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First published online August 17, 2007; 10.1104/pp.107.103432 Plant Physiology 145:426-436 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
AtHIPM, an Ortholog of the Apple HrpN-Interacting Protein, Is a Negative Regulator of Plant Growth and Mediates the Growth-Enhancing Effect of HrpN in Arabidopsis1,[C],[OA]Department of Plant Pathology, Cornell University, Ithaca, New York 14853
HrpN (harpin) protein is critical to the virulence of the fire blight pathogen Erwinia amylovora in host plants like apple (Malus x domestica). Moreover, exogenous treatment of Arabidopsis (Arabidopsis thaliana), a nonhost plant, with partially purified HrpN enhances growth. To address the bases of the effects of HrpN in disease, we sought a HrpN-interacting protein(s) in apple, using a yeast two-hybrid assay. A single positive clone, designated HIPM (HrpN-interacting protein from Malus), was found. HIPM, a 6.5-kD protein, interacted with HrpN in yeast and in vitro. Deletion analysis showed that the N-terminal 198 of 403 amino acids of HrpN are required for interaction with HIPM. HIPM orthologs were found in Arabidopsis (AtHIPM) and rice (Oryza sativa; OsHIPM). HrpN also interacted with AtHIPM in yeast and in vitro. In silico analyses revealed that the three plant proteins contain putative signal peptides and putative transmembrane domains. We showed that both HIPM and AtHIPM have functional signal peptides, and green fluorescent protein-tagged HIPM and AtHIPM associated, in clusters, with plasma membranes. Both HIPM and AtHIPM are expressed constitutively; however, they are expressed more strongly in apple and Arabidopsis flowers than in leaves and stems. The size of AtHIPM knockout mutant plants of Arabidopsis was slightly larger than the wild-type plants. Interestingly, the knockout mutant did not exhibit enhanced plant growth in response to treatment with HrpN. Overexpression of AtHIPM conversely resulted in smaller plants. These results indicate that AtHIPM functions as a negative regulator of plant growth and mediates enhanced growth that results from treatment with HrpN.
Harpins are proteins produced by Gram-negative plant-pathogenic bacteria; they share the following distinctive characteristics: heat stability, richness in Gly, no Cys, and few aromatic amino acids. Since HrpN of Erwinia amylovora was characterized as the first cell-free elicitor of the hypersensitive response (HR) in plants (Wei et al., 1992  ), several other harpins have been characterized from various Gram-negative plant-pathogenic bacteria: HrpN and HrpW of Erwinia spp.; HrpZ, HrpW, HopPtoP, and HopPmaHPto of Pseudomonas syringae; PopA1 of Ralstonia solanacearum; and HpaG and its orthologs of Xanthomonas campestris like XopA (He et al., 1993; Arlat et al., 1994; Bauer et al., 1995; Charkowski et al., 1998; Kim and Beer, 1998; Kim et al., 2004; Ramos, 2004). Harpins are secreted through the Hrp type III secretion system like avirulence (Avr) proteins of plant pathogenic bacteria, which directly or indirectly interact with corresponding resistance (R) proteins (Alfano and Collmer, 2004). However, unlike Avr proteins, which mostly are delivered to the plant cytoplasm, harpins are localized to the plant apoplast.
Some harpins have virulence functions in host plants. Mutation in hpaG by transposon insertion or mutation of its ortholog, xopA, by deletion resulted in reduced symptoms and reduced bacterial growth in host plants (Noel et al., 2002
In addition to virulence activity in host plants and avirulence activity to induce HR in nonhost plants, the HrpN protein induces several beneficial effects when applied to plants. First, HrpN of E. amylovora induces systemic acquired resistance, resulting in resistance to pathogens in Arabidopsis (Arabidopsis thaliana; Dong et al., 1999 In this study, we hypothesized that harpins interact with plant protein(s) to increase susceptibility in host plants and that the interacting protein(s) may also be involved in induction of beneficial effects like growth enhancement in Arabidopsis. As a first step, we identified a HrpN-interacting protein(s) from apple, using a yeast two-hybrid assay. One protein, designated HIPM, (HrpN-interacting protein from Malus), was found from this assay. Based on the amino acid sequence of HIPM, we found an ortholog in Arabidopsis, AtHIPM. Both HIPM and AtHIPM interacted with HrpN in yeast and in vitro. Both proteins have functional signal peptides (SPs) and associate, in clusters, with plasma membranes. For functional analysis, we chose AtHIPM in Arabidopsis and determined if this protein is required for growth enhancement by treatment with HrpN. We found that AtHIPM is needed for Arabidopsis to exhibit enhanced growth in response to HrpN, and AtHIPM functions as a negative regulator of plant growth.
HIPM and Its ortholog, AtHIPM, Interact with HrpN, a Harpin of E. amylovora Apple proteins that interact with HrpN, the archetype harpin of E. amylovora, were screened with a yeast two-hybrid system. A cDNA prey library from ‘Gala’ apple was screened with the full-length HrpN protein as bait. Of the more than 106 primary yeast transformants screened, 24 positive clones were selected initially. Those 24 prey clones were isolated from yeast, sequenced, and retransformed into yeast harboring the hrpN gene cloned in a bait vector. On retesting, 23 of the clones exhibited negative phenotypes for interaction, while one positive clone was found; it was designated HIPM (HrpN-interacting protein from Malus; Fig. 1 ).
Two proteins, LexA-lamin and DspA/E4.7, encoded by the 4.67-kb 5'-end portion of the dspA/E gene of E. amylovora, were chosen to determine whether HrpN-HIPM interaction is specific. HIPM did not interact with either protein (Fig. 1), suggesting that HrpN-HIPM interaction is specific. The positive clone contained 314 bp of cDNA of the HIPM transcript, which may encode only the 53 C-terminal amino acids of HIPM. Because the positive clone did not contain the full sequence of the HIPM gene, the 5' end of the gene was amplified and cloned using the 5'-RACE kit. The HIPM gene from apple encodes a protein of approximately 6.5 kD, which contains 60 amino acids (Fig. 2 ). Two orthologs, At3g15395-1 and XP_464477, were found in the genome databases of Arabidopsis and rice (Oryza sativa); these were designated AtHIPM and OsHIPM, respectively. AtHIPM and OsHIPM consist of 59 and 61 amino acids, respectively. At the amino acid level, HIPM is 66% identical to AtHIPM and 65% identical to OsHIPM, while AtHIPM is 58% identical to OsHIPM (Fig. 2).
To determine if AtHIPM also interacts with HrpN in yeast, the part of the AtHIPM gene encoding 52 amino acids, which is the orthologous region to the original HIPM prey clone, was cloned into the prey vector and cotransformed with the hrpN gene as bait. AtHIPM also interacted with HrpN in yeast (Fig. 1). In addition, whether HrpN, HIPM, or AtHIPM self-interacts was determined in yeast. However, no self-interaction of HIPM and AtHIPM was detected, but HrpN showed strong self-interaction (Fig. 1). An in vitro pull-down assay was carried out to confirm the interaction of HrpN and HIPM or AtHIPM in vitro. Purified HrpN fused with the T7 tag was pulled down with T7 tag antibody-linked agarose beads after mixing with HIPM-FLAG or AtHIPM-FLAG protein. HIPM-FLAG or AtHIPM-FLAG was detected only when it was pulled down with T7-HrpN, indicating positive interaction of HrpN with HIPM and AtHIPM in vitro (Fig. 3 ).
The N-Terminal 198 Amino Acids of HrpN Are Required for Interaction with HIPM To identify the domain of HrpN that interacts with HIPM, nine truncated derivatives of HrpN were generated as shown in Figure 4 . First, their expression or stability in yeast was determined using the LexA antibody. Seven of the nine derivatives were expressed in yeast (Fig. 4). Second, whether interaction with HIPM occurred was determined in yeast. The N-terminal 198 amino acids of HrpN, HrpN1–198, were needed for full interaction with HIPM, although there was very weak interaction with HrpN50–403 and HrpN101–403.
To determine whether the N-terminal 198 amino acids of HrpN are sufficient for the wild-type level of virulence in host plants, the plasmid carrying DNA encoding the N-terminal 198 amino acids of HrpN with its indigenous hrp promoter was transformed into the hrpN deletion mutant of E. amylovora strain Ea273; virulence of the strain and appropriate control strains was determined in immature pear (Pyrus communis) fruits. This complemented strain failed to restore virulence in immature pear fruits (data not shown), indicating that the N-terminal 198 amino acids of HrpN are not sufficient for virulence activity of HrpN.
A domain search of two databases, the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/) for SP prediction and the EXPASy server (http://us.expasy.org/) for transmembrane (TM) domain prediction, resulted in the identification of putative SP and TM domains in HIPM, AtHIPM, and OsHIPM (Fig. 2). To determine if the putative SPs in both HIPM and AtHIPM are functional, we employed the yeast-based SP trap method, which is based on lack of growth of the yeast suc2 mutant in Suc medium. The HIPM and AtHIPM genes were fused with the suc2 gene lacking the DNA region encoding its own SP, and those constructs were put into the yeast suc2 mutant. The suc2 mutant grew in Suc medium with the full-length cDNAs of both full-length HIPM (HIPM1–60) and AtHIPM (AtHIPM1–59), but not with HIPM21–60 and AtHIPM21–59, which lack the N-terminal 20 amino acids (Fig. 5 ). These results indicate that HIPM and AtHIPM have functional SPs in their N termini, and as a consequence the proteins may be secreted or embedded in plasma membranes.
Both HIPM and AtHIPM Associate, in Clusters, with Plasma Membranes Although we showed that both HIPM and AtHIPM have SPs that are functional in a yeast system, where HIPM and AtHIPM are located in plant cells was not clear. To address the question of location, the GFP gene, whose expression was driven by the 35S promoter, was fused with HIPM and AtHIPM. HIPM-GFP, AtHIPM-GFP, and GFP itself (as a control) were transiently expressed in leaves of Nicotiana benthamiana by agroinfiltration. Green fluorescence was observed with confocal microscopy 24 h after agroinfiltration. Green autofluorescence was observed in the intercellular space of untransformed plants (Fig. 6A ). Unlike the GFP control, in which green fluorescence was seen throughout the cytoplasm (Fig. 6B), green fluorescence from both HIPM-GFP (Fig. 6, C and E, top) and AtHIPM-GFP (Fig. 6D) accumulated as large spots coincident with plasma membranes (Fig. 6). In addition, in 0.8 M mannitol solution, cell shapes were irregular due to plasmolysis (Fig. 6F, top) unlike the round shape in water (Fig. 6E, bottom), and green fluorescence from HIPM-GFP remained coincident along with plasma membranes (Fig. 6F, top) and did not appear to be cell wall or plasmodesmata localized. These results indicate that both HIPM and AtHIPM have functional SPs and both proteins ultimately localize to plasma membranes.
Both HIPM and AtHIPM Are Expressed More Strongly in Flowers Than in Stems and Leaves
HIPM was found in apple, which is a host of E. amylovora. Flowers, vigorously growing young leaves, and shoot tips are the important infection courts for E. amylovora (Vanneste, 2000 Because there were scant indications of HIPM expression based on northern hybridization (data not shown), the more sensitive semiquantitative reverse transcription (RT)-PCR technique was used. Based on the RT-PCR results shown in Figure 7A , expression of the HIPM gene was very low, and HIPM was expressed constitutively in leaves and shoots. However, HIPM was expressed more strongly in flowers than in leaves and shoots. Interestingly, HIPM expression was relatively strong in the TC, P, and F stages of flower development, but, as petals started to fall, the expression level decreased to the same level as seen in leaves and shoots.
The expression of HIPM also was determined following inoculation of apple leaves with E. amylovora strain Ea273. Total RNA was isolated 6, 12, 22, and 45 h after inoculating greenhouse-grown trees with E. amylovora or buffer. The level of HIPM expression did not change as a result of inoculation with E. amylovora, based on RT-PCR results (Fig. 7B). To determine the expression pattern of AtHIPM in Arabidopsis, total RNAs isolated from leaves, inflorescent shoots, closed flowers, open flowers, and siliques were analyzed by the same methods as used with HIPM from apple. Like HIPM in apple, AtHIPM expression levels were low, and expression was strongest in closed and open flowers, relative to other plant parts, as determined by RT-PCR (Fig. 7C).
To determine the biological significance of the interaction of HrpN-HIPM and HrpN-AtHIPM in plants, we took advantage of Arabidopsis for its availability of mutant lines, short life cycle, and ease of transformation (http://www.arabidopsis.org). One T-DNA knockout (KO) line was obtained from the Arabidopsis stock center (Columbus, OH). In this line T-DNA was inserted in the 5'-untranslated region of the AtHIPM gene. Only one copy of T-DNA was present in the homozygous KO line, based on the 3:1 (kanamycin resistant:kanamycin sensitive) segregation ratio of progenies from a backcross with the wild-type plant (data not shown). Expression of AtHIPM was first tested in the KO line by RT-PCR to ascertain whether the line would be useful for loss-of-function tests. As shown in Figure 8A , no AtHIPM transcripts were detected in the preparation from the T-DNA KO line, relative to the wild type, confirming its value for use in further experiments.
Under normal growing conditions, the KO line of Arabidopsis grew like the wild type; all developmental stages were normal. However, the size of aerial parts (top growth) was approximately 9% larger than that of the wild-type plants, although root length did not differ significantly from that of the wild type (Fig. 8, B and C). We examined the effects of the AtHIPM mutation in Arabidopsis on enhanced plant growth in response to HrpN, using the KO line, compared to the wild-type Arabidopsis. First, root growth was determined after seeds grew for 10 d in Murashige and Skoog (MS) medium. Seeds of the KO line and the wild type were treated with 15 µg/mL HrpN or water. Root growth of the wild type increased by approximately 9% after treatment with HrpN (Fig. 8B). However, in the KO line, treatment with HrpN did not enhance plant growth. Instead, treatment with HrpN reduced root growth by approximately 6% (Fig. 8B). Second, the top growth of Arabidopsis was determined 1 week after spraying 3-week-old plants with 15 µg/mL HrpN. Consistently, the top growth of the wild-type plants increased by approximately 10% following treatment with HrpN; however, treatment of the KO line with HrpN reduced top growth (Fig. 8C). Interestingly, top growth of the KO line that was treated with water was similar to that of the wild type treated with HrpN. These results indicate that AtHIPM is needed for Arabidopsis to respond to treatment with HrpN with enhanced growth.
We generated two AtHIPM-overexpressing lines and two vector-transformed lines, using pBI121-AtHIPM and pBI121 construct, respectively. The level of AtHIPM expression was first determined in these lines by RT-PCR as shown in Figure 9A . The level of AtHIPM expression in lines transformed with pBI121-AtHIPM was much higher than in lines transformed with the pBI121 vector. We examined both root and top growth of these lines under normal growing conditions. As shown in Figure 9, B and C, both root and leaf length in the AtHIPM-overexpressing lines were smaller than those of vector-transformed plants, indicating that AtHIPM functions as a negative regulator of plant growth. This finding is consistent with the evidence that AtHIPM mutation results in larger plants.
Mutation in AtHIPM Does Not Disrupt Responses to 2,4-Dichlorophenoxyacetic Acid, Methyl Jasmonate, and Ethylene in Arabidopsis
Since AtHIPM was shown to function as a negative regulator of plant growth, we examined whether AtHIPM function is connected to effects of several plant hormones that are involved in the inhibition of plant growth. Both methyl jasmonate (MeJA) and auxin inhibit root growth in plants (Chadwick and Burg, 1967
In the dark, treatment with ethylene causes the triple response, which is characterized by swelling of the hypocotyls and inhibition of root and hypocotyl growth in plants (Guzman and Ecker, 1990
HrpN-Interacting Proteins from Apple and Arabidopsis
We searched for HrpN-interacting proteins from apple using a yeast two-hybrid assay and found a single small protein, HIPM. We studied HIPM and its ortholog AtHIPM, and here present evidence that their SPs are functional and that both proteins associate with plasma membranes of plant cells. Harpins, including HrpN of E. amylovora, are not translocated into plant cells, but they are secreted from bacteria and localize outside plant cells (Hoyos et al., 1996 HIPM orthologs were found in three different plant species: apple, Arabidopsis, and rice. These plants represent diverse plant classes; two are dicots, one woody and one herbaceous, and one is a monocot. These findings suggest that the HIPM gene is conserved among plant species. However, the possible presence of HIPM orthologs in many other plant species remains to be determined.
We showed that HIPM and AtHIPM associate with plasma membranes of plant cells. Unlike other membrane proteins, HIPM and AtHIPM were not uniformly distributed among plasma membranes, but appeared to localize to specific positions in plasma membranes. Furthermore, GFP signals fused with HIPM were coincident with plasma membranes under high osmotic conditions. This rules out cell wall and plasmodesmata as possible target sites of HIPM or AtHIPM in plant cells.
One possible specific position in plasma membranes for HIPM and AtHIPM is a lipid raft, although GFP spots on plasma membranes from HIPM-GFP and AtHIPM-GFP fusion proteins are larger than the lipid rafts generally seen. Plasma membrane microdomains containing distinct molecular compositions such as lipid rafts have been reported mostly in mammalian cells, but recently they also were reported in plant cells (Bhat and Panstruga, 2005
Based on RT-PCR data, both HIPM and AtHIPM are weakly and constitutively expressed in leaves and stems. Interestingly, both genes were more strongly expressed in flowers than in leaves and stems of apple and Arabidopsis. Expression levels were reduced coincidently with the formation of fruiting structures. In apple, flowers are important infection sites for E. amylovora (Vanneste, 2000
We showed that the N-terminal 198 amino acids of HrpN are required for full interaction with HIPM. Although the N-terminal 198 amino acids of HrpN were sufficient for interaction with HIPM, we found that this portion was not sufficient for virulence in immature pear fruits. This suggests that virulence requires portions of the C terminus of HrpN in addition to the N-terminal 198 amino acids necessary for interaction with HIPM. It is not clear how the N-terminal portion and the C-terminal portion of HrpN function together to facilitate virulence of E. amylovora, but we propose two hypothetical models. After HIPM interacts with the N-terminal portion of HrpN, the C-terminal portion of HrpN may block interaction of HIPM with a second host protein, which may be located in plasma membranes like receptor kinases. Under normal conditions, interaction of HIPM and a second host protein may increase disease resistance, but if HrpN is present this interaction would be interrupted, resulting in inhibition of defense responses and subsequent disease. Alternatively, interaction of the N-terminal portion of HrpN with HIPM may allow the C-terminal portion of HrpN to interact with a second host protein. This interaction may block a positive regulatory function of a second host protein that functions in disease resistance.
HrpN is required for development of the fire blight disease in apple, and it also induces several beneficial effects in plants such as growth enhancement. In this study, top growth of an AtHIPM KO mutant line treated with water was similar to the growth of the wild-type plant treated with HrpN. In addition, overexpression of AtHIPM resulted in smaller plants. These observations support the notion that AtHIPM acts as a negative regulator of plant growth. A negative regulatory function of AtHIPM may explain why plants exhibit enhanced growth after treatment with HrpN; when HrpN is present, it may intercept AtHIPM by protein-protein interaction, resulting in the inhibition of its negative regulatory function. This inhibition may lead to larger plants, as does mutation of AtHIPM.
Interestingly, treatment of the AtHIPM KO mutant line with HrpN reduced plant growth, as compared to the water-treated control. This indicates that HrpN itself may inhibit growth of plant cells in the absence of AtHIPM. Previously, HrpN was shown to cause ion leakage by regulating ion channels in Arabidopsis suspension cells (El-Maarouf et al., 2001 Our findings that HrpN interacts with HIPM and AtHIPM and that AtHIPM functions as a negative regulator of plant growth may shed light on how HrpN contributes to development of fire blight disease in host plants. Apple transgenic plants, in which the HIPM gene is silenced, will likely provide more direct evidence as to whether or not HIPM is important for development of fire blight disease. In addition, our findings concerning HIPMs may provide some clues about how growth-enhancing activity of HrpN can be connected to its virulence activity, even though stronger evidence is needed for iron-clad conclusions. This connection could be confirmed if site-directed mutants of HrpN protein lacking growth-enhancing activity were discovered. It would be intriguing to investigate whether HrpN mutant proteins without growth-enhancing activity still contain virulence activity by determining whether those proteins restore virulence of E. amylovora hrpN mutants in host plants.
Our results clearly show that HrpN of E. amylovora interacts with HIPM and its ortholog, AtHIPM, of Arabidopsis. The N-terminal portion of HrpN is essential to interaction with the HIPMs. Both plant genes are small, but contain functional SPs and associate with plasma membranes in clusters. We also show that AtHIPM functions as a negative regulator of growth and its knockout mutant abolishes the enhanced growth that generally results from treatment of plants with HrpN.
Plant Material ‘Gala’ apple (Malus x domestica) was used to generate a cDNA prey library for yeast two-hybrid assay, and the HIPM gene was characterized from it. Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used as the source of the AtHIPM gene and for transformation of the AtHIPM-overexpressing construct. A T3 line, in which T-DNA was inserted in 5'-untranslated region of the AtHIPM gene, was obtained from the Arabidopsis Biological Resource Center (Columbus, OH), and T4 seeds with homozygosity of the T-DNA-inserted locus, which was determined by PCR with two gene-specific primers, 5'-TTAGATATCCACATAACATGTGC-3' and 5'-TTCACAAACATAGCATGACAGG-3', and one primer from T-DNA, 5'-TGGTTCACGTAGTGGGCCATCG-3', were used for further experiments. Nicotiana benthamiana was used for transient expression experiments.
E. amylovora strain Ea273 and its hrpN deletion mutant Ea273
Total RNA was isolated from several parts of Arabidopsis and apple, using the RNeasy kit (Qiagen) and the protocol described previously (Komjanc et al., 1999
General DNA manipulations, including cloning and plasmid construction, were performed as described (Sambrook et al., 1989
A 5' RLM-RACE kit (Ambion) was used to clone and sequence the 5' end of the HIPM transcript. Ten micrograms of total RNA from ‘Gala’ apple was used to make cDNA. As gene-specific primers, 5'-ACAGCACTTCCAATTGCACACG-3' and 5'-CTTTAGTTGTCTGTCCACAGCA-3' were used for amplifying the 5' end of the HIPM transcript.
The Matchmaker LexA two-hybrid system (CLONTECH) was used for screening HrpN-interacting protein(s) in apple. Briefly, bait in the pGilda vector and prey in the pB42AD vector were cotransformed into Saccharomyces cerevisiae EGY48 (ura3, his3, trp1, LexAop-LEU2, p8op-lacZ) by the LiAc/PEG transformation method (Yeast Protocols Handbook; CLONTECH). Transformants were selected on minimal synthetic dropout (SD) medium containing Glc, but lacking His, Trp, and uracil (SD/glu-HTU). To identify yeast transformants with a positive interaction, the transformants were screened on SD medium with Gal, but without His, Trp, uracil, and Leu (SD/gal-HTUL). Yeast colonies with positive interaction grow in this medium. A yeast strain transformed with p53 as bait and large antigen T as prey was used as a positive control. LexA-lamin and DspA/E4.7 from the 4.67-kb 5'-end portion of dspA/E (Meng et al., 2006
The full-length hrpN gene was cloned in the pGilda vector by PCR with EcoRI added to the forward primer 5'-AGGAATTCATGAGTCTGAATACAAGTGC-3' and with BamHI added to reverse primer 5'-GCGGATCCAAGCTTAAGCCGCGCCCAG-3'. This clone was called pGilda-hrpN and was used as bait. A cDNA prey library was generated from total RNA isolated from the ‘Gala’ apple in the pB42AD vector with added EcoRI and XhoI sites. For cloning of hrpN and HIPM into pB42AD and pGilda, pGilda-hrpN and pAD-HIPM were digested with EcoRI and XhoI, and ligated to pB42AD and pGilda, respectively. AtHIPM was amplified by PCR with the forward primer 5'-CGGAATTCAACGATAAGTGGTCAATGAG and two reverse primers, 5'-CGGGATCCTTAGACATTATCACCATCACCTTG and 5'-GCCGCTCGAGGTATTCAACTGAGCACTACTTG, for cloning into pGilda and pB42AD, respectively. The full-length hrpW gene was amplified by PCR and cloned into pGilda and pB42AD vectors. To remove portions of the HrpN protein from both the N terminus and the C terminus, four more forward primers and five more reverse primers were used. For deleting 50, 100, 150, 200, 250, and 300 amino acids from the C terminus, the forward primer with added EcoRI, 5'-AGGAATTCATGAGTCTGAATACAAGTGC-3', was used. The reverse primers with added BamHI, 5'-CGGGATCCTTACTTGGCTTTGTTGAACTGCTC-3', 5-CGGGATCCTTAGAACTGACCGATTTCCTTCGC-3', 5'-CGGGATCCTTACAGGTTTTGCAGCCCTTTGC-3', 5'-CGGGATCCTTAATAGGCGTTCTGCTCGCCTTCG-3', and 5'-CGGGATCCTTAGGACGTTGAGTTAATACCCAGC-3', were used for PCR. For deleting 50, 100, 150, and 200 amino acids from N terminus, the reverse primer with added BamHI, 5'-GCGGATCCAAGCTTAAGCCGCGCCCAG-3', and the forward primers with added EcoRI, 5'-CGGAATTCGATACCGTCAATCAGCTG-3', 5-CGGAATTCCTGAACGATATGTTAGGC-3', 5'-CGGAATTCCAGCTGCTGAAGATGTTCAGC-3', and 5'-CGGAATTCAATGCTGGCACGGGTCTTGACG-3', were used for PCR. These PCR products were cloned into the pGilda vector.
To determine interaction between HrpN and HIPM and AtHIPM in vitro, HrpN was tagged with T7 tag in the pET-24a vector, overexpressed in Escherichia coli BL21(DE3), and purified with T7 Tag affinity purification kit (Novagen). Both HIPM and AtHIPM were tagged with FLAG in the pFLAG-CTC vector (Scientific Imaging Systems) and overexpressed in E. coli BL21(DE3). Cell lysate with 5 µg of T7-HrpN was incubated with 50 µL of T7 tag antibody agarose beads at room temperature for 30 min with shaking. Beads were washed thrice more with T7 Tag bind/wash buffer, then incubated with cell lysate with 5 µg of HIPM-FLAG or AtHIPM-FLAG at room temperature for 1 h, and washed four times more with T7 Tag bind/wash buffer. One hundred fifty microliters of elution buffer was added in the pellet and proteins were eluted from the T7 tag antibody agarose beads. This step was repeated once more, and 45 µL of neutralization buffer was added in the protein solution. Eighteen microliters of protein solution was resuspended in 6 µL of 4x SDS-PAGE loading buffer.
Protein samples were denatured by holding in boiling water for 5 min, electrophoresed on a 4% to 20% gradient of SDS-PAGE gel (Gradipore), and transferred to PVDF (Immobilon-P; Millipore) by a semidry electroblotting method (Gravel and Golaz, 1996
This system was used to determine whether putative SPs are functional as first described (Yamane et al., 2005
The full lengths of the HIPM and AtHIPM genes were fused in frame with soluble-modified GFP (smGFP) in pCAMBIA2300-smGFP (Davis and Vierstra, 1998
Leaf discs were harvested 24 h after agroinfiltration, and GFP signals were observed with a Bio-Rad Leica MRC-600 confocal microscope (Bio-Rad Biosciences) using an HC PL APO 20x oil immersion objective and the 488 nm and 543 nm lines generated by argon lasers at the Cornell Biotechnology Resource Center. Emission window ranges for GFP and chlorophyll were 500 to 580 nm and 634 to 718 nm, respectively. After GFP signals were captured, three sections were overlaid in a single image, and images were saved with a resolution of 512 x 512 pixels.
To make AtHIPM-overexpressing Arabidopsis plants, the full length of the AtHIPM gene was cloned into the pBI121 vector, named pBI121-AtHIPM. The floral dipping method was used for Arabidopsis transformation (Clough and Bent, 1998
For measurement of root growth, approximately 30 seeds of Arabidopsis, previously held at 4°C for 5 d, were lined up on two plates of 0.5x MS medium containing 3% Suc. For determining the effect of HrpN, seeds were soaked in 0.075% agarose solution with 0.5 mg/mL Messenger (15 µg/mL HrpN; Eden Bioscience). The seeded plates were placed vertically in a growth room at 22°C and illuminated for 14 h per day. After 10 d, root length was measured. For top growth, cold-treated seeds were planted in 2.5-inch (6.3-cm) rectangular pots and grown for 3 weeks in a growth room at 22°C and illuminated for 14 h per day. Messenger (0.5 mg/mL) and water, as the control treatment, were sprayed once on the plants to runoff. The lengths of the three longest leaves per plant were measured 1 week after spraying. All data were analyzed statistically with Duncan's multiple range test according to the general linear model procedure of the SAS (version 9.0; SAS Institute). Duncan's groupings are indicated in the figures.
For MeJA and auxin, approximately 30 cold-treated seeds of Arabidopsis were lined up on two plates of 0.5x MS medium containing 1% Suc and MeJA or 2,4-dichlorophenoxyacetic acid at 0, 1, or 5 µM. The plates were placed vertically in a growth room at 22°C with 14 h of light per day. Root length was measured after 10 d. 1-Aminocyclopropane-1-carboxylic acid (10 µM) was added to 0.5x MS medium to simulate the presence of ethylene. Plates were kept vertically in the dark for 3 d, and the triple response was determined.
The GenBank accession number for HIPM gene described in this study is EU078328.
We are grateful to Xiangdong Meng for making the cDNA prey library used in this study, to Jean Bonasera for providing apple RNA samples, to Jian Hua for providing critical review, and to Sara Carpenter for editorial suggestions. We thank Jocelyn Rose and Sang-Jik Lee for providing the yeast-based SP trap system. We thank Kent Loeffler for expert photography. Received June 8, 2007; accepted July 30, 2007; published August 17, 2007.
1 This work was supported in part by the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service (special grant no. 2003–34367–13158) and by the Eden Bioscience. 
2 Present address: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Steven V. Beer (svb1{at}cornell.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.103432 * Corresponding author; e-mail svb1{at}cornell.edu.
Abramovitch RB, Kim YJ, Chen S, Dickman MB, Martin GB (2003) Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. EMBO J 22: 60–69[CrossRef][Web of Science][Medline] Alfano JR, Collmer A (2004) Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu Rev Phytopathol 42: 385–414[CrossRef][Web of Science][Medline] Arlat M, Van Gijsegem F, Huet JC, Pernollet JC, Boucher CA (1994) PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum. EMBO J 13: 543–553[Web of Science][Medline] Barny MA (1995) Erwinia amylovora hrpN mutants, blocked in harpin synthesis, express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco. Eur J Plant Pathol 101: 333–340[CrossRef] Bauer DW, Wei Z-M, Beer SV, Collmer A (1995) Erwinia chrysanthemi harpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis. Mol Plant Microbe Interact 8: 484–491[Web of Science][Medline] Bhat RA, Panstruga R (2005) Lipid rafts in plants. Planta 223: 5–19[CrossRef][Web of Science][Medline] Chadwick AV, Burg SP (1967) An explanation of the inhibition of root growth caused by indole-3-acetic acid. Plant Physiol 42: 415–420 Chapman PJ, Catlin GA (1976) Growth stages in fruit trees—from dormant to fruit set. N Y Food Life Sci Bull 58: 1–12 Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A (1998) The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J Bacteriol 180: 5211–5217 Chen CH, Lin HJ, Feng TY (1998) An amphipathic protein from sweet pepper can dissociate harpinPss multimeric forms and intensify the harpinPss-mediated hypersensitive response. Physiol Mol Plant Pathol 52: 139–149[CrossRef] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Davis SJ, Vierstra RD (1998) Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 36: 521–528[CrossRef][Web of Science][Medline] Dong H, Delaney TP, Bauer DW, Beer SV (1999) Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J 20: 207–215[CrossRef][Web of Science][Medline] Dong HP, Peng J, Bao Z, Meng X, Bonasera JM, Chen G, Beer SV, Dong H (2004) Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense. Plant Physiol 136: 3628–3638 El-Maarouf H, Barny MA, Rona JP, Bouteau F (2001) Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells. FEBS Lett 497: 82–84[CrossRef][Web of Science][Medline] Gravel P, Golaz O (1996) Protein blotting by the semidry method. In JM Walker, ed, The Protein Protocols Handbook. Humana Press, Totowa, NJ, pp 249–260 Guzman P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell 2: 513–523 He SY, Huang H-C, Collmer A (1993) Pseudomonas syringae pv. syringae harpinPss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell 73: 1255–1266[CrossRef][Web of Science][Medline] Hoyos ME, Stanley CM, He SY, Pike S, Pu XA, Novacky A (1996) The interaction of harpinPss, with plant cell walls. Mol Plant Microbe Interact 9: 608–616[Web of Science] Kim JF, Beer SV (1998) HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J Bacteriol 180: 5203–5210 Kim JG, Jeon E, Oh J, Moon JS, Hwang I (2004) Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants. J Bacteriol 186: 6239–6247 Kim JG, Park BK, Yoo CH, Jeon E, Oh J, Hwang I (2003) Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island. J Bacteriol 185: 3155–3166 Komjanc M, Festi S, Rizzotti L, Cattivelli L, Cervone F, De Lorenzo G (1999) A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus x domestica by Venturia inaequalis infection and salicylic acid treatment. Plant Mol Biol 40: 945–957[CrossRef][Web of Science][Medline] Lee J, Klusener B, Tsiamis G, Stevens C, Neyt C, Tampakaki AP, Panopoulos NJ, Noller J, Weiler EW, Cornelis GR, et al (2001) HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro. Proc Natl Acad Sci USA 98: 289–294 Meng X, Bonasera JM, Kim JF, Nissinen RM, Beer SV (2006) Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen. Mol Plant Microbe Interact 19: 53–61[CrossRef][Web of Science][Medline] Noel L, Thieme F, Nennstiel D, Bonas U (2002) Two novel type III-secreted proteins of Xanthomonas campestris pv. vesicatoria are encoded within the hrp pathogenicity island. J Bacteriol 184: 1340–1348 Oh C-S, Kim JF, Beer SV (2005) The Hrp pathogenicity island of Erwinia amylovora and the identification of three novel genes required for systemic infection. Mol Plant Pathol 6: 125–138[CrossRef] Oh J, Kim J-G, Jeon E, Yoo C-H, Moon JS, Rhee S, Hwang I (2007) Amyloidogenesis of type III-dependent harpins from plant pathogenic bacteria. J Biol Chem 282: 13601–13609 Peng J-L, Dong H-S, Dong H-P, Delaney TP, Bonasera JM, Beer SV (2003) Harpin-elicited hypersensitive cell death and pathogen resistance requires the NDR1 and EDS1 genes. Physiol Mol Plant Pathol 62: 317–326[CrossRef] Perino C, Gaudriault S, Vian B, Barny MA (1999) Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora. Cell Microbiol 1: 131–141[CrossRef][Web of Science][Medline] Racape J, Belbahri L, Engelhardt S, Lacombe B, Lee J, Lochman J, Marais A, Nicole M, Nurnberger T, Parlange F, et al (2005) Ca2+-dependent lipid binding and membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in tobacco. Mol Microbiol 58: 1406–1420[Web of Science][Medline] Ramos AR (2004) Hrp proteins and harpins: defining their roles in the type III protein secretion system in Pseudomonas syringae. PhD thesis. Cornell University, Ithaca, NY Rosenberger CM, Brumell JH, Finlay BB (2000) Microbial pathogenesis: lipid rafts as pathogen portals. Curr Biol 10: R823–R825[CrossRef][Web of Science][Medline] Sambrook J, Fritsch EF, Maniatis TE (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Staswick PE, Su W, Howell SH (1992) Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc Natl Acad Sci USA 89: 6837–6840 Strobel NE, Ji C, Gopalan S, Kuc JA, He SY (1996) Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 HrpZPss protein. Plant J 9: 431–439[CrossRef][Web of Science] Vanneste JL (2000) Fire Blight; The Disease and Its Causative Agent, Erwinia amylovora. CAB International, Wallingford, UK Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257: 85–88 Wilson PA, Lagna G, Suzuki A, Hemmati-Brivanlou A (1997) Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1. Development 124: 3177–3184[Abstract] Yamane H, Lee SJ, Kim BD, Tao R, Rose JK (2005) A coupled yeast signal sequence trap and transient plant expression strategy to identify genes encoding secreted proteins from peach pistils. J Exp Bot 56: 2229–2238
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ABSTRACT
RESULTS
-HrpN) and FLAG tag M2 antibody (
hrpN were used to assay virulence of strains in immature pear (Pyrus communis) fruits as described (Oh et al., 2005