Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.


Introduction
Proteinase inhibitors (PIs) are commonly found in the plant kingdom and have been identified mainly in plant shoots and storage tissues [1]. At least 74 PI families have been reported based on structural homology, topological relationships, and relative site [2][3][4]. Plant PIs (PPIs) are involved in flowering, seed germination, protein storage, and programmed cell death. PPIs are also induced in plants in responses to wounding and pathogen infections [5]. Several studies have showed that PPIs are effective against predators or pathogens such as insects, nematodes, fungal, and viruses [6,7]. For example, introduction and expression of soybean Kuntiz trypsin inhibitor and Bowman-Birk inhibitor in sugarcane can confer resistance to sugarcane borer Diatraea saccharalis [8]. In Arabidopsis, overexpression of rice cysteine PI or cowpea serine PI can reduce Rotylenchulus reniformis female fecundity and density [9]. Constitutive expression of rice cysteine proteinase oryzacystatin I was effective against Tobacco etch virus (TEV) and Potato virus Y (PVY) in transgenic N. benthamiana plants [10].
Cucumis metuliferus (horned melon) is native to South Africa, is a highly nutritious source and is also reported to have resistance against many pathogens [11]. C. metuliferus line PI 292190 is immune to Papaya ringspot virus (PRSV), a member of the genus Potyvirus of the family Potyviridae, but C. metuliferus line Acc. 2459 is susceptible [12]. C. metuliferus line PI 292190 has a single dominant resistant gene, Wmv, against PRSV and has been used for selecting attenuated strains of PRSV [13]. This has made C. metuliferus an ideal model in studying plant-virus interaction and pathogenicity of PRSV in plants. In a previous study, we identified several transcript derived fragments (TDFs) from PI 292190 using cDNA-amplified fragment length polymorphism (cDNA-AFLP) [14]. One of these TDFs showed sequence similarity to serine proteinase inhibitor genes. This TDF was induced at 48 hour post inoculation in PRSV-resistant line PI 292190 but much later in PRSV-susceptible line Acc. 2459 (21 dpi) after PRSV infection. To test the function of C. metuliferus serine proteinase inhibitor (CmSPI), RNAi (RNA interference) was conducted in this study. RNAi initiated by double stranded RNA (dsRNA) modulates gene expression in eukaryotes via small interference RNA (siRNA) and microRNA (miRNA) [15,16]. RNAi is a powerful tool to study gene expression and analyze gene function [17]. The dsRNA can be formed in vivo by transforming plants with a construct that encodes hairpin RNA (hpRNA). Hairpin RNAi (hpRNAi) approach can lead to off target effects such as down-regulation of endogenous genes sharing sequence similarity with the hpRNAi construct. Moreover, an intron or inter-space contained self-complementary hpRNA (ihpRNA) construct could be more effective in its silencing ability [18].
This study has successfully obtained CmSPI RNAi transgenic lines in the PI 292190 genetic background, which showed a down regulation of CmSPI and suppression of the anti-PRSV resistance in C. metuliferus line PI 292190. However, transformation on the PRSV-susceptible C. metuliferus line Acc. 2459 was shown to be difficult. Therefore a full length genomic CmSPI fragment was cloned and used to transform N. benthamiana. Since PRSV could not infect N. benthamiana, the testing for resistance in transgenic N. benthamiana plants was conducted using a related potyvirus, potato virus Y (PVY), which is capable of infecting N. benthamiana. Two of the N. benthamiana transgenic lines were shown to be resistant to PVY infection. This study has provided evidence that the function of C. metuliferus CmSPI, a serine proteinase inhibitor gene, plays an important role in potyvirus resistance in both C. metuliferus and N. benthamiana.

Cloning CmSPI Gene cDNA and Full Length Genomic Fragment of Cucumis metuliferus
The C. metuliferus (PI 292190) cDNA fragment was identified using CapFishing Full-length cDNA Premix Kit (Seegene, Inc., Seoul, Korea) and the full length genomic fragment was identified by cassette ligation-mediated PCR genome walking [19]. In order to synthesize CmSPI cDNA, total RNA was isolated using previously described methods [20]. Three micrograms of RNA extracted from C. metuliferus line PI 292190 at 48 h post PRSV inoculation (hpi) were used for first strand cDNA synthesis, and the complementary strand was produced using the oligo-dT adaptor primer and the 5 1 end of cDNA was ligated with CapFishing adaptor using reverse transcriptase. The mixture was then used for 3 1 and 5 1 rapid amplification of cDNA end (RACE) PCR reaction using specific primers HMK2007-14 and HMK2007-15 (Table 1). The PCR products were ligated into the cloning vector yT & A (Yeastern Biotech Co., Taipei, Taiwan) for sequencing. To identify full length CmSPI genomic fragment, 5 µg of DNA extracted from C. metuliferus line PI 292190 were digested with restriction enzymes (BamHI, PstI, EcoRI, EcoRV, HpaI, NcoI, BstXI, HindIII, KpaI, SalI, and XbaI) and then ligated with adaptors HMK2010-35 and HMK2010-36. PCR primers (HMK2010-37, HMK2010-38, HMK2011-118, HMK2011-119, HMK2011-127, and HMK2011-128) were used for genome walking (shown in Table 1) and the PCR products were then ligated into cloning vector yT & A for sequencing.

Construction of Binary Vector and Bacterial Strain
The cDNA fragment of CmSPI gene for the RNAi construct was amplified by PCR with the primers, HMK2007-93 and HMK2007-94 (Table 1), and ligated into the yT & A vector. Sense and antisense CmSPI sequences were digested with BamH I-Nco I and BsrG I-Xho I individually and ligated into the pEPJ86i plasmid vector [25], in which the sense and antisense fragments were located in tandem with an inter-space sequence between them, and this ihpRNA construct was placed behind the cauliflower mosaic virus (CaMV) 35S promoter. The entire RNAi construct was then subcloned into the binary vector pGA482G and introduced into Agrobacterium tumefaciens strain LBA4404 for Agrobacterium-mediated transformation in C. metuliferus.
The full length genomic sequence of CmSPI gene was amplified from C. metuliferus resistant line PI 292190 with the primers, HMK2012-6 and HMK2012-23 ( Table 1). The PCR reaction was conducted using the proof-reading DNA polymerase, Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA). The resulting PCR product was digested with Kpn I and Hpa I and ligated into the binary vector pGA482G and introduced into Agrobacterium tumefaciens strain LBA4404 for Agrobacterium-mediated transformation into N. benthamiana plants.

Plant Material and Transformation
Transformation of C. metuliferus resistant line PI 292190 was conducted according to a previously described protocol [26]. In brief, cotyledon explants of the resistant line PI 292190 were infected with A. tumefaciens strain LBA4404 harboring the CmSPI RNAi binary vector construct. The resulting calli were screened for kanamycin resistance and the regenerated T 0 plants were grown in soil-vermiculite mixture in pots. These T 0 transgenic plants were tested by Southern hybridization and then self-pollinated to harvest T 1 seed for RNA and protein analysis and virus inoculation test. The transformation protocol for N. benthamiana was performed as previously described [27] with minor modifications. Young leaves harvested from two-month-old N. benthamiana plants were infected with A. tumefaciens strain LBA4404 carrying the full length genomic CmSPI binary vector construct. The resulting calli were screened for kanamycin resistance and regenerated T 0 plants were grown in soil-vermiculite mixture in pots to harvest T 1 and T 2 seed. The T 1 and T 2 transgenic N. benthamiana lines were used for the virus inoculation test.

Inoculation of Plant Viruses
PRSV was obtained from C. metuliferus susceptible line Acc. 2459 leaves after virus inoculated. PVY obtained from infected N. benthamiana leaves was inoculated to transgenic N. benthamiana lines. Virus inoculation was conducted using a 1:50 (w/v) dilution of the homogenized leaves infected with the virus in 10 mM phosphate buffer (0.033 M K 2 H 2 PO 4 , 0.067 M K 2 HPO 4 at pH 7.0).

Genomic PCR
Genomic DNA was isolated from leaves of the transgenic C. metuliferus and N. benthamiana plants by cetyltrimethyl ammonium bromide (CTAB) method as previously described [28]. The presence of the transgene was detected by PCR with primer set HMK2011-102/HMK2011-103 for the intron sequence and HMK2013-60/HMK2013-61 primers for the CmSPI gene. PCR analysis was performed in a reaction mixture containing 10 µM specific primer pairs, 1ˆTaq DNA polymerase reaction buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100 and 1.75 mM MgCl 2 ), 1U Taq DNA polymerase and 2.5 mM dNTP in a final volume of 20 µL.

Southern Hybridization
Ten micrograms of genomic DNA from transgenic plants were digested with Nco I to completion, subjected to 1% agarose gel electrophoresis and transferred onto a nylon membrane (Perkin Elmer Life Science, Waltham, MA, USA). To detect the CmSPI RNAi construct in transgenic C. metuliferus, the membrane was hybridized with α 32 P-labeled intron sequence probe (generated by PCR with primer set HMK2011-102/HMK2011-103), and for detection of the full length genomic CmSPI construct in transgenic N. benthamiana, the membrane was hybridized with α 32 P-labeled CmSPI gene probe (generated by PCR with primer set HMK2013-60/HMK2013-61).

RT-PCR (Reverse Transcription PCR)
The reverse transcription reaction was used Superscript III (Invitrogen TM ) with HMK2013-61 as a primer for cDNA synthesis. The PCR using the specific primer set HMK2013-60/HMK2013-61was used for CmSPI gene expression detection.

Northern Hybridization
Ten micrograms of total RNA from C. metuliferus line PI 292190 infected with PRSV at 48 hpi were subjected to denaturing electrophoresis in a 1% agarose gel containing 5% MOPS and then transferred onto a nylon membrane. The membrane was hybridized with α 32 P-labeled CmSPI gene probe (generated by PCR with primer set HMK2013-60/HMK2013-61 for CmSPI gene expression detection.

Western Hybridization
Total protein was extracted from transgenic plants subjected to 16% poly-acrylamide gel electrophoresis, and transferred onto PVDF blotting membrane (GE Healthcare, UK). Immunostaining used anti-CmSPI polyclonal antibodies (CmSPI anti rabbit) and the one-step BCIP/NBT detection system (Pierce, Rockford, IL, USA).
2.6.6. Detection of Small Interfering RNA Ten micrograms of total RNA extracted from PRSV-infected C. metuliferus line PI 292190 at 48 hpi were subjected to 15% poly-acrylamide gel electrophoresis, and transferred onto a nylon membrane. The membrane was hybridized with α 32 P-labeled CmSPI gene probe (generated by PCR with primer set HMK2013-60/HMK2013-61) to detect CmSPI gene expression.

Cloning and Functional Characterization of C. metuliferus CmSPI Gene
To identify full length cDNA and genomic fragments of CmSPI, RACE and genome walking were conducted. The ORF of CmSPI (KR012492) was identified as 219 bp in length, and the coding region of genomic fragment including 5 1 and 3 1 UTR was identified as 888 bp, containing two exons and one intron as shown in Figure 1. CmSPI sequence was analyzed in plant cis-acting regulatory DNA elements database, and the predicted reactive site of CmSPI was located at Lys48 (K48) and Asp49 (D49).
In addition, several conserved sequences of cis elements were identified in CmSPI promoter or upstream region. The "ACGT" sequence, a core abscisic acid response element [29], was found at 917 bp on the promoter region. The T/GBOXATPIN2 element "AACGTG" (identified in Arabidopis thaliana [30]) was present at´89 to´94 of CmSPI. Another element "CCATAA" (´42 to´47), a wound response element, is present on the nopaline synthae promoter [31] (Figure 1).
Phylogenetic analysis of amino acid sequence data from plant serine type PIs was conducted by neighbor-joining methods with the HKY85 genetic distance model as shown in Figure 2. Two distinct subtypes, potato I type and potato II type, were identified and CmSPI was grouped into potato I type which is a small size protein (8 kDa) containing two reactive sites.

. Generation of CmSPI RNAi Transgenic Plant
To study CmSPI gene function by reverse genetics, an RNAi construct was generated for ihpR nstitutively expressing an inverted repeat of 203 bp CmSPI fragment (from +46 to +682 nt e ron sequence) under the control of the cauliflower mosaic virus (CaMV) 35S prom metuliferus PRSV-resistant line PI292190 was transformed by Agrobacterium-med nsformation with the CmSPI ihpRNA construct (Figure 3). Southern hybridization (Figure nfirmed that eight out of ten kanamcyin resistant plants (T0 generation) included the transgen

Generation of CmSPI RNAi Transgenic Plant
To study CmSPI gene function by reverse genetics, an RNAi construct was generated for ihpRNA, constitutively expressing an inverted repeat of 203 bp CmSPI fragment (from +46 to +682 nt except intron sequence) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. C. metuliferus PRSV-resistant line PI292190 was transformed by Agrobacterium-mediated transformation with the CmSPI ihpRNA construct (Figure 3). Southern hybridization ( Figure 4A) confirmed that eight out of ten kanamcyin resistant plants (T 0 generation) included the transgene and harbored the same size, indicating that these transgenic plants might be generated from the same calli. Two of these transgenic plants, T 0 H-1 and T 0 H-3, were self-pollinated to generate T 1 seeds and the T 1 progeny were then screened by genomic PCR (Figure 4B). Both T 1 H-1 line (47 plants, χ 2 = 0.211, p = 0.487) and T 1 H-3 line (48 plants, χ 2 = 1.778, p value = 0.317) showed goodness of fit to a 3:1 ratio, a segregation pattern indicative of a single dominant transgene inheritance in both cases. The resistant control line PI292190, susceptible control line Acc. 2459, and two T 1 transgenic lines (T 1 H-1-2 and T 1 H-3-1) were then analyzed by RT-PCR, Northern hybridization for detecting high molecular weight or low molecular weight (small) RNAs, and Western hybridization. A decreased expression of CmSPI transcripts relative to the PRSV-resistant line PI292190 was evident ( Figure 4C,D). For small interfering RNA (siRNA) analysis, the abundant accumulation of low molecular weight siRNA in the transgenic lines, a hallmark of RNA silencing, was detected ( Figure 4E). To examine protein level in the RNAi transgenic lines, Western hybridization was conducted with a polyclonal antibody against CmSPI. A clear band (8 kDa) was detected in individual wild type controls (PRSV-resistant line PI 292190 and PRSV-susceptible line Acc. 2459) but a very faint band was shown in each individual transgenic lines (T 1 H-1-2 and T 1 H-3-1) ( Figure 4F). In summary, both RNA and protein levels of CmSPI expression were down regulated greatly by the silencing effect of CmSPI ihpRNA construct.

Alteration of Virus Resistance in CmSPI RNAi Transgenic C. metuliferus
To examine the effect of silencing CmSPI in PRSV-resistance C. metuliferus, a total of 95 T 1 transgenic plants (47 T 1 H-1 and 48 T 1 H-3 plants) along with three C. metuliferus resistant control plants (PI292190), and three C. metuliferus susceptible control plants (Acc. 2459) were inoculated with PRSV. The susceptible control Acc. 2459 showed typical symptoms of PRSV infection at 7 day post inoculation (dpi), the development of prominent mosaicism and chlorosis of leaves, water soaked streaking on the petiole and upper stem, and the distortion of young leaves ( Figure 5A,C). The resistant control PI292190 showed immunity against PRSV and no symptoms were detected ( Figure 5A,B). After silencing CmSPI on the resistant line PI292190, CmSPI RNAi T 1 transgenic plants were losing immunity to PRSV and showed PRSV infection symptoms on the topmost leaves after PRSV inoculation at 21 dpi ( Figure 5D,E). This indicated that silencing of CmSPI gene could break down the resistance to PRSV in these RNAi transgenic lines.

Alteration of Virus Resistance in CmSPI RNAi Transgenic C. metuliferus
o examine the effect of silencing CmSPI in PRSV-resistance C. metuliferus, a total of 95 sgenic plants (47 T1H-1 and 48 T1H-3 plants) along with three C. metuliferus resistant control pl 92190), and three C. metuliferus susceptible control plants (Acc. 2459) were inoculated with PR susceptible control Acc. 2459 showed typical symptoms of PRSV infection at 7 day post inocula ), the development of prominent mosaicism and chlorosis of leaves, water soaked streaking on ole and upper stem, and the distortion of young leaves ( Figure 5A,C). The resistant control PI292 ed immunity against PRSV and no symptoms were detected ( Figure 5A,B). After silencing Cm he resistant line PI292190, CmSPI RNAi T1 transgenic plants were losing immunity to PRSV ed PRSV infection symptoms on the topmost leaves after PRSV inoculation at 21 dpi (Fig  ). This indicated that silencing of CmSPI gene could break down the resistance to PRSV in th i transgenic lines.

Resistance to Virus in Transgenic N. benthamiana
To confirm the function of CmSPI in potyviruses resistance, the full length CmSPI genomic fragment with size 2858 bp (from´1457 to +1411 nt) was cloned and transformed into N. benthamiana by Agrobacterium-mediated transformation. All transgenic plants showed no obvious morphological alterations as compared to wild type N. benthamiana. Eleven T 0 transgenic plants were tested by PCR analysis and nine of them showed CmSPI gene positive signal (data not shown). The T 0 lines with transgenes were selected and self-pollinated to harvest seeds. T 1 plants derived from individual T 0 transgenic lines along with wild type N. benthamiana control plants were inoculated with PVY. As shown in Table 2, two of these transgenic lines, T 1 F-21 and T 1 F-51, showed some degree of PVY resistance. The symptoms of PVY infection were observed on those non-transformed N. benthamiana plants at 7 dpi. Compared to wild type N. benthamiana control plants, these two lines showed no symptoms at 14 dpi ( Figure 6). Two lines (T 1 F-21-2 and T 1 F-51-10) selected from the T 1 population were then self-pollinated to generated T 2 plants. Using Southern blot, two T 2 lines, T 2 F-21-2-5 and T 2 F-51-10-8, showed one copy of the CmSPI insertion ( Figure 7A). In RNA level, CmSPI gene expression was observed after virus inoculation at 2 dpi in T 2 transgenic plants in which RNA expression levels were lower than those in C. metuliferus control PI 29219 ( Figure 7B). Furthermore, T2 plants generated from independent T 1 lines (T 1 F-21-2 and T 1 F-51-10) and three wild type N. benthamiana control plants were inoculated with the sap obtained from wild type N. benthamiana plants infected with PVY. Different levels of viral resistance were observed in these two lines. A total of two out of 44 plants of T 2 F-51-10 line showed PVY infected symptoms, but 33/44 of T 2 F-21-2 plants showed PVY infected symptoms at 21 dpi (Table 3). This result indicated the resistant function of CmSPI expression against PVY was identified in N. benthamiana transgenic plants. The      The values indicate the number of plants with symptoms of disease (left of slash) and total number of inoculated plants (right of slash). Plants were observed for up to 21 days post-inoculation.

CmSPI Is Involved in Plant Physiological Responses and Plant Defense
In our previous study, TDFs isolated from C. metuliferus were shown to be related to PRSV resistance using cDNA-AFLP analysis, and one of the candidate genes, CmSPI, was strongly induced after PRSV inoculation at 48 hpi [14]. In this study, we cloned and identified the full length sequence of the CmSPI gene from PRSV resistant line PI 292190. The ORF of CmSPI was identified as 219 bp in length and codes for a small protein (8 kDa) classified as a serine type proteinase inhibitor belonging to the serpin (serine PI) family, the largest and most widespread superfamily of PIs [32]. Plant PIs are active against different classes of proteinases and have been classified into different families [6]. They have been reported to interact with a variety of factors involving developmental and protein storage signals, wounding responses, and abiotic and biotic stress responses [33][34][35][36][37]. For example, peanut Bowman-Birk inhibitor genes, AhBBI, encode serine protease inhibitors that respond to drought stress, exogenous jasmonic acid (JA), and abscisic acid (ABA) [38]. The expression of soybean cysteine PI genes, R1 and N2, was induced by wounding and methyl jasmonate treatments, indicating a role for RI and N2 in plant defense [39]. The present study also detected several cis elements in the promoter region of the C. metuliferus CmSPI including a core abscisic acid response element [29], a T/GBOXATPIN2 element [30], and a wound response element indicating the CmSPI gene is involved in the responses to ABA, JA, and wounding [31].

Silencing of CmSPI Alters PRSV Resistance in C. metuliferus
To confirm the antiviral function of CmSPI in PRSV resistance, RNAi was performed using an inter-space or intron contained self-complementary hpRNA construct to enhance the silencing efficiency. In the present study, the immune response against PRSV in C. metuliferus resistant line PI 292190 was broken down after silencing CmSPI transgene expression in CmSPI RNAi transgenic plants. However, the mosaic symptoms on the upper leaves of transgenic lines were observed in a delayed fashion, after 21 dpi, compared to the severe symptoms at 7 dpi in PRSV susceptible control Acc. 2459. This could be explained by the fact that the expression of CmSPI was knocked down instead of completely knocked out. This virus has to replicate and accumulate enough to overcome the threshold of basic defense to display symptoms in CmSPI RNAi transgenic lines. Alternatively, P1/Hc-Pro of PRSV is a strong virus silencing suppressor by binding to CmSPI-derived siRNAs [40], which might reduce the RNAi efficiency of CmSPI RNAi transgenic plants.

Expression of CmSPI in N. benthamiana Plants Can Supply Resistance for PVY
Plant viruses such as potyviruses, tymoviruses, nepoviruses, comoviruses, and closteroviruses would require proteinases to process their own proteins for replication and propagation [41]. Hence plant PIs might play a role in limiting proteinase function in these viruses. In a previous report, constitutive expression of rice cysteine PIs (oryzacytatin I) in transgenic N. benthamiana lines induced resistance against both Tobacco etch virus (TEV) and PVY infections [10]. Interestingly, a serine PI instead of cysteine PIs, CmSPI, identified in this study, was also shown to provide resistance function in N. benthamiana against PVY, a member of the potyvirus group. Potyviruses contains three proteinases (P1, HC-Pro, and NIa ) which have been reported in regulation of viral gene expression, replication and infection cycle [42,43]. It has been reported that a serine proteinase encoded by P1 gene can self-cleave at its C terminus for its function and the reactive sites of P1 proteinase included Asp233, Gly-Ser-Ser256-Gly [43]. In this study, CmSPI is a serine type PI and its reactive site was located at the 48th (K48) and 49th (D49) amino acid residues which might obstruct the function of P1 serine proteinase of potyviruses. This suggests that plant PIs both cysteine or serine type potentially have a role in defending against different viruses. Interestingly, the T 2 plants derived from two resistant T 1 transgenic N. benthamiana lines showed variations (42/44 in T 2 F-51-10 and 11/44 in T 2 F-21-2) with regard to their resistance to PVY. It is not uncommon that variations in resistance occur among different generations of transgenic lines. For example, a previous study reported that R 1 plants expressing N gene segments of Tomato spotted wilt virus (TSWV) showed variations in their resistance to viruses [44]. However, further experiments would be required to understand the association between the inheritance of the transgene to its target traits.

Conclusions
We have demonstrated that the C. metuliferus CmSPI gene encodes a serine proteinase inhibitor. Knocking down CmSPI gene expression in the PRSV-resistant C. metuliferus line PI 292190 broke down its resistance against PRSV. Furthermore, heterologous expression of the C. metuliferus CmSPI gene in N. benthamiana provided resistance to PVY, another Potyvirus, and indicated its important role in the mechanism of potyviruses resistance.