Genome-wide identification of the Sec-dependent secretory protease genes in Erwinia amylovora and analysis of their expression during infection of immature pear fruit

Genome-wide identification of the Sec-dependent secretory protease genes in Erwinia amylovora and analysis of their expression during infection of immature pear fruit 梨火疫菌Sec依赖分泌蛋白酶的全基因组鉴定及其在侵染幼梨过程中的基因表达分析 1 2 first-author 1 3 1 3 3 3 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:wenjunzhao@188.comliweimin01@caas.cn †E-mail:wenjunzhao@188.comliweimin01@caas.cn wenjunzhao@188.com 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:wenjunzhao@188.comliweimin01@caas.cn †E-mail:wenjunzhao@188.comliweimin01@caas.cn wenjunzhao@188.com College of Plant Science, Tarim University, Alar 843300, China College of Plant Science, Tarim University, Alar 843300, China Southern Xinjiang Key Laboratory of Integrated Pest Management, Tarim University, Alar 843300, China Southern Xinjiang Key Laboratory of Integrated Pest Management, Tarim University, Alar 843300, China Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China Chinese Academy of Inspection and Quarantine, Beijing 100176, China Chinese Academy of Inspection and Quarantine, Beijing 100176, China The general secretory (Sec) pathway represents a common mechanism by which bacteria secrete proteins, including virulence factors, into the extracytoplasmic milieu. However, there is little information about this system, as well as its associated secretory proteins, in relation to the fire blight pathogen Erwinia amylovora . In this study, data mining revealed that E. amylovora harbors all of the essential components of the Sec system. Based on this information, we identified putative Sec-dependent secretory proteases in E. amylovora on a genome-wide scale. Using the programs SignalP, LipoP, and Phobius, a total of 15 putative proteases were predicted to contain the N-terminal signal peptides (SPs) that might link them to the Sec-dependent pathway. The activities of the predicted SPs were further validated using an Escherichia coli -based alkaline phosphatase (PhoA) gene fusion system that confirmed their extracytoplasmic property. Transcriptional analyses showed that the expression of 11 of the 15 extracytoplasmic protease genes increased significantly when E. amylovora was used to inoculate immature pears, suggesting their potential roles in plant infection. The results of this study support the suggestion that E. amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants, and shed new light on the interaction of E. amylovora with host plants. 目的 2 鉴定参与梨火疫菌侵染的重要Sec依赖分泌蛋白酶. 创新点 2 构建了梨火疫菌Sec依赖分泌蛋白酶编码基因在侵染寄主植物过程中的时序表达图谱. 方法 2 利用生物信息学与大肠杆菌PhoA检测体系两者结合,在全基因组水平筛选并鉴定梨火疫菌的Sec依赖分泌蛋白酶;利用逆转录实时定量聚合酶链反应(RT-qPCR),分析Sec依赖分泌蛋白酶编码基因在梨火疫菌侵染寄主植物过程中转录表达的时序变化. 结论 2 梨火疫菌含有完整的Sec分泌系统,可由此分泌至少15种蛋白酶,其中11种蛋白酶可能在病原菌侵染寄主植物过程中发挥功能. Erwinia amylovora Sec-dependent pathway Protease Gene expression Plant infection 梨火疫菌 Sec依赖分泌系统 蛋白酶 基因表达 侵染 Introduction 1 Fire blight is a destructive and highly infectious disease of apple, pear, quince, blackberry, raspberry, and many rosaceous ornamentals (Eastgate, 2000 2000 ; Malnoy et al., 2012 2012 ). The disease has spread to more than 50 countries around the world since it was first reported in North America in the late 1700s (Malnoy et al., 2012 2012 ). The causative agent of fire blight has long been known to be Erwinia amylovora , a Gram-negative enterobacterium (Eastgate, 2000 2000 ; Malnoy et al., 2012 2012 ), although some studies showed that Erwinia pyrifoliae (Kim et al., 1999 1999 , 2001 2001 ) and Erwinia piriflorinigrans (López et al., 2011 2011 ), two closely related bacterial species, could also cause fire blight symptoms in pear. In nature, E. amylovora cells are disseminated among host plants via wind, rain, and insects (Eastgate, 2000 2000 ; Malnoy et al., 2012 2012 ). By using natural openings and wounds, particularly in flowers, as infection routes, E. amylovora enters the host and causes the initial symptoms of water soaking, followed by the wilting and necrosis that result in the scorched and blackened appearance of infected tissue. The symptoms are generally localized to the blossom bracts or young shoots. However, in a highly susceptible host, E. amylovora can systemically migrate into mature tissues, leading to the death of an entire tree (Eastgate, 2000 2000 ; Malnoy et al., 2012 2012 ). In recent decades there has been remarkable progress toward elucidating the virulence mechanisms of E. amylovora in host plants (Eastgate, 2000 2000 ; Malnoy et al., 2012 2012 ; Piqué et al., 2015 2015 ; Castiblanco et al., 2018 2018 ; Lee et al., 2018 2018 , 2019 2019 ; Emeriewen et al., 2019 2019 ; Schachterle and Sundin, 2019 2019 ). Many factors, such as exopolysaccharide (EPS), amylovoran, levan, lipopolysaccharide (LPS), biofilm formation, and motility, have been determined to play roles in the pathogenicity of E. amylovora or host colonization (Koczan et al., 2009 2009 ; Piqué et al., 2015 2015 ; Kharadi et al., 2018 2018 ). Moreover, it has been found that E. amylovora has evolved diverse secretion systems, including types I, II, III, and VI (T1SS, T2SS, T3SS, and T6SS, respectively), to promote the infection of plants (Bogdanove et al., 1996 1996 ; Zhang et al., 1999 1999 ; Oh and Beer, 2005 2005 ; Cianciotto and White, 2017 2017 ; Kamber et al., 2017 2017 ; Tian et al., 2017 2017 ). Among them, T3SS is one of the most remarkably versatile pathogenic factors used by numerous Gram-negative bacterial pathogens of animals and plants to inject proteins (also called effectors) directly into host cells (Tang et al., 2006 2006 ; McCann and Guttman, 2008 2008 ). E. amylovora uses T3SS to secrete several effector proteins (Zhao et al., 2006 2006 ; Nissinen et al., 2007 2007 ): DspA/E is essential for the multiplication and survival of the bacterium in host plants (Gaudriault et al., 1997 1997 ) and non-host plants (Oh et al., 2007 2007 ; Degrave et al., 2013 2013 ); HrpN contributes to the elicitation of callose (Boureau et al., 2011 2011 ); Eop1 acts as a host-range limiting factor (Asselin et al., 2011 2011 ); and, AvrRpt2 EA causes symptoms that mimic those of fire blight and induces a salicylic acid-dependent defense response when expressed transgenically in apple (Schröpfer et al., 2018 2018 ). Both DspA/E and HrpN are required to trigger defense responses and cell death in host and non-host plants (Gaudriault et al., 1997 1997 ; Boureau et al., 2006 2006 ; Degrave et al., 2008 2008 ). Protein secretion in bacterial pathogens is one of the most high-profile research topics in relation to the virulence of these organisms. The general protein secretory (Sec) pathway, in combination with the twin-arginine translocation (Tat) pathway, accounts for most bacterial protein transport across cytoplasmic membranes (Holland, 2010 2010 ; Segers and Anné, 2011 2011 ; Tsirigotaki et al., 2017 2017 ). The Sec-dependent pathway is found widely in bacteria and includes three transmembrane proteins SecY, SecE, and SecG that are assembled into channel SecYEG to translocate proteins with the aid of the peripheral membrane ATPase SecA. In addition, the Sec complex has at least three other membrane proteins (SecD, SecF, and YajC) that are involved in efficient protein export (Tsirigotaki et al., 2017 2017 ; Cranford-Smith and Huber, 2018 2018 ; Smets et al., 2019 2019 ). A protein secreted via the Sec system is synthesized at the ribosome as an unfolded preprotein with an N-terminal signal peptide (SP) fused to the mature domain. Once the preprotein is translocated into the periplasm, the SP is cleaved and removed by exocytoplasmic signal peptidase I (LepB) or lipoprotein signal peptidase II (LspA). This results in the release and folding of a mature domain that is either retained in the periplasm or secreted as an outer membrane or extracellular protein via secondary secretion pathways, such as T5SS, the localization of lipoprotein (Lol) pathway, or T2SS (Tsirigotaki et al., 2017 2017 ; Cranford-Smith and Huber, 2018 2018 ; Smets et al., 2019 2019 ). Many proteases in Gram-negative bacteria have been found to play key roles during the infection process of pathogens. Those that are most well-known include the two ATP-dependent serine proteases, Lon and Clp, which serve as the major proteolytic components in the cytosol and contribute to pathogenesis by their timely degradation of the transcriptional regulators that control expression of T3SS (Frees et al., 2013 2013 ; Figaj et al., 2019 2019 ). HtrA/DegP is another highly characterized protease. As a non-ATP-dependent serine protease, HtrA/DegP is localized in the periplasm and involved in the turnover and export of extracellular virulence factors (Frees et al., 2013 2013 ; Figaj et al., 2019 2019 ). Recent studies have shown that HtrA/DegP of two species of human pathogenic bacteria, Helicobacter pylori and Chlamydia trachomatis , can be secreted into either the extracellular environment or host cell cytosol (Löwer et al., 2008 2008 ; Hoy et al., 2010 2010 ; Wu et al., 2011 2011 ), indicating its involvement in diverse mechanisms of bacterial virulence. In contrast to cytoplasmic and periplasmic proteases, proteases anchored to the bacterial surface, secreted into the extracellular environment, or injected into the host cells interact closely with the host components, and thus may serve directly as virulence factors (Figaj et al., 2019 2019 ). In this study, we first mined the annotated genome data of E. amylovora in the GenBank public database and confirmed that the bacterium possesses all of the essential components of the Sec system. Based on this information, we identified the Sec-dependent secretory proteases from the whole genome of E. amylovora using web-based algorithms coupled with a laboratory-based Escherichia coli alkaline phosphatase (PhoA) assay (Liu et al., 2019 2019 ). Further, we examined gene expression levels of the putative Sec-dependent secretory proteases when E. amylovora infected immature pears. Materials and methods 1 In silico analysis 2 A total of 3393 putative proteins, including 3368 chromosomal proteins (GenBank No. NC_013961) and 25 proteins encoded by plasmids (GenBank No. NC_013957), were predicted in E. amylovora CFBP1430. Using SignalP Version 4.0 with default settings for Gram-negative bacteria (Petersen et al., 2011 2011 ), the 3393 E. amylovora proteins were screened for those bearing N-terminal SPs that could conceivably link them to the Sec pathway. The obtained Sec-dependent secretory proteins were filtered further to select putative proteases according to protein annotations previously designated (Smits et al., 2010 2010 ). The presence of the N-terminal SPs in putative extracytoplasmic proteases was re-examined with the additional programs LipoP Version 1.0 (Juncker et al., 2003 2003 ) and Phobius (Käll et al., 2004 2004 , 2007 2007 ). PhoA assay 2 The SP of each putative Sec-dependent secretory protease was tested using an E . coli -based PhoA assay as described previously (Liu et al., 2019 2019 ). Briefly, based on the E. amylovora CFBP1430 genomic sequence (GenBank No. NC_013961.1), specific forward and reverse primers (Table S1 S1 ) were first designed to amplify the DNA sequence of each putative SP. The resulting polymerase chain reaction (PCR) product was then ligated into Nde I- Hin dIII-digested pET-mphoA, generating a construct with an in-frame gene fusion between the SP and the mphoA gene (see the schematic diagram of vectors in Fig. 1a 1a ). The construct was subsequently transformed into chemically competent cells of E. coli BL21, followed by the detection of PhoA activity of transformants on indicator LB agar containing 90 µg/mL 5-bromo-4-chloro-3-indoyl phosphate (BCIP), 100 mmol/L isopropy-β- D -thiogalactoside (IPTG), and 75 mmol/L Na 2 HPO 4 . After 12 h of incubation at 37 °C, a transformant that remained white indicated a lack of PhoA, while a colony that turned blue denoted PhoA activity. The BL21 cells that harbored pET-phoA were used as positive controls and those that harbored pET-mphoA as negative controls. (a) Diagrams of the expression cassettes for the phoA gene. pET-phoA harboring the full length Escherichia coli phoA gene was used as a positive control, while pET-mphoA harboring mphoA lacking its native SP was used as a negative control. The encoding sequences of the putative signal peptides in the 15 proteases were designated Prt n SP (where “ n ” represents the numbers ranging from 1 to 15) and fused with the mphoA gene, producing pET-Prt n SP-mphoA. (b) The putative signal peptides of the 15 Sec-dependent secretory proteases directed the extracytoplasmic export of mphoA moiety. After 12 h of incubation on LB media containing isopropy-β- D -thiogalactoside (IPTG, 100 mmol/L), 5-bromo-4-chloro-3-indoyl phosphate (BCIP, 90 μg/mL), and Na 2 HPO 4 (75 mmol/L) at 37 °C, the E. coli BL21 cells that harbored pET-Prt n SP-mphoA turned blue, while the cells that harbored pET-mphoA remained white Immature pear infection and total RNA extraction 2 Immature pear fruits were inoculated with cells of E. amylovora DSM17948 as previously described with some modifications (Pletzer and Weingart, 2014 2014 ; Pletzer et al., 2015 2015 ). The cells were grown overnight in LB broth at 28 °C, harvested by centrifugation at 3000 g for 10 min at 4 °C, and resuspended to an OD 600 (optical density at 600 nm) of about 2 in sterile distilled water. Four two-month-old pears ( Pyrus bretschneideri L. cv. Dangshan Su) were surface-sterilized with 0.02 g/mL sodium hypochlorite for 20–30 min. Following six rinses with sterile distilled water, the pears were cut in half, and each cut surface was immediately inoculated with two 100-μL drops of cell suspension and incubated in a humidified chamber at 28 °C. At 8 and 24 h post-inoculation (hpi), the pear tissues at the inoculation site were excised with a sterilized blade and stored at −80 °C. The experiment was carried out three times. The start culture (designated 0 h) used to infect the immature pear fruits, and the tissues collected at 8 and 24 hpi were individually subjected to total RNA extraction using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative reverse transcription PCR and data analysis 2 By using the primers listed in Table S2 S2 , quantitative reverse transcription PCR (RT-qPCR) analysis was performed as descried previously (Hong et al., 2017 2017 ). The E. amylovora recA (recombinase A) gene (GeneID: 8914602) was included as an internal reference. Each experiment was performed with three technical replicates. The 2 −ΔΔ C T method was used to calculate relative gene expression values (Livak and Schmittgen, 2001 2001 ), which were transformed to fold-change and plotted in figures. Statistical analyses were conducted using Student’s t -test (SPSS 10.0, Chicago, IL, USA). Results 1 In silico analyses of the Sec system and Sec-dependent secretory proteases in E . amylovora 2 To date, there are three E. amylovora strains, CFBP1430 (Smits et al., 2010 2010 ), ATCC 49946 (Sebaihia et al., 2010 2010 ), and E-2 (Lagonenko et al., 2008 2008 ), whose complete genomic sequences are available in the GenBank database. Data mining of their chromosome-encoded proteins (GenBank Nos. NC_013961.1, NC_013971.1, and NZ_CP024970.1) showed that each strain had the essential components of the Sec pathway, including SecA, SecY, SecE, SecG, SecD, SecF, and YajC, as well as LepB and LspA, two exocytoplasmic signal peptidases (Table 1 1 ), according to the protein annotations previously designated (Smits et al., 2010 2010 ). These findings indicate that E. amylovora can secrete proteins using the Sec-dependent secretion process. Therefore, the Sec-dependent secretory proteins of E. amylovora were predicted on the basis of the CFBP1430 genome using SignalP 4.0 (Petersen et al., 2011 2011 ). As a result, we identified 168 E. amylovora proteins bearing N-terminal SPs that could potentially be secreted via the Sec system (Table S3 S3 ). Based on the protein annotations previously designated (Smits et al., 2010 2010 ), these proteins included 15 proteases (Table 2 2 ). Given the general significance of bacterial proteases in virulence (Xia, 2004 2004 ; Figaj et al., 2019 2019 ), the experiments described below focused on these putative extracytoplasmic proteases. The genes referred to in this study were identified from three E . amylovora strains, CFBP1430 (GenBank No. NC_013961.1), ATCC 49946 (GenBank No. NC_013971.1), and E-2 (GenBank No. NZ_CP024970.1), whose complete genomic sequences are available 2 Gene 3 Locus_tag CFBP1430 ATCC 49946 E-2 SecA EAMY_RS30645 EAM_RS03465 AD997_RS03640 SecD EAMY_RS21905 EAM_RS04720 AD997_RS04940 SecF EAMY_RS21910 EAM_RS04725 AD997_RS04945 SecE EAMY_RS18480 EAM_RS01205 AD997_RS01265 YajC EAMY_RS21900 EAM_RS04715 AD997_RS04935 SecG EAMY_RS19030 EAM_RS15075 AD997_RS15760 SecY EAMY_RS32865 EAM_RS15600 AD997_RS16320 LepB EAMY_RS29460 EAM_RS12265 AD997_RS12890 LspA EAMY_RS30890 EAM_RS03220 AD997_RS03390 The protein annotations referred to in this study are based on E . amylovora CFBP1430 (GenBank No. NC_013961.1). The putative proteases were defined as serine, cysteine, and metalloproteases based on the MEROPS protease database (Rawlings et al., 2018 2018 ), and their cleavage sites were predicted using SignalP 4.0 (Petersen et al., 2011 2011 ), LipoP 1.0 (Juncker et al., 2003 2003 ), and Phobius (Käll et al., 2004 2004 , 2007 2007 ) Protein GenBank No. Signal peptidase Cleavage site Protease type  EaPrt1 WP_004155063.1 LepB 24–25 Serine protease  EaPrt2 WP_004155111.1 LepB 20–21 Serine protease  EaPrt3 WP_004155489.1 LepB 22–23 Cysteine protease  EaPrt4 WP_004155561.1 LepB 22–23 Metalloprotease  EaPrt5 WP_013035939.1 LepB 29–30 Serine protease  EaPrt6 WP_004156768.1 LepB 25–26 Serine protease  EaPrt7 WP_004156843.1 LepB 30–31 Serine protease  EaPrt8 WP_004157647.1 LepB 20–21 Serine protease  EaPrt9 WP_004157888.1 LepB 21–22 Serine protease  EaPrt10 WP_004158423.1 LepB 19–20 Serine protease  EaPrt11 WP_004158446.1 LspA 21–22 Cysteine protease  EaPrt12 WP_004159031.1 LepB 27–28 Metalloprotease  EaPrt13 WP_004159492.1 LepB 26–27 Serine protease  EaPrt14 WP_004161039.1 LepB 23–24 Metalloprotease  EaPrt15 WP_004158249.1 LepB 24–25 Serine protease The presence of N-terminal SPs in the putative proteases was subjected to additional verification using LipoP (Juncker et al., 2003 2003 ) and Phobius (Käll et al., 2004 2004 , 2007 2007 ). Notably, among the 15 extracytoplasmic proteases, only EaPrt11 contained a lipoprotein SP that was cleaved by LspA, while all the others had SPs that were processed by LepB (Table 2 2 ). According to the MEROPS protease database (Rawlings et al., 2018 2018 ), the 15 putative proteases were members of the serine, cysteine, or metalloprotease classes (Table 2 2 ), the three most abundant types of proteolytic enzymes in bacteria (Supuran et al., 2001 2001 ). A BLAST search of the GenBank database showed that all the putative proteases were highly conserved among strains of the genus Erwinia (data not shown). However, most have no orthologs in the well-studied bacterial plant pathogens of the genera Xanthomonas , Pseudomonas , or Agrobacterium , with the exception of EaPrt1, EaPrt9, EaPrt14, and EaPrt15, which share 40%–50% similarity with the Do family serine endopeptidase (GenBank No. WP_169707362) of Xanthomonas campestris , the carboxy terminal-processing peptidase (GenBank No. WP_170826269), M20 family metallopeptidase (GenBank No. WP_055001508) of Pseudomonas syringae , and the α/β fold hydrolase (GenBank No. WP_174031637) of Agrobacterium rhizogenes , respectively. Validation of the Sec-dependent signal peptides via the PhoA assay 2 Next, we used the phoA gene fusion assay (Liu et al., 2019 2019 ) to determine whether the putative extracytoplasmic proteases could be exported via the Sec translocon. The DNA sequence that encoded the putative SP of each protein was cloned and fused in-frame with mphoA of pET-mphoA (Fig. 1a 1a ), resulting in 15 constructs that were transformed individually into E. coli BL21. After 12 h of incubation on the indicator LB agar, the transformants that expressed Prt4SP-mphoA and Prt11SP-mphoA turned pale blue, and those that harbored the other 13 constructs turned dark blue, while the negative control (BL21 cells with pET-mphoA) remained white (Fig. 1b 1b ). These results indicated that although the SPs tested displayed different activities in the E. coli -based PhoA assay, each could translocate the mphoA moiety from the cytoplasm to periplasm, suggesting that the 15 putative proteases that contained the cognate SPs would also be secreted via the E. amylovora Sec pathway. Gene expression analysis of the Sec-dependent secretory proteases during plant infection 2 To determine the potential involvement of the 15 extracytoplasmic proteases in host plant infection, we examined their levels of gene expression during E. amylovora infection of the immature pear fruit using two-step RT-qPCR. Of the 15 proteases, the expression of EaPrt4 , 5 , 8 , 14 , and 15 steadily increased during the course of a 24-h infection (Fig. 2a 2a ), while the expression of EaPrt6 , 11 , and 13 continuously decreased (Fig. 2b 2b ). In addition, the messenger RNAs (mRNAs) of EaPrt1 , 2 , 3 , 7 , 9 , 10 , and 12 peaked at 8 hpi, but decreased at 24 hpi, displaying an “up-down” expression pattern (Fig. 2c 2c ). Only EaPrt2 showed levels of expression at both 8 and 24 hpi that were not significantly different from that at 0 h. Note that compared with its level of expression at 0 h, the expression of EaPrt7 showed about 37-fold increase at 8 hpi, but declined sharply to about 6-fold increase at 24 hpi. This was the most significantly altered expression pattern among all the genes tested. Total RNA was prepared from the immature pears infected by E. amylovora at 8 and 24 h post inoculation (hpi) and from the start culture used to infect the immature pear fruits (0 h). Two-step RT-qPCR was used to assess the gene expression of the putative proteases relative to the recA gene. Over the course of a 24-h infection, the levels of expression of EaPrt4 , 5 , 8 , 14 , and 15 steadily increased (a), while the expression of EaPrt6 , 11 , and 13 continuously decreased (b), and EaPrt1 , 2 , 3 , 7 , 9 , 10 , and 12 exhibited an “up-down” expression pattern (c). The experiment was performed with three technical replicates. The mean relative expression levels and the standard errors are indicated for each gene. The asterisks denote significant differences according to a Student’s t -test ( ** P < 0.01 and * P < 0.05, vs. 0 h) Discussion 1 Bacterial pathogens have evolved multiple sophisticated systems to secrete proteins that help enable them to use an environmental niche or act as pathogens (Stavrinides et al., 2008 2008 ; Green and Mecsas, 2016 2016 ). Most secretory proteins in bacteria are translocated across the cytoplasmic membrane via the Sec pathway (Holland, 2010 2010 ; Segers and Anné, 2011 2011 ; Tsirigotaki et al., 2017 2017 ). However, there is little information about the Sec system in E. amylovora . In this study, data mining of the E. amylovora -annotated proteins showed that the bacterium possesses the full components of the Sec pathway, which could thereby enable the E. amylovora proteins, including potential virulence factors, to be secreted into the extracytoplasmic environment. Thus, we combined the bioinformatics screen and the E. coli -based alkaline PhoA assay to further define 15 Sec-dependent secretory proteases from the whole genome of E. amylovora . Secretory proteins previously identified from E. amylovora have almost all been Type III effectors (Nissinen et al., 2007 2007 ; Schröpfer et al., 2018 2018 ), which are directly injected into the host cells and thus closely interact with the host components. Based on the properties of Sec-dependent secretory proteins (Tsirigotaki et al., 2017 2017 ; Smets et al., 2019 2019 ), once the 15 Sec-dependent E. amylovora proteases have crossed the inner plasma membrane, we inferred that they could either remain in the periplasm or be further translocated through the outer membrane as membrane bound or free proteins. Nevertheless, in view of the gene expression profiles during the infection of immature pears, 11 of the 15 proteases tested showed significantly increased levels of gene expression, implying their potential roles in the context of plant infection. In particular, there were six (EaPrt1, 3, 7, 9, 10, and 12) that could conceivably function during the early stages of E. amylovora infection, since they showed the highest fold increase in gene expression at 8 hpi. In contrast, EaPrt2, 6, 11, and 13, whose gene expression profiles showed either a lack of significant variation or a continuous decrease, might have no or only minor roles in plant infection. While the involvement of proteases in the virulence of phytopathogenic bacteria is less well understood than their involvement in the virulence of animal and human bacterial pathogens, several studies indicate that phytopathogenic bacteria have evolved extracellular metalloproteases to invade host plants (Figaj et al., 2019 2019 ). One well-studied example is found in species of Pseudomonas , which secretes the metalloprotease AprA into the plant apoplast to specifically recognize and degrade the bacterial flagellin monomer, a strong inducer of innate immune responses, thus enabling the bacterium to evade host immune defense mechanisms (Bardoel et al., 2011 2011 ; Pel et al., 2014 2014 ). Moreover, the bacterial extracellular metalloproteases are presumed to participate in the degradation of the plant cell wall, the first barrier against plant pathogens (Figaj et al., 2019 2019 ). Pectobacterium carotovorum produces an extracellular metalloprotease that catalyzes the degradation of plant cell wall structural proteins in vitro (Feng et al., 2014 2014 ), as does X. campestris pv. campestris (Dow et al., 1998 1998 ). In this study of 11 Sec-dependent E. amylovora proteases potentially involved in host infection, three (EaPrt4, 12, and 14) were designated metalloproteases based on the MEROPS protease database (Rawlings et al., 2018 2018 ), and merit further investigation. Of particular interest was EaPrt7, a putative serine protease that showed the most significantly upregulated level of gene expression during the infection of immature pears, suggesting its significance during host plant infection. EaPrt13, an ortholog of HtrA/DegP, was not included in the 11 proteases with potential roles in host infection. In contrast to the well-known role of HtrA/DegP in the virulence of medically significant plant pathogenic bacteria (Yorgey et al., 2001 2001 ; Frees et al., 2013 2013 ; Figaj et al., 2019 2019 ), EaPrt13 seemed not to be implicated in plant infection because its gene expression decreased continuously over the course of a 24-h infection. Conclusions 1 In this study, a total of 15 putative Sec-dependent secretory proteases, including 2 cysteine proteases, 3 metalloproteases, and 10 serine proteases, were identified from E. amylovora on a genome-wide scale. Of these putative extracytoplasmic proteases, 11 showed significantly increased levels of gene expression when E. amylovora interacted with mature pears, and were purported to be engaged in plant infection. While the extracytoplasmic localization site, as well as the specific biological function of each putative protease, remains to be determined, the data from this study suggest that E. amylovora has evolved a suite of Sec-dependent extracytoplasmic proteases to disrupt host homeostasis via their proteolytic activities, thereby promoting plant infection. References JE Asselin , JM Bonasera , JF Kim , 等 . Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor . Phytopathology , 2011 . 101 ( 8 ): 935 - 944 . 21469934 DOI: 10.1094/PHYTO-12-10-0339 http://doi.org/10.1094/PHYTO-12-10-0339 . Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor BW Bardoel , S van der Ent , MJC Pel , 等 . Pseudomonas evades immune recognition of flagellin in both mammals and plants . PLoS Pathog , 2011 . 7 ( 8 ): e1002206 21901099 DOI: 10.1371/journal.ppat.1002206 http://doi.org/10.1371/journal.ppat.1002206 . Pseudomonas evades immune recognition of flagellin in both mammals and plants AJ Bogdanove , ZM Wei , L Zhao , 等 . Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of yopN of Yersinia spp . J Bacteriol , 1996 . 178 ( 6 ): 1720 - 1730 . 8626302 DOI: 10.1128/jb.178.6.1720-1730.1996 http://doi.org/10.1128/jb.178.6.1720-1730.1996 . Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of yopN of Yersinia spp T Boureau , H ElMaarouf-Bouteau , A Garnier , 等 . DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants . Mol Plant Microbe Interact , 2006 . 19 ( 1 ): 16 - 24 . 16404949 DOI: 10.1094/MPMI-19-0016 http://doi.org/10.1094/MPMI-19-0016 . DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants T Boureau , S Siamer , C Perino , 等 . The HrpN effector of Erwinia amylovora , which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves . Mol Plant Microbe Interact , 2011 . 24 ( 5 ): 577 - 584 . 21463207 DOI: 10.1094/MPMI-09-10-0212 http://doi.org/10.1094/MPMI-09-10-0212 . The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves LF Castiblanco , LR Triplett , GW Sundin . Regulation of effector delivery by type III secretion chaperone proteins in Erwinia amylovora . Front Microbiol, 9:146 , 2018 . DOI: 10.3389/fmicb.2018.00146 http://doi.org/10.3389/fmicb.2018.00146 . Regulation of effector delivery by type III secretion chaperone proteins in Erwinia amylovora NP Cianciotto , RC White . Expanding role of type II secretion in bacterial pathogenesis and beyond . Infect Immun , 2017 . 85 ( 5 ): e00014 - 17 . 28264910 DOI: 10.1128/IAI.00014-17 http://doi.org/10.1128/IAI.00014-17 . Expanding role of type II secretion in bacterial pathogenesis and beyond T Cranford-Smith , D Huber . The way is the goal: how SecA transports proteins across the cytoplasmic membrane in bacteria . FEMS Microbiol Lett , 2018 . 365 ( 11 ): fny093 DOI: 10.1093/femsle/fny093 http://doi.org/10.1093/femsle/fny093 . The way is the goal: how SecA transports proteins across the cytoplasmic membrane in bacteria A Degrave , M Fagard , C Perino , 等 . Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana . Mol Plant Microbe Interact , 2008 . 21 ( 8 ): 1076 - 1086 . 18616404 DOI: 10.1094/MPMI-21-8-1076 http://doi.org/10.1094/MPMI-21-8-1076 . Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana A Degrave , M Moreau , A Launay , 等 . The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen . Mol Plant Pathol , 2013 . 14 ( 5 ): 506 - 517 . 23634775 DOI: 10.1111/mpp.12022 http://doi.org/10.1111/mpp.12022 . The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen JM Dow , HA Davies , MJ Daniels . A metalloprotease from Xanthomonas campestris that specifically degrades proline/hydroxyproline-rich glycoproteins of the plant extracellular matrix . Mol Plant Microbe Interact , 1998 . 11 ( 11 ): 1085 - 1093 . 9805395 DOI: 10.1094/MPMI.1998.11.11.1085 http://doi.org/10.1094/MPMI.1998.11.11.1085 . A metalloprotease from Xanthomonas campestris that specifically degrades proline/hydroxyproline-rich glycoproteins of the plant extracellular matrix JA Eastgate . Erwinia amylovora : the molecular basis of fireblight disease . Mol Plant Pathol , 2000 . 1 ( 6 ): 325 - 329 . 20572979 DOI: 10.1046/j.1364-3703.2000.00044.x http://doi.org/10.1046/j.1364-3703.2000.00044.x . Erwinia amylovora: the molecular basis of fireblight disease OF Emeriewen , T Wöhner , H Flachowsky , 等 . Malus hosts- Erwinia amylovora interactions: strain pathogenicity and resistance mechanisms . Fron Plant Sci, 10:551 , 2019 . DOI: 10.3389/fpls.2019.00551 http://doi.org/10.3389/fpls.2019.00551 . Malus hosts-Erwinia amylovora interactions: strain pathogenicity and resistance mechanisms T Feng , C Nyffenegger , P Højrup , 等 . Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1 . Appl Microbiol Biotechnol , 2014 . 98 ( 24 ): 10077 - 10089 . 24946865 DOI: 10.1007/s00253-014-5877-2 http://doi.org/10.1007/s00253-014-5877-2 . Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1 D Figaj , P Ambroziak , T Przepiora , 等 . The role of proteases in the virulence of plant pathogenic bacteria . Int J Mol Sci , 2019 . 20 ( 3 ): 672 DOI: 10.3390/ijms20030672 http://doi.org/10.3390/ijms20030672 . The role of proteases in the virulence of plant pathogenic bacteria D Frees , L Brøndsted , H Ingmer . Dougan DA . Bacterial proteases and virulence . Regulated Proteolysis in Microorganisms , 2013 . Springer, Dordrecht , 161 - 192 . DOI: 10.1007/978-94-007-5940-4_7 http://doi.org/10.1007/978-94-007-5940-4_7 . Bacterial proteases and virulence S Gaudriault , L Malandrin , JP Paulin , 等 . DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae , is secreted via the Hrp secretion pathway in a DspB-dependent way . Mol Microbiol , 1997 . 26 ( 5 ): 1057 - 1069 . 9426142 DOI: 10.1046/j.1365-2958.1997.6442015.x http://doi.org/10.1046/j.1365-2958.1997.6442015.x . DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way ER Green , J Mecsas . Bacterial secretion systems: an overview . Microbiol Spectr , 2016 . 4 ( 1 ): VMBF - 0012-2015 . DOI: 10.1128/microbiolspec.VMBF-0012-2015 http://doi.org/10.1128/microbiolspec.VMBF-0012-2015 . Bacterial secretion systems: an overview IB Holland . Economou A . The extraordinary diversity of bacterial protein secretion mechanisms . Protein Secretion , 2010 . Humana Press, New York , 1 - 20 . DOI: 10.1007/978-1-60327-412-8_1 http://doi.org/10.1007/978-1-60327-412-8_1 . The extraordinary diversity of bacterial protein secretion mechanisms L Hong , JL Liu , SZ Midoun , 等 . Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2017 . 18 ( 10 ): 833 - 844 . DOI: 10.1631/jzus.B1700088 http://doi.org/10.1631/jzus.B1700088 . Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina B Hoy , M Löwer , C Weydig , 等 . Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion . EMBO Rep , 2010 . 11 ( 10 ): 798 - 804 . 20814423 DOI: 10.1038/embor.2010.114 http://doi.org/10.1038/embor.2010.114 . Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion AS Juncker , H Willenbrock , G von Heijne , 等 . Prediction of lipoprotein signal peptides in Gram-negative bacteria . Protein Sci , 2003 . 12 ( 8 ): 1652 - 1662 . 12876315 DOI: 10.1110/ps.0303703 http://doi.org/10.1110/ps.0303703 . Prediction of lipoprotein signal peptides in Gram-negative bacteria L Käll , A Krogh , ELL Sonnhammer . A combined transmembrane topology and signal peptide prediction method . J Mol Biol , 2004 . 338 ( 5 ): 1027 - 1036 . 15111065 DOI: 10.1016/j.jmb.2004.03.016 http://doi.org/10.1016/j.jmb.2004.03.016 . A combined transmembrane topology and signal peptide prediction method L Käll , A Krogh , ELL Sonnhammer . Advantages of combined transmembrane topology and signal peptide prediction–the Phobius web server . Nucleic Acids Res , 2007 . 35 ( S2 ): W429 - W432 . 17483518 DOI: 10.1093/nar/gkm256 http://doi.org/10.1093/nar/gkm256 . Advantages of combined transmembrane topology and signal peptide prediction–the Phobius web server T Kamber , JF Pothier , C Pelludat , 等 . Role of the type VI secretion systems during disease interactions of Erwinia amylovora with its plant host . BMC Genomics, 18:628 , 2017 . DOI: 10.1186/s12864-017-4010-1 http://doi.org/10.1186/s12864-017-4010-1 . Role of the type VI secretion systems during disease interactions of Erwinia amylovora with its plant host RR Kharadi , LF Castiblanco , CM Waters , 等 . Phosphodiesterase genes regulate amylovoran production, biofilm formation, and virulence in Erwinia amylovora . Appl Environ Microbiol , 2018 . 85 e02233 - 18 . 30366999 DOI: 10.1128/AEM.02233-18 http://doi.org/10.1128/AEM.02233-18 . Phosphodiesterase genes regulate amylovoran production, biofilm formation, and virulence in Erwinia amylovora WS Kim , L Gardan , SL Rhim , 等 . Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees ( Pyrus pyrifolia Nakai) . Int J Syst Bacteriol , 1999 . 49 ( 2 ): 899 - 906 . 10319516 DOI: 10.1099/00207713-49-2-899 http://doi.org/10.1099/00207713-49-2-899 . Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai) WS Kim , S Jock , JP Paulin , 等 . Molecular detection and differentiation of Erwinia pyrifoliae and host range analysis of the Asian pear pathogen . Plant Dis , 2001 . 85 ( 11 ): 1183 - 1188 . 30823165 DOI: 10.1094/PDIS.2001.85.11.1183 http://doi.org/10.1094/PDIS.2001.85.11.1183 . Molecular detection and differentiation of Erwinia pyrifoliae and host range analysis of the Asian pear pathogen JM Koczan , MJ McGrath , YF Zhao , 等 . Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: implications in pathogenicity . Phytopathology , 2009 . 99 ( 11 ): 1237 - 1244 . 19821727 DOI: 10.1094/PHYTO-99-11-1237 http://doi.org/10.1094/PHYTO-99-11-1237 . Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: implications in pathogenicity AL Lagonenko , VS Komardina , YA Nikolaichik , 等 . First report of Erwinia amylovora fire blight in Belarus . J Phytopathol , 2008 . 156 ( 10 ): 638 - 640 . DOI: 10.1111/j.1439-0434.2008.01420.x http://doi.org/10.1111/j.1439-0434.2008.01420.x . First report of Erwinia amylovora fire blight in Belarus JH Lee , V Ancona , YF Zhao . Lon protease modulates virulence traits in Erwinia amylovora by direct monitoring of major regulators and indirectly through the Rcs and Gac-Csr regulatory systems . Mol Plant Pathol , 2018 . 19 ( 4 ): 827 - 840 . 28509355 DOI: 10.1111/mpp.12566 http://doi.org/10.1111/mpp.12566 . Lon protease modulates virulence traits in Erwinia amylovora by direct monitoring of major regulators and indirectly through the Rcs and Gac-Csr regulatory systems JH Lee , V Ancona , T Chatnaparat , 等 . The RNA-binding protein CsrA controls virulence in Erwinia amylovora by regulating RelA, RcsB, and FlhD at the posttranscriptional level . Mol Plant Microbe Interact , 2019 . 32 ( 10 ): 1448 - 1459 . 31140921 DOI: 10.1094/MPMI-03-19-0077-R http://doi.org/10.1094/MPMI-03-19-0077-R . The RNA-binding protein CsrA controls virulence in Erwinia amylovora by regulating RelA, RcsB, and FlhD at the posttranscriptional level XL Liu , YY Fan , C Zhang , 等 . Nuclear import of a secreted “ Candidatus Liberibacter asiaticus” protein is temperature dependent and contributes to pathogenicity in Nicotiana benthamiana . Front Microbiol, 10:1684 , 2019 . DOI: 10.3389/fmicb.2019.01684 http://doi.org/10.3389/fmicb.2019.01684 . Nuclear import of a secreted “Candidatus Liberibacter asiaticus” protein is temperature dependent and contributes to pathogenicity in Nicotiana benthamiana KJ Livak , TD Schmittgen . Analysis of relative gene expression data using real-time quantitative PCR and the 2 −∆∆ C T Method . Methods , 2001 . 25 ( 4 ): 402 - 408 . 11846609 DOI: 10.1006/meth.2001.1262 http://doi.org/10.1006/meth.2001.1262 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT Method MM López , M Roselló , P Llop , 等 . Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms . Int J Syst Evol Microbiol , 2011 . 61 ( 3 ): 561 - 567 . 20382791 DOI: 10.1099/ijs.0.020479-0 http://doi.org/10.1099/ijs.0.020479-0 . Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms M Löwer , C Weydig , D Metzler , 等 . Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA . PLoS ONE , 2008 . 3 ( 10 ): e3510 18946507 DOI: 10.1371/journal.pone.0003510 http://doi.org/10.1371/journal.pone.0003510 . Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA M Malnoy , S Martens , JL Norelli , 等 . Fire blight: applied genomic insights of the pathogen and host . Annu Rev Phytopathol , 2012 . 50 475 - 494 . 22702352 DOI: 10.1146/annurev-phyto-081211-172931 http://doi.org/10.1146/annurev-phyto-081211-172931 . Fire blight: applied genomic insights of the pathogen and host HC McCann , DS Guttman . Evolution of the type III secretion system and its effectors in plant-microbe interactions . New Phytol , 2008 . 177 ( 1 ): 33 - 47 . 18078471 DOI: 10.1111/j.1469-8137.2007.02293.x http://doi.org/10.1111/j.1469-8137.2007.02293.x . Evolution of the type III secretion system and its effectors in plant-microbe interactions RM Nissinen , AJ Ytterberg , AJ Bogdanove , 等 . Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels . Mol Plant Pathol , 2007 . 8 ( 1 ): 55 - 67 . 20507478 DOI: 10.1111/j.1364-3703.2006.00370.x http://doi.org/10.1111/j.1364-3703.2006.00370.x . Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels CS Oh , SV Beer . Molecular genetics of Erwinia amylovora involved in the development of fire blight . FEMS Microbiol Lett , 2005 . 253 ( 2 ): 185 - 192 . 16253442 DOI: 10.1016/j.femsle.2005.09.051 http://doi.org/10.1016/j.femsle.2005.09.051 . Molecular genetics of Erwinia amylovora involved in the development of fire blight CS Oh , GB Martin , SV Beer . DspA/E, a type III effector of Erwinia amylovora , is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death . Mol Plant Pathol , 2007 . 8 ( 3 ): 255 - 265 . 20507497 DOI: 10.1111/j.1364-3703.2007.00390.x http://doi.org/10.1111/j.1364-3703.2007.00390.x . DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death MJC Pel , AJH van Dijken , BW Bardoel , 等 . Pseudomonas syringae evades host immunity by degrading flagellin monomers with alkaline protease AprA . Mol Plant Microbe Interact , 2014 . 27 ( 7 ): 603 - 610 . 24654978 DOI: 10.1094/MPMI-02-14-0032-R http://doi.org/10.1094/MPMI-02-14-0032-R . Pseudomonas syringae evades host immunity by degrading flagellin monomers with alkaline protease AprA TN Petersen , S Brunak , G von Heijne , 等 . SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods , 2011 . 8 ( 10 ): 785 - 786 . 21959131 DOI: 10.1038/nmeth.1701 http://doi.org/10.1038/nmeth.1701 . SignalP 4.0: discriminating signal peptides from transmembrane regions N Piqué , D Miñana-Galbis , S Merino , 等 . Virulence factors of Erwinia amylovora : a review . Int J Mol Sci , 2015 . 16 ( 6 ): 12836 - 12854 . 26057748 DOI: 10.3390/ijms160612836 http://doi.org/10.3390/ijms160612836 . Virulence factors of Erwinia amylovora: a review D Pletzer , H Weingart . Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora . BMC Microbiol, 14:185 , 2014 . DOI: 10.1186/1471-2180-14-185 http://doi.org/10.1186/1471-2180-14-185 . Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora D Pletzer , A Stahl , AE Oja , 等 . Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora . Arch Microbiol , 2015 . 197 ( 6 ): 761 - 772 . 25837473 DOI: 10.1007/s00203-015-1109-0 http://doi.org/10.1007/s00203-015-1109-0 . Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora ND Rawlings , AJ Barrett , PD Thomas , 等 . The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database . Nucleic Acids Res , 2018 . 46 ( D1 ): D624 - D632 . 29145643 DOI: 10.1093/nar/gkx1134 http://doi.org/10.1093/nar/gkx1134 . The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database JK Schachterle , GW Sundin . The leucine-responsive regulatory protein Lrp participates in virulence regulation downstream of small RNA ArcZ in Erwinia amylovora . mBio , 2019 . 10 ( 3 ): e00757 - 19 . 31138749 DOI: 10.1128/mBio.00757-19 http://doi.org/10.1128/mBio.00757-19 . The leucine-responsive regulatory protein Lrp participates in virulence regulation downstream of small RNA ArcZ in Erwinia amylovora S Schröpfer , C Böttcher , T Wöhner , 等 . A single effector protein, AvrRpt2 EA , from Erwinia amylovora can cause fire blight disease symptoms and induces a salicylic acid-dependent defense response . Mol Plant Microbe Interact , 2018 . 31 ( 11 ): 1179 - 1191 . 30204065 DOI: 10.1094/MPMI-12-17-0300-R http://doi.org/10.1094/MPMI-12-17-0300-R . A single effector protein, AvrRpt2EA, from Erwinia amylovora can cause fire blight disease symptoms and induces a salicylic acid-dependent defense response M Sebaihia , AM Bocsanczy , BS Biehl , 等 . Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946 . J Bacteriol , 2010 . 192 ( 7 ): 2020 - 2021 . 20118253 DOI: 10.1128/JB.00022-10 http://doi.org/10.1128/JB.00022-10 . Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946 K Segers , J Anné . Traffic jam at the bacterial Sec translocase: targeting the SecA nanomotor by small-molecule inhibitors . Chem Biol , 2011 . 18 ( 6 ): 685 - 698 . 21700205 DOI: 10.1016/j.chembiol.2011.04.007 http://doi.org/10.1016/j.chembiol.2011.04.007 . Traffic jam at the bacterial Sec translocase: targeting the SecA nanomotor by small-molecule inhibitors D Smets , MS Loos , S Karamanou , 等 . Protein transport across the bacterial plasma membrane by the Sec pathway . Protein J , 2019 . 38 ( 3 ): 262 - 273 . 31134461 DOI: 10.1007/s10930-019-09841-8 http://doi.org/10.1007/s10930-019-09841-8 . Protein transport across the bacterial plasma membrane by the Sec pathway THM Smits , F Rezzonico , T Kamber , 等 . Complete genome sequence of the fire blight pathogen Erwinia amylovora CFBP 1430 and comparison to other Erwinia spp . Mol Plant Microbe Interact , 2010 . 23 ( 4 ): 384 - 393 . 20192826 DOI: 10.1094/MPMI-23-4-0384 http://doi.org/10.1094/MPMI-23-4-0384 . Complete genome sequence of the fire blight pathogen Erwinia amylovora CFBP 1430 and comparison to other Erwinia spp J Stavrinides , HC McCann , DS Guttman . Host-pathogen interplay and the evolution of bacterial effectors . Cell Microbiol , 2008 . 10 ( 2 ): 285 - 292 . 18034865 DOI: 10.1111/j.1462-5822.2007.01078.x http://doi.org/10.1111/j.1462-5822.2007.01078.x . Host-pathogen interplay and the evolution of bacterial effectors CT Supuran , A Scozzafava , A Mastrolorenzo . Bacterial proteases: current therapeutic use and future prospects for the development of new antibiotics . Expert Opin Ther Pat , 2001 . 11 ( 2 ): 221 - 259 . DOI: 10.1517/13543776.11.2.221 http://doi.org/10.1517/13543776.11.2.221 . Bacterial proteases: current therapeutic use and future prospects for the development of new antibiotics XY Tang , YM Xiao , JM Zhou . Regulation of the type III secretion system in phytopathogenic bacteria . Mol Plant Microbe Interact , 2006 . 19 ( 11 ): 1159 - 1166 . 17073299 DOI: 10.1094/MPMI-19-1159 http://doi.org/10.1094/MPMI-19-1159 . Regulation of the type III secretion system in phytopathogenic bacteria YL Tian , YQ Zhao , LY Shi , 等 . Type VI secretion systems of Erwinia amylovora contribute to bacterial competition, virulence, and exopolysaccharide production . Phytopathology , 2017 . 107 ( 6 ): 654 - 661 . 28421913 DOI: 10.1094/PHYTO-11-16-0393-R http://doi.org/10.1094/PHYTO-11-16-0393-R . Type VI secretion systems of Erwinia amylovora contribute to bacterial competition, virulence, and exopolysaccharide production A Tsirigotaki , J de Geyter , N Šoštarić , 等 . Protein export through the bacterial Sec pathway . Nat Rev Microbiol , 2017 . 15 21 - 36 . 27890920 DOI: 10.1038/nrmicro.2016.161 http://doi.org/10.1038/nrmicro.2016.161 . Protein export through the bacterial Sec pathway X Wu , L Lei , SQ Gong , 等 . The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol . BMC Microbiol, 11:87 , 2011 . DOI: 10.1186/1471-2180-11-87 http://doi.org/10.1186/1471-2180-11-87 . The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol YJ Xia . Proteases in pathogenesis and plant defence . Cell Microbiol , 2004 . 6 ( 10 ): 905 - 913 . 15339266 DOI: 10.1111/j.1462-5822.2004.00438.x http://doi.org/10.1111/j.1462-5822.2004.00438.x . Proteases in pathogenesis and plant defence P Yorgey , LG Rahme , MW Tan , 等 . The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice . Mol Microbiol , 2001 . 41 ( 5 ): 1063 - 1076 . 11555287 DOI: 10.1046/j.1365-2958.2001.02580.x http://doi.org/10.1046/j.1365-2958.2001.02580.x . The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice YX Zhang , DD Bak , H Heid , 等 . Molecular characterization of a protease secreted by Erwinia amylovora . J Mol Biol , 1999 . 289 ( 5 ): 1239 - 1251 . 10373365 DOI: 10.1006/jmbi.1999.2846 http://doi.org/10.1006/jmbi.1999.2846 . Molecular characterization of a protease secreted by Erwinia amylovora YF Zhao , SY He , GW Sundin . The Erwinia amylovora avrRpt2 EA gene contributes to virulence on pear and AvrRpt2 EA is recognized by Arabidopsis RPS2 when expressed in Pseudomonas syringae . Mol Plant Microbe Interact , 2006 . 19 ( 6 ): 644 - 654 . 16776298 DOI: 10.1094/MPMI-19-0644 http://doi.org/10.1094/MPMI-19-0644 . The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in Pseudomonas syringae Compliance with ethics guidelines Wang-bin ZHANG, Hai-lin YAN, Zong-cai ZHU, Chao ZHANG, Pei-xiu DU, Wen-jun ZHAO, and Wei-min LI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B2000281 Project supported by the Key Technologies R&D Program of Xinjiang Production and Construction Corps (No. 2018AB038) and the National Natural Science Foundation of China (No. 31970126) 2020-05-28 2020-07-21 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 21