Abstract:
The invention relates to a gDNA and corresponding cDNA sequence comprising pathogenesis related protein-10 (CbPR-10) gene in  Capsicum baccatum . The polypeptide encoded by the DNA sequence has ribonuclease activity which makes the enzyme highly effective in inhibiting the growth of fungal pathogen. The use of the DNA sequence disclosed herein contains the gDNA, cDNA, protein and a genetic construct for the production of transgenic plants, especially genetically transformed pepper plants, with an enhanced resistance against fungal pathogens.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the DNA sequences encoding a pathogenesis related-10 (CbPR-10) protein from  Capsicum baccatum . The gDNA contains a novel nucleic acid sequence for a promoter. The polypeptide of CbPR-10 shows weak ribo-nuclease activity, which becomes stronger after fungal induced phosphorylation of the protein. The invention also relates to the sequences comprising the cDNA sequence fused with invented or other appropriate promoter and terminator in a genetic construct for transformation of plants or industrial use. Such an expression of the genetic construct in plant results in an increased resistance to phytopathogenic fungi, especially anthracnose fungus. 
     2. Description of Prior Art 
     Plants respond to the invasion of phyto-pathogens by coordinated and integrated set of metabolic alterations. To resist the pathogen, various genes are induced at the infection site and distal part of the plant in association with the development of hypersensitive reaction (HR) and systemic acquired resistance (SAR), respectively. These genes include pathogenesis-related (PR) and antibiotic proteins, etc. 
     The PR proteins are a class of proteins that are synthesized in plants in response to pathogenic infection. Usually, PR proteins are not present in healthy plants, but are synthesized in response to pathological or related stresses. In the latest proposition, PR proteins are divided into 14 families based on their sequences, serological properties, and biological activities. When most PRs are extra-cellular proteins, PR-10 is the first described intracellular protein in cultured parsley cell upon elicitor treatment. It has been supposed that the intracellular PR-10 is capable of cleaving viral RNA or other pathogen&#39;s RNA in the plant cells. According to Park et al., (2004), CaPR-10 protein, isolated from  Capsicum annuum , is phosphorylated upon TMV-P 0  inoculation and functions as a kind of RNase being able to cleave viral RNA. Also, recombinant CaPR-10 inhibited oomycete growth as well as viral infection. So, it has been suggested that cytosolic phosphorylated PR-10 protein is an active component of an inducible-defense mechanism against pathogen infection. 
     A majority of cultivated pepper lines have been developed from  C. annuum  species which are very susceptible to anthracnose fungus. A pathogenic agent of anthracnose disease,  Colletotrichum acutatum , causes the most destructive disease in widely cultivated pepper varieties and results in serious economical loss. By anthracnose fungus, most serious damage occurs in the fruit of pepper. This pathogen becomes necrotrophic after penetration into the epidermal cells, so that fungal hyphae colonize intracellularly in subcuticular tissues of the fruit. Since PR-10 localizes in the cytoplasm of the plant cell, it will be valuable to examine the inhibitory effect of PR-10 protein during the fungal infection. So, we isolated and characterized a PR-10 gene from  C. baccatum  that showed incompatible interaction with anthracnose fungus. Also, relative ribonuclease and antifungal activity were compared with previously reported CaPR-10 protein of the susceptible species,  C. annuum.    
     There are a few  Capsicum  accessions, such as  C. baccatum  and  C. chinense  species, showing resistance to anthracnose fungus. If defense related genes of resistant species are available, it will provide opportunities to transfer the genes into susceptible species. Recently, genetic engineering techniques are offering relief from the destructive disease in cultivated pepper lines through the development of fungus control systems using resistance related genes isolated from fungal resistant species. Ultimately, biotechnological applications of CbPR-10 protein may provide agronomically relevant level of disease control on pepper cultivation without harmful side effects. 
     SUMMARY OF THE INVENTION 
     This invention relates to a cDNA clone of SEQ ID NO: 1 from  Capsicum baccatum , designated as pathogenesis related protein 10 (CbPR-10) of SEQ ID NO: 2. The fruits of  C. baccatum  are resistant to the anthracnose fungus,  Colletotrichum acutatum . However, most commercial pepper varieties are developed from  C. annuum , which is very susceptible to the fungus. Until now, no commercial cultivars resistant to the anthracnose have been developed. In this work, since anthracnose fungi colonize in the cuticular cells of the fruit, an intracellular pathogenesis related protein, PR-10, was cloned from resistant pepper species to clarify the functional relationship in the incompatible reaction of the fruit with the fungus. 
     Deduced amino acid sequence of CbPR-10 showed 96% identity with that of CaPR-10 from  C. annumm  species and contained P-loof conserved among PR-10 proteins. But, a few putative phosphorylation sites are differed from that of CaPR-10. The phosphorylated CbPR-10 protein with a predicted size of 19 kDa was clearly detectable in coincidence with 18 kDa in infected fruits of  C. baccatum , while only 18 kDa is detectable in that of  C. annuum . Immunohistochemical examination revealed that PR-10 accumulation was localized in epidermal cells and a few layer of subcuticular cells of the unripe fruits. After infection, protein accumulation markedly increased in both cells layers, especially in the fruits of  C. baccatum . The expression of CbPR-10 genes was induced by fungal infection, but expression level was much higher in  C. baccatum . Characterization of the enzymatic properties indicated that the recombinant CbPR-10 protein exhibits stronger ribonucleolytic activity rather than CaPR-10 protein and also showed better antifungal activity to anthracnose fungus. 
     Regulatory sequences may be useful in controlling temporal and/or spatial expression of a gene. So, identification of promoters which are active in infection site is of interest. A potential source for pathogen inducible promoters is the pathogenesis-related (PR) family of defense-related genes. Typically, PR proteins are expressed the infection site of plant tissues in response to the pathogen. In this experiment, nucleic acid sequences comprising the regulatory region of pathogenesis related protein 10 was isolated and characterized by the ability to regulate expression of CbPR10 gene. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Anthracnose symptoms developed on susceptible and resistant pepper fruits,  Capsicum annuum  cv. Yeo-ju and  Capsicum baccatum  cv. PBC80, respectively, at 7 days after inoculation with KSCa-1 isolate of  Colletotrichum acutatum.    
         FIG. 2 . Deduced amino acid sequences of SEQ ID NO: 2 and nucleotide sequence of gDNA of pathogenesis related 10 protein (CbPR-10) isolated from  Capsicum baccatum  (SEQ ID NO: 9). 
         FIG. 3 . Alignment of the deduced amino acid sequences of CbPR-10 of SEQ ID NO: 2 with CaPR-10 of SEQ ID NO: 4. The CbPR-10 encoded polypeptide is aligned to pathogenesis related 10 protein (CaPR-10) from  Capsicum annuum  (GeneBank No. AF244121). P-loop motif is underlined and putative phosphorylation sites are enclosed by square. Sequence variations between two species are aligned and marked. 
         FIG. 4 . Gene expression (A) and protein accumulation (B) of CaPR-10 and CbPR-10 in the fruits from their respective pepper species inoculated with anthracnose fungus. In A, total RNA was prepared from fruits at 0, 24, 48, and 72 HAI and 100 ng of total RNA was used as template for RT-PCR analysis. In B, immunoblot analysis of PR-10 protein (2 μg) was conducted in infected pepper fruits. Soluble proteins were extracted from infected fruits with anthracnose fungus at 0, 24, 48, and 72 HAI. The polyclonal antibody raised against the recombinant CaPR-10 was used at 1:1500 dilution and HRP-conjugated anti-rat IgG antibody was used as the secondary antibody at 1:5000 dilution. 
         FIG. 5 . Immunolocalization of CaPR-10 and CbPR-10 proteins in infected fruits of susceptible and resistant species,  C. annuum  and  C. baccatum , respectively. The pepper fruits were inoculated with  C. acutatum  and prepared samples from the inoculation sites at 0, 24, 48, 72 HAI. The sections were incubated with polyclonal antibody against the recombinant CaPR-10 and detected with peroxidase labeled secondary antibody. The localization of PR-10 protein is detected by a red color. Control experiments using pre-immune serum did not show any reactions (data not shown). A bar represents 100 μm. 
         FIG. 6 . Expression and purification of recombinant CaPR-10 and CbPR-10 proteins. SDS-PAGE analysis of the recombinant PR-10 proteins produced in  E. coli  at different steps of purification. Lane M, Protein molecular weight markers (size: kDa). From the left, lane 1, soluble fraction of  E. coli  BL21 cells carrying pGEX6p-1 plasmid that codes for GST protein; lane 2, soluble fraction of  E. coli  with pGEX6P-1/CaPR-10 codes for GST-CaPR-10 protein, grown without IPTG; lane 3; same as lane 2, but induced with IPTG; lane 4, soluble fraction of  E. coli  with pGEX6P-1/CbPR-10 codes for GST-CbPR-10 protein, grown without IPTG; lane 5, same as lane 4, but induced with IPTG; lane 6, purified GST/CaPR-10 fusion protein; lane 7, purified GST/CbPR-10 fusion protein; lane 8, cleavaged CaPR-10 protein; lane 9, cleavaged CbPR-10 protein. 
         FIG. 7 . Ribonucleolytic activity of recombinant PR-10 proteins against pepper total RNA. Each reaction mixture contained 5 μg of total RNA from pepper fruits and incubated for 3 hours at 56° C. Hydrolyzed RNAs were separated in 1.0% agarose gels. Lane 1, RNA+distilled water; lane 2, RNA+protein elution buffer; lane 3, RNA+CaPR10 protein (2 μg); lane 4, RNA+CbPR10 protein (2 μg); lane 5, RNA+RNase A (SIGMA, USA). Relative levels of RNA were shown in the bar graphs underneath. 
         FIG. 8 . Time course of RNA degradation by ribonucleolytic activity of recombinant PR-10 proteins against pepper total RNA. Each reaction mixture contained 5 μg of total RNA from pepper fruits and incubated for 3 hours at 56° C. Hydrolyzed RNAs were separated in 1.0% agarose gels. Upper gel, RNA+CaPR-10 (2 μg); lower gel, RNA+CbPR-10 (2 μg). Relative levels of RNA were shown in the bar graphs underneath. 
         FIG. 9 . Microscopical observation of an in vitro fungal inhibition assay at 24 hours after addition of recombinant PR-10 proteins to the spore of anthracnose fungus. Viability of the fungus was determined by using Live/Dead bacterial viability kit (Molecular probe, USA). Left panel: recombinant CaPR-10 protein was added at 0, 0.05, and 0.5 mg/L. Right Panel: recombinant CbPR-10 protein was added at 0, 0.05, and 0.5 mg/L. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a cDNA clone, designated as CbPR-10, which was isolated from infected unripe fruits of  C. baccatum  showing incompatible interaction with  C. acutatum  ( FIG. 1 ). Thus, gDNA sequence corresponding CbPR-10 was cloned from  C. baccatum . The sequence was comprised of the promoter of SEQ ID NO: 3 and coding region of CbPR-10 gene. The promoter contains transcriptional initiation site, which favor the initiation of transcription in infected tissues ( FIG. 2 ). Then, full-length of cDNA was cloned of which contains an open reading frame of 480 bp from the the first translation start (ATG) at nucleotide position 1 to a translational stop (TGA) at position 478. The nucleotide sequences of CaPR-10 encode a polypeptide of 159 amino acids with an estimated molecular mass of 17.3 kDa. The amino acid sequence of the cDNA is highly homologous to the genes encoding pathogenesis related protein 10 (PR-10) found in other plants. Especially, the nucleotide sequence is almost identical to that of CaPR-10 from  C. annuum  ( FIG. 3 ). So, the clone was named as CbPR-10 for pathogenesis related protein 10 of  C. baccatum . Based on the nucleotide sequence, the CbPR-10 gene belongs to the PR-10 family. Sequence identity is highest at 98%, 90%, and 83% with PR-10 from  C. annuum, C. chinense , and  Solanum virginianum , respectively. 
     The expression pattern of CbPR-10 gene was investigated in the infected fruits of  C. baccatum  with anthracnose fungus and compared with that of CaPR-10 gene in  C. annuum  showing susceptible reaction. RT-PCR analysis was performed with total RNA extracted from infected fruits of both species at various time points ( FIG. 4A ). CbPR-10 in  C. baccatum  was induced by fungal infection on the first day, peaked on the second day and then declined after inoculation. However, the induced level of CaPR10 in  C. annuum  showed dual expression pattern on the first and third day at much less level. 
     We examined whether the time-courses of PR-10 accumulation are correlated with anthracnose symptom development on infected fruits. Western blot analysis was performed with soluble proteins extracted from the fruits of both species. Using anti-CaPR-10 antibody, an 18 kDa protein corresponding to PR-10 was detected in the unripe fruits ( FIG. 4B ). In resistant reaction of  C. baccatum , immunoblot analysis indicated that the level of 18 kDa increased slightly, 19 kDa band appeared on the first day after the infection with anthacnose fungus and these bands were maintained further in the fruits. In the susceptible reaction of  C. annuum , however, basal level of CaPR-10 protein decreased significantly on the first day and then increased slightly. Interestingly, 19 kDa band was clearly detectable only in the infected fruits of  C. baccatum  while undetectable in those of  C. annuum . These data suggest that the phosphorylation of CbPR-10 protein in 19 kDa is related to the resistance response of the fruits of  C. baccatum  against anthracnose fungus. 
     In previous studies, no lesion formed on the resistant fruit of  C. baccatum  in planta inoculation, but typical anthracnose symptom developed in the infected fruit of  C. annuum  with anthracnose fungus. So, immunohistochemical study was conducted to clarify the accumulation and location of PR-10 proteins in infected fruits of both species. PR-10 proteins are located in the cytosol as expected from the sequence analysis, showing no signal peptide ( FIG. 5 ). Higher accumulation of CbPR-10 was detected in the fruit epidermis of  C. baccatum  and fungal induced accumulation was also much higher in both epidermis and sub-epidermal layers in  C. baccatum  during infection stage. 
     The recombinant PR-10 proteins expressed in  E. coli  were shown to cleave pepper total RNA, but relative activity of CbPR-10 was much stronger than CaPR-10 ( FIGS. 7 ,  8 ). To clarify the biological properties of CbPR-10, antifungal activity of recombinant CbPR-10 was investigated. Normal development of the fungus was seriously impaired by the application of the proteins so that fungal hypha tended to elongate and branch and, particularly, appressorium formation was blocked during the fungal development. There was no significant difference in the antifungal activity between CaPR-10 and CbPR-10, but fungal viability was effectively damaged by CbPR-10 than CaPR-10 ( FIG. 9 ). 
     This invention includes the expression vectors comprising the nucleotide sequence of CbPR-10 associated with a regulatory nucleotide sequence that controls expression of CbPR-10 in a host cell. The host expression vector systems include  E. coli  or plants transformed with recombinant expression vectors. 
     EXAMPLES 
     Materials and Methods 
     Plant materials.  Capsicum baccatum  cv. PBC80, which is identified to be a resistance to  Colletotrichum acutatum , was used. As a susceptible plant,  C. annuum  cv. Yeo-ju was used. Plants were grown under protected green house conditions at 25° C. Fully grown unripe green fruits of eight month-old pepper plants were used for nucleic acid extraction and pathogen inoculation.
 
Fungal pathogen and inoculation. Inoculum preparation and artificial inoculation procedures were followed by the methods of Kim et al., (2004) with slight modifications. The Korean isolate of  Colletotrichum acutatum , KSCa-1, was used as anthracnose pathogen. The isolate was grown on potato dextrose agar (PDA) medium (Sigma, USA) at 25° C. under an alternative 16 h fluorescent light and 8 h dark. The 7-day-old PDA plates were flooded with distilled water and fungal colonies were gently scraped from the plates. Then the suspension was filtered through four layers of cheesecloth to remove mycelial debris. Inoculum concentration was adjusted to 5×10 5  conidia/mL with a hemacytometer. As a wounding inoculation method, microinjection specialized with a needle with an accurately adjustable wounding depth was used. The detached fruits were washed once with distilled water and were injected with 2 μL of the conidial suspension at usually three sites of fruit epidermis and at a depth of 0.8 mm. The inoculated fruits were placed in an acryl box moistened with four layers of wet kitchen towel. The acryl boxes were tightly sealed with wrap of vinyl chloride to maintain near 100% of relative humidity and incubated at 25° C.
 
Gene cloning, sequence and gene expression analysis. Total RNA was extracted from infected unripe fruits using RNeasy Plant Kit (Quiagene, Germany) according to the manufacturer&#39;s instructions. A forward primer, 5′ATGGGTGCTTATACCT3′ (SEQ ID NO: 5) and a reverse primer, 5′TTAAACATAGACAGAAGGAT3′ (SEQ ID NO: 6), were used for RT-PCR. The PCR product of a full-length cDNA was cloned in pGEMT-easy vector (Promega, USA) and sequenced. cDNA sequencing was performed with an ALFexpress automated DNA seqencer (Amersham, UK). Analyses of nucleotide and amino acid sequences were performed with the DNASIS sequence analysis software for Windows, version 2.1 (Hitachi, USA). For a homology search, the cDNA sequences were compared to the sequences in the NCBI nonredundant database using the BLAST electronic server. To determine expression level of PR-10 genes, RT-PCR analysis was conducted using 100 ng of total RNA extracted from infected fruits as template.
 
Cloning of gDNA by PCR. Coding region of CbPR-10 gene was amplified by PCR from gDNA of  C. baccatum  as template. Primers used were 5′ATGGGTGCTTATACCT3′ (SEQ ID NO: 7) and 5′TTAAACATAGACAGAAGGAT3′ (SEQ ID NO: 8). Then, to clone upstream region of CbPR-10 gene using inverse PCR, pepper genomic DNA was digested with HindIII, and self-ligated to form circular DNA fragments. These were amplified by PCR using LA or Ex Taq polymerase (TaKaRa, Japan) with a set of primers corresponding to the N-terminal of the PR-10 sequence. Temperature cycles were as follows: 94° C. for 1 min, followed by 30 cycles of 94° C. for 1 min, 60° C. for 3 min, and 70° C. for 1 min. A 1.2 kb PCR product containing parts of the CbPR-10 sequence, was sub-cloned into Topo vector (Invitrogen, USA) and sequenced.
 
SDS-PAGE, Western blot analysis, and immunohistochemistry. SDS-PAGE was performed with total proteins separated on 12% polyacrylamide gels according to Laemmli (1970) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. For immunoblot analysis, primary antibody was used at a 1:1500 dilution. A goat anti-rat antibody coupled to alkaline phosphatase was used as secondary antibody at a 1:5000 dilution. The secondary antibody was visualized with luminol (ECL, USA).
 
For immunolocalization study, pepper fruits were fixed in 1% glutaraldehyde/3% paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.0, dehydrated in ethanol, and embedded in paraffin. Tissues were transverse-sectioned into slices 10 μm in thickness. For immunolabeling, deparaffinized sections were incubated with primary antibody for 12 hours at 12° C. Polyclonal antibody against recombinant CaPR-10 protein was used at a dilution of 1:2000. Control tissues were incubated with pre-immune serum. Then the sections were incubated with biotinylated secondary antibody of goat anti-rat (DAKO, USA). For detection, the secondary antibody was colorized with 3-amino-9-ethylcarbazole according to the manufacturer&#39;s instructions (DAKO, USA).
 
Recombinant CaPR-10 and CbPR-10 proteins. The open reading frame of CaPR-10 or CbPR-10 cDNA were amplified by polymerase chain reaction and inserted in-frame with the glutathione-S-transferase (GST) coding sequence in expression vector pGEX6p-1 (Pharmacia Biotech, Sweden) between an EcoRI and XhoI site. Each GST fusion protein was expressed in  E. coli  and purified according to the manufacturer&#39;s instructions. The concentration of protein was determined by the Bradford method (Bradford, 1976). The pure CaPR-10 or CbPR-10 proteins after PreScision Pretease digestion were used for the analyses of ribonuclease and antifungal activities.
 
Ribonuclease activity of recombinant CbPR-10 protein. RNA degradation assay was carried out according to the method described by Bantignies et al., (2000).
 
Antifungal activity of CbPR-10 protein. A monoclonal KSCa-1 isolate of  C. acutatum  was cultured on potato dextrose agar (Difco, USA) for 7 days in the dark at 28° C. Spores were harvested and suspended in sterile distilled water. Ten microliters of spore suspension (5×10 5  spores per milliliter) was used in drop culture amended with 10 μl of recombinant protein or PBS buffer and applied to cover glasses. The cover glasses were incubated in humidified chamber at 25° C. in the dark for 24 hours. To observe fungal viability, germinated spores were stained with Live/Dead bacterial viability kit (Molecular probe, USA).
 
Cloning and Sequence Analysis of CbPR-10 cDNA
 
     By using RT-PCR, a cDNA fragment was cloned from the unripe fruits of  Capsicum baccatum  infected with anthracnose fungus,  Colletotrichum acutatum . A full-length cDNA clone was designated as CbPR-10 ( Capsicum baccatum  pathogenesis related protein 10). The CbPR-10 cDNA is 480 base pairs (bp) long and encodes a predicted protein of 159 amino acids ( FIG. 2 ) with an estimated molecular mass of 18 kilodaltons (kDa). The CbPR-10 protein contains ‘P-loof’ that is conserved among PR-10 proteins and possesses several putative phosphorylation sites predicted by NetPhos WWW server ( FIG. 3 ). The CbPR-10 protein shares very high homology of 95% identity with CaPR-10 (Park et al., 2004) from  C. annuum  and 61% identity with STH-10 protein from potato (Matton and Brisson, 1989). 
     Cloning and Sequence Analysis of CbPR-10 gDNA 
     In internal region of a CbPR-10 gene was cloned by PCR method. The primers were designed based on the nucleotide sequence of CaPR-10 gene because the sequence of CbPR-10 showed 96% of identity with that of CaPR-10 gene. A 1025 bp of amplification product was composed of two exons intervening with an intron sequence and 3′ UTR region. Then, inverse PCR (iPCR) was conducted to clone the regulatory region of the gene. A product of 1.3 kb was amplified in the PCR reaction containing  C. baccatum  gDNA digested with HindIII ( FIG. 2 ). The promoter sequence was determined for a region of 1154 bp upstream of the ATG initiation codon. A number of sequences with significant similarity to previously characterized elements in inducible genes were identified such as the SA-responsive element (TCA), elicitor-responsive element (ELI box), heat shock element (HSE), the wound responsive elements (WUN), TATA box, etc. 
     Induction of PR-10 Gene by Fungal Infection 
     To assess the expression of PR-10 during fungal infection, spore suspension of anthracnose fungus was inoculated to elicit resistant and susceptible reactions in the unripe fruit of  C. baccatum  and  C. annuum , respectively. Semi-quantitative reverse polymerase chain reaction (RT-PCR) was conducted in both fruits showing different disease symptom ( FIG. 4A ). The result shows that the mRNA level of CbPR-10 in  C. baccatum  was induced on the first day, peaked on the second day and then declined after inoculation. However, the induced level of caPR10 in  C. annuum  showed dual expression pattern on the first and third day at much less level. 
     Induction and Phosphorylation of PR-10 Protein by Fungal Infection 
     Using anti-PR-10 antibody, an 18 kDa protein corresponding to PR-10 with a predicted size 18 kDa was detected in pepper fruit infected with anthracnose fungus ( FIG. 4B ). In resistant reaction, immunoblot analysis indicated that the level of CbPR-10 protein increased slightly at the first day after the infection with anthacnose fungus and maintained further in the fruit of  C. baccatum . In the susceptible reaction, however, basal level of CaPR-10 protein was significantly decreased at the first day and then slightly increased in that of  C. annuum . The phosphorylated PR-10 protein with a predicted size 19 kDa was clearly detectable in infected fruits of  C. baccatum  while undetectable in those of  C. annuum . An unknown weak band with a molecular weight of about 20 kDa was cross-reacted with the anti-CaPR-10 antibody in both  C. baccatum  and  C. annuum . These data suggest that the phosphorylation of CbPR-10 protein is probably related to the resistance response of the fruits of  C. baccatum  against anthracnose fungus. 
     Immunolocalization of PR-10 Protein in Infected Pepper Fruits 
     To clarify the localization and accumulation of PR-10 protein during fungal infection, immunohistochemical examination of the protein was conducted with transverse-sections of the fruits of both species. The sections were prepared from infected unripe fruits with anthracnose fungus at 0, 24, 48, and 72 HAI, respectively ( FIG. 5 ). The accumulation of PR-10 protein was localized rarely in outer epidermal cells in both fruits at 0 HAI. After fungal infection, PR-10 accumulation was gradually increased in epidermal cells and sub-epidermal layers of the fruits. However, the amount of the proteins was significantly higher in the fruits of  C. baccatum  than that of  C. annuum . The results suggest that CbPR-10 protein is localized in the epidermal cell of pepper fruits during normal development. Fungal induced CbPR-10 accumulation was occurred massively in cortical parenchyma cells as well as epidermal cells of the fruits of  C. baccatum , while much less amount of CaPR-10 protein was observed in the normal or infected fruits of  C. annuum.    
     Ribonuclease Activity of Recombinant CbPR-10 Protein 
     To access the functional activity of CbPR-10 protein, recombinant CbPR-10 protein was expressed in  E. coli , purified ( FIG. 6 ), and tested for the ribonuclease activity according to the method of Bantignies et al., (2000). The total RNA extracted from the unripe pepper fruits was incubated with recombinant CbPR-10 as well as CaPR-10 or RNase A (SIGMA, USA). RNase A was used as positive control and recombinant CaPR-10 protein was used for the comparison of relative enzyme activities between  C. annuum  and  C. baccatum . In  FIG. 7 , relatively stronger enzyme activity of the CbPR-10 was shown as faster migration of degradation products in the agarose gel rather than that of CaPR-10. As another control, RNase A degrades pepper RNA completely under the same condition.  FIG. 8  shows relative enzyme activities were compared between CaPR-10 and CbPR-10 during a time course. The result showed that CbPR-10 had much stronger ribonuclease activity than CaPR-10. 
     Antifungal Activity Between Recombinant CbPR-10 and CaPR-10 Protein 
     Antifungal activity of recombinant CbPR-10 was investigated to clarify the biological properties of CbPR-10 in plant. To examine the effect of CbPR-10 on the growth of anthracnose fungus, recombinant CbPR-10 protein was amended with the fungal spores. Related activity of CaPR-10 protein was compared as positive control. The spores of fungus start to germinate at 1 hr and develop first appressorium at 6 hrs after deposition of the surface of cover glass. However, normal development of the fungus was severely impaired by the application of recombinant PR-10 protein. At low concentration of the protein, the tip of hypha tended to elongate and branched without developing appressorium ( FIG. 9 ). The germination of the spores was completely blocked at 0.5 mg/ml of both CaPR-10 and CbPR-10 proteins. In addition, the viability of the fungus showing an abnormal growth was examined by staining with Live/Dead bacterial viability kit (Molecular probe, USA). The results showed that viability of the fungus was severely affected as low as 0.05 mg/L of PR-10 protein. 
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