An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Journal of Integrative Agriculture 2019, 18(6): 1189–1198 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae XU Jin-bo*, ZHANG Cui-ping*, WUNIERBIEKE Mei-li, YANG Xiao-fei, LI Yi-lang, CHEN Xiao-bin, CHEN Gong-you, ZOU Li-fang Department of Environment and Resource, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R.China

Abstract Xanthomonas oryzea pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, which is a significant threat to many of rice-growing regions. The type III secretion system (T3SS) is an essential virulence factor in Xoo. Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium. The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo. Thus, an efficient and reliable genetic tool system is required for detection of the T3SS proteins. In this study, we constructed a protein expression vector pH3-flag based on the backbone of pHM1, a most widely used vector in Xoo strains, especially a model strain PXO99A. This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site (MCS), containing a modified pUC18 polylinker, and Flag as a C-terminal tag. The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression. We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo. This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp. Keywords: Xanthomonas oryzae pv. oryzae, broad-host range vector, expression vector, T3SS genes

1. Introduction Xanthomonas oryzea pv. oryzae (Xoo) causes bacterial blight of rice, the most important bacterial disease of rice in Received 26 September, 2018 Accepted 7 December, 2018 XU Jin-bo, E-mail: [email protected]; ZHANG Cui-ping, E-mail: [email protected]; Correspondence ZOU Li-fang, Tel: +86-21-34206149, E-mail: [email protected] * These authors contributed equally to this study.

some major rice-growing countries (Nino-Liu et al. 2006).

© 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(19)62606-7

of extracellular polysaccharide (EPS) (Nino-Liu et al. 2006;

The pathogens enter rice leaves through water pores or wounds and then spread systemically through the xylem tissues, where they multiply and produce a large amount Han et al. 2008). The increased bacterial cells and EPS block the vascular eventually causing the whole leaf of

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rice whitish and grayish, a typical symptom of bacterial blight (Nino-Liu et al. 2006; Buttner and Bonas 2010). The successful pathogenesis by Xoo in host rice, therefore, relies on a controlled global virulence regulation network (Mole et al. 2007; Buttner and Bonas 2010). Elucidating the molecular mechanisms that control expression of vital virulence associated genes in Xoo require some efficient molecular tools such as protein expression vectors. The protein expression vectors usually have a common motif in which a tag encoded DNA fragment is present upstream or downstream of the multiple cloning site (MCS) (Dammeyer et al. 2013). Known open reading frame (ORF) sequences with or without their native promoters can be ligated into the MCS to produce recombinant proteins, and then the tagged proteins can easily be detected by specific antibodies (Prior et al. 2010). Generally, Xoo strains have strict restriction modification systems which merely allow replication of some broad-host-range plasmids in their cells (Choi et al. 1998; Prior et al. 2010). Most of the existing protein expression vectors that were suitable for use in Escherichia coli could not be maintained in many Xoo cells. Some broad-host-range protein expression vectors reported to date in Gram-negative bacteria, either contain the constitutive lacIq-Ptrc promoter or a arabinose inducing PBAD promoter or are unstable enough to be used in vivo for gene regulation analysis. Some vectors wildly used in Xoo strains include pHM1, pML123 and pUFR034 and pBBR1 derivative vectors (DeFeyter et al. 1990; Labes et al. 1990; Kovach et al. 1995). However, these vectors usually are cloning vectors lacking the essential tag motifs for protein detection. The first challenge to design a new expression vector is selecting an appropriate cloning vector as the backbone. Ideally, this backbone vector must be stable and capable of being taken up with high efficiency by the Xoo cells. In order to increase the accuracy of protein detection in the experiments of gene regulation analysis, one or more transcriptional terminators upstream of the MCS should be incorporated to prevent read-through transcription. It’s also important to consider the sensitivity of the protein tags. 3×cMyc, a triple tandem cMyc tag, is substantially more sensitive than Flag tag. The weak to moderate differences of some target proteins that have been fused to 3×cMyc in the wild type and it’s relative mutants are often confounded by the strong immunoblotting signals in some experiment conditions. However, this interference is largely reduced by using the Flag tag instead of 3×cMyc tag. Some Xanthomonas cells, for instance, a Xoo model strain PXO99A and a X. oryzae pv. oryzicola typical strain RS105 have native His-antibody affinity (unpublished data). In our previous study, we tested the stability of pHM1, pUFR034, pML123 and pPROBE-AT in the cells of PXO99A using the hrpG::gusA transcriptional fusion system and

found that GUS activities of the hrpG promoter are more stable when the promoter fragment was cloned in pHM1 than any other three vectors (Wang et al. 2015). In this study, we constructed an new protein expression vector pH3-flag specific for gene regulation analysis based on the backbone of pHM1 (Hopkins et al. 1992). This vector combines all of the important features listed above containing a synthesized MCS-FLAG cassette which is bound by the (T1)4 and T0T1 transcriptional terminators. We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo. This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

2. Materials and methods 2.1. Bacterial strains, plasmids, and culture conditions The wild-type Xanthomonas oryzae pv. oryzae PXO99A and its derived mutants PΔhrpG, PΔhrpX and PΔtrh were grown in NA (0.5% peptone, 0.1% yeast, 1% sucrose, 0.3% beef extract, and 1.5% agar) or NB (NA without agar) medium at 28°C. E. coli DH5a strains were grown in Luria-Bertani (LB) medium at 37°C. The hrp-inducing medium for Xoo strains is XOM3 (D-xylose, 1.8 g L–1; L-methionine, 670 μmol L–1; sodium L-glutamate, 10 mmol L–1; NaFe2+-EDTA, 240 μmol L–1; MgCl2, 5 mmol L–1; KH2PO4, 14.7 mmol L–1; MnSO4, 40 μmol L–1 (pH 6.0)) (Xiao et al. 2007). Antibiotics were used at the following concentrations: kanamycin (Km), 40 μg mL–1; spectinomycin (Sp), 40 μg mL–1. The primers used in this study are listed in Appendix A.

2.2. Construction of vectors and plasmids The expression vector was constructed in three steps. First, the complete MCS-FLAG DNA cassettes were synthesized by OGENE BIOTEK (Shanghai, China) through synthetic biology, then was ligated into the EcoRV site of the pUC57simple vector by the company creating pSV-flag. Second, the fragment containing four T1 terminators amplified from plasmid pPROBE-NT (Miller et al. 2000) using primers T14-F and T14-R was fused with the fragment containing a bacteriophage lambda T0 terminator and a E. coli rrnB T1 amplified from plasmid pPROBE-NT using primers T01-F and T01-R by the HindIII site. Then, the fusion was cloned into the BamHI site of pHM1 (GenBank accession number: EF059993) to create plasmid pH3. Finally, the HindIIIdigested MCS-FLAG DNA cassette from pSV-flag was cloned into the HindIII site of pH3 to create the expression

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vector pH3-flag. The complete nucleotide sequences of all related vectors have deposited in GenBank. The accession numbers are as follows: pH3, MH445408; pH3flag, MH445409. The promoter regions of hrpG, hrpX, hrpF, hrpB1, hrcQ, hrcU, hrpE, hpa1, hpa2 and hrpD6 were amplified from PXO99A genomic DNA (GenBank: NC_010717.1) using primers listed in Appendix A. The PCR product was cloned via SalI and EcoRI into pNG (Wang et al. 2015), a promoter probe vector, to create the construct of hrp/hrc/hpa::gusA fusions. The recombinant hrp/hrc/hpa::gusA digested by HindIII was ligated into the HindIII site of pHG2 (Wang et al. 2015) to create plasmid pHG2-hrpG, pHG2-hrpX, pHG2hrpF, pHG2-hrpB1, pHG2-hrcQ, pHG2-hrcU, pHG2-hrpE, pHG2-hpa1, pHG2-hpa2 and pHG2-hrpD6, respectively. The fragment containing the hrpG ORF and its native promoter was amplified from PXO99 A genomic DNA (GenBank: NC_010717.1) using primers hrpG-F and hrpG-R. The PCR product was cloned via SalI and KpnI into pH3-flag to create plasmid pH3-hrpG::flag. Following the same procedure, the plasmid pH3-hrcC::flag were constructed using the primers hrcC-F and hrcC-R. The functional fragment of hrpX was amplified using primers hrpX-F and hrpX-R. Firstly, the resulting PCR product was cloned into pSV-flag via XbaI and EcoRI to create plasmid pSV-hrpX::flag. Secondly, the recombinant hrpX::flag digested by HindIII was ligated into the HindIII site of pH3 and pHM1, respectively, to create plasmid pH3-hrpX::flag and pHM1-hrpX::flag. As the same way, the plasmid pH3-hrpB1::flag and pHM1-hrpB1::flag were constructed using the primers hrpB1-F and hrpB1-R. Primers for PCR were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The enzymes involved in PCR, digestion and ligation reactions were purchased from TaKaRa Biomedical Technology Co., Ltd. (Dalian, China). DNA extraction and gel purification were performed as the instruction manual of GBS Biotechnology (Nanjing, China).

2.3. Western blot assay Fresh Xoo strains were grown overnight in NB medium at 28°C, and harvested by centrifugation at 8 000 r min–1 for 3 min. The harvested bacterial cells were washed twice, and the OD600 was adjusted to 1.0 with XOM3 and then induced in XOM3 medium for 3–12 h at 28°C. The induced bacterial cells were washed twice with PBS and the OD600 was adjusted to 1.0 with PBS. Took 80 µL sample mixed with 20 µL 5× loading buffer, then the mixture was boiled for 10 min and put on ice during loading. The Flag-labeled antibody was used to detect the expression of target genes in Xoo strains. After running two protein gels, the proteins were transferred to PVDF membranes (Merk Millipore Ltd.,

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Shanghai), one protein membrane was probed with the antibody corresponding to the tag, while another one with reference antibody to serve as a control. The membranes were first blocked with PBST buffer (PBS buffer with 5% skim milk and 0.1% Tween 20) and then incubated with primary antibodies diluted with PBST buffer (1:5 000) for 1 h on shaking at room temperature. Membranes were washed for three times and incubated with secondary antibody diluted with PBST buffer (1:5 000). After final thorough washing of the membrane with PBST buffer, the proteins were detected using the EasySee Western Kit (Transgene, Beijing).

3. Result 3.1. Construction of the protein expression vector Many phytopathogenic Xoo strains have strict DNA restriction modification systems and only allow replication of some broad-range-host vectors in their cells (Choi et al. 1998). pHM1, a mobilizable cosmid vector with the oriV and pSa replicons (Fig. 1-A), is the most commonly used vector in Xoo cells because its genetically stable replication and delivery efficiency is much higher when compared to other vectors such as pUFR034 and some pBBR-derived vectors (Innes et al. 1988; Fujikawa et al. 2006; Makino et al. 2006). The potential of pHM1 as an important genetic tool in Xoo strains could be further exploited. Therefore, we constructed a new protein expression vector pH3-flag using it as the backbone. Firstly, we constructed pH3 derived from pHM1 by insertion of the four tandem copies of T1 terminators (T1)4 and two strong transcriptional terminators T0T1 from pPROBE-AT into the BamHI sites to prevent read-through transcription effect from the native lac operon (Fig. 1-B). Secondly, we artificially synthesized the DNA fragment of a MCS-FLAG cassette containing a multiple cloning site (MCS) with eight unique restriction enzyme sites (from SphI to EcoRI) and the Flag tag encoded sequence which were flanked by two HindIII restriction sites (Fig. 1-C). Lastly, the protein expression vector pH3-flag was created by insertion of the MCS-FLAG cassette into the HindIII site of pH3 in the order of MCS downstream of the (T1)4 terminators (Fig. 1-D). The pH3-flag not only retains the genetic advantages of pHM1, but also contains five unique restriction sites, which facilitates the cloning of the interested genes.

3.2. Development of the T3SS protein detection system The core hrp cluster of Xoo contains nine hrp genes, nine hrc genes and eight hpa genes referred to as T3SS genes which are not constitutively expressed (Fig. 2-A) (Zou et al.

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Fig. 1 Construction of protein expression vector pH3-flag. A, map of the cosmid vector pHM1 according to the sequence deposited in GenBank. B, map of the vector pH3. C, sequence of the multiple cloning site (MCS)-FLAG DNA cassette. A modified pUC18 polylinker, in which the SmaI site has been deleted. Flag, a FLAG tag encoded sequence. D, map of the protein expression vector pH3-flag. Details are given in Materials and methods. rrnB T1, transcription terminator T1 from the Escherichia coli rrnB gene; lambda T0, transcription terminator T0 from phage lambda; parB, encoding partition protein, a repressor and autoregulator of partition operon; parA, encoding partition protein ATPase; oriV, RK2 origin of replication; cos, cos region from phage lambda; traJ, encoding oriT-recognizing protein; oriT, incP origin of transfer; traK, encoding oriT-binding protein; SmR, aadA gene encoding spectinomycin resistance regions, pSa ori, origin of replication from bacterial plasmid pSa; repA, encoding trans-acting replication function of pSa. Some restriction enzyme sites were indicated in the maps.

2006), but are activated when the bacteria enter the plant or are cultivated in a hrp-inducing medium XOM3, whereas are

repressed in a nutrition rich medium NA (Xiao et al. 2007). Our previous studies on X. oryzae pv. oryzicola (Xoc) T3SS

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genes indicated that expression of T3SS genes in different hrp operons might exhibit different levels and proteins encoded by the low expressed T3SS genes were hardly to be detected by Western blot assay (Li et al. 2011; Cui et al. 2013). For this reason, we sought to determine which T3SS genes are ideal candidates for protein detection. Therefore, a GUS activity assay firstly was performed to determine the promoter activities of nine T3SS genes hpa2, hpa1, hrcC, hrpB1, hrcU, hrcQ, hrpD6, hrpE and hrpF in the addition of two major T3SS regulatory genes hrpG and hrpX in the background of the wild type strain. We found that the GUS activities of the promoters of hrpB1, hrcU, hrcQ, hrpF and hrpG were higher when compared to those of hpa1, hpa2 and hrpE (Fig. 2-B). We cloned the functional fragments of hrpG, hrpX, hrpB1, hrcU, hrcQ and hrpF containing their native promoters into the pH3-flag vector to obtain the HrpFlag or Hrc-Flag fusion proteins (Fig. 3-A). Finally, HrpG, HrpX and HrpB1 protein expression in the wild type was verified by immunoblotting with an anti-FLAG antibody. We found that the expression of HrpG-Flag, HrpX-Flag and HrpB1-Flag could be detected in the wild type cells at 3 h after induction both in the rich medium NB and in the hrp-inducing medium XOM3, however, these three protein expression levels in XOM3 were significantly higher than that in NB (Fig. 3-B). To examine whether the (T1)4 terminators in pH3-flag is effective to prevent the read-through transcription effect that originates upstream of the lac operon of pHM1, we also A

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constructed the HrpX-Flag and HrpB1-Flag fusion proteins in pHM1 without the (T1)4 terminators (Fig. 3-A). The HrpXFlag and HrpB1-Flag expression levels in absence of the (T1)4 terminators were higher than that in presence of the (T1)4 terminators, whereas this expression pattern was evidently observed in XOM3 medium when compared to in NB medium (Fig. 3-B). This result indicated that the (T1)4 terminators in pH3-flag vector reduced the background level of protein expression which is a huge interference in the study of gene regulation network. In order to test whether this system is effective for T3SS protein detection in hrp-inducing condition, the HrpG, HrpX and HrpB1 proteins levels in the wild type cells were monitored at different induction time points. We found that HrpG-Flag, HrpX-Flag and HrpB1-Flag expression were significantly increased with the induction time between 6 and 12 h (Fig. 4). In the meantime, we found that the amount of viable bacteria was reduced between 6 and 12 h after induction in XOM3 medium when the probability of protein detection errors was significantly increased. Therefore, we recommend the optimal induction time of Xoo cells in XOM3 medium is 3 to 6 h, and T3SS proteins could be detected effectively by Western blot assay.

3.3. Application of the protein expression system in gene regulation analysis Regulation of T3SS genes in Xoo depends on two master

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Fig. 2 Relative promoter activity of the T3SS genes in the wild type strain PXO99A. A, genetic organization map of the core hrp cluster in Xanthomonas oryzea pv. oryzae (Xoo). Open arrows indicate the positions and orientations of the hrp, hrc and hpa genes. Black rectangles above open arrows indicate the positions of the PIP-Box, a conserved plant inducible promoter (PIP) box sequence (TTCGC-N15-TTCGC) can be bound by HrpX. B, promoter GUS activities of the hrpG, hrpX, hrpF, hrpB1, hrcQ, hrcU, hrpE, hpa1, hpa2 and hrpD6. Data are mean±SD (n=3).

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Fig. 3 HrpG, HrpX and HrpB1 expression levels in the wild-type induced in XOM3 and NB media by Western blot assay. A, sketch map of the constructions of fusion expression vector. B, comparison of HrpG, HrpX, HrpB1 proteins in the wild-type induced in XOM3 with in NB. Cells were cultured in XOM3 for 3 h at 28°C, then the total protein extracts were analyzed by immunoblotting using anti-FLAG antibodies. RNAP, RNA polymerase subunit alpha from Escherichia coli, was used as a loading control. Value is the relative protein abundance which was calculated by ImageJ Software. Similar results were observed in two independent experiments. ND, not detected.

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Fig. 4 Monitoring of the HrpG, HrpX and HrpB1 expression levels in the wild type cells at different induction time points. The wild type cells containing the constructs of pH3-hrpG::flag, pH3-hrpX::flag and pH3-hrpB::flag were cultured in XOM3 at 28°C, then the total protein extracts were analyzed by immunoblotting using anti-FLAG antibodies. Samples were taken at the time points (h) on the top. RNAP, RNA polymerase subunit alpha from Escherichia coli was used as a loading control. Value is the relative protein abundance which was calculated by ImageJ Software. Similar results were observed in two independent experiments.

regulators HrpG and HrpX (Buttner and Bonas 2010). HrpG, a OmpR transcriptional regulator, activates the transcriptional expression of hrpX which encodes an AraCtype transcriptional activator and binds to a conserved plant

inducible promoter (PIP) box sequence (TTCGC-N15TTCGC) present in the promoter regions of most of hrp/hrc/hpa genes, activating T3SS expression (Furutani et al. 2006; Li et al. 2012; Xue et al. 2014). The expression of hrpG is

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the shielding of the (T1)4 terminators (Fig. 5-C); however, the HrpB1-Flag expression levels were dramatically reduced in the hrpG and hrpX mutants compared to the wild type when the (T1)4 terminators were added upstream the construct of hrpB1::flag fusion (Fig. 5-C). This suggests that there is an interference by read-through transcription on the expression of hrpB1::flag in the construct of pHM1-hrpB1::flag, however, the interference was significantly reduced in the presence of the (T1)4 terminators in the construct of pH3-hrpB1::flag. The improved protein expression system in this study is an effective tool to accurately and truly reflect the expression levels of T3SS proteins.

controlled by multiple regulators including the H-NS protein XrvA and Trh, a member of the GntR regulator family (Tsuge et al. 2006; Feng et al. 2009). Here we demonstrated the application of this protein expression system for the study of regulation of T3SS genes expression in Xoo. The HrpGFlag expression level in the trh mutant was compared to the wild type, and the HrpX-Flag and HrpB1-Flag expression levels in the presence and absence of the (T1)4 terminators were detected in the hrpG and hrpX mutants compared to the wild type. Protein hybridization assays showed that the HrpG-Flag level was significantly lower in the trh mutant than that in the wild type (Fig. 5-A), HrpX-Flag could not be detected in the hrpG mutant with or without the control of the (T1)4 terminators (Fig. 5-B), which is consistent with the previous findings that Trh is a positive regulator of the expression of hrpG and HrpG positively regulates the expression of hrpX (Tsuge et al. 2006). Unexpectedly, the HrpB1-Flag expression levels were not significantly different among the hrpG, hrpX mutants and the wild type without

3.4. HrcC expression depends on HrpG but not HrpX hrcC is the sole gene in the hrpA operon of the genomic core hrp cluster of Xoo , whereas, in the promoter region of which, the PIP-box is not found (Fig. 2-A). We speculate that the hrcC expression could not be regulated by HrpX, however,

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Fig. 5 Application of pH3-flag in the regulative analysis of HrpG, HrpX and HrpB1 proteins. A, comparison of HrpG expression levels in the wild type (WT) with the trh mutant. B, comparison of HrpX expression levels with or without terminator motifs in the wild type and the hrpG mutant by Western blot assay. C, comparison of HrpB1 expression levels with or without terminator motifs in the wild type, the hrpG mutant and the hrpX mutant by Western blot assay. Cells were cultured in XOM3 for 3 h at 28°C, then the total protein extracts were analyzed by immunoblotting using anti-FLAG antibodies. RNAP, RNA polymerase subunit alpha from Escherichia coli was used as a loading control. Value is the relative protein abundance which was calculated by ImageJ Software. Similar results were observed in two independent experiments. ND, not detected.

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no evidence was provided to support the hypothesis. To confirm this hypothesis, we tested the HrcC-Flag expression levels in the hrpG and hrpX mutants compared to the wild type using our newly developed protein expression system, we found that the HrcC-Flag expression levels in the hrpG mutant was significantly lower than that in the wild type (Fig. 6). However, this expression pattern was not observed in the hrpX mutant when compared to the wild type (Fig. 6). This result supports our notion that the hrcC expression depends on HrpG but not HrpX.

4. Discussion In this study, a broad-host-range protein expression vector pH3-flag derived from pHM1 was constructed. Using this vector, we analyzed the endogenous expression of HrpG, HrpX and HrpB1 in the hrp-inducing conditions compared to the nutrition-rich conditions, as well as in the mutant strains compared to the wild type and established a standardized procedure to detect the expression levels of T3SS proteins. Most existing bacterial protein expression systems have a narrow host range because they are usually based on pET vector series which couldn’t replicate in Xoo strains (Liu and Yang 2012). Whereas most broad-host-range plasmids which can replicate in Xoo strains are merely cloning vectors lack of essential motifs for protein detection. Some wildly used plasmids in Xoo strains include pHM1, pML123 and pUFR034 and pBBR1 series vectors (DeFeyter et al. 1990; Labes et al. 1990; Kovach et al. 1995). We preferred to use pHM1 than any others as the backbone of our new expression vector because it can overcome many hurdles. First, it can be stably maintained in cells of some Xoo strains such as PXO99A, a typical Xoo strain, which is a very important prerequisite for a protein expression HrcC POX99A

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Fig. 6 HrcC expression is dependent on the hrpG gene but not on the hrpX gene. Abundance of HrcC proteins in the wild type and the hrpG and hrpX mutants were detected by Western blot assay. Cells were cultured in XOM3 for 3 h at 28°C, then the total protein extracts were analyzed by immunoblotting using anti-FLAG antibodies. RNAP, RNA polymerase subunit alpha from Escherichia coli was used as a loading control. Value is the relative protein abundance which was calculated by ImageJ Software. Similar results were observed in two independent experiments.

vector. Second, it is a low copy number cosmid, which renders a desired expression levels of the T3SS proteins in our planned experiments. A possible drawback of this vector includes its low copy number make it harder for DNA manipulation (Innes et al. 1988). The new protein expression vector pH3-flag described here provides high-level versatility in terms of high electroporation delivery yields, carefully engineered MCSFLAG fusion and (T1)4 terminators to avoid interference of read-through transcription. We almost combined all essential motifs of an ideal protein expression vector into this vector. In our previous work, we also constructed a 3Myc-tag expression vector pH3-3myc based on the same backbone of pH3 vector, however, we found that it was not effectively applied in sensitively differentiate the protein expression levels in mutant strains compared to the wild type (data not shown). According to our experiences, we recommend using Flag as the tag to construct a protein expression vector if which will be applied in gene regulation analysis in Xoo strains. We did not detect the signals for HrpF, HrcU and HrcC proteins in the Western blot assay. We speculate that HrpF, HrcU and HrcQ are membrane proteins which are difficult to be obtained using our methods in Western blot assay. The biggest improvement in our protein expression system is the use of (T1)4 terminators which usually exist in certain promoter-prove vectors to prevent the effect of read-through transcription caused by vector background. No hybridization signals were observed in the wild type containing pH3-flag (Fig. 3-B), which indicates that (T1)4 terminators have reduced interferes from vector background to zero. In the case of induced expression, the HrpB1-Flag expression levels were not obviously different in the hrpG and hrpX mutants compared to the wild type when the hrpB1::flag fusion was constructed in pHM1 without the (T1)4 terminators added (Fig. 5-C). However, the HrpB1Flag expression levels were dramatically reduced in the hrpG and hrpX mutants when the (T1)4 terminators were constructed upstream the fusion (Fig. 5-C). This suggests that there is an interference of read-through transcription which may cause constitutive expression of the hrpB1::flag fusion masking the correct regulatory pattern. However, this interference was significantly reduced when the (T1)4 terminators was used. As the influence of read-through transcription did not appear in the case of the hrpX::flag fusion (Fig. 5-B), we speculate that this difference might be related to the cloning sequences of the functional genes such as hrpB1 and hrpX. Taken together, the terminator motif plays a very important role in constructing a protein expression vector to be used in research fields of gene regulation.

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5. Conclusion In summary, we constructed a protein expression vector pH3-flag which could be very effectively used as a molecular tool in understanding T3SS genes expression and regulation in Xoo strains. This vector will be very useful for gene complementation and gene regulation in other bacteria where the oriV and pSa origins of replication can replicate. This protein expression system may be potential for further exploiting as a transcriptional expression system if the MCS-FLAG motif is replaced by a promoter-probe cassette.

Acknowledgements This work was supported by the National Key R&D Program of China (2017YFD0200400) and the National Natural Science Foundation of China (31772122 and 31470235). We thank Dr. Fazal Haq, Shanghai Jiao Tong University, China, for critical reading of the manuscript and Jan Leach (Colorado State University, USA) for providing the pHM1 vector. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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