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. 2015 Dec 4;17(5):691–702. doi: 10.1111/mpp.12324

Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora

Jae Hoon Lee 1, George W Sundin 2, Youfu Zhao 1,✉
PMCID: PMC6638409  PMID: 26440313

Summary

The type III secretion system (T3SS) is a key pathogenicity factor in E rwinia amylovora. Previous studies have demonstrated that the T3SS in E . amylovora is transcriptionally regulated by an RpoN–HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant‐pathogenic bacteria. Complementation of the hrp L mutant with promoter deletion constructs of the hrp L gene and promoter activity analyses using various lengths of the hrp L promoter fused to a promoter‐less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between −138 and −125 nucleotides (TGCAA‐N4‐TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrp L gene among plant enterobacterial pathogens. Results of quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) and electrophoresis mobility shift assay coupled with site‐directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E . amylovora was characterized by complementation of the hrp S mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E . amylovora.

Keywords: fire blight, HrpL, RpoN, sigma factor, T3SS, virulence

Introduction

Like many other Gram‐negative plant‐pathogenic bacteria, the hypersensitive response and pathogenicity type III secretion system (hrp‐T3SS) is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight on apples and pears (Khan et al., 2012; Zhao, 2014). The hrp‐T3SS functions in the delivery of effector proteins into plant cells, which eventually interfere with plant innate immune systems and cellular metabolism during pathogenesis (Büttner, 2012). Molecular genetic studies of E. amylovora pathogenesis have demonstrated that the expression of hrp‐T3SS genes is activated by the master regulator HrpL, a member of the exocytoplasmic functions (ECF) subfamily of sigma factors (Wei and Beer, 1995). The promoter region of genes regulated by HrpL possesses the hrp box (GGAACC‐N16‐CCACNNA), in which HrpL binds and directs RNA polymerase (RNAP) for transcription initiation (McNally et al., 2012). In E. amylovora, hrpL transcription is, in turn, controlled by another alternative sigma factor, sigma factor 54 (RpoN), which, together with its modulation protein YhbH, interacts with enhancer binding protein (EBP) HrpS to trigger the onset of T3SS (Ancona et al., 2014). The interaction between HrpS and RpoN also requires the function of integration host factor (IHF) in E. amylovora (Lee and Zhao, 2015). Thus, E. amylovora can rapidly activate the hrp‐T3SS in response to inducing signals through this RpoN–HrpL sigma factor cascade (Ancona et al., 2014; Lee and Zhao, 2015). However, the exact role of YhbH in hrpL transcription remains unknown. Furthermore, it has been reported recently that the linear nucleotide (nt) second messengers, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), are also essential for the expression of T3SS and virulence by activation of the RpoN–HrpL alternative sigma factor cascade (Ancona et al., 2015).

In bacteria, gene expression is mainly regulated at the level of transcription initiation, and core RNAP requires sigma factors for promoter recognition and initiation. RpoN directs RNAP to the consensus −24 (GG) and −12 (TGC) promoter regions, and the RpoN‐RNAP holoenzyme forms a highly stable, closed complex with DNA which is unable to spontaneously isomerize into an open complex (Buck et al., 2000; Guo et al., 2000). To initiate transcription, specific EBPs must be present to remodel the RpoN‐RNAP holoenzyme (Cannon et al., 2001). With the assistance of IHF, RpoN contacts EBPs at the conserved motif (GAFTGA) (Jovanovic et al., 2011; Zhang et al., 2009) and, using the energy provided by ATP hydrolysis, the RpoN‐RNAP–promoter complex undergoes a conformational change to initiate transcription (Bush and Dixon, 2012; Schumacher et al., 2006). Thus, this type of gene regulation is evolutionarily advantageous as the resulting transcription is tightly and specifically regulated. Consequently, RpoN‐dependent gene expression is considered as the second paradigm of bacterial transcription and is often responsible for the creation of precise responses to environmental changes by rapid regulation of stress response genes (Buck et al., 2006).

EBPs are members of the functionally versatile AAA+ (ATPase associated with various cellular activities) family of proteins, which can couple chemical energy derived from ATP hydrolysis to a mechanical action (Schumacher et al., 2004; Wang, 2004). In order to initiate RpoN‐dependent transcription, EBPs must recognize and bind to a specific upstream activator sequence (UAS) near the promoter of the target gene. As the UAS is commonly located at 80–150 bp upstream of the transcription start site, interactions between the RpoN‐RNAP holoenzyme and EBPs are often achieved by IHF‐induced DNA bending (Carmona and Magasanik, 1996; Hoover et al., 1990; Huo et al., 2006). So far, most UASs reported have a dyad symmetry sequence in which the inactive state of EBPs in a dimeric form binds (Bush and Dixon, 2012) and promotes oligomerization for increased ATPase activity (De Carlo et al., 2006; Rombel et al., 1998). However, the UAS for HrpS remains unidentified.

One structurally conserved motif within the AAA+ domain of EBPs is the GAFTGA motif. The surface‐exposed loop of the GAFTGA motif enables EBPs to interact with RpoN, leading to conformational changes in the AAA+ domain and substrate remodelling on ATP hydrolysis (Bordes et al., 2003; Zhang et al., 2002). All six residues of the GAFTGA motif are essentially required for full transcriptional activity (Bush and Dixon, 2012). However, about 7% of the EBPs, including HrpS proteins in both E. amylovora and Pseudomonas syringae pv. tomato DC3000, contain tyrosine (Y) instead of phenylalanine (F) in the GAFTGA motif (Zhang et al., 2009). Interestingly, it has been reported recently that substitution of Y with F in HrpS of P. syringae pv. tomato DC3000 increases hrpL promoter activity by about 1.5‐fold, suggesting a regulatory reason for the natural variants (Jovanovic et al., 2011). This led us to ask whether an F to Y substitution in the GAYTGA motif of HrpS would also affect hrpL gene expression in E. amylovora.

The precise binding site of HrpS within the hrpL promoter is currently unknown in plant‐pathogenic bacteria. Our main goal in this study was to address this knowledge gap by identification of the HrpS binding site within the E. amylovora hrpL promoter. In addition, we wished to determine the effect of amino acid substitution in the conserved motif of HrpS on T3SS gene expression and virulence. Our results demonstrated that a 14‐bp dyad symmetry sequence (TGCAA‐N4‐TTGCA) in the hrpL promoter, conserved among enterobacterial plant pathogens, is the UAS for HrpS binding. Our results also showed that Y and F can compensate functionally for each other in the GAYTGA motif of HrpS in E. amylovora.

Results

UAS of the hrp L promoter is necessary for its transcription

The hrpL promoter of E. amylovora contains the conserved binding site for RpoN (−12 nt, −24 nt) and IHF (−43 nt) in close proximity to the transcription start site. In contrast, EBPs bind at a UAS generally located distant (−80 to −150 nt) from the gene (Bush and Dixon, 2012). On the basis of these observations, we made nine constructs containing different lengths of the hrpL promoter (within −398 to +86 nt) fused to a promoter‐less green fluorescent protein (gfp) reporter gene, and their promoter activities were determined by flow cytometry (Fig. 1). Constructs containing the hrpL promoter up to −128 nt (pZW2‐1 to pZW2‐4) exhibited basal levels of GFP intensity relative to the vector control (pFPV25), whereas constructs containing the hrpL promoter region from −153 nt to −398 nt (pZW2‐5 to pZW2‐9) exhibited a greater than two‐fold increase in GFP intensity (Fig. 1). This indicates that the hrpL promoter region up to −153 nt is required for the activation of hrpL gene expression. In addition, an approximately 10% increase in GFP intensity was observed between strains containing plasmids pZW2‐6 and pZW2‐7, suggesting that an unknown factor with a potential role in enhancing hrpL gene expression may bind to −215 and −153 nt of the hrpL promoter, or the presence of small regulatory RNA and other regulatory elements may contribute to this increase.

Figure 1.

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Schematic diagram of the hrp L promoter and promoter–gfp fusion constructs, and green fluorescent protein (GFP) intensities in E rwinia amylovora. Series deletions of the hrp L promoter–gfp fusion constructs were generated in pFPV25 and introduced into E . amylovora strain Ea273. The 5′ end of each construct relative to the putative transcription start site of the hrp L gene is indicated. Bacterial strains were grown in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 18 h. GFP activity refers to a geometric mean of three replicates ± standard deviations. GFP activities marked with the same letter were not significantly different (P < 0.05). Black arrow, hrp L gene and its promoter; dashed line, putative HrpS binding site; open rectangle, RpoN binding site; filled rectangle, integration host factor (IHF) binding site.

The dyad symmetry sequence in the hrp L promoter is required for virulence

It has been reported that most UASs identified so far contain a dyad symmetry sequence (Bush and Dixon, 2012). To further delineate the potential HrpS binding site, sequence analysis was conducted between −153 and −104 nt of the hrpL promoter, and a 14‐bp dyad symmetry sequence (TGCAA‐N4‐TTGCA) spanning from −138 to −125 nt of the hrpL promoter was identified. To verify the results obtained by reporter gene‐based promoter activity assay, various 5′‐deletion constructs of the hrpL promoter were generated (Fig. 2), including constructs containing no, half and full dyad symmetry sequence, which were designated as pHrpL2, pHrpL3 and pHrpL4, respectively.

Figure 2.

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Schematic diagram of constructs for complementation of the hrp L mutant, virulence on pears and apple shoots, and hypersensitive response (HR) assay on tobacco. Series constructs containing the hrp L gene with different lengths of the hrp L promoter were cloned into pWSK29 and introduced into the hrp L mutant. The 5′ end of each construct relative to the putative transcription start site of hrp L is indicated. + represents disease severity on pears at 8 days post‐inoculation and HR severity on tobacco at 24 h post‐infiltration, respectively. Numbers represent average necrosis length on apple shoots at 7 days post‐inoculation in centimetres ± standard deviation. Necrosis lengths followed by the same letter were not significantly different (P < 0.05).

Different hrpL complementation strains were first tested for virulence on immature pear fruits (Fig. 2). Pear fruits infected by strain hrpL(pHrpL) showed water soaking symptoms at 2 days post‐inoculation (DPI), necrotic lesions with visible bacterial ooze at 4 DPI and black lesions covering the entire surface of pear fruits at 8 DPI. Similar disease was observed for the hrpL mutant complemented with constructs carrying a half or full dyad symmetry sequence (pHrpL3, pHrpL4 and pHrpL5). Interestingly, construct pHrpL2, which does not contain the dyad symmetry sequence, partially complemented the hrpL mutant, and resulted in strongly reduced disease. No disease was observed for strain hrpL(pHrpL1) and the vector control strain hrpL(pWSK29).

Virulence assays were also carried out on apple shoots (Fig. 2). Visible necrosis around the inoculation site was observed for the hrpL mutant complemented with the full length of the promoter construct (pHrpL) at 3 DPI, and the length of the necrotic lesion reached 18.25 cm at 7 DPI. No disease symptoms were observed for strain hrpL(pHrpL1) and the vector control strain hrpL(pWSK29), whereas similar disease was observed for strain hrpL(pHrpL5) relative to strain hrpL(pHrpL). The lengths of the necrotic lesions were slightly reduced for strain hrpL(pHrpL4) (14.4 cm) and about 50% reduced for strain hrpL(pHrpL3) (9.9 cm) relative to strain hrpL(pHrpL). Similar to the results obtained from immature pear fruits, construct pHrpL2 barely rescued the hrpL mutant, and the length of the necrotic lesion was reduced significantly (2.0 cm) at 7 DPI.

The hrpL complementation strains were further tested for their ability to elicit the hypersensitive response (HR) on tobacco leaves (Fig. 2). Consistent with the disease‐causing ability, the hrpL mutant complemented with pHrpL, pHrpL4 and pHrpL5 induced strong HR, whereas hrpL(pHrpL1) did not result in HR at 24 h post‐infiltration. A gradually reduced HR was observed for the hrpL mutant complemented with constructs pHrpL3 and pHrpL2 (Fig. 2).

Together, these results indicate that the complete dyad symmetry sequence of the hrpL promoter is required for full virulence of E. amylovora. However, the construct containing half of the dyad symmetry sequence is sufficient to complement the hrpL mutant and to cause disease. Furthermore, our results suggest that the hrpL promoter region spanning −121 to −104 nt may also influence either HrpS binding or an unknown factor involved in the activation of hrpL transcription. This region contains an 11‐bp mirror sequence (TTTGG‐N‐GGTTT).

Intact dyad symmetry sequence in the hrp L promoter is critical for the transcription of the hrp L and hrp A genes

To evaluate the role of the dyad symmetry sequence in the hrpL promoter in T3SS gene expression, the relative expression of the hrpL and hrpA genes was determined by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). Consistent with virulence and HR assays, expression of the hrpL gene in strains hrpL(pHrpL4) and hrpL(pHrpL5) was similar to or slightly higher than that of strain hrpL(pHrpL) in both in vitro (Fig. 3A) and in vivo (Fig. 3B) conditions. Expression of the hrpL gene was about 5–10‐fold and 25–50‐fold lower in strains hrpL(pHrpL3) and hrpL(pHrpL2), respectively, whereas it was barely detected in strain hrpL(pHrpL1). The expression trend of the hrpA gene in all strains tested was similar to that of the hrpL gene (Fig. 3A,B). These results indicate that the intact dyad symmetry sequence in the hrpL promoter is required for full expression of hrpL and hrpA genes. As the expression of T3SS genes was not completely abolished in strain hrpL(pHrpL2), this further suggests that the 11‐bp mirror sequence might contribute to hrpL gene expression.

Figure 3.

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Expression of type III secretion system (T3SS) genes in the hrp L mutant complemented with various constructs in vitro and in vivo. Relative expression of the hrpL and hrp A genes in the hrp L mutant complemented with constructs (pHrpL1–pHrpL5) relative to construct pHrpL by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) In vitro in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 6 h. (B) In vivo on immature pear fruits for 6 h. The mutant strain complemented with a vector (pWSK29) was used as a negative control. The rpo D gene was used as an endogenous control, and the values of the relative fold change are the means of three replicates. Error bars indicate standard deviation. Fold changes marked with the same letter were not significantly different (P < 0.05).

Next, site‐directed mutagenesis (SDM) was performed to generate base substitution constructs to further evaluate the role of the dyad symmetry sequence and the mirror sequence in T3SS gene expression (Fig. 4). Constructs pHrpL3 and pHrpL2 were chosen to generate mutant constructs as they fully and partially complement, respectively, the hrpL mutant in virulence (Fig. 2). Three pHrpL3‐based mutant constructs, pHrpL3‐Mut1, pHrpL3‐Mut2 and pHrpL3‐Mut3, contained 3‐, 3‐ and 1‐nt substitution(s), respectively, at the half dyad symmetry sequence (Fig. 4). These mutant constructs partially complemented the hrpL mutant in causing disease on immature pears to a similar degree to the pHrpL2 construct (Fig. S1, see Supporting Information). Expression of the hrpL and hrpA genes was about 5–10‐fold lower in strain hrpL(pHrpL3‐Mut1), 2.5–5‐fold lower in strain hrpL(pHrpL3‐Mut2) and 1.5–2.5‐fold lower in strain hrpL(pHrpL3‐Mut3) relative to strain hrpL(pHrpL3) (Fig. 5). However, one pHrpL2‐based mutant construct, pHrpL2‐Mut4, contained three base substitutions at the mirror sequence. The virulence and T3SS gene expression of strain hrpL(pHrpL2‐Mut4) were similar to those of strain hrpL(pHrpL2) (Figs 6, S1). These results suggest that the dyad symmetry sequence, but not the mirror sequence, is required for full expression of the hrpL gene.

Figure 4.

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Schematic diagram of nucleotide replacement constructs for complementation of the hrp L mutant and virulence assay on immature pear fruits. Constructs containing mutation(s) on the dyad symmetry sequence and the mirror repeat were generated on the basis of plasmids pHrpL3 and pHrpL2, respectively, and were introduced into the hrp L mutant. The 5′ end of each construct relative to the putative transcription start site of the hrp L gene is indicated, and base substitution(s) are indicated in red. + represents the disease severity on pears at 8 days post‐inoculation. The dyad symmetry sequence is underlined, and the mirror sequence is indicated with italic letters.

Figure 5.

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Expression of type III secretion system (T3SS) genes in the hrp L mutant complemented with constructs containing mutation(s) in the dyad symmetry sequence in vitro and in vivo. Relative expression of the hrp L and hrp A genes in the hrp L mutant complemented with mutant constructs (pHrpL3‐Mut1–pHrpL3‐Mut 3) relative to its original construct by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) In vitro in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 6 h. (B) In vivo on immature pear fruits for 6 h. The hrp L mutant strain complemented with a vector (pWSK29) was used as a negative control. The rpo D gene was used as an endogenous control, and the values of the relative fold change are the means of three replicates. Error bars indicate standard deviation. Fold changes marked with the same letter were not significantly different (P < 0.05).

Figure 6.

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Expression of type III secretion system (T3SS) genes in the hrp L mutant complemented with constructs containing mutations on the mirror repeat sequence in vitro and in vivo. Relative expression of the hrp L and hrp A genes in the hrp L mutant complemented with mutant construct (pHrpL2‐Mut4) relative to its original construct (pHrpL2) by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) In vitro in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 6 h. (B) In vivo on immature pear fruits for 6 h. The mutant strain complemented with a vector (pWSK29) was used as a negative control. The rpo D gene was used as an endogenous control, and the values of the relative fold change are the means of three replicates. Error bars indicate standard deviation. Fold changes marked with the same letter were not significantly different (P < 0.05).

HrpS 250–325 binds to the dyad symmetry sequence in the hrp L promoter

In order to demonstrate that the HrpS protein binds to the dyad symmetry sequence, full‐length HrpS protein was first over‐expressed. However, despite many attempts to optimize the purification conditions, purification of the full length of the HrpS protein was not successful. Therefore, a truncated HrpS protein containing only the DNA binding domain (HrpS250–325) was over‐expressed and successfully purified for subsequent electrophoresis mobility shift assay (EMSA). To assess the specific binding of truncated HrpS250–325 protein to the dyad symmetry sequence, two different 26‐bp probes containing the intact dyad symmetry sequence and the mutated version of the dyad symmetry sequence were subjected to EMSA (Fig. 7A). HrpS250–325 bound to the intact dyad symmetry sequence in a concentration‐dependent manner, whereas no shift was observed for the mutated sequence (Fig. 7A), suggesting that HrpS specifically binds to the dyad symmetry sequence of the hrpL promoter.

Figure 7.

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Electrophoretic mobility shift assay (EMSA) and sequence alignment of the dyad symmetry sequence on the hrp L promoter in plant enterobacterial pathogens. (A) A 26‐bp sequence of the hrp L promoter region (−144 to −119 nucleotides) containing the original or mutated dyad symmetry sequence was tested for binding to truncated HrpS 250–325 protein. The dyad symmetry sequence is underlined, and mutated nucleotides are indicated in red (not all the 26‐bp sequence is shown). Black arrows indicate the unshifted DNA (free probe) and the protein–DNA complex (HrpS 250–325‐bound probe). The concentration of purified HrpS250–325 is indicated above each lane. (B) Sequence alignment of the dyad symmetry sequences, indicated in red; numbers refer to the nucleotide position relative to the putative transcription start site of the hrp L gene.

In addition, sequence analysis of the hrpL promoter in other enterobacterial plant pathogens, including Pectobacterium atroseptica, Dickeya dadantii and Pantoea stewartii, showed that the identical dyad symmetry sequence exists in the hrpL promoter at a similar position relative to the putative transcription start site in all three pathogens (Fig. 7B), further indicating that the dyad symmetry sequence is well conserved among them.

Y and F function interchangeably in the conserved GAYTGA motif of HrpS

It has been reported previously that a single amino acid substitution from Y to F within the GAYTGA motif of HrpS in P. syringae pv. tomato DC3000 leads to a 1.5‐fold increase in hrpL promoter activity (Jovanovic et al., 2011). To investigate the effect on T3SS gene expression of a similar substitution within the GAYTGA motif of HrpS in E. amylovora, three single amino acid substitution mutant variants (Y100F, Y100A and T101A) were constructed and introduced into the hrpS mutant. The results of virulence assay on immature pears and HR assay on tobacco indicated that the hrpS mutant could be rescued by the Y100F variant, but not by either Y100A or T101A variants (Fig. 8). Although the expression of the hrpS gene was identical in the hrpS mutant complemented by the three mutant variants and wild‐type HrpS, expression of the hrpL and hrpA genes was abolished in the hrpS mutant complemented with the Y100A and T101A mutant variants of HrpS, but was not affected by the Y100F variant (Fig. S2, see Supporting Information). These results indicate that the Y100F substitution in the GAYTGA motif does not affect the function of HrpS in E. amylovora.

Figure 8.

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Virulence and hypersensitive response (HR) assays of E rwinia amylovora wild‐type (WT), hrp S mutant and different complementation strains of the hrp S mutant. (A) Symptoms caused by WT, hrp S mutant and its complementation strains on immature pear fruits. Immature pears were surface sterilized, pricked with a sterile needle and inoculated with 2 μL of bacterial suspension. Symptoms were recorded and photographs were taken at 4 and 8 days post‐inoculation (DPI). (B) HR assay on tobacco leaves. E rwinia amylovora  WT, hrpS mutant and its complementation strains were infiltrated into 8‐week‐old tobacco leaves. Phosphate‐buffered saline (PBS) was used as a negative control. Photographs were taken at 24 h post‐infiltration.

Discussion

In most Gram‐negative plant‐pathogenic bacteria, T3SS plays a central role in the colonization of host plants. T3SS‐secreted effectors impede host immunity, enabling pathogens to overcome host defence barriers and to establish successful infection (Büttner, 2012). Therefore, a comprehensive understanding of the function and regulation of T3SS is critical in the study of plant–bacterium interactions (Büttner and Bonas, 2006). The current model of T3SS regulation in Pseudomonas, Erwinia and Pectobacterium indicates that HrpS acts as an essential activator of RpoN‐dependent hrpL transcription (Ancona et al., 2014; Chatterjee et al., 2002; Grimm et al., 1995; Wei et al., 2000; Yap et al., 2005). Extrapolating from the function of EBPs, HrpS might play an essential role in the sensing of environmental cues and activation of T3SS. In this study, for the first time, we identified the binding site of HrpS on the hrpL promoter in plant‐pathogenic bacteria.

Like many other EBPs, the binding site of HrpS exhibits perfect dyad symmetry and is located between 80 and 150 bp upstream of the transcription start site. Interestingly, the HrpS binding site reported in this study is similar to that of EBP NtrC (TGCACC‐N5‐GGTGCA), but is different from those reported for EBPs PspF (GTGAA‐N1‐TTCAC) and NorR (AGTCAA‐N1‐TTGACT) in Escherichia coli (Bush et al., 2004; Jovanovic and Model, 1997; Reitzer and Magasanik, 1986; Reitzer and Schneider, 2001). The nitrogen regulatory protein C (NtrC) activates a variety of genes involved in nitrogen utilization (Reitzer and Schneider, 2001). Earlier studies have reported that amino acids, including aspartic and glutamic acids, together with fructose or mannitol, strongly induce T3SS gene expression in Pseudomonas and Erwinia (Ancona et al., 2015; Anderson et al., 2014; Chatnaparat et al., 2015). It is tempting to speculate that there might be a connection between nitrogen metabolism and T3SS gene activation in plant‐pathogenic bacteria. Therefore, it could not be ruled out that EBPs such as NtrC and HrpS could bind to and regulate each other's target genes, which deserves further investigation.

It is reasonable to expect that genes regulated directly by HrpS in E. amylovora should also contain binding sites for RpoN and IHF (Ancona et al., 2014; Lee and Zhao, 2015). A hidden Markov model predicted 38 genes containing a potential RpoN binding site with a bit‐score classifier threshold of >13 (Table S1, see Supporting Information). However, promoters of these potential RpoN‐regulated genes do not contain the full dyad symmetry sequence, except for the promoter of the hrpL gene. In addition, the construct that contains half of the dyad symmetry sequence was able to complement the hrpL mutant, but expression of the hrpL gene was reduced. It is tempting to speculate that, as HrpS forms a homohexamer and binds to DNA, it is possible that the HrpS homohexamer could still loosely bind to the half dyad symmetry sequence, but the binding may not be as firm or as strong as with the full dyad symmetry sequence, resulting in weak interaction with RpoN and thus reduced activation of the hrpL gene. It is also possible that genes containing the half dyad symmetry sequence could be regulated by HrpS, but this needs to be further explored.

Furthermore, the Pe. atroseptica, D. dadantii and Pa. stewartii genomes contain the identical dyad symmetry sequence in the hrpL promoter (Fig. 7B), further corroborating our findings. However, this dyad symmetry sequence is not present in the hrpL promoter of P. syringae. Unlike Erwinia and other related plant enterobacteria, in which HrpS forms a homohexameric complex to activate RpoN‐dependent transcription, HrpS of P. syringae forms a heterohexamer via interaction with another EBP HrpR (Hutcheson et al., 2001). Although hrpS and hrpR of P. syringae are believed to arise from gene duplication events, they share about 60% sequence identity and 75% sequence similarity to each other (Jovanovic et al., 2011), presumably leading to a non‐dyad symmetry binding sequence on the hrpL promoter.

In addition to the dyad symmetry sequence, an unusual mirror sequence downstream of the HrpS binding site on the hrpL promoter in E. amylovora may also play a role in the activation of hrpL expression. Construct pHrpL2, containing only the mirror sequence but not the dyad symmetry sequence, could still partially complement the hrpL mutant in virulence. A comparison of the hrpL promoter sequence between E. amylovora and other plant enterobacteria revealed that the mirror sequence is only present in E. amylovora. However, the exact role of the mirror sequence and/or its surrounding region in the regulation of T3SS gene expression remains unclear. Furthermore, T3SS gene expression and virulence on apple shoots were increased when the hrpL mutant was complemented with constructs containing the promoter further upstream of the dyad symmetry sequence. This suggests that we still cannot rule out the possibility that other unknown factors or elements may also be involved in the activation of hrpL gene expression.

The importance of the conserved GAFTGA motif for the activation of RpoN‐dependent transcription has been demonstrated through amino acid substitution analyses on several different EBPs. All six residues of the GAFTGA motif are essential for full EBP activity, and mutation in the F residue has been shown to adversely affect ATPase activity, RpoN contact and oligomerization of EBPs (Bush and Dixon, 2012). It has been reported that, in about 7% of EBPs, including HrpS, F of the GAFTGA motif is replaced with Y (Zhang et al., 2009). Although both amino acids share a similar aromatic ring structure, substitution of F with Y within the GAFTGA motif of NifA and PspF results in significantly decreased EBP activities (Gonzalez et al., 1998; Zhang et al., 2009). In contrast, substitution of Y with F in the GAYTGA motif of HrpS in P. syringae pv. tomato DC3000 increases its activity by 50% (Jovanovic et al., 2011). In this study, substitution of Y with F within the GAYTGA motif of HrpS had no effect on its function in E. amylovora, suggesting that these two amino acids can function interchangeably in the GAYTGA motif of HrpS in E. amylovora.

In general, EBPs utilize the N‐terminal regulatory domain to sense environmental cues through various signal transduction intermediates, including the phosphoryl group, ligands and anti‐activator proteins (Bush and Dixon, 2012). However, HrpS lacks this N‐terminal regulatory domain, and thus may have evolved to regulate its activity differently. In P. syringae, HrpS activity is regulated through protein–protein interactions, including Lon‐dependent degradation and interplay with HrpV and HrpG (Ortiz‐Martín et al., 2010). HrpV inhibits the formation of HrpRS heterohexamer via direct interaction with HrpS, whereas HrpG suppresses the negative regulation of HrpV on HrpRS via direct interaction with HrpV (Jovanovic et al., 2011; Preston et al., 1998; Wei et al., 2005). A recent study has shown that HrpV and HrpG of E. amylovora also interact with each other (Gazi et al., 2015), although their roles in the regulation of HrpS activity and T3SS gene expression remain unknown. In Pectobacterium, Dickeya and Pantoea, HrpS is regulated at the transcriptional level by the HrpX/HrpY two‐component system. However, in E. amylovora, expression of hrpS is not dependent on HrpX/HrpY (Zhao et al., 2009). Future work should be focused on the molecular mechanisms underlying the regulation of hrpS gene expression and HrpS protein stability.

In summary, our results demonstrate that a 14‐bp dyad symmetry sequence between −138 and −125 nt (TGCAA‐N4‐TTGCA) on the hrpL promoter is the binding site for HrpS. Our results also show that Y and F function interchangeably in the GAYTGA motif of HrpS in E. amylovora. In addition to RpoN, HrpS, IHF and YhbH, it is believed that other unknown transcription factors may also interact directly with the hrpL promoter for the activation of hrpL gene expression. The identification of novel regulators will further our understanding of the function and regulation of T3SS, and bacterial pathogenesis.

Experimental Procedures

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Luria–Bertani (LB) medium was used for the routine culture of E. amylovora and Es. coli strains. Bacteria were also grown in hrp‐inducing minimal medium (HMM) [1 g (NH4)2SO4, 0.246 g MgCl2.6H2O, 0.099 g NaCl, 8.708 g K2HPO4, 6.804 g KH2PO4] containing 10 mm galactose as carbon source to induce T3SS gene expression (Ancona et al., 2014; Yang et al., 2014). When required, antibiotics were added to the medium at the following concentrations: 10 μg/mL chloramphenicol (Cm), 100 μg/mL ampicillin (Ap) and 20 μg/mL kanamycin (Km). The primer sequences used for cloning, qRT‐PCR and SDM in this study are listed in Table S2 (see Supporting Information).

Table 1.

Bacterial strains and plasmids used in this study

Strains, plasmids Description Reference or source
Strains
Erwinia amylovora
Ea1189 Wild‐type, isolated from apple Burse et al. (2004)
Ea273 Wild‐type, isolated from apple Wang et al. (2010)
ΔhrpL hrpL::Km; KmR‐insertional mutant of hrpL of Ea 1189, KmR Ancona et al. (2014)
ΔhrpS hrpS::Km; KmR‐insertional mutant of hrpS of Ea 1189, KmR Ancona et al. (2014)
Escherichia coli
DH10B F− mcrA Δ(mrr‐hsdRMS‐mcrBC) Φ80/acZ ΔM15 ΔlacX74 recA1 endA1 araΔ139 Δ(ara, leu)7697 galU galK rpsL nupG λ Invitrogen, San Diego, CA, USA
XL10‐Gold TetR Δ(mcrA)183 Δ(mcrCB‐hsdSMR‐mrr)173 endA1 supE44 thi‐1 recA1 gyrA96 relA1 lac Hte Stratagene, San Diego, CA, USA
BL21 (DE3) F− ompT hsdS B (rB − mB −) gal dcm (DE3) Novagen, San Diego, CA, USA
Plasmids
pFPV25 ApR, GFP based promoter trap vector containing a promoter‐less gfpmut3a gene Valdivia and Falkow (1997)
pWSK29 ApR, cloning vector, low copy number Wang and Kushner (1991)
pET28a(+) KmR, T7 expression vector carrying an N‐terminal His‐Tag/thrombin/T7 Tag configuration plus an optional C‐terminal His‐Tag sequence Novagen
pZW2 608‐bp DNA fragment of hrpL gene (−398–+210) in pFPV25 Wang et al. (2010)
pZW2‐1 144‐bp DNA fragment containing hrpL gene (−58–+86) in pFPV25 This study
pZW2‐2 180‐bp DNA fragment containing hrpL gene (−94–+86) in pFPV25 This study
pZW2‐3 190‐bp DNA fragment containing hrpL gene (−104–+86) in pFPV25 This study
pZW2‐4 214‐bp DNA fragment containing hrpL gene (−128–+86) in pFPV25 This study
pZW2‐5 239‐bp DNA fragment containing hrpL gene (−153–+86) in pFPV25 This study
pZW2‐6 263‐bp DNA fragment containing hrpL gene (−177–+86) in pFPV25 This study
pZW2‐7 301‐bp DNA fragment containing hrpL gene (−215–+86) in pFPV25 This study
pZW2‐8 350‐bp DNA fragment containing hrpL gene (−264–+86) in pFPV25 This study
pHrpL 1.317‐kb DNA fragment containing hrpL gene (−398–+919) in pWSK29 Ancona et al. (2014)
pHrpL1 1.023‐kb DNA fragment containing hrpL gene (−104–+919) in pWSK29 This study
pHrpL2 1.040‐kb DNA fragment containing hrpL gene (−121–+919) in pWSK29 This study
pHrpL3 1.050‐kb DNA fragment containing hrpL gene (−131–+919) in pWSK29 This study
pHrpL4 1.060‐kb DNA fragment containing hrpL gene (−141–+919) in pWSK29 This study
pHrpL5 1.072‐kb DNA fragment containing hrpL gene (−153–+919) in pWSK29 This study
pHrpL3‐Mut1 1.050‐kb DNA fragment containing hrpL gene (−131–+919) with a mutation at position −128 to −126 in pWSK29 This study
pHrpL3‐Mut2 1.050‐kb DNA fragment containing hrpL gene (−131–+919) with a mutation at position −124 to −122 in pWSK29 This study
pHrpL3‐Mut3 1.050‐kb DNA fragment containing hrpL gene (−131–+919) with a mutation at position −127 in pWSK29 This study
pHrpL2‐Mut4 1.040‐kb DNA fragment containing hrpL gene (−121–+919) with a mutation at position −115 to −113 in pWSK29 This study
pHrpS 1.81‐kb DNA fragment containing hrpS gene in pWSK29 Ancona et al. (2014)
Y100F 1.81‐kb DNA fragment containing hrpS gene with a mutation (Y100F) in pWSK29 This study
Y100A 1.81‐kb DNA fragment containing hrpS gene with a mutation (Y100A) in pWSK29 This study
T101A 1.81‐kb DNA fragment containing hrpS gene with a mutation (T101A) in pWSK29 This study
pHrpS250–325‐His 238‐bp DNA fragment containing hrpS gene in pET28a This study
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Construction of plasmids

For the generation of transcriptional gfp fusions, different lengths of the hrpL upstream sequence were amplified by PCR, restriction enzyme digested and ligated into the pFPV25 plasmid using standard molecular biology protocols (Sambrook and Russel, 2001). For the complementation of the hrpL mutant, different 5′ deletions of the hrpL gene were cloned into the pWSK29 plasmid as described above. SDM was performed using the QuickChange XL site‐directed mutagenesis kit (Stratagene, San Diego, CA, USA) according to the manufacturer's instructions. The resulting plasmids were verified by sequencing at the UIUC Core Sequencing Facility and transformed into E. amylovora strains by electroporation.

GFP reporter assays by flow cytometry

Overnight cultures of E. amylovora Ea273 strains carrying different gfp‐promoter fusion plasmids were harvested and washed with 0.5 × phosphate‐buffered saline (PBS). The bacterial suspensions were adjusted to an optical density at 600 nm (OD600) of 0.2 in HMM and incubated at 18 °C for 18 h. GFP intensities were measured for a total of 100 000 events by a BD FACSCanto flow cytometer (BD Bioscience, San Jose, CA, USA) and analysed by flow cytometry software FCS Express V3 (De Novo Software, LA, CA, USA). The experiments were repeated at least twice.

Virulence assays on immature pear fruits and apple shoots

Virulence assays were performed as described previously (Wang et al., 2011). Overnight cultures of E. amylovora WT, mutants and complementation strains were harvested and resuspended in 0.5 × PBS. Immature Bartlett pears (Pyrus communis L. cv. Bartlett) were surface sterilized with 10% bleach and rinsed with sterile distilled water. After drying, pears were pricked with a sterile needle, inoculated with 2 μL of cell suspensions at OD600 = 0.001 for each strain and incubated in a humidified chamber at 28 °C. Symptoms were recorded at 4 and 8 DPI, and pears were assayed in triplicate for each strain tested. Young ‘Gala’ apple shoots (about 22–25 cm in length) were also pricked with a sterile needle at the shoot tip and inoculated with 5 μL of cell suspensions at OD600 = 0.1. Plants were kept at 25 °C for a 16 h light photoperiod in a glasshouse. The length of necrotic symptoms from the inoculation site was measured at 8 DPI, and the average value was considered as the disease severity. For each strain tested, six to seven shoots were assayed. The experiments were repeated at least twice.

HR assay on tobacco

Bacterial strains grown overnight in LB medium with appropriate antibiotics were harvested and resuspended in 0.5 × PBS to OD600 = 0.1. Eight‐week‐old tobacco (Nicotiana tobacum) leaves were infiltrated with bacterial suspension using a needleless syringe and kept in a humidified chamber at 28 °C. HR symptoms were recorded at 24 h post‐infiltration. The experiments were repeated at least twice.

RNA isolation

To isolate RNA for in vitro T3SS gene expression, bacterial cultures grown in LB medium with appropriate antibiotics were harvested, washed twice and adjusted to OD600 = 0.2 in 5 mL of HMM. After 6 h of incubation at 18 °C, 4 mL of RNA protect reagent (Qiagen, Hilden, Germany) was added to 2 mL of bacterial cell cultures, mixed by vortex and incubated for 5 min at room temperature. For in vivo T3SS gene expression, overnight bacterial cultures were harvested and resuspended in 0.5 × PBS at OD600 = 0.2–0.3. Surface‐sterilized immature Bartlett pears were cut in half and inoculated with bacterial suspension (Wang et al., 2012). After 6 h of incubation at 28 °C in a moist chamber, bacterial cells were collected by washing pear surfaces with a solution containing 2 mL of RNA protect reagent (Qiagen) and 1 mL of water. Cells collected for both in vivo and in vitro T3SS gene expression were harvested by centrifugation. RNA was extracted using an RNeasy Mini kit (Qiagen) and the eluted total RNA was DNase treated using Turbo DNA‐freeTM (Ambion, Foster City, CA) according to the manufacturer's instructions. The quality and quantity of RNA were assessed using a Nano‐Drop ND‐100 spectrophotometer (Nano‐Drop Technologies, Wilminton, DE, USA).

qRT‐PCR

Reverse transcription for cDNA synthesis was performed using a Superscript® VILOTM cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) on 1 μg of total RNA according to the manufacturer's instructions. One hundred nanograms of reverse transcription product were used for qRT‐PCR analysis in an amplification mixture containing Power SYBR® Green PCR master mix (Applied Biosystems, Foster City, CA, USA). The primers for qRT‐PCR were designated using Primer3 software. The qRT‐PCR cycle was 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and a final dissociation curve analysis step from 65 to 95 °C. Gene expression levels were analysed using the relative quantification (ΔΔCt) method, and an rpoD gene was used as an endogenous control to normalize gene expression data.

Expression and purification of HrpS 250–325‐6His protein

As the over‐expression of E. amylovora full‐length HrpS protein led to the formation of insoluble aggregates (inclusion bodies) in Es. coli BL21 (DE3) strain, truncated HrpS250–325 protein containing only the DNA binding domain was expressed and purified. The corresponding part of the hrpS gene (748–975 nt) was cloned into the pET28a expression vector (Novagen, San Diego, CA, USA). The plasmid was designated as pHrpS250–325‐6His and introduced into Es. coli BL21 (DE3). For protein over‐expression, the resulting transformant was subcultured in 500 mL of fresh LB medium for 2–3 h and induced by 0.1 mm Isopropyl b‐D‐1‐thiogalactopyranoside (IPTG) at 18 °C overnight. Cells were harvested by centrifugation, washed with cell wash buffer (50 mm 3‐(N‐morpholino)propanesulfonic acid (MOPS), 150 mm NaCl) and resuspended [1 : 10 ratio (w/v)] in cell wash buffer. The cell suspensions were frozen at −80 °C until further use. Thawed cell suspensions were lysed by treatment of 250 μg/mL lysozyme (Promega, Madison, WI, USA) and sonication. Cell lysates were centrifuged, and Nickel‐nitrilotriacetic acid (Ni‐NTA) agarose resin (Qiagen) was added to the supernatants. Columns were washed with equilibration/wash buffer (50 mm MOPS, 300 mm NaCl, 60 mm imidazole), and the proteins were eluted with elution buffer (50 mm MOPS, 300 mm NaCl, 500 mm imidazole). Samples were dialysed overnight against buffer containing 20 mm MOPS and 1 mm dithiothreitol (DTT). The protein concentration was measured using Qubit protein assays (Invitrogen).

EMSA

Complementary oligonucleotides comprising the HrpS binding site from the hrpL promoter region of E. amylovora or mutated version of the same sequence were 3′ biotinylated using the biotin 3′ end DNA labelling kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. For annealing, equal amounts of the end‐labelled, complementary oligonucleotides were mixed together, denatured at 100 °C for 1 min and incubated at room temperature for 1 h before use. Protein–DNA binding assays were performed using a lightshift® chemiluminescent EMSA kit (Pierce). Increasing amount of HrpS250–325‐6His protein (0–2.05 μm) were added in reaction volumes of 10 μL containing 20 fmol of labelled oligonucleotides, 1 × binding buffer, 50 ng/μL Poly(dI·dC), 0.5 mm MgCl2, 0.1% Nonidet P‐40, 0.05 mg/mL bovine serum albumin (BSA) and 5% glycerol. Reaction mixtures were incubated at room temperature for 20 min, mixed with 2.5 μL of 5 × loading buffer and resolved into a 6% native polyacrylamide gel in 0.5 × TBE buffer [44.5 mm Tris‐base, 44.5 mm boric acid and 1 mm ethylenediaminetetraacetic acid (EDTA)]. Resolved binding reactions were transferred to a positively charged nylon membrane and cross‐linked using a UV‐light cross‐linking instrument. The chemiluminescent signals were developed according to the manufacturer's instructions and visualized using an ImageQuant LAS 4010 CCD camera (GE Healthcare, Little Chalfont, UK).

Statistical analysis

Gene expression and virulence data means were compared using a one‐way analysis of variance (ANOVA) and Student–Newman–Keuls test to determine differences in means (P = 0.05) using the SAS program, Gary, NC, USA.

Supporting information

Fig. S1 Virulence assays of Erwinia amylovora wild‐type (WT) and different complementation strains of the hrpL mutant on immature pears. 1, WT Ea1189; 2, hrpL(pHrpL3); 3, hrpL(pHrpL3‐Mut1); 4, hrpL(pHrpL3‐Mut2); 5, hrpL(pHrpL3‐Mut3); 6, hrpL(pHrpL2); 7, hrpL(pHrpL2‐Mut4); 8, hrpL(pHrpL); 9, hrpL(pWSK29), DPI, days post‐inoculation.

Fig. S2 Expression of type III secretion system (T3SS) genes in the hrpS mutant complemented with mutant constructs in vitro and in vivo. Relative expression of the hrpS, hrpL and hrpA genes in the hrpS mutant complemented with mutant variants (Y100F, Y100A and T101A) relative to its original construct (pHrpS) by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) In vitro in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 6 h. (B) In vivo on immature pear fruits for 6 h. The rpoD gene was used as an endogenous control, and the values of the relative fold change are the means of three replicates. Error bars indicate standard deviation. Fold changes marked with the same letter are not significantly different (P < 0.05).

Table S1 RpoN‐dependent enhancer binding proteins (EBPs) in Erwinia amylovora and their potential targets.

Table S2 Primers used in this study.

Click here for additional data file. (3MB, zip)

Acknowledgements

This project was supported by the Agriculture and Food Research Initiative Competitive Grants from the US Department of Agriculture (USDA) National Institute of Food and Agriculture and USDA‐Hatch Project ILLU‐802‐913 (YFZ). The authors have no conflicts of interest to declare.

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Associated Data

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Supplementary Materials

Fig. S1 Virulence assays of Erwinia amylovora wild‐type (WT) and different complementation strains of the hrpL mutant on immature pears. 1, WT Ea1189; 2, hrpL(pHrpL3); 3, hrpL(pHrpL3‐Mut1); 4, hrpL(pHrpL3‐Mut2); 5, hrpL(pHrpL3‐Mut3); 6, hrpL(pHrpL2); 7, hrpL(pHrpL2‐Mut4); 8, hrpL(pHrpL); 9, hrpL(pWSK29), DPI, days post‐inoculation.

Fig. S2 Expression of type III secretion system (T3SS) genes in the hrpS mutant complemented with mutant constructs in vitro and in vivo. Relative expression of the hrpS, hrpL and hrpA genes in the hrpS mutant complemented with mutant variants (Y100F, Y100A and T101A) relative to its original construct (pHrpS) by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) In vitro in hypersensitive response and pathogenicity (hrp)‐inducing minimal medium (HMM) at 18 °C for 6 h. (B) In vivo on immature pear fruits for 6 h. The rpoD gene was used as an endogenous control, and the values of the relative fold change are the means of three replicates. Error bars indicate standard deviation. Fold changes marked with the same letter are not significantly different (P < 0.05).

Table S1 RpoN‐dependent enhancer binding proteins (EBPs) in Erwinia amylovora and their potential targets.

Table S2 Primers used in this study.

Click here for additional data file. (3MB, zip)

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