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Physiological and Molecular Plant Pathology 74 (2009) 27–33

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier .com/locate/pmpp

Inhibition of a Hevea brasiliensis protease by a Kazal-like serine proteaseinhibitor from Phytophthora palmivora

Dutsadee Chinnapun a, Miaoying Tian b, Brad Day b, Nunta Churngchow a,*

a Department of Biochemistry, Faculty of Science, Prince of Songkla University, Kanchanawanich Street, Hat-Yai, Songkhla 90112, Thailandb Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA

a r t i c l e i n f o

Article history:Accepted 24 August 2009

Keywords:Serine protease inhibitorKazal familyPhytophthora palmivoraHevea brasiliensisSubtilisin A

* Corresponding author. Tel.: þ66 74 288261; fax:E-mail address: nunta.c@psu.ac.th (N. Churngchow

0885-5765/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.pmpp.2009.08.005

a b s t r a c t

Protease inhibitors have been implicated in virulence of the oomycete plant pathogen Phytophthorainfestans. Phytophthora palmivora, the causative agent of ‘‘leaf fall’’ and ‘‘black stripe’’ in the rubber plant(Hevea brasiliensis), belongs to the same genus as P. infestans and likely shares conserved pathogenesismechanism. Based on the sequences of the Kazal-like serine protease inhibitor EPI10 from P. infestans andits ortholog from Phytophthora ramorum, we designed a pair of primers to amplify the potential homologfrom P. palmivora. A full-length cDNA was isolated using reverse transcription polymerase chain reaction(RT-PCR) followed by rapid amplification of cDNA ends (RACE), and designated Ppepi10. Ppepi10 encodesa 222 amino acid protein containing three putative Kazal domains, designated Kazal1, Kazal2 and Kazal3.In vitro protein expression and protease inhibition analyses revealed that both rKazal1 and rKazal2domains inhibited the activity of subtilisin A but neither had an effect on the proteases chymotrypsin andtrypsin. Moreover, both of them interacted with a 95 kDa protease from H. brasiliensis leaf extracts,suggesting a role for Ppepi10 in pathogenicity through suppression of host plant defenses.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Protease inhibitors are important natural tools for regulating theproteolytic activity of their targets. For example, inhibitors canblock the activity of a protease to regulate signaling mechanismsthrough receptor interactions [2]. By definition, protease inhibitorsfunction by impairing the proteolytic activity of target proteins. Inthe case of host–microbe interactions, the functions of host plantproteases are impacted through the specific recognition and activityof their cognate pathogen inhibitors. Based on the catalytic types ofthe inhibited proteases, protease inhibitors may be classified into atleast 6 types: cysteine protease inhibitors, serine protease inhibi-tors, threonine protease inhibitors, aspartic protease inhibitors,glutamic protease inhibitors and metalloprotease inhibitors.

The protease inhibitors directed against serine proteases can bedivided into at least 20 different families based on sequence simi-larity, topology, and mechanism of binding [13]. Among these, theKazal family is one family of serine protease inhibitors that haspreviously been characterized in plant–pathogen interactions[24,25]. This family is named after L. Kazal, who discovered thepancreatic secretory trypsin inhibitors, PSTI, present in all verte-brates [23]. Recently, they have been found in many organisms,

þ66 74 446656.).

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such as Neospora caninum which secretes a single domain Kazalinhibitor called NcPI-S [16]. This inhibitor was found to be highlyinhibitory to subtilisin, yet has little or no activity against elastaseor chymotrypsin. Serine protease inhibitors EPI1 and EPI10, whichalso belong to the Kazal family have been reported to be secreted byPhytophthora infestans [24,25]. The two-domain EPI1 and the three-domain EPI10 proteins were shown to inhibit and interact with thepathogenesis-related protein P69B a subtilase of tomato [24,25].

Phytophthora palmivora is an oomycete that is the causativeagent of ‘‘leaf fall’’ and ‘‘black stripe’’ in the rubber plant. It attacksthe petioles, causing mature leaves to fall prematurely and attacksthe tapping surface resulting in poor latex production. Although itis a pathogen of great economic importance in Thailand, little isknown about the molecular mechanisms involved in the pathoge-nicity and host specificity of P. palmivora. P. palmivora belongs tothe same genus as P. infestans and likely shares some conservedpathogenesis mechanisms. Based on previous work by Tian et al.[24,25], we sought to identify potential inhibitors of defenseresponse in the Hevea brasiliensis–P. palmivora interaction. In short,we hypothesized that protease inhibitors from P. palmivora mightalso play a role in the suppression of plant defense responses. Tothis end, we focused on identifying protease inhibitors fromP. palmivora with similarity to EPI10 from P. infestans because it wasshown to have specific activity as well as play a role in suppressionplant defense [24] and it has highly conserved sequences with

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other Phytophthora species such as Phytophthora ramorum andPhytophthora sojae.

Previous work has described that the full-length protein, EPI10,from P. infestans has protease inhibitor activity; however, functionalactivity of individual Kazal domains has not been fully characterized[24]. In this study, we describe the isolation, cloning and functionalcharacterization of a Kazal-like extracellular serine proteaseinhibitor gene from P. palmivora, Ppepi10. This is the first serineprotease inhibitor gene that has been cloned and characterizedfrom P. palmivora. The Ppepi10 gene was produced using RT-PCR andRACE-PCR, and was identified to contain 3 Kazal domains, desig-nated Kazal1, Kazal2 and Kazal3. Moreover, we describe the inhibi-tory activity against proteins from rubber leaf using a zymogrambuffer system and co-immunoprecipitation to identify the specificprotease targeted by rKazal1 and rKazal2 of rPpEPI10.

2. Materials and methods

2.1. Plant growth and protein extraction

Rubber plants (RRIM600 cultivar) were grown in a growthchamber with a photoperiod of 12 h of light and 12 h of dark at 25 �C.Leaves (5 g) of a 12 week-old rubber plant were homogenized withliquid nitrogen using a mortar and pestle. Total protein was extractedwith 10 ml of 100 mM Tris–HCl buffer pH 7.5 followed by precipi-tation with 90% ammonium sulfate saturation at 4 �C. The pellet wascollected by centrifugation at 12,000 � g for 20 min at 4 �C and thendissolved in sterile distilled water. The solution was then desalted byloading onto a PD-10 column and eluted with distilled water. Elutedfractions from the PD-10 column were monitored for protein contentat a wavelength of 280 nm. The eluted fractions with high proteincontent were pooled and analyzed further, as described below.

2.2. Phytophthora strain and culture condition

P. palmivora, isolated from a diseased H. brasiliensis plant wasmaintained on potato dextrose agar (PDA) medium at 25 �C. ForRNA extraction, P. palmivora was grown in Henninger medium [6]on a rotary shaker at 100 rpm and 25 �C for 15 days.

2.3. Primers design for P. palmivora epi10 (Ppepi10)

DNA primers for isolation of P. palmivora epi10 (Ppepi10) weredesigned based on alignments using the program CLUSTAL-X ofEPI10 from P. infestans (GenBank accession no. AY586282) andPramEPI10 from P. ramorum (Trace identified no. 303447516) [25].These sequences were obtained from the NCBI databases (www.ncbi.nlm.nih.gov). The conserved regions of epi10 from P. infestansand P. ramorum were used to create the primers for Ppepi10.

2.4. RNA isolation, RT-PCR and RACE-PCR

Mycelium from P. palmivora was ground in liquid nitrogen, and totalRNA was extracted using the RNeasy plant mini kit (Qiagen). For RT-PCR, total RNA was treated with DNA-Free (Ambion, Austin, TX), andfirst-strand cDNAs were synthesized using the SuperScript III reversetranscriptase RT-PCR system (Invitrogen) from 3 mg of total RNA. Theforward primer (50-TTTGGATGCCTCGACGTGTA-30) and degeneratereverse primer (50-CGGAGCCGCACACAGGRGCATAGTTGTC-30) wereused for RT-PCR. RACE-PCR was employed to obtain the full-lengthPpepi10 cDNA sequence, using Smart RACE cDNA amplification kitfollowing the instructions of the manufacturer (Clontech). The forwardprimer (50-ATGCTACTTGCGCCTTGCGTCTTGC-30) and reverse primer(50-CACAGGGGCATAGTTGTCGGGACAC-30) were used respectively for30-RACE and 50-RACE in the first round of RACE-PCR reactions. The PCR

products from the first RACE-PCR reaction were used as the DNAtemplate in the nested PCR reactions using the oligonucleotides (50-TGCCTGGACGTGTACGACCCGGTG-30) and (50-GACCCTCGTTCGAG-TATTCCTTTCCA-30) as nested 30-RACE and nested 50-RACE primers,respectively. The primers for RACE-PCR reactions were designed basedon the obtained partial Ppepi10 sequence.

2.5. Cloning and sequencing RACE products

The PCR products from RACE-PCR were cloned into the TOPOPCR 2.1 Vector (Invitrogen) and transformed into Escherichia coliTOP10 cells, according to the manufacturer’s instructions. Plasmidsfor sequencing were extracted using the QIAprep spin miniprep kit(Qiagen).

2.6. Sequence analyses

The signal peptide of Ppepi10 was predicted using SignalP 3.0[7]. The Kazal domains of Ppepi10 were identified by searching withthe InterPro database (http://www.ebi.ac.uk/tool/InterProScan).Multiple alignments of the Kazal domains from P. palmivora(Ppepi10), P. infestans (EPI1 and EPI10) [24,25], the crayfish Paci-fastacus leniusculus (PAPI-1) [9], and the apicomplexan Toxoplasmagondii (TgPI1) [18] were conducted using the program CLUSTAL-X.The sequences were obtained from the NCBI database (www.ncbi.nlm.nih.gov). The P. palmivora sequence described in this paperwas deposited in GenBank under accession no. FJ643536.

2.7. Plasmid and bacterial strain used for production of rKazal1,rKazal2 and rKazal3 proteins

Plasmid pFLAG-Kazal1, pFLAG-Kazal2 and pFLAG-Kazal3 wereconstructed by cloning the PCR-amplified DNA fragments of Kazal1,Kazal2 and Kazal3, respectively into the EcoRI and KpnI sites of pFLAG-ATS (Sigma), a vector allowing secreted expression of N-terminal FLAGfusion proteins. The oligonucleotides Kazal1-F (50-GCGGAATTCCATC-GACGACGACAAGTGCTCAT TC-30), Kazal1-R (50-GGGGTACCCTAGTCTGCGGGGCCGCTGG-30), Kazal2-F (50-GGGAATTCCATGTGCCCGGACGCTTGCCTG-30), Kaza2-R (50-GGGGTACCCTACGGTGGTCCCGTGTAGCC-30),Kazal3-F (50-GGGAATTCCATGTGCGCTGACATGTTGTGTCC-30) andKazal3-R (50-GCGGGTACCTTACAGATTTAAAGTTTGAGAATAGGTC-30)were used to amplify the fragments. The introduced EcoRI and KpnIrestriction site are underlined. The transformation of pFLAG-Kazal1,pFLAG-Kazal2 and pFLAG-Kazal3 into the E. coli strain BL21 wasperformed using electroporator at 2500 volts.

2.8. Expression and purification of rKazal1, rKazal2 and rKazal3

Cultures of E. coli BL21 containing pFLAG-Kazal1, pFLAG-Kazal2and pFLAG-Kazal3 were grown overnight on a rotary shaker at250 rpm and 37 �C. Overnight cultures were diluted (1:100) in LBmedium containing carbenicillin (50 mg/ml) and incubated on a rotaryshaker at 250 rpm and 37 �C until an optical density of OD600¼ 0.3, atwhich time isopropyl-b-D-thiogalactopyranoside (IPTG) was added toa final concentration of 0.4 mM. The cultures were further incubatedovernight on a rotary shaker at 250 rpm and 28 �C. The culturesupernatants of pFLAG-Kazal1, pFLAG-Kazal2 and pFLAG-Kazal3 wereused to purify rKazal1, rKazal2 and rKazal3 with anti-FLAG M2 affinitygel column (Sigma). After loading each sample, the column waswashed with TBS buffer. Then the protein was eluted with 0.1 Mglycine (pH 3.5), and equilibrated to a neutral pH with 20 ml of 1 M Tris(pH 8.0), for each 1 ml eluted fraction. Protein concentration wasmeasured at 280 nm and calculated using an extinction coefficient of6147 M�1 cm�1 for rKazal1 and 7575 M�1 cm�1 for rKazal2 deter-mined with the approach of Gill and von Hippel [5].

D. Chinnapun et al. / Physiological and Molecular Plant Pathology 74 (2009) 27–33 29

2.9. SDS-PAGE and western blot analyses

SDS-PAGE was performed using 15% (w/v) polyacrylamide gels.After electrophoresis, gels were stained with silver nitrate followingthe method of Merril [17], or stained with Coomassie Brilliant Blue[20]. A value for the relative molecular mass (Mr) of the protein bandwas estimated by comparison with a BenchMark pre-stained proteinladder (6–180 kDa; Invitrogen). For Western blot analyses, proteinswere transferred to nitrocellulose membranes using a Mini trans-blotapparatus. Detection of antigen–antibody complexes was carried outwith anti-Flag M2-peroxidase (Sigma) and Super Signal West Picochemiluminescent substrate (Pierce).

2.10. Protease inhibition assays

Protease inhibition analysis of commercial serine proteases againstrKazal1 and rKazal2 was performed using the colorimetric Quanti-cleave protease assay kit (Pierce). Twenty pmol of rKazal1 or rKazal2was incubated with 20 pmol of chymotrypsin (Sigma), subtilisinA (Sigma), or trypsin (Pierce), in a volume of 50 ml for 30 min at 25 �C.After 30 min, 100 ml of succinylated casein (2 mg/ml in 50 mM Trisbuffer, pH 8) was added and incubated at room temperature for20 min. After that, 50 ml of chromogenic reagent 2,4,6-trini-trobenzenesulfonic acid was added and incubated for 20 min at roomtemperature before measuring protease activity at 450 nm byspectrophotometry.

The BioRad zymogram buffer system was used to test for inhibi-tion of plant protease by rKazal1 and rKazal2. Both 20 and 80 pmol ofrKazal1 or rKazal2 were incubated with 8 ml (0.08 mg) of rubber leafproteins for 30 min at 25 �C before mixing with zymogram samplebuffer. The 8% zymogram gel was prepared as standard SDS-poly-acrylamide gel with 0.1% gelatin added in both separating gel andstacking gel. The samples were then loaded onto the gel withoutboiling or addition of reducing reagents. After electrophoresis, the gel

Fig. 1. Alignment of epi10 sequences from P. infestans and P. ramorum using CLUSTAL-X. Theprimer sequences used for RT-PCR. F, forward primer; R, reverse primer.

was incubated in 1�zymogram renaturation buffer, 2.5% (v/v) TritonX-100, for 30 min at room temperature. Then it was equilibrated in1�zymogram developing buffer containing 50 mM Tris: 0.2 M NaCl:5 mM CaCl2 and 0.02% Brij 35 for another 30 min at room tempera-ture, followed by a 4 h incubation at 37 �C in fresh 1�zymogramdeveloping buffer before staining with 0.5% Coomassie brilliant blue.After the gel was destained with solution containing methanol: aceticacid: water, 50:10:40, areas of protease activity appear as clear bandagainst a blue background where the protease has digested thegelatin substrate.

2.11. Co-immunoprecipitation

The FLAG-tagged protein immunoprecipitation kit (Sigma) wasused for testing for the co-immunoprecipitation of rKazal1 andrKazal2 with protein extracts from rubber leaf. A total of 800 pmolof purified rKazal1 or rKazal2 were incubated with 200 ml (2 mg) ofrubber leaf proteins for 30 min at 25 �C. Forty ml of anti-FLAG M2resin was added and incubated at 4 �C, overnight with gentleshaking. The bound protein complexes were eluted with 60 ml ofFLAG peptide solution (150 ng/ml). The analysis was performedusing an 8% gel for SDS-PAGE and silver nitrate staining.

3. Results

3.1. Isolation of a full-length Ppepi10 cDNA

CLUSTAL-X alignment of sequences of EPI10 from P. infestans(GenBank accession no. AY586282) and PramEPI10 from P. ramo-rum (Trace identified no. 303447516) was used to generate thePpepi10 primer set. As shown in Fig. 1, the alignment of epi10sequences from two Phytophthora species revealed a highlyconserved basic sequence structure. Two relatively long conservedstretches were chosen for the forward primer and the degenerate

conserved sequences are highlighted in black. The underlined sequences indicated DNA

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reverse primer (Fig. 1). We obtained the partial Ppepi10 sequence(Fig. 2) by sequencing a cDNA fragment amplified by RT-PCR.

The full-length Ppepi10 sequence was obtained using RACE-PCRas described in Materials and methods. The gene specific primersfor both 50-RACE and 30-RACE reactions were designed based on thepartial Ppepi10 sequence (Fig. 2). The sequences from two RACE-PCRreactions were assembled into the full-length Ppepi10 sequence,which was deposited at the National Center for BiotechnologyInformation GenBank under accession no. FJ643536.

3.2. Ppepi10 encodes a putative extracellular Kazal-likeserine protease inhibitor

From the full-length Ppepi10 cDNA sequence, we identified anopen reading frame of 669 bp that corresponded to a predictedtranslated product of 222 amino acids. SignalP 3.0 analysis of theputative protein identified a 19-amino acid signal peptide witha significant mean S value of 0.931. Using the InterPro database(http://www.ebi.ac.uk/tool/InterProScan), three domains of Ppepi10that were similar to Kazal inhibitors (InterPro IPR002350) weredetected (Fig. 3A).

Multiple sequence alignment of the Kazal domains from P. pal-mivora (PpEPI10; this study), P. infestans (EPI1 and EPI10), thecrayfish P. leniusculus (PAPI-1), and the apicomplexan T. gondii(TgPI1) produced by CLUSTAL-X, illustrate the signature sequenceof the Kazal-like serine protease inhibitor family (Fig. 3B). The firstdomain of PpEPI10 (PpEPI10a) represented a typical Kazal domain,which contains six Cys residues forming 3 disulfide bridges ofCys1–5, Cys2–4 and Cys3–6. For the second (PpEPI10b) and thethird domains (PpEPI10c), the Cys residues at position 6 and/or 3were missing, representing atypical Kazal domains which are verycommon in Kazal-like inhibitors of plant pathogenic oomycetes[26]. For all three domains, the predicted P1 residues, which playcentral roles in determining the specificity of Kazal inhibitors, areAsp, as are also found in the Kazal domains of P. infestans EPI1 andEPI10 (Fig. 3).

3.3. Expression and purification of PpEPI10 Kazal domains

rKazal1, rKazal2 and rKazal3 were cloned into the pFLAG-ATSprotein expression vector (Sigma) in order to express and purifyrecombinant proteins fused with the FLAG epitope (N-terminalfusion). After performing Western blot analyses of rKazal1, rKazal2

Fig. 2. Partial Ppepi10 sequence obtained from sequencing of the Ppepi10 cDNA frag-ment amplified by RT-PCR. The underlined nucleotides indicate the primer sequencesused for RACE-PCR.

and rKazal3 with FLAG antibody, we found that only rKazal1 andrKazal2 were expressed (data not shown). Additional proteinexpression constructs (e.g., pGEX 4T-1; GE Healthcare Life Sciences)were made for expressing and purifying Kazal3, however,none expressed the Kazal3 domain, suggesting a possible level oftoxicity in E. coli that prevented us from moving forward with thisconstruct. Consequently only rKazal1 and rKazal2 were purified byimmunoaffinity using a gravity column packed with anti-FLAG M2affinity gel. The purified rKazal1 and rKazal2 were eluted from thecolumn and analyzed by SDS-PAGE and staining with CoomassieBrilliant Blue. The apparent molecular weights were identified as17 kDa for rKazal1 (Fig. 4A) and 15 kDa for rKazal2 (Fig. 4B).

3.4. rKazal1 and rKazal2 inhibit the serine protease subtilisin A

The rKazal1 and rKazal2 proteins were tested for the inhibitionof several commercially available serine proteases using the color-imetric Quanti-cleave protease assay kit. Three serine proteaseswere used for identifying the inhibitor specificity: chymotrypsin,subtilisin A and trypsin. In three independent experiments, wefound that rKazal1 and rKazal2 proteins did not inhibit chymo-trypsin or trypsin. However, both of them inhibited the activity ofsubtilisin A, with inhibition values of approximately 48% for rKazal1and 26% for rKazal2 (Fig. 5).

These results indicate that the Kazal1 and Kazal2 domains ofPpepi10 encoded a functional protease inhibitor that specificallytargets the subtilisin class of serine proteases. Moreover, our dataindicates that the rKazal1 domain has a higher activity than rKazal2.

3.5. rKazal1 and rKazal2 inhibit a protease from rubber leaf

rKazal1 and rKazal2 were tested for inhibition of proteases presentin rubber leaf using the zymogram buffer system. A protein extractfrom rubber leaves was incubated with or without rKazal1 and rKa-zal2, and the protease activity was monitored using the gel proteaseassay, described above. Interestingly, we found that 20 pmol of rKazal1completely inhibited the protease present in rubber leaf extracts(Fig. 6A); however, rKazal2 required a higher amount (80 pmol) toinhibit the protease (Fig. 6B). The zymogram gels showed that therubber leaf extract had at least two proteases, but only one of themwas inhibited by rKazal1 and rKazal2 (Fig. 6). This result demonstratesthat rKazal1 and rKazal2 could inhibit a protease (likely a serineprotease) from rubber leaf, and the rKazal1 was more active.

3.6. rKazal1 and rKazal2 interact with protease from rubber leaf

rKazal1 and rKazal2 were further tested for co-immunoprecip-itation with rubber tree proteins using FLAG antibody covalentlylinked to agarose beads. As shown in Fig. 7, both rKazal1 and rKa-zal2 co-precipitated a 95 kDa rubber tree protein, consistent withour results of protease inhibition, described above.

4. Discussion

Plant pathogens produce effector proteins that can eitherpromote infection (virulence proteins) or trigger defense responses(avirulence proteins) in their host plants [4,10,11,15,22]. Severaloomycete pathogens produce elicitor proteins as avirulence prod-ucts which in turn induce plant defense responses and pro-grammed cell death referred to as the ‘‘hypersensitive response’’(HR). Recently, many effector molecules from oomycete pathogenshave been reported which function to inhibit plant protein func-tions and in term, presumably shut down plant defense responses.For example, P. sojae secretes glucanase inhibitor proteins (GIPs)that specifically inhibit the endoglucanase activity of soybeans.

Fig. 3. PpEPI10 is predicted to be a member of the Kazal family of serine protease inhibitors. (A) Schematic representation of PpEPI10 structure. SP, signal peptide. The three Kazaldomains are designated as PpEPI10a, PpEPI10b and PpEPI10c. The positions of the amino acid residues starting from the N terminus are indicated by numbers. The predicted P1residues, which play central roles in determining the specificity of Kazal inhibitors, are Asp. C is the cysteine residues and the disulfide linkages predicted based on the structure ofother Kazal domains are shown. (B) Sequence alignment of the Kazal inhibitor domains. Protease inhibitor PpEPI10a–c, FJ643536 from P. palmivora, EPI10a–c, AY586282 and EPI1a–b,AY586273 from P. infestans, PAPI-1a–d, CAA56043 from the crayfish Pacifastacus leniusculus and TgPI-1a–d, AF121778 from the apicomplexan T. gondii. The asterisks indicate amino acidresidues that define the Kazal family protease inhibitor domains. The arrows indicate the positions of the Cys residues that are missing in PpEPI10b and PpEPI10c. The P6, P5, P4, P3, P2,P1, P10, P20 , P30 , P140 , P150 and P180 are contact positions of Kazal domains to interact with their serine proteases.

D. Chinnapun et al. / Physiological and Molecular Plant Pathology 74 (2009) 27–33 31

Furthermore, GIPs and soybean endoglucanases interact in vivoduring pathogenesis in soybean roots [8]. EPI1 and EPI10 of theKazal family are secreted from P. infestans during pathogenesis andboth the two-domain EPI1 and the three-domain EPI10 proteinswere shown to inhibit and interact with the pathogenesis-relatedprotein P69B a subtilase of tomato [24,25].

P. palmivora, the causative agent of leaf fall and black stripe in therubber plant (H. brasiliensis) produces the avirulence moleculeselicitin, as well as a 75 kDa elicitor. Both of them trigger defensereactions, inducing accumulation of scopoletin, peroxidase, phenoliccompounds and local resistance in H. brasiliensis [3]. To date,

Fig. 4. SDS-PAGE analysis of purified rKazal1 and rKazal2 proteins from anti-FLAG M2affinity column stained with Coomassie brilliant blue. (A) Lane M indicates proteinstandard. Lane ‘‘rKazal1’’ represents purified rKazal1 from the anti-FLAG M2 affinitycolumn. (B) Purified rKazal2-Flag, as described in ‘‘A’’, above.

however, no protease inhibitor has been identified from P. palmivora,thus making the identification of the specific protease inhibitordescribed herein an important advance in understanding the viru-lence strategies of P. palmivora.

In the present study, we describe the isolation of a proteaseinhibitor gene from P. palmivora, Ppepi10, and identified it as a memberof the Kazal family of serine protease inhibitors (Fig. 3B). Furthermore,we showed that Kazal1 and Kazal2 of PpEPI10 encoded functionalprotease inhibitor domains that specifically targeted the subtilisin classof serine proteases using the colorimetric Quanti-cleave protease assaykit. The rKazal1 and rKazal2 from PpEPI10 inhibited only the activity ofsubtilisin A but did not inhibit chymotrypsin and trypsin (subtilisin A,

Fig. 5. Protease inhibition assay of rKazal1 and rKazal2 using the colorimetric Quanti-cleave protease assay kit. Standard error of deviation was calculated from the threeindependent replications. The experiment was repeated three times with similarresults.

Fig. 6. Protease inhibition assay using the zymogram buffer system. ‘‘Control’’ is the rubber leaf proteins without incubation with rKazal1 or rKazal2. The ‘‘rKazal1 þ plant proteins’’are rubber leaf proteins incubated with rKazal1. The ‘‘rKazal2 þ plant proteins’’ are rubber tree proteins incubated with rKazal2. (A) The zymogram buffer gel using 20 pmol ofrKazal1 or rKazal2 incubated with 8 ml (0.8 mg) of rubber leaf proteins. The arrow indicates the band inhibited by rKazal1. (B) The zymogram buffer gel using 80 pmol of rKazal1 orrKazal2 incubated with 8 ml (0.8 mg) of rubber leaf proteins. The arrow indicates the band inhibited by rKazal1 or rKazal2.

D. Chinnapun et al. / Physiological and Molecular Plant Pathology 74 (2009) 27–3332

chymotrypsin and trypsin, represent three major classes of serineproteases) (Fig. 5).

The Ppepi10 gene shares approximately 72% sequence similarityto P. infestans epi10, and like epi10, encodes for a protein containingthree Kazal domains (Fig. 3B). The first domain (PpEPI10a) is similarto the Kazal domains from previously identified oomycete inhibi-tors (Fig. 3B). The second domain (PpEPI10b) and the third domain(PpEPI10c) are atypical, lacking the third and sixth Cys for PpEPI10band the sixth Cys for PpEPI10c. Nonetheless, PpEPI10b exhibits thesame pattern of abnormal sequences to that of the EPI10b domainof P. infestans. That is, EPI10b from P. infestans also lacked both thethird and sixth Cys but retained the other four Cys (Fig. 3B).

Inhibitors of the Kazal-type typically exhibit two or more inhibi-tory domains that may be specific for different protease substrates[12]. For example, seven Kazal-type domains of ovoinhibitor fromavian egg white have multiple targets, and at least five active sites:two for trypsin, two for chymotrypsin and one for porcine pancreaticelastase [21]. Another well-characterized member of the Kazal-typeinhibitors is the thrombin inhibitor rhodniin, isolated from the

Fig. 7. Co-immunoprecipitation of rKazal1 (A) or rKazal2 (B) with rubber leaf proteins usingnitrate. The various inputs are shown below the gel image. Lane ‘‘Plant proteins’’ representinput with purified rKazal1 or rKazal2. Lane ‘‘Plant protein þ rKazal1/rKazal2’’ represents thprotein band pulled down with the FLAG antibody in the presence of rubber leaf proteins

assassin bug Rhodnius prolixus. Rhodniin is composed of two Kazal-type domains, however, only one of which displays the typical activityof Kazal-type inhibitors. Interestingly, the second, ‘‘inactive’’ domainhas been shown to function independently through binding to thefibrinogen recognition exosite of thrombin by electrostatic interaction[1]. In the current study, we demonstrate that both rKazal1 andrKazal2 specifically targeted the subtilisin class of serine proteases.

Protein inhibitors of serine proteases are well studied and much isknown about the interactions between an inhibitor and its cognateenzyme, as most of these inhibitors act through a common mecha-nism [14]. In short, this interaction is characterized by a tight asso-ciation between the enzyme and substrate (low Km) and thehydrolysis reaction is very slow [19]. The higher activity of rKazal1 onthe enzyme activity of subtilisin A shown in Fig. 5 infers that the Ki ofrKazal1 is lower than for rKazal2. However, the lack of the third andsixth Cys for rKazal2 should not affect the Ki of rKazal2 because thecontact residues of Kazal domains to interact with their serineproteases are not the third and sixth Cys position. The contact posi-tions of Kazal domains presented by Stephen et al. [23], P6, P5, P4, P3,

FLAG antibody. The elutes were run on SDS-PAGE gel followed by staining with silvers the input with rubber leaf proteins alone. Lane ‘‘rKazal1’’ or ‘‘rKazal2’’ represents thee input with the mixture of rubber leaf proteins incubated with rKazal1 or rKazal2. Theand rKazal1/rKazal2 is indicated by the arrow. M, protein standard.

D. Chinnapun et al. / Physiological and Molecular Plant Pathology 74 (2009) 27–33 33

P2, P1, P10, P20, P30, P140, P150 and P180 were shown in Fig. 3B.Nevertheless, the P3 (the second conserved cysteine residue) andP150 (a conserved asparagine residue) of contact positions show a lowvariation in each Kazal domain then the other 10 contact positionswere usually used for calculation the Ki of Kazal domains [23].

The amount of rKazal2 (80 pmol) that completely inhibiteda protease from rubber leaf was 4 times higher than that for rKazal1(20 pmol) and yet only one band was removed by these two inhibi-tors (Fig. 6). We also performed co-immunoprecipitation on therubber tree proteins incubated with rKazal1 and rKazal2 to identifythe rubber tree protease targeted by rKazal1 and rKazal2 fromrPpEPI10, and found that both rKazal1 and rKazal2 co-precipitatedwith the protein from the rubber leaf. Therefore, it is expected thatthis protein should be a rubber leaf protease (Fig. 7). As a conse-quence, rKazal1 and rKazal2 should interact with the same rubberleaf protease because the proteins from our co-immunoprecipitationof rKazal1 and rKazal2 with rubber leaf protein each had a molecularweight of about 95 kDa (Fig. 7) which concurs with the resultobtained in Fig. 6. We hypothesize that these 95 kDa proteins arePR-proteins found in H. brasiliensis, and as such, serve a primary rolein plant defense. At present, we are pursuing the identification of the95 kDa protein(s), however, this advance is limited in the face of a lackof genomic sequence data from H. brasiliensis.

In summary, the work presented herein leads us to concludethat PpEPI10 from P. palmivora is a Kazal-like extracellular serineprotease inhibitor. It contains three Kazal domains, of which, Kazal1and Kazal2 were demonstrated to be important functional domainscapable of inhibiting a protease from rubber leaf.

Acknowledgements

This research was carried out in the laboratory of Brad Day atMichigan State University and supported by the Thailand ResearchFund (TRF) through the Royal Golden Jubilee Ph.D. Program (RGJ-PHD) to Ms. Dutsadee Chinnapun (PHD/0235/2548), a GraduateResearch Fund from the Prince of Songkla University, Thailand andin part by a National Science Foundation CAREER Award (B. Day;IOS-0641319), as well as support from the Michigan AgriculturalExperiment Station (MAES). We thank the Research TechnologySupport Facility, Michigan State University for help with DNAsequencing and Prof. Dr. Brian Hodgson, Prince of Songkla Univer-sity for revision the manuscript and valuable comments.

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