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Genome-Wide Identification of Plant-Upregulated Genes of Erwinia Chrysanthemi 3937 Using a GFP-Based IVET Leaf Array

Posted on: Saturday, 4 September 2004, 06:00 CDT

A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (pwrM[four dots above]Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB[four dots above]Tn5, hrcJ[four dots above]Tn5, and hrpD[four dots above]Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT[four dots above]Tn5), and an eutR homolog from Salmonella typhimurium (eutR[four dots above]Tn5) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB[four dots above]Tn5, hrcC[four dots above]Tn5, and hrpD[four dots above]Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.

Additional keywords: oxidative stress, phosphotransferase system, transport of oligogalacturonides, type III secretion system.

Erwinia chrysanthemi is an opportunistic necrotrophic pathogen that causes soft rot, wilts, and blight diseases on a wide range of plant species. Like other many enteric pathogens, such as Escherichia coli O157:H7 and Yersinia peslis, Envinia chrysanthemi cells do not appear to invade host cells internally in the pathogenic phase. They remain in the intercellular spaces of infected plant tissue and use several secretion systems to inject virulence factors into host cells. In addition to causing local disease, the bacteria may enter vascular elements of infected plants, thereby moving rapidly through the host (Chatterjee et al. 2000; Collmer and Keen 1986; Expert 1999; Hugouvieux-Cotte-Pattat et al. 1996; Perombelon and Kelman 1980).

Many virulence determinants have been discovered in E. chrysanthemi, including the well-studied extracellular enzymes such as pectate lyase, pectinase, and cellulase; siderophore-dependent iron uptake systems, sap, and msrA gene (Expert 1999; Franza and Expert 1991; Franza et al. 1999; Hassouni et al. 1999; Lopez- Solanilla et al. 2001; Tardy et al. 1997). Virulence gene expression in E. chrysanthemi is a highly regulated phenomenon affected by a variety of parameters, including temperature, pH, iron levels, growth phase, and population density. This organism uses complex regulation strategies for virulence gene expression and possesses wellknown sophisticated sensing systems such as ExpI/ExpR quorum sensing and two-component signal transduction pathways to sense and respond to environmental signals. It also utilizes transcriptional regulators such as cyclic AMP receptor protein (CRP), plant- inducible regulator (Pir), KdgR and PecS, and the RsmA/rsmB post- transcriptional regulatory system to coordinate virulence gene expression (Chatterjee et al. 2000; Franza et al. 1999; Hugouvieux- Cotte-Pattat et al. 1996; Nachin and Barras 2000; Nassar and Reverchon 2002; Nasser et al. 1994, 1998). Recently, the type III secretion system also has been reported to play a role in pathogenicity (Bauer et al. 1994, 1995; Lopez-Solanilla et al. 2001; Yang et al. 2002).

Virulence genes of pathogens can be divided into class I, class II, and class III categories according to Wassenaar and Gaastra (2001). Class I genes are called "true virulence genes", class II genes include global or specific regulators that regulate the activities of the class I genes, and class III virulence genes compromise host defense, cytoskeletal structure, and intracellular signaling, or encode other devices required for the pathogen to cope effectively with the nutrition-limited and generally hostile environment found within hosts. The distinction between class III virulence genes and housekeeping genes may be blurred. One major distinction is that, unlike true housekeeping genes, class III virulence genes are expected to be upregulated in the host environment compared with growth of the pathogen on laboratory culture media.

Unlike other host-specific plant bacterial pathogens, E. chrysanthemi is an opportunistic plant pathogen and attacks many different species of hosts. It is especially important to identify class III virulence genes in this unique bacterium because class III genes may facilitate A. chrysanthemi infection and colonization in a wide variety of plant hosts (Okinaka et al. 2002; Yang et al. 2002). Our results with microarray and mutagencsis experiments to study host upregulated genes of E. chrysanthemi 3937 on African violet have revealed several new class III virulence genes in the bacterium (Okinaka et al. 2002). Bacterial genes involved in virulence are likely to be plant inducible; therefore, instead of simply looking at type III secretion genes by sequence analysis and hrp functional assays (Boch et al. 2002; Pouts ct al. 2002; Okinaka et al. 2002), we intend to identify diverse classes of virulence gene by using green fluorescent protein (GPP)-based in vivo expression technology (IVET) leaf array.

Beyond identification of virulence genes in E. chrysanthemi 3937, a broader understanding of host-regulated genes in this bacterium during the infection process is needed. Until now, various plant- regulated virulence genes of E. chrysanthemi 3937 have been identified (Expert 1999; Franza and Expert 1991; Franza el al. 1999; Hassouni ct al. 1999; Lopez-Solanilla et al. 2001; Tardy et al. 1997), but little information is available on general host upregulated genes of the bacterium at a genomic level. In many infected plant tissues, a common shift in metabolic pattern, including activation of peroxidase and accumulation of an array of secondary metabolic substances (phenolics, flavonoids, coumarins, steroids, and so on) was observed (Goodman et al. 1986). As a consequence, many bacterial genes involved in growth inside of the plants might not necessarily be virulence genes, and altered global gene expression patterns are expected as the bacteria tailor their gene expression to adapt themselves inside of host plants (Okinaka ct al. 2002). To broaden our understanding in this area, a stable GPP-bascd IVET leaf assay technique was introduced in this study to identify genes in E. chrysanthemi 3937 that are upregulated in a spinach host plant. Combining with the genome analysis database ASAP (a systematic genome package for community analysis of genomes) (Glasner et al. 2003), 10,000 individual IVET clones of E. chrysanthemi 3937 were screened for host upregulated genes and the identified genes were assigned to seven categories on the basis of their predicted cellular or physiological functions. Several plant upregulated genes were further selected and their roles in virulence on a plant host were investigated by two different assays, local leaf maceration and systemic invasion, on African violet. Several mutants of E. chrysanthemi 3937 caused local maceration but had reduced ability to cause systemic infection of the plant host.

RESULTS

IVET screening plant-upregulated genes.

The validity of our promoter-probe screening was tested with promoters of known virulence genes ArpG, ArpW, and pelD). Using a promoter-probe vector, pPROBE-AT (Miller and Lindow 1997), as a transcriptional reporter, a weak GPP fluorescence was observed from wild-type E. chrysanthemi 3937 cells containing promoter hrpN (pPROBE-AT[four dots above]PhrpN) and pelD (pPROBE-AT[four dots above]PpelD) grown in Luria-Bertani (LB) broth (Table 1). No to very weak expression of GFP was observed from E. chrysanthemi 3937 cells containing plasmid pPROBE-AT[four dots above]PhrpG grown in LB broth. All three promoters of hrpG, hrpN, and pelD were found to be upregulaled and produced elevated green fluorescent light in spinach leaves. No GFP expression was observed in wild-type E. chrysanlhemi 3937 cells that contained the pPROBE-AT vector alone, or constructs pPROBE-AT[four dots above]PhrpGop, pPROBE-AT[four dots above]PhrpNop, and pPROBE-AT[four dots above]PpelDop, in which the promoter sequences were in the opposite orientation with respect to the promoter-less GFP promoter. Further verification of the IVET screening system was done by constructing a microarray clone ECH205 (Bacillus subi His pcptide synlhase homolog, plant/culture ratio 5.4) (Okinaka et al. 2002)promoter region into pPROBE-AT vector as described above, and upregulated expression of GFP was observed in spinach leaves.

Plant upregulated clones of E. chrysanthemi were visually distinguished by a fluorescent microscope. To insure the consistency of our IVET screening, only the E. chrysanthemi library clones leading to bacterial fluorescence of similar or higher intensity as pPROBE-AT[four dots above]PhrpG, and pPROBE-AT[four dots above]PhrpN in leaves and no or low fluorescence in LB broth, were recognized as plant upregulated clones. The library clones showed minor differences in fluorescence signal within LB broth and plant leaf discs were eliminated because of the limitation of this method.

A library of 1.5-kb fragments of genomic DNA of wild-type E. chrysanthemi 3937 was ligated into the promoter-probe vector, pPROBE- AT. From 10,000 clones screened in spinach leaves, 61 plant-up- regulated clones of E. chrysanthemi 3937 were identified (Table 2). These upregulated clones were divided into seven functional categories, including metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%). The function of the remaining 30% is unknown.

Among these plant-uprcgulaled genes, several known or new putative virulence factor homologues of Erwinia spp. and other pathogens were identified. These include the type III secretion genes hrpA and hrpB, which also have been found in Pseudomonas syringae by the IVET method (Boch et al. 2002). The dspE homologue, observed for the first time in E. chrysanthemi 3937, is an essential pathogenicity factor that is secreted through the type III secretion pathway in E. amylovora (Bogdanove et al. 1998; Gaudriault et al. 1997). A potential hrp box, the consensus binding site sequence of the alternative sigma factor hrpL, was found upstream of the hrpA (5'-GGAACCACCTCGCATTATCTCCTACTTA-3') and dspE genes (5'- GGAACCGGCGGCGGCGGCATACCACTCA-3') of E. chrysanthemi 3937. In addition, genes involved in cell wall degradation and sugar transport also were identified as upregulated in spinach. These included type II secretory component (out genes), oligogalacturonate lyase (ogl), and a rhamnogalacturonide transporter (rhiT). Peptide methionine sulfoxide reductasc (msrA), which repairs oxidized proteins and protects E. chrysanthemi cells from oxidative stress in host environments (Hassouni et al. 1999), also was found to be upregulated in our assay. In addition, we found that the indigoidine gene (indA), conferring resistance to oxidative stress (Reverchon et al. 2002), also was upregulated in the plant hosts.

Several regulatory genes were found to be upregulated in the host plant from our IVET assay. These include the LysR-family lranscriptional regulatory protein MetR, the AraC-family regulatory proteins EutR and XyIR, the IclR-family transcriptional regulator SrpS, the phosphate rcgulon transcriptional regulatory protein PhoB, the transcription factor ReIB, and the cyclic AMP receptor protein CRP. CRP is a main activator of pectinolysis genes in E. chrysanthemi 3937 (Reverchon et al. 1997). Although we failed to recover iron uptake genes previously reported in E. chrysanthemi 3937 (Expert 1999; Franza et al. 1999), several new putative iron uptake genes (yfeA, hmuS/hmuT/hmuU) were identified from our IVET assay. Several putative xenobiotic resistance genes (bacA and crcB), chemotaxis signaling related genes (cheR and trg), and a phosphotransfera.e system (PTS) gene (nagE) were found to be plant upregulated. In addition, we discovered several putative exported proteins with unknown function that were plant upregulated in the IVET assay. Five genes (ASAP IDs 14870, 16412, 18371, 19359, and 19592) were chosen as the representative of transporter, metabolism, regulator, and type III secretion pathway, respectively, for the future study.

Plant-upregulated gene knockout and virulence assays.

Using the in vitro Tn5 insertional mutagenesis strategy, we constructed mutations in five plant upregulated genes and five adjacent open reading frames (ORFs) that might contribute to virulence. ASAPIDs 16412, 14870, 18371, 19592, and 19359 were plant upregulated genes identified in IVET assay. ASAP IDs 18369 and 18373 were genes flanking 18371. ASAP IDs 19590, 19588, and 19586 were genes flanking 19592. Mutants of E. chrysanthemi 3937 were considered to have reduced local leaf maceration ability when their pathogenicity index (PI) values were <1. Mutants hrpBr.TnS, hrcJ[four dots above]Tn5, and hrpD[four dots above]Tn5 of E. chrysanthemi 3937, with PI values of 0.019, 0, and 0.016, respectively, exhibited lower local leaf maceration in African violet (Fig. 1; Table 3). In addition, they showed reduced ability to cause systemic invasion in African violet (Fig. 2). Although the ability to produce local maceration was not significantly altered for the rhiT[four dots above]Tn5 and eutR[four dots above]Tn5 mutants of E. chrysanthemi 3937, systemic invasiveness was impaired in both. Seven days after inoculation, 8 of 10 plants inoculated with wild-type E. chrysanthemi 3937 developed systemic invasion, while 3 and 4 of 10 plants developed similar symptoms in the eutR[four dots above]Tn5 and rhiT[four dots above]Tn5 mutants, respectively. Only 1 of 10 African violet plants developed systemic maceration symptoms in the hrpB[four dots above]Tn5 mutant. Using the statistical Fisher's exact test, there was a significant difference in systemic maceration capacity between the wild-type bacterium and the eutR[four dots above]Tn5 (P = 0.032), hrpB[four dots above]Tn5 (P = 0.02), and rhiT[four dots above]Tn5 (P = 0.075) mutants. The hrpB[four dots above]Tn5 mutant is an auxotroph and was not able to grow on M9 minimum medium. No significant reduction of virulence was observed in the other Tn5 mutants constructed.

Table 1. Strains, plasmids and DNA primers used in this study

DISCUSSION

Recently, microarray gene expression technology was used to identify gene expression profiles of organisms in different environments (DeLisa et al. 2001; Okinaka et al. 2002; Wei et al. 2001). However, several limitations (e.g., low amount of available RNA from target cells and the presence of contaminating organisms in samples) still deter the usage of this modern technology (Marco et al. 2003). A variety of IVET strategies, including the differential fluorescence induction (DFI) of GFP in cells, host-specific complementation of nutritional auxotrophies, and expression of selectable antibiotic resistance genes, remained the most comprehensive approaches to identify bacterial genes induced in a host- or habit-specific environment (Heithoff et al. 1997; Mahan et al. 1993, 1995; Rainey 1999; Valdivia and Falkow 1996, 1997; Wang et al. 1996), Recently, a novel IVET screening strategy termed habitat- inducible rescue of survival (HIRS) was further developed to identify genes of P. syringae that were weakly expressed on plants (Marco et al. 2003). Several limiting factors (e.g., the choice of baseline for genes to be considered 'off' in vitro) also have been reported that influenced the type and number of genes identified from this promoter trap strategy. In addition, the level of induction in vivo also was affected by the location, extent, and duration of these environment-inducible genes in IVET assays (Boch et al. 2002; Marco et al. 2003).

Table 2. Plant-upregulaled genes of Erwinia chrysanthemi 3937 identified from a green fluorescent protein-based in vivo expression technology leaf array assay using spinach as a host plant

Table 2. Plant-upregulated genes of Erwinia chrysanthemi 3937 identified from a green fluorescent protein-based in vivo expression technology leaf array assay using spinach as a host plant

E. chrysanthemi 3937 is an opportunistic necrotrophic bacterial pathogen that attacks a wide range of plant species (Chatterjee et al. 2000; Collmer and Keen 1986; Hugouvieux-Cotte-Pattat et al. 1996; Perombelon and Kelman 1980). Until now, limited information was available on gene expression profiles of E. chrysanthemi 3937 during host-microbe interactions (Aguilar et al. 2002; Okinaka et al. 2002). To discover plant upregulated genes of E. chrysanthemi 3937 in a genome-wide level, we combined the genome analysis database ASAP with a GFP-based IVET leaf array to do a mass screening of bacterial genes that are specifically upregulated in spinach. Compared with the plant-upregulated genes of E. chrysanthemi identified from the previous microarray analysis (Okinaka et al. 2002), only a few identical genes (e.g., yfe and hmuU) were identified in our IVET assay. However, many different genes with similar functions were identified in microarray and IVET studies. The major plant-upregulated genes with similar functions identified in both assays include chemotaxis genes; genes homologous to PTS; genes related to xenobiotic resistance; putative oxidoreductases; genes involved in transposition, recombination, or DNA repair; putative iron acquisition genes; and other transporters. Using the microarray assay, RNA expression profiles were determined at the instant of sampling time. On the other hand, using a GFP- based reporter, an accumulation of the upregulated signals from bacterial inoculation until harvest was recognized. Different host plants were used in our microarray and IVET assays. These might explain why, in many cases, different plant-upregulated bacterial genes with similar functions were identified in microarray and IVET assays.

With the advantage of the stability of the long-life GFP, host- upregulated genes of E. chrysanthemi 3937 during the 18-h infection period in spinach were recognized and divided into different functional categories. Our study indicated that 70% of these plant- upregulated genes in E chrysanthemi 3937 could be assigned to functional categories. During disease development, a similar ratio of genes involved in regu\lation (11%), transport (11%), and cell process (11%) were upregulated in E. chrysanthemi 3937. It is possible that the induction of transposase genes (2%) in the plant host may have occurred from the stress in the plant environment (lives et al. 2001; Okinaka et al. 2002).

Several genes were identified with probable roles in stress response to reactive oxygen species. These included peptide methionine sulfoxide reductase (msrA) and the indA. IVET work done in Streptococcus gordonii also identified the msrA gene induced in endocarditis of the New Zealand white rabbit (Kilic et al. 1999). MsrA can repair oxidatively damaged proteins and is required for full virulence of E. chrysanthemi (Hassoouni et al. 1999). IndA is involved in biosynthesis of the blue pigment indigoidine and protects the bacteria against the reactive oxygen species generated during the plant defense response (Reverchon et al. 2002). This might indicate that active oxygen species were produced from the host plant to defend against the invading pathogen and the pathogens make use of different products to protect themselves from the oxidative stresses.

Six transcriptional factors (PhoB, MetR, CRP, EutR, XylR, and SipS) and one unknown regulator (ASAP ID 18849) were found to be upregulated in spinach in this study. PhoB is a component of a two- component signal transduction pathway. MetR, CRP, EutR, XylR, SrpS, and the unknown regulator contained a helix-turn-helix (HTH) motif either at the N-terminus (MetR, SrpS, and unknown regulator) or at the C-terminus (CRP, EutR, and XyIR). PUP-25 contained an EAL domain (pfam00563), which is a good candidate for a diguanylate phosphodiesterase function. In E. chrysanthemi, several transcriptional factors related to pathogenicity have been investigated intensely by other researchers. These include the positive regulators CRP, Pir, and ExpR, and negative regulators, such as KdgR, PecS, and PecT (Chatterjee et al. 2000; Franza et al. 1999; Hugouvieux-Cotte-Pattat et al. 1996; Nassar and Reverchon 2002; Nasser et al. 1994, 1998). The recruitment of many regulators may indicate that successful pathogenicity requires complex gene regulation and expression for rapid adaptation to the complicated and diverse plant host environment (Buell et al. 2003). Unraveling the secrets of the temporal and spatial expression pattern in E. chrysanthemi 3937 regulators may help us understand its adaptation and fitness in host environments.

Iron is in short supply in numerous environments, and many bacteria secrete siderophores specifically to chelate iron. In our studies, various putative iron-uptake genes, including hmuSTU and yfeA homologues, were recognized as plant upregulated. Similarly, many genes related to iron metabolism were found to he induced with the IVET approach in other bacterial pathogens. These include entF and fhuA in Klebsiella murium, fur and fptA in P. aeruginasa, and fepA in Klebsiella pneumoniae (Heithoff et al. 1997; Lai et al. 2001; Wang et al. 1996). In E. chrysobactin, two structurally unrelated siderophores, chrysobactin and achromobactin, were identified. Chrysobactin is required for the bacterium to cause systemic infection in African violet. Plant hosts inoculated with an E. chrysanthemi achromobactin mutant exhibit delayed symptoms (Expert 1999; Franza et al. 1999).

Two redundant transport systems, TogMNAB and TogT, have been identified in E. chrrysanthemi 3937 and are able to independently mediate the transport of oligogalacturonides through the inner membrane of this bacterium. Inactivation of both systems is necessary to give a clear phenotype of transportation deficiency (Hugouvieux-Cotte-Pattat and Reverchon 2001; Hugouvieux-Cotte- Pattat et al. 2001). There is strong evidence that the newly identified rhiT plays a role in the uptake of rhamnogalacluronides (Hugouvieux-Cotte-Pattat 2004). Rhamnogalacturonides are generated by the extracellular enzyme RhiE, a 62-kDa protein that has rhamnogalacturonatelyase activity on rhamnogalacturonan I (RG-I). The reduced virulence of the rhiE mutant indicates that degradation of the RG-I region of pectin is important for full virulence of E. chrysanthemi (Laatu and Condemine 2003). In this study, a disruption of rhiT in E. chrysanthemi 3937 did not significantly alter local maceration, but systemic invasion capability was compromised. The mechanism of reduced systemic invasion ability of the rhiT mutant in the host plant is unclear. One possibility is that a deletion of the rhiT gene in E. chrysanthemi 3937 could disrupt the production of other cxoenzymes and pathogenicity factors regulated by degraded pectin and pectate intermediates (Toth et al. 2003). The precise production of these virulence factors during the infection process might be required for a systemic invasion of E. chrysanthemi 3937 in host plants. In any event, the rhiT mutant further reveals the importance of pectin utilization for E. chrysanthemi pathogenicity. Similarly, eutR[four dots above]Tn5 mutants of E. chrysanthemi 3937 had a reduced capacity to invade systcmically. Gathering more information from further local and systemic infection assays may perhaps help us explain the mechanism of delayed systemic infection.

Fig. 1. Local maceration lesions caused by wild-type Erwinia chrysanthemi 3937 (W) and mutantspup2D[four dots above]Tn5 (A), hrpD[four dots above]Tn5 (B), hrcJ[four dots above]Tn5 (C1 and C2), and hrpB[four dots above]Tn5 (D) un leaves of African violet cv. Katja. Bacterial cells were inoculated in the middle of each half side of the same leaf. Phosphate buffer (pH 7.4, 50 mM) were used to suspend the bacterial cells and a volume of 0.05 ml of bacterial suspension with a bacterial concentration of 10^sup 6^ CPU/ml was used. P indicates leaves infiltrated with phosphate buffer alone. Both C1 and C2 are hrcJ mutants with Tn5 transposon insertion at different locations of hrcJ gene.

Several plant-upregulated genes of E. chrysanthemi 3937 related to phosphate metabolism, transportation, and regulation were identified through the IVET assay. They include ppK, phoB, pstB, phnC, phnD, and a potential Pi ABC transporter periplasmic substrate- binding component homologue. Inorganic polyphosphate (Poly P) is a chain of many hundreds of phosphates (Pi) linked by high-energy phosphoanhydride bonds and widespread in all life forms. PPK is a bifunctional polyphosphate kinase that synthesizes polyphosphates from ATP or converts poly P and ADP to ATP. There are several potential functions for cellular Poly P, including a phosphate and energy reservoir, a chelator of metal ions, a buffer against alkali, and a channel for DNA entry (Kim et al. 2002; Kornberg et al. 1999; Shiba et al. 2000). Some evidence has been gathered for the role of ppK in virulence. For example, inorganic polyphosphate was required for motility of bacterial pathogens, and ppK of P. aeruginosa is responsible for biofilm development, quorum sensing, and virulence (Rashid and Kornberg 2000; Rashid et al 2000a and b). Von Kruger and associates (1999) verified that a Vibrio cholerae phoB mutant was less able to colonize rabbit intestines than the wild-type bacterium. It will be interesting to further investigate whether or not the ppK and phoB of E. chrysanthemi 3937 are important for bacterial response to Pi starvation, virulence gene expression, and survival.

Table 3. Local lesion maceration by wild-type Erwinia chrysanthemi 3937 and mutants on African violet cv. Kalja

Fig. 2. Development of symptoms induced by Erwinia chrysanthemi 3937 wild type and rhiT[four dots above]Tn5, eutR[four dots above]Tn5, hrpB[four dots above], and pup[four dots above]Tn5 mutant strains on African violet cv. Katja plants. A systemic invasion assay was used as described by Nachin and associates (2001) with minor modification. For each strain, 10 plants (one leaf per plant) were inoculated. Response was considered as systemic when at least one leaf and its petiole were macerated.

The bacterial PTS catalyzes sugar phosphorylation and dephosphorylation, and regulates a variety of bacterial physiological processes. It plays important roles in chemotaxis, intermediary metabolism, gene transcription, and virulence (Saier and Reizer 1994). In our IVET assay, a nagE homologue in E. chrysanthemi 3937 was upregulated in the plant host. NagE is a component of an N-acetylglucosamine-specific IIABC PTS system, containing a PTS-EIIC conserved domain at the N-terminus and a PTS- EIIB conserved domain at the C-terminus.

Bacteria use chemotaxis to respond to microenvironments by swimming toward or away from stimulants to optimize their growth and survival. Although a lot of work has been carried out on the role of motility as virulence factor for bacteria (Gosink et al. 2002; Larsen and Boesen 2001; O'Toole et al. 1996), little has been done on E. chrysanthemi. This study identified the cheR and trg homologues in E. chrysanthemi 3937 as upregulated in the plant host. An additional investigation about the interactions among chemotaxis, PTS, and Pi systems identified in the IVET assay may lead to an understanding of the regulatory mechanisms involved in the bacterial infection process. In addition, the upregulation of nagE (N- acetylglucosamine-specific PTS system component), b1329 (ABC transporter, periplasmic murein peptide-binding protein), and bacA (undecaprenol kinase, bacilracin resistance protein) all may be related to the increased need for cell wall growth during bacteria proliferate within the plant environment.

Various genes related to purinc and pyrimidinc biosynthesis have been reported in IVET and signature-tagged mutagenesis (STM) work. These include purCELK in Streptococcus spp.; purDHK, purKLFC, and carBA in V. cholerae; and purHLD in P. cteriiginosa. The interruption of these genes may reduce the virulence of these pathogens in their hosts (Chiang and Mekalanos 1998; Lehoux et al. 2002; Merrell et al. 200\1; Polissi et al. 1998). Although the pur M homolog mutant (purM[four dots above]Tn5) of E. chrysanthemi 3937 showed reduced virulence on African violet, it was unable to grow on M9 minimum medium. The cause of reduction in virulence of this mutant may be due to its auxotrophic phenotype. Other genes found in this IVET study were the genes involved in xenobiotic resistance, as well as several transposase genes that also were reported by other groups using IVET or STM approaches (Boch et al. 2002; Coulter et al. 1998; Kilic et al. 1999; Merrell et al. 2002; Wang et al. 1996; Wu et al. 2002).

Although the type III secretion system has been identified in E. chrysanthemi, relatively little is known about its role in pathogenicity (Bauer et al. 1994, 1995; Lopez-Solanilla et al. 2001 ; Yang et al. 2002). The mutants of hrpB, hrcJ, and hrpD homologues from the hrpA operon of E. chrysanthemi 3937 had reduced local leaf maceration and reduced systemic invasion abilities, which further confirmed our previous result that hrp genes in E. chrysanlhemi 3937 are crucial for full virulence (Yang et al. 2002).

The rhiT and eutR genes of E. chrysanthemi 3937 identified in this study can be classified as class III virulence genes. However, a significant proportion of bacterial genes upregulated in plant hosts in this study are not necessarily for its pathogenicity. E. chrysanthemi is a bacterial pathogen that can infect a wide range of plant species. In this study, we identified a broad spectrum of genes in E. chrysanthemi 3937 that were upregulated in spinach. With the completion of E. chrysanthemi 3937 genome sequencing, it will be interesting to further compare arrays of genes in the bacterium that are regulated in different plant hosts and study how the bacterium coordinates gene expression to adapt to different host environments.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media.

The bacterial strains and plasmids used in this study are listed in Table 1. Wild-type E. chrysanthemi 3937 and mutant strains were stored at -80C in 15% glycerol and grown on LB agar. Antibiotics used were kanamycin at 50 g/ml and ampicillin at 100 g/ml. Primers used for polymerase chain reaction (PCR) in this report also are listed in Table 1.

pPROBE-AT:PpelD, pPROBE-AT:PhrpG, and pPROBE-AT:PhrpN promoter vector construction.

DNA fragments of pelD, hrpG, and hrpN promoter regions were PCR amplified from E. chrysanlhemi 3937 chromosomal DNA. The amplified DNA fragments were gel purified with QIAquick gel extraction kit (Qiagen, Valencia, CA, U.S.A.) and ligated into the pGEM-T Easy vector system containing 3'T overhangs at the insertion side (Promega Corp., Madison, Wl, U.S.A.). The plasmid DNA was digested with EcoRI to release the pelD, hrpG, and hrpN promoter fragments which were ligated into the promoter-probe vector pPORBE-AT at the EcoRI site. The orientation of the inserted DNA fragments in pPROBE- AT was determined by PCR. Plasmids containing DNA inserts with the correct (promoter transcribes toward GFP promoter-probe cassettes) and the opposite orientations were selected. These plasmids were designated as pPROBE-AT[four dots above]PpelD, pPROBE-AT[four dots above]PhrpG, pPROBE-AT[four dots above]PhrpN (promoter with the transcription orientation toward the promoter-less GFP reporter of pPROBE-AT), pPROBE-AT[four dots above]PpelDop, pPROBE-AT[four dots above]PhrpGop, and pPROBE-AT[four dots above]PhrpNop (promoter with the transcription orientation opposite to promoterless GFP reporter). Seven different plasmids, pPROBE-AT[four dots above] PpelD, pPROBE-AT[four dots above]PhrpG, pPROEE-AT[four dots above]PhrpN, pPROBE-At[four dots above]PpelDop, pPROBE-AT[four dots above]PhrpGop, pPROBE-AT[four dots above]PhrpNop, and the pPROBE-AT vector alone, were electroporated individually into wild-type E. chrysanthemi 3937. Plasmid-containing bacteria were inoculated into spinach leaves and LB media. The GFP protein expression from bacteria was observed using an Olympus fluorescent microscope.

GFP-based IVET leaf array screening strategy.

A library of 1.5-kb fragments of Sau3A1 partially digested gcnomic DNA of wild-type E. chrysanthemi 3937 was cloned by ligation into the BamHl site of the promoter-probe vector, pPROBE-AT. The 1.5- kb chromosomal fragments were size fractionated through agarose gel elctrophoresis and further purified through QIAquick gel extraction kit. The pPROBE-AT plasmid contained a broad host-range replicon from pBBR1 and a promoterless GFP reporter. Random library clones constructed in pPROBE-AT were electroporated into wild-type E. chrysanthemi 3937 as described (Yang et al. 2002). The plasmid- containing bacteria were grown on LB agar at 28C overnight and transferred into microtitcr plates containing 3-mm discs cut from market-purchased spinach leaves. Microtitcr plates containing plant leaves and bacteria were vacuum infiltrated in a closed chamber to introduce bacteria into the leaf discs (with a vacuum pressure of 500 mmHg for 3 min, repeated six times). All leaf discs were incubated at 28C for 18 h with moderate shaking. As controls, we used bacteria carrying the pPROBE-AT vector alone and plasmids pPROBE-AT[four dots above]PpelD, pPROBE-AT[four dots above]PhrpG, and pPROBE-AT[four dots above]PhrpN that contained known E. chrysanthemi plant-inducible promoters of hrpG, pelD, and hrpN. After incubation, the leaf discs were mechanically macerated to release bacterial cells. The cell suspension was spotted on glass plates by using a hand arrayer and observed under a fluorescent microscope to monitor relative GFP fluorescence. Fluorescent bacteria were selected and rescreened on LB liquid medium and in spinach leaf discs. Bacterial clones leading to fluorescence of similar intensity between host leaves and LB broth were assumed to contain constitutive promoters and were discarded. Bacteria carrying plant upregulated promoters (no or lower fluorescence on LB medium compared with host leaf) were confirmed by rescreening on leaves and LB media. The consistent clones were end sequenced to identify the cloned genes or promoter regions.

Sequencing and annotating candidate clones.

Initially, sequencing primers were designed for primer walking to complete the full length of the insert DNA fragments in pPROBE-AT if end sequencing did not give the full length of the insert DNA fragments. The vector sequences were trimmed and remaining DNA sequences were assembled in Vector NTI (InforMax Inc., Bethesda, MD, U.S.A.). The ORF prediction and homologue search of ORFs were analyzed by Vector NTI through NCBI Blast searches. After the preliminary E. chrysanthemi 3937 genome sequence became available, the candidate clones were end sequenced from both ends and compared in ASAP to obtain the full-length sequence of insert DNA. The functional groups of plant-upregulated genes from our IVET assay were categorized accordingly (Glasner et al. 2003; Serres and Riley 2001). Several other bioinformatics tools were used to analyze their conserved motifs and putative functions, such as NIH NCBI Conserved Domain Database and Search Service (CDD), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Escherichia coli genome and proteome databases (GenProtEC).

Exploring the potential functions of plant-upregulated genes by Tn5 mutagenesis and virulence assays.

Initially, a pooled library screening strategy was used to identify Erwinia chrysanthemi 3937 library clones containing target genes for mutation. Specific PCR primers were designed for a 7- to 8- kb DNA insert library pool (48 library clones were pooled as one screening unit) of E. chrysanthemi 3937. Once an expected PCR DNA fragment was observed in E. chrysanthemi library pools, 48 insert library clones were amplified individually to obtain the library clone containing a target gene. Selected ORFs on plant-upregulated plasmid clones were mutagenized with Tn5 transposon using an EZ[four dots above]TN insertion kit with the procedures suggested by the manufacturer (Epicentre Technologies, Madison, WI, U.S.A.). Tn5 insertions were physically mapped and precisely located by sequencing with transposon primers IR-F or IR-R. The plasmid constructs then were electroporated into E. chrysanthemi 3937 and plasmid curing and marker exchange were accomplished by growth of kanamycin-resistant transformants in low-phosphate medium broth (Ried and Collmer 1987; Yang et al. 2002). Mutations in the resultant kanamycin-resistant and ampicillin-sensitive colonies then were confirmed by PCR, using transposon and target gene-specific primers.

Subsequently, PCR primers were designed for plant-upregulated clones or genes based on the available E. chrysanthemi 3937 genome data. DNA fragments were amplified using a proofreading DNA polymerase (FailSafe PCR system; Epicentre), cloned into pGEM-Teasy vector, and mutated with Tn5, after which marker exchange mutagenesis was performed as described above.

Two different virulence assays, local leaf maceration and systemic invasion, were conducted on African violet cv. Katja leaves. In the local maceration assay, a volume of 0.05 ml with a bacterial concentration of 106 CFU/ml of wild-type E. chrysanthemi 3937 and its mutants was inoculated in the middle of each half side of the same leaf. Phosphate buffer (pH 7.4, 50 mM) was used to suspend the bacterial cells. The inoculated plants were placed in a growth chamber under the conditions described previously (Yang et al. 2002). Three replicate plants with a total of 12 leaves were inoculated for each of the wild-type bacterium and its mutants. The maceration area of plant leaves caused by the bacterial strains was precisely measured with an image analysis software, ASSESS (The American Phytopathological Society, St. Paul, MN, U.S.A.). The PI for each bacterial mutant was calculated according to the equation PI = [Sigma](lesion size of leaves from the bacterial mutant/ lesi\on size of leaves from the wild-type bacterium)/number of leaves used for measurement.

In the systemic invasion assay, the pathogenicity of E. chrysanthemi 3937 was evaluated as described by Nachin and associates (2001) with minor modification. A volume of 0.05 ml of bacterial suspension with an optical density at 600 nm of 0.3 was inoculated into the front edge of the African violet cv. Katja leaf. Inoculated plants were kept in a growth chamber with regular water misting (5 min in every 4 h) to provide humid conditions.

ACKNOWLEDGMENTS

We gratefully acknowledge M. L. P. Collins for a critical review of this manuscript. We thank Patricia Steen and Peter W. Hickmott for valuable assistance with microscopic analyses. This project was supported by USDA-NRICGP award no. 2001-35319-10015 and NSF award no. MCB-0211750.

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Shihui Yang,1 Nicole T. Perna,2 Donald A. Cooksey,1 Yasushi Okinaka,3 Steven E. Lindow,4 A. Mark Ibekwe,5 Noel T. Keen,1 and Ching-Hong Yang6

1 Department of Plant Pathology, University of California, Riverside 92521, U.S.A.; 2 Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison 53706, U.S.A.; 3 Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-hiroshima, 739-8528, Japan; 4 Department of Plant and Microbial Biology, University of California, Berkeley 94720, U.S.A.; 5 United States Department of Agriculture- Agricultural Research Service George E. Brown Jr. Salinity Lab., Riverside, CA 92507, U.S.A., 6 Department of Biological Sciences, University of Wisconsin-Milwaukee 53211, U.S.A.

Submitted 22 December 2003. Accepted 3 May 2004.

Corresponding author: Ching-Hong Yang; Telephone: +1-414-229- 6331; Fax: +1-414-229-3926; E-mail: chyang@uwm.edu

N. T. Keen passed away on April 18, 2002.

Copyright American Phytopathological Society Sep 2004

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