Physiological and Molecular Plant Pathology 74 (2009) 27–33 Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp Inhibition of a Hevea brasiliensis protease by a Kazal-like serine protease inhibitor 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, Thailand b Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA article info abstract Article history: Protease inhibitors have been implicated in virulence of the oomycete plant pathogen Phytophthora Accepted 24 August 2009 infestans. 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 pathogenesis Keywords: mechanism. Based on the sequences of the Kazal-like serine protease inhibitor EPI10 from P. infestans and Serine protease inhibitor its ortholog from Phytophthora ramorum, we designed a pair of primers to amplify the potential homolog Kazal family from P. palmivora. A full-length cDNA was isolated using reverse transcription polymerase chain reaction Phytophthora palmivora (RT-PCR) followed by rapid amplification of cDNA ends (RACE), and designated Ppepi10. Ppepi10 encodes Hevea brasiliensis Subtilisin A a 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 rKazal2 domains inhibited the activity of subtilisin A but neither had an effect on the proteases chymotrypsin and trypsin. 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. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction such as Neospora caninum which secretes a single domain Kazal inhibitor called NcPI-S [16]. This inhibitor was found to be highly Protease inhibitors are important natural tools for regulating the inhibitory to subtilisin, yet has little or no activity against elastase proteolytic activity of their targets. For example, inhibitors can or chymotrypsin. Serine protease inhibitors EPI1 and EPI10, which block the activity of a protease to regulate signaling mechanisms also belong to the Kazal family have been reported to be secreted by through receptor interactions [2]. By definition, protease inhibitors Phytophthora infestans [24,25]. The two-domain EPI1 and the three- function by impairing the proteolytic activity of target proteins. In domain EPI10 proteins were shown to inhibit and interact with the the case of host–microbe interactions, the functions of host plant pathogenesis-related protein P69B a subtilase of tomato [24,25]. proteases are impacted through the specific recognition and activity Phytophthora palmivora is an oomycete that is the causative of their cognate pathogen inhibitors. Based on the catalytic types of agent of ‘‘leaf fall’’ and ‘‘black stripe’’ in the rubber plant. It attacks the inhibited proteases, protease inhibitors may be classified into at the petioles, causing mature leaves to fall prematurely and attacks least 6 types: cysteine protease inhibitors, serine protease inhibi- the tapping surface resulting in poor latex production. Although it tors, threonine protease inhibitors, aspartic protease inhibitors, is a pathogen of great economic importance in Thailand, little is glutamic protease inhibitors and metalloprotease inhibitors. known about the molecular mechanisms involved in the pathoge- The protease inhibitors directed against serine proteases can be nicity and host specificity of P. palmivora. P. palmivora belongs to divided into at least 20 different families based on sequence simi- the same genus as P. infestans and likely shares some conserved larity, topology, and mechanism of binding [13]. Among these, the pathogenesis mechanisms. Based on previous work by Tian et al. Kazal family is one family of serine protease inhibitors that has [24,25], we sought to identify potential inhibitors of defense previously been characterized in plant–pathogen interactions response in the Hevea brasiliensis–P. palmivora interaction. In short, [24,25]. This family is named after L. Kazal, who discovered the we hypothesized that protease inhibitors from P. palmivora might pancreatic secretory trypsin inhibitors, PSTI, present in all verte- also play a role in the suppression of plant defense responses. To brates [23]. Recently, they have been found in many organisms, this end, we focused on identifying protease inhibitors from P. palmivora with similarity to EPI10 from P. infestans because it was * Corresponding author. Tel.: þ66 74 288261; fax: þ66 74 446656. shown to have specific activity as well as play a role in suppression E-mail address: [email protected] (N. Churngchow). plant defense [24] and it has highly conserved sequences with 0885-5765/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2009.08.005 28 D. Chinnapun et al. / Physiological and Molecular Plant Pathology 74 (2009) 27–33 other Phytophthora species such as Phytophthora ramorum and products from the first RACE-PCR reaction were used as the DNA Phytophthora sojae. template in the nested PCR reactions using the oligonucleotides (50- Previous work has described that the full-length protein, EPI10, TGCCTGGACGTGTACGACCCGGTG-30)and(50-GACCCTCGTTCGAG- from P. infestans has protease inhibitor activity; however, functional TATTCCTTTCCA-30)asnested30-RACE and nested 50-RACE primers, activity of individual Kazal domains has not been fully characterized respectively. The primers for RACE-PCR reactions were designed based [24]. In this study, we describe the isolation, cloning and functional on the obtained partial Ppepi10 sequence. characterization of a Kazal-like extracellular serine protease inhibitor gene from P. palmivora, Ppepi10. This is the first serine 2.5. Cloning and sequencing RACE products protease inhibitor gene that has been cloned and characterized from P. palmivora. The Ppepi10 gene was produced using RT-PCR and The PCR products from RACE-PCR were cloned into the TOPO RACE-PCR, and was identified to contain 3 Kazal domains, desig- PCR 2.1 Vector (Invitrogen) and transformed into Escherichia coli nated Kazal1, Kazal2 and Kazal3. Moreover, we describe the inhibi- TOP10 cells, according to the manufacturer’s instructions. Plasmids tory activity against proteins from rubber leaf using a zymogram for sequencing were extracted using the QIAprep spin miniprep kit buffer system and co-immunoprecipitation to identify the specific (Qiagen). protease targeted by rKazal1 and rKazal2 of rPpEPI10. 2.6. Sequence analyses 2. Materials and methods The signal peptide of Ppepi10 was predicted using SignalP 3.0 2.1. Plant growth and protein extraction [7]. The Kazal domains of Ppepi10 were identified by searching with the InterPro database (http://www.ebi.ac.uk/tool/InterProScan). Rubber plants (RRIM600 cultivar) were grown in a growth Multiple alignments of the Kazal domains from P. palmivora chamber with a photoperiod of 12 h of light and 12 h of dark at 25 C. (Ppepi10), P. infestans (EPI1 and EPI10) [24,25], the crayfish Paci- Leaves (5 g) of a 12 week-old rubber plant were homogenized with fastacus leniusculus (PAPI-1) [9], and the apicomplexan Toxoplasma liquid nitrogen using a mortar and pestle. Total protein was extracted gondii (TgPI1) [18] were conducted using the program CLUSTAL-X. with 10 ml of 100 mM Tris–HCl buffer pH 7.5 followed by precipi- The sequences were obtained from the NCBI database (www.ncbi. tation with 90% ammonium sulfate saturation at 4 C. The pellet was nlm.nih.gov). The P. palmivora sequence described in this paper collected by centrifugation at 12,000 Â g for 20 min at 4 Candthen was deposited in GenBank under accession no. FJ643536. dissolved in sterile distilled water. The solution was then desalted by loading onto a PD-10 column and eluted with distilled water. Eluted 2.7. Plasmid and bacterial strain used for production of rKazal1, fractions from the PD-10 column were monitored for protein content rKazal2 and rKazal3 proteins at a wavelength of 280 nm. The eluted fractions with high protein content were pooled and analyzed further, as described below. Plasmid pFLAG-Kazal1, pFLAG-Kazal2 and pFLAG-Kazal3 were constructed by cloning the PCR-amplified DNA fragments of Kazal1, 2.2. Phytophthora strain and culture condition Kazal2 and Kazal3, respectively into the EcoRI and KpnIsitesofpFLAG- ATS (Sigma), a vector allowing secreted expression of N-terminal FLAG P. palmivora, isolated from a diseased H. brasiliensis plant was fusion proteins. The oligonucleotides Kazal1-F (50-GCGGAATTCCATC- maintained on potato dextrose agar (PDA) medium at 25 C. For GACGACGACAAGTGCTCAT TC-30), Kazal1-R (50-GGGGTACCCTAGTCTG RNA extraction, P. palmivora was grown in Henninger medium [6] CGGGGCCGCTGG-30), Kazal2-F (50-GGGAATTCCATGTGCCCGGACGC on a rotary shaker at 100 rpm and 25 C for 15 days. TTGCCTG-30), Kaza2-R (50-GGGGTACCCTACGGTGGTCCCGTGTAGCC-30), Kazal3-F (50-GGGAATTCCATGTGCGCTGACATGTTGTGTCC-30) and 2.3. Primers design for P. palmivora epi10 (Ppepi10) Kazal3-R (50-GCGGGTACCTTACAGATTTAAAGTTTGAGAATAGGTC-30) were used to amplify the fragments. The introduced EcoRI and KpnI DNA primers for isolation