes and eukaryotes and in the urea cycle in most terrestrial vertebrates (Holden et al. 1999; Raushel et al. 1999). This step is particular to eukaryotes because CPS has evolved critical features that allow it to remove excess and potentially neurotoxic ammonia via the urea cycle, including the use of only free ammonia as a nitrogen donor (Ahuja and Powers-Lee 2008). CPSI deficiency is a rare inborn error in the urea cycle that leads to life threatening hyperammonemia (Ono et al. 2009). CPSII knock-out mutants invade host plants but do not replicate in vivo, possessing a unique and potential ability to induce a strong and long-lasting host protective immune response (Dzierszinshi and Hunter 2008). Most prokaryotes carry one form of CPS that participates in both arginine and pyrimidine biosynthesis. However, certain bacteria can have separate forms. In these bacteria, CPSI and CPSII usually work by combining with each other and form a tetrameric (αβ)4 protein in the Escherichia coli model. The synthesis catalyzed by the tetramer would then take part in the metabolism of arginine and pyrimidine. CPS heterodimers from E. coli contain two molecular tunnels: an ammonia tunnel and a carbamate tunnel. The expression of CPS is subject to the complex regulation of various metabolites on the pathways for pyrimidine and arginine synthesis (Bouvier et al. 1984). The catalytic mechanism of CPS requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain (Thoden et al. 1999; Kim et al. 2002). carA and carB form a carAB operon, and its transcription is under the control of two promoters (Bouvier et al. 1984; Piette et al. 1984). The upstream promoter, P1, responds to pyrimidine and is under the control of at least five transcription factors, PepA (Charlier et al. 1995), integration host factor (IHF) (Charlier et al. 1993), PyrH (Kholti et al. 1998), PurR (Devroede et al. 2004) and RutR (Shimada et al. 2007). The downstream promoter, P2, responds to arginine and is regulated by the arginine sensor ArgR (Devroede et al. 2004). These transcription factors include four repressors (IHF, PepA, PurR, and ArgR) that bind directly to the regulation sequence in the carAB

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operon. The combinatorial disruption of three repressors increased the carA expression levels in accordance with the degree of disruption, which positively correlated with thymidine production (Koo et al. 2011). The molecular function of CPS in prokaryotes has not been well characterized to date. The disruption of CPS in a CPS-negative mutant Streptococcus thermophilus A17 (ΔcarB) was investigated in the previous study by cultivating the strain in a chemically defined medium, diverse gas compositions and milk (Arioli et al. 2009). The results revealed that carB gene inactivation determined the auxotrophy of S. thermophilus for arginine and uracil, which resulted in slow growth in milk. Another functional proteomics study indicated that CPS may play a role in mediating nitrogen homeostasis in Pseudomonas fluorescens (Han et al. 2012). In this study, we generated deletion mutants of CPS in X. citri subsp. citri, and found that the deficiency in CPSII affected bacterial growth when adding several substrates as the sole carbon and nitrogen source. Most importantly, the mutant showed both T2SS- and T3SS-deficiency phenotypes. These findings help us to elucidate the role of CPS in plant pathogenic bacteria.

2. Results 2.1. The deletion of carB gene led to the complete loss of pathogenicity in Citrus paradise and a hypersensitive response in non-host plants The small subunit of carbamoyl phosphate synthase CarA was coded by XAC29_09385, and the large subunit CarB was coded by XAC29_09390 in the Xac 29-1 genome. To uncover the role of either subunit in bacterial pathogenicity, deletion mutants were constructed using two-step homology recombination. Each mutant was confirmed by PCR and Southern blot to ensure that the target gene was deleted from the genomic DNA (Appendixes A and B). Duncan leaves were inoculated with the mutants ΔcarA, ΔcarB and ΔcarAB to examine the changes in canker development. Seven days after inoculation, the non-polar defection mutants ΔcarB and ΔcarAB did not show canker symptoms, while the deletion mutation of carA did not affect canker development (Fig. 1-A). Two days after the inoculation of the non-host plants, ΔcarB and ΔcarAB lost the ability to induce a hypersensitive response in Nicotiana benthamina and tomato. In contrast, mutant ΔcarA retained the ability to induce the hypersensitive response as the wild type. The complemented strains CΔcarA and CΔcarAB restored the phenotypes to wild type (Fig. 1-B). These findings indicated that only the large subunit CarB of carbamoyl-phosphate synthase was required for canker development in X. citri subsp. citri.

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A Xac29-1

Xac29-1

ΔcarA CΔcarA

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ΔcarB

ΔcarAB

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CΔcarAB

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ΔcarB CΔcarB ΔcarAB CΔcarAB

ΔcarB Mock

amylase activities, the plates containing carboxymethyl cellulose or starch were inoculated with the X. citri subsp. citri strains. Two days after cultivation, the degradation of carboxymethyl cellulose and starch was evaluated according to the degradation haloes under and around the cell colonies. The diameters of both hydrolysis haloes produced by ΔcarA did not distinctly differ from those observed of the wild type for all repeated experiments. In contrast, the hydrolysis haloes produced by ΔcarB and ΔcarAB remarkably reduced (Figs. 2 and 3). For the extracellular cellulose activity, the diameter of the wild-type degradation halo was approximately 5.0 cm, while the diameters of the ΔcarB and ΔcarAB mutants were only 2.0 cm (Fig. 2). For the extracellular amylase activity, the wild type produced a 0.4 cm degradation halo, and the ΔcarB and ΔcarAB strains only produced 0.1 cm degradation haloes (Fig. 3). The deletion of carB reduced the extracellular cellulase activity by 60% and the amylase activity by 80%.

2.3. Utilization of carbon and nitrogen resources Fig. 1 The phenotypes of carbamoyl-phosphate synthase mutants on plants. A, canker development on host plants. B, hypersensitive response in non-host tobacco and tomato plants.

2.2. carB influenced extracellular enzyme activities To assess the changes in the extracellular cellulase and A

CPS catalyzes glutamate to generate glutamine and ammonia, resulting in arginine and pyrimidine from a series of metabolic processes. ΔcarA, ΔcarB and ΔcarAB were grown in minimal media MMX supplemented with pyrimidine, glutamic acid, glutamine or 2-katoglutaric acid to assess the effect of carbamoyl-phosphate synthase on bacterial growth. The mutation in carA did not affect bacterial growth in MMX media. Eighty-four hours post inoculation, the OD600 value B

Xac 29-1

0.6

Mutant

Complementation

*

*

*

0.3

**

0.2 **

**

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*

0.4

**

ΔcarB

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Xac 29-1

ΔcarA

*

Xac 29-1

Haloes in diameter (cm)

0.5

0.1

Xac 29-1

ΔcarAB

CΔcarAB

0 ΔcarA

ΔcarB

ΔcarAB

Fig. 2 Extracellular celluase activity. A, the degradation haloes on carboxymethyl cellulose media. B, the diameters of the degradation haloes. The tests were repeated three times; the data in the figure are the mean values±SD. One and two asterisks in each horizontal data column indicate significant differences at P=0.05 and P=0.01 by t test, respectively. The same as below.

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B Xac 29-1 *

Xac 29-1

ΔcarA

ΔcarB

CΔcarA

CΔcarB

*

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Complementation

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0.4 0.3 *

0.2

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Haloes in diameter (cm)

0.5

**

0.6

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0.1

Xac 29-1

ΔcarAB

CΔcarAB

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ΔcarB

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Fig. 3 Extracellular amylase activity. A, the degradation halos on NA media added with starch. B, the diameter of the degradation halos.

reached 0.03 for the wild type (Fig. 4). In contrast, the mutants ΔcarB and ΔcarAB greatly reduced their growth in MMX. When the substrates were added, the growth rates of ΔcarB and ΔcarAB improved but remained slower than that of the wild type (Fig. 4). The complementation strains partially restored the growth speed to that of the wild type. This finding suggested that the mutation in carB influenced the utilization of diverse carbon and nitrogen substrates by X. citri subsp. citri.

2.4. hrp genes were suppressed in ΔcarB mutant Because the mutation in carB impaired the pathogenicity in citrus plants and the ability to induce a hypersensitive response in non-host plants, we investigated the hrp gene expression pattern in a carB deletion genetic background. After the inoculation of citrus plants, qRT-PCR was applied to evaluate the transcription level of all 26 hrp genes. Fig. 5 shows that the expression level of hrpX was reduced to only 40% of that of the wild type. Four hrpB operon genes (hrpB7, hrpN, hrpB5 and hrpB4), two hrpC operon genes (hrcU and hpaP), three hrpD operon genes (hrcQ, hrcR and hrcS) and one hrpF operon gene (hpaF) were suppressed in ΔcarB (Fig. 5). The left 15 hrp genes did not distinctly differ from the wild type (data not shown).

3. Discussion The small and large subunits of CPS in X. citri subsp. citri play diverse roles in bacterial pathogenicity. CPS cata-

lyzes the formation of carbamoyl phosphate, an unstable and high-energy phosphate compound required for the biosynthesis of arginine and pyrimidine in ureotelic vertebrates (Stapleton et al. 1996; Holden et al. 1999). CPSI functions in mitochondria and catalyzes synthesis of carbamoyl phosphate from ammonia and bicarbonate (McGivan et al. 1976; Crouser et al. 2006). This reaction is the first committed step of the urea cycle, which is important in the removal of excess urea from cells (Jones and Spector 1960). CPSII catalyzes the reactions that produce carbamoyl phosphate in the cytosol (Holden et al. 1998). In the prokaryote Escherichia coli, CPS exhibits a double function, as it acts in both biosynthetic routes. The small subunit CarA and large subunit CarB combine to form a tetrameric (αβ)4 (Kim et al. 2002). The deletion mutation of CPSI in X. citri subsp. citri did not affect the bacterial pathogenicity, while mutation in CPSII rendered multiple phenotypic alterations. We proposed that CPSII plays predominant roles in CPS function in X. citri subsp. citri. Nevertheless, CPSI and CPSII may also form a tetramer, since their amino acid sequences share 62 and 70% identities with the homologies in E. coli, respectively. CPSII affects the phenotypes due to auxotrophy. The catalysant of CPS in prokaryotes is not involved in the urea cycle, but it does produce the products arginine and pyrimidine (Beckwith et al. 1962). Furthermore, the intermediate product glutamate is the raw material for α-ketoglutaric acid, which participates in the TCA cycle (Peng et al. 1993). This finding suggests that CPS functions in several parts of the carbon and nitrogen metabolic cycles. Therefore,

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Xac 29-1

OD600 values

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

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0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0 12 24 36 48 60 72 84

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0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

ΔcarAB MMX

0 12 24 36 48 60 72 84 MMX+Glutamic acid

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CΔcarAB 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0 12 24 36 48 60 72 84

0 12 24 36 48 60 72 84

MMX+Glutamine

0 12 24 36 48 60 72 84

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0 12 24 36 48 60 72 84

MMX+2-ketoglutaric acid

0 12 24 36 48 60 72 84

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0 12 24 36 48 60 72 84

Time post inoculation (h)

Fig. 4 The utilization of carbon and nitrogen substrates. The Xanthomonas strains were cultured in MMX medium. The applied substrates were 5% glutamic acid, glutamine and 2-katoglutaric acid. The OD600 values were tested every 12 h post inoculation.

the deletion of the CPSII coding gene carB greatly affected the bacterial growth in NA and MMX media (Figs. 3 and 5). The carbon and nitrogen substrate utilizations of mutants ΔcarB and ΔcarAB were impaired. Despite the lack of direct evidence, CPSII is presumed essential for X. citri subsp. citri to multiply in citrus tissue because the bacterium

needs to acquire carbon and nitrogen nutrients from host plants during colonization. We have successfully restored the growth of ΔcarB in NA to some extent by adding citrus leaf extracts (data not shown) but did not identify the type of nutrient substrate that was involved in. Further studies are needed to identify the type of substrate that compensates

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Xac 29-1

Relative expression level

2.0

CΔcarB

ΔcarB

1.5

1.0

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hrpB7

hrcN

hrpB5

hrpB4

hrcU

hpaP

hrcQ

hrcR

hrcS

hpaF

hrpX

Fig. 5 hrp gene expression in the ΔcarB mutant. The total RNA was extracted from inoculated plant leaves 24 h post inoculation using Trizol reagent. The expression of gyrA was used as the internal control to verify significant variations at the cDNA level via qRT-PCR.

the phenotypic alteration of ΔcarB. CPSII deficiency affected the ability of bacteria to activate the type III system in plants. In X. citri subsp. citri, the type III secretion system is encoded by a gene cluster composed of 24 hrp genes, which are organized into six transcription operons, hrpA–F (Alegria et al. 2004; Dunger et al. 2005). The hrpB, hrpD and hrpF operons are essential for the pathogenicity on host plants and HR induction in non-host plants. Like other Xanthomonas strains, the hrp genes are controlled by the OmpR family regulator HrpG and the AraCtype transcriptional activator HrpX (Guo et al. 2011). HrpX is controlled by HrpG and responsible for the activation hrpB-F operons (Wengelnik and Bonas 1996). The expressions of hrp genes are induced in plant tissue or in specific minimal media that are likely mimic plant conditions (Schulte and Bonas 1992). In our study, the expressions of hrpX, hrpB7, hrpN, hrpB5, hrpB4, hrcQ, hrcR, hrcS and hpaF were suppressed in the ΔcarB mutant, while the expression of hrpX reduced by 60%. The decrease in CPSII seemingly blocked the expression of hrpX and thus led to the suppression of the expression of the eight hrp genes. This suppression led to the loss of pathogenicity in citrus plants, as well as the induction of the hypersensitive response in non-host tobacco and tomato plants.

4. Conclusion In this study, we constructed the deletion mutants of two subunit coding genes of carbamoyl-phosphate synthase in X. citri subsp. citri and confirmed that the deletion of the large subunit gene carB led to multiple phenotypic alterations. The mutant ΔcarB and double mutant ΔcarAB lost pathogenicity in host plant citrus and the ability to induce a hypersensitive response in non-host plants. The deletion

of the carB gene also affected the utilization of nitrogen and carbon substrates and extracellular enzyme activities. When interacting with the host plant, the hrp regulator gene hrpX and the hrp genes in the hrpB, hrpC, hrpD and hrpF operons were suppressed in the ΔcarB mutant. These data have advanced our understanding of the roles of CPS in the plant pathogenic bacterium.

5. Materials and methods 5.1. Bacterial strains, plasmids and culture conditions The bacterial strains and plasmids used in this study are listed in Table 1. The Xanthomonas citri subsp. citri strains 29-1 (Xac 29-1) were cultivated in nutrient broth (NB) medium or NB supplemented with 1.5% agar (NA) at 28°C. During the construction of non-polar mutants, NAN, which was similar to NA but did not contain sucrose, and NAS, which was similar to NA but contained 10 times the level of sucrose, were used to cultivate X. citri. subsp. citri. Escherichia coli strains were cultured in Luria-Bertani medium (LB) at 37°C. Antibiotics were applied at the following concentrations: ampicillin (Ap), 100 μg mL–1; kanamycin (Km), 50 μg mL–1 and gentamycin (Gm), 10 μg mL–1.

5.2. Construction of non-polar deletion mutant The non-polar mutants of ΔcarA, ΔcarB and double mutant ΔcarAB were constructed using the suicide vector pKMS1 via twice homologous recombination in the wild type. The upside and downside DNA fragments of related genes were PCR amplified from Xac 29-1 genomic DNA using the primer pairs carA1.F/carA1.R, carA2.F/carA2.R, carB1.F/carB1.R

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and carB2.F/carB2.R (Appendix C). Two corresponding flanking fragments of related genes were ligated together into the vector pKMS1 at the XbaI and PstI sites, resulting in pKMS-carA and pKMS-carB (Table 1). The recombinants of plasmid were individually introduced into the wild-type strain via electroporation. The recovered cells were then plated on NAN medium containing Km and incubated at 28°C for 4–7 d until single colonies formed. The single colonies were transferred to a NBN broth, cultured for approximately 12 h and then plated on NAS plates. Within 3 d, the single colonies that emerged were transferred to both NA plus Km and NA plates. The Km-sensitive colonies were the potential desired deletion mutants. The combination sets of primers carA1.F/carA2.R and carB1.F/carB2.R were applied to amplify the genomic DNA fragment from deletion mutants (Appendix C). The DNA productions were sequenced to ensure that the target genes were deleted from the genome sequence. A 420-bp upside fragment of carA and 686 bp carB DNA fragments were amplified using a Southern blot probe and the primers ScarA.F/ScarA.R and ScarB.F/ScarB.R (Appendix C). The double-deletion mutant ΔcarAB was then generated by introducing the recombinant plasmid pKMS-carB into the mutant ΔcarA genetic background.

5.3. Complementation analysis Three DNA fragments containing the opening-reading fragments of carA, carB and carAB, were amplified by PCR with primers carA.F/carA.R and carB.F/carB.R (Appendix C). To constitutively express the gene in the mutant, the promoter region of wxacO was cloned into the vector pBBR1MCS-5

at XhoI and HindIII using the primers wxaco.p.F and wxaco.p.R (Appendix C). The carA gene was inserted into the HindIII and XbaI sites with the primers carA.F and carA.R (Appendix C). The carB gene was inserted into the XbaI and SacI sites with the primers carA.F and carA.R (Appendix C). For the complementation analysis of the double mutant, carA and carB were inserted into the HindIII and SacI sites together, resulting in pCcarAB. The plasmid recombinants were individually introduced into the corresponding mutant strain via electroporation.

5.4. Pathogenicity and hypersensitive response (HR) assays The X. citri. subsp. citri strains were cultured in NB broth and grown at 28°C for 24–36 h until OD600=0.8. The cells were sub-cultured (1:100) in 40 mL fresh NB and incubated for another 16 h until the OD600 reached 0.6. After centrifugation at 6 000 r min–1 for 10 min at 4°C, the cell pellets were washed twice with sterile water and then re-suspended with sterile water to a final concentration of 108 CFU mL–1 (OD600=0.3). Bacterial suspensions were injected into fully expanded grapefruit (Citrus paradise Macf. cv. Duncan) leaves with needleless syringes. Disease symptoms were scored and photographed 7 d after inoculation. For the HR assay, the bacteria were infiltrated into Nicotianna benthamina and tomato leaves. The plant responses were assessed 2 d after inoculation. Each test was repeated at least three times.

5.5. Extracellular enzymes assays Skim milk (2%) was added to NA plates to examine the

Table 1 Strains and plasmids used in this study Strain or plasmid Strains Escherichia coli DH5α Xanthomonas citri subsp. citri Xac 29-1 ΔcarA ΔcarB ΔcarAB CΔcarA CΔcarB CΔcarAB Plasmids pBBR1MCS-5 pCcarA pCcarB pCcarAB pKMS-carA pKMS-carB pKMS1

Relevant characteristics

Φ901acZΔm15, recA1 Wild type A carA knock-out mutant of strain Xac 29-1 A carB knock-out mutant of strain Xac 29-1 A carA and carB knock-out mutant of strain Xac 29-1 Gmr, ΔcarA harboring pCcarA Gmr, ΔcarB harboring pCcarB Gmr, ΔcarAB harboring pCcarAB Gmr, mob, broad host range cloning vector pBBR1MCS-5 expressing carA gene under wxacO promoter pBBR1MCS-5 expressing carB gene under wxacO promoter pBBR1MCS-5 expressing carA and carB genes with native promoter Kmr, a 1 014 bp fusion cloned in pKMS1 for a 1 129 bp deletion in carA Kmr, a 2 267 bp fusion cloned in pKMS1 for a 1 730 bp deletion in carB Kmr, suicide vector, mob+

Source

Invitrogen, USA This lab This work This work This work This work This work This work Kovach et al. (1994) This work This work This work This work This work This lab

GUO Jing et al. Journal of Integrative Agriculture 2015, 14(7): 1338–1347

extracellular protease activity (Zou et al. 2012). The cellulase activity was assessed as described previously (Yan and Wang 2012). In each assay, 1.5 µL X. citri subsp. citri cells were spotted. The halos surrounding the colonies represented the secretion of extracellular protease and cellulose. The hydrolytic activity was calculated by subtracting the diameters of colonies from halos. The tests were repeated three times and the data shown in the figure was the mean of value.

5.6. Determination of bacterial growth in minimal media added with diverse substrates The cultured cells were washed twice with sterilized water and then re-suspended with sterilized water to OD600=1.0. The suspended cells were sub-cultured (1:100) in MMX medium (glucose 5.0 g, (NH4)2SO4 2.0 g, MgSO4·7H2O 0.2 g, K2HPO4 4.0 g, KH2PO4 6.0 g, Na2C6H5O7·2H2O 6.0 g, NH4NO3 1.0 g, per 1 000 mL) supplemented with 1‰ pyrimidine, 5% glutamic acid, glutamine or 2-katoglutaric acid. The OD600 values were tested every 12 h post inoculation. All experiments were repeated at least three times.

5.7. RnA isolation and qRT-PCR The total RNA was extracted from inoculated plants 24 h after inoculation using Trizol reagent as recommended by the manufacturer (Invitrogen, USA). The potential trance of genomic DNA was removed by RNase-free DNase I (TaKaRa, Dalian, China) before synthesis of first-strand cDNA. All primers used for qRT-PCR are listed in Appendix D. PCR reaction was performed on the Applied Biosystems 7500 Real-Time PCR System using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Dalian, China). The PCR thermal cycle conditions were defined as follows: denature at 95°C for 30 s and 41 cycles for 95°C, 5 s; 60°C, 34 s. The expression of gyrA was used as the internal control to verify the significant variation at the cDNA level. The comparative threshold method was used to calculate the relative mRNA level. All RT-PCRs were performed in triplicate.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31171832) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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