Patent Abstract:
The present invention related to two cDNA clones, designated to PepDef (pepper defensin protein gene) and PepThi (pepper thionin-like protein gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof.

Full Description:
[0001]    This is a division of application Ser. No. 09/442,631 filed Nov. 18, 1999. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    The present invention related to two cDNA clones, designated to PepDef (pepper defensin protein gene) and PepThi (pepper thionin-like protein gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof.  
           [0003]    Plants have developed defense mechanisms to defend themselves against phytopathogens. Plants&#39; first responses to pathogen infection include fortification of cell walls for physical barriers by deposition of lignin (Dean and Kuc, 1988) and by oxidative cross-linking (Brisson et al., 1994) as well as the hypersensitive reaction (HR). HR causes a rapid cell death of infected tissues to halt further colonization by pathogens (Goodman and Novacky, 1994). The next array of defense strategies includes the production of antimicrobial phytoalexins (van Etten et al., 1989), pathogenesis-related (PR) proteins (Linthorst, 1991; Ponstein et al., 1994), and cysteine (Cys)-rich proteins, such as lipid transfer protein (Garcia-Olmedo et al., 1995) and thionins (Bohlmann, 1994).  
           [0004]    Thionins are small, highly basic, Cys-rich proteins that show antimicrobial activity and seem to have a role in plant defense against fungi and bacteria. The overexpression of the THI2. 1 thionin in Arabidopsis enhanced resistance to a phytopathogenic fungus (Epple et al., 1997). The overexpression of a-hordothionin in tobacco also enhanced resistance to a phytopathogenic bacterium (Carmona et al., 1993). In addition, during barley and powdery mildew interactions, the accumulation of thionins was higher in the incompatible interaction than in the compatible one (Ebrahim-Nesbat et al., 1993).  
           [0005]    The thionins contain a signal sequence, the thionin domain and an acid polypeptide domain as well as the conserved Cys residues (Bohlmann et al., 1994). A new class of Cys-rich antimicrobial protein, γ-thionin, has a similar size (5 kD) and the same number of disulfide bridges as thionins. However, since γ-thionins do not have significant sequence homologies with thionins, they have been described as plant defensins (Terras et al., 1995). Both defensin and thionin genes in Arabidopsis are inducible via a salicylic acid-independent pathway different from that for PR proteins (Epple et al., 1995; Penninckx et al., 1996).  
           [0006]    Fruit ripening represents a genetically synchronized process that involves developmental events unique to plant species. Generally, ripe fruits are susceptible to pathogen attack (Swinburne, 1983; Prusky et al., 1991). Therefore, fruit as one of the reproductive organs of the plants must be protected from pathogens to maintain their integrity and seed maturation. Several antifungal proteins that are responsible for protection against pathogens during fruit ripening have been identified (Fils-Lycaon et al., 1996; Meyer et al., 1996; Salzman et al., 1998). Also, PR proteins are developmentally expressed during the formation of flowers (Lotan et al., 1989; Cote et al., 1991).  
           [0007]    [0007] Colletotrichum gloeosporioides  (Penz.) causes anthracnose diseases in many plant species (Daykin, 1984; Dodds et al., 1991; Prusky et al., 1991).  C. gloeosporioides  is the most prevalent species among  C. acutatum, C. coccodes, C. dematium, C. gloeosporioides,  and  G. cingulata  to cause anthracnose diseases on pepper ( Capsicum annuum  L.) (Kim et al., 1986; Manandhar et al., 1995). In previous study, we found that the unripe-mature-green fruit of pepper cv. Nokkwang interacted compatibly with  C. gloeosporioides,  whereas the interaction of the ripe-red fruits with fungus was incompatible (Oh et al., 1998). To investigate the activation of defense-related genes from the incompatible-pepper fruit upon  C. gloeosporioides  infection, we isolated a defensin gene and a thionin-like gene by using mRNA differential display. The regulation of these Cys-rich protein genes was studied during fruit ripening and in the initial infection process during the compatible and incompatible interactions. We report here what appears to be the first case of a defensin gene and a thionin-like gene induced via different signal transduction pathways in a plant and fungus interaction.  
         SUMMARY OF THE INVENTION  
         [0008]    The present invention relates to two cDNA clones, designated to a defensin gene, PepDef, and a thionin-like gene, PepThi, the sequences of which are depicted in SEQ ID No. 1 and No. 3, respectively. The anthracnose fungus,  C. gloeosporioides,  interacts incompatibly with ripe fruits of pepper ( Capsicum annuum ). It interacts compatibly with the unripe-mature fruits. We isolated PepDef and PepThi expressed in the incompatible interaction by using mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. The expression of PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungal-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruits, it was induced by wounding, but not by jasmonic acid. The expression of PepDef gene is enhanced in the unripe fruit by jasmonic acid, while suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses. The PepDef and PepThi car be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepDef and PepThi genes of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]    [0009]FIG. 1. Alignment of the deduced amino acid sequences from PepDef (GenBank accession number X95363) and PepThi cDNAs (AF112443) of pepper (Meyer et al., 1996) with other thionins from tomato ( Lycopersicon esculentum,  U20591; Milligan and Gasser, 1995),  Nicotiana excelsior  (AB005266), tobacco ( N. tabacum,  Z 11748; Gu et al., 1992), and  N. paniculata  (AB005250). The conserved cysteine arrangement —C( . . . )C—X—X—X—C( . . . )G-X—C( . . . )C—X—C— is indicated by arrows.  
         [0010]    [0010]FIG. 2. Expression and induction of PepDef and PepThi genes from various organs of pepper by  Colletotrichum gloeosporioides  infections and wounding. RNAs were isolated from ripe fruit (R), unripe fruit (U), leaf, stem, and root at 24 h after the treatments of fungal infection (FI) and wounding (W). In addition, RNAs of both ripe and unripe fruits at 48 h after wounding (R48 and U48) were isolated. Ten μl at 5×10 5  conidialml of  C. gloeosporioides  was used for the inoculation of various pepper organs. Organs treated with 10 μl sterile-water except fungal spores for 24 h were used as the controls (C).  
         [0011]    [0011]FIG. 3. Differential induction of PepDef and PepThi genes from both ripe and unripe fruits of pepper by  Colletotrichum gloeosporioides  infections. RNAs were isolated from both ripe (incompatible interaction) and unripe fruits (compatible interaction) after the fungal infection with time course. Time is indicated in h after infection.  
         [0012]    [0012]FIG. 4. Induction and suppression of PepDef and PepThi genes from both ripe and unripe fruits of pepper by exogenous salicylic acid (SA) and jasmonic acid (JA) treatments. RNAs were isolated from both ripe (R) and unripe fruits (U) treated with SA (1=0.5 mM, 2=5 mM) and JA (3=4 μM, 4=40 μM) for 24 h. Fruits treated with 10 μl sterile-water except fungal spores for 24 h were used as the control (C). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0013]    The present invention has identified two cDNA clones, designated to PepDef and PepThi, from the incompatible interaction between pepper and the pepper anthracnose fungus  Colletotrichum gloeosporioides  using MRNA differential display and cDNA library screening.  
         [0014]    The PepThi cDNA is 506 bp in length with 9 bp of 5′-untranslated region and 245 bp of 3′-untranslated region including the poly(A) tail (GenBank AF 112443). The PepThi clone represented a full-length cDNA of the 0.5 kb transcript identified by RNA gel blot analysis. The cDNA contained one open reading frame encoding a polypeptide of 9.5 kDa with 84 amino acids. The deduced amino acid sequence of PepThi(SEQ ID No. 4) contained an N-terminal secretory signal peptide that was cleaved after glycine at position 25 (FIG. 1). PepThi is a Cys-rich polypeptide containing the consensus Cys arrangement —C( . . . )C—X—X—X—C( . . . )G-X—C( . . . )C—X—C—.  
         [0015]    The PepDef cDNA is 225 bp except 5′-untranslated region and 3′-untranslated region including the poly(A) tail (X95363). The PepDef clone represented a full-length cDNA of the 0.45 kb transcript identified by RNA gel blot analysis. The cDNA contained one open reading frame encoding a polypeptide of 8.5 kDa with 75 amino acids. The deduced amino acid sequence of PepDef(SEQ ID No. 3) contained an N-terminal secretory signal peptide that was cleaved after alanine at position 27 (FIG. 1). PepDef is also a Cys-rich polypeptide containing the consensus Cys arrangement —C( . . . )C—X—X—X—C( . . . )G-X—C( . . . )C—X—C—.  
         [0016]    The expression of PepThi gene was observed in ripe fruits, leaves, stems, and roots of pepper, respectively. The basal and non-induced level of PepThi gene was higher in the leaves and roots than in the fruits and stems. In the fruits, the PepThi mRNA was highly induced by fungal infection and wounding. Also, the accumulation of the PepThi mRNA increased in the stems with fungal infection and wounding. However, the level of PepThi mRNA was not significantly changed in the leaves and roots by the treatments.  
         [0017]    The PepDef mRNA was not detected in leaves, stems, and roots even after fungal infection and wounding. However, the basal level of PepDef gene was very high in the ripe fruit, and undetectably low in the unripe fruit. Interestingly, the level of PepDef mRNA was reduced in the ripe fruit by fungal infection and wounding. This phenomenon was also observed in the ripe fruit by JA treatment. The accumulation of PepDef mRNA was not significantly induced in the unripe fruit by fungal infection and wounding for 24 h or 48 h. These results suggest that PepDef and PepThi genes are developmentally and organ-specifically regulated, and the induction by fungal infection and wounding is also subject to developmental regulation.  
         [0018]    To examine the time course of the induction of PepDef or PepThi mRNAs in response to the fungal infection, RNA gel blot analysis was performed with the ripe and unripe fruits at 0, 3, 6, 12, 24, 48, and 72 h after inoculation (HAI) using PepDef and PepThi cDNAs as probes. The uninoculated incompatible-ripe fruit contained a basal level of PepThi mRNA. However, the expression of PepThi was rapidly induced in the ripe fruit upon fungal infection and reached a maximum at 48 and 72 HAIs. In compatible-unripe fruits, the accumulation of PepThi mRNA was late, at 12 HAI, and reached its maximum level at 72 HAI.  
         [0019]    Accumulation of PepDef mRNA in the unripe fruit was very low. PepDef expression was suppressed by fungal infection in the ripe fruit. The transcript levels dropped until 48 HAI, and had begun to increase again 72 HAI. Since PepDef gene was highly expressed in the ripe fruit and PepThi gene was induced in the ripe fruit by the fungal infection, these genes may be involved in the defense mechanism during fruit ripening against the phytopathogen.  
         [0020]    To identify inducers of PepDef and PepThi gene expression from fruits, RNA gel blot analysis was performed with unripe and ripe fruits treated with exogenous jasmonic acid (JA) and salicylic acid (SA) for 24 h. The PepThi mRNA was highly accumulated in the unripe fruit compared to in the ripe fruit by SA at 5 MM (FIG. 4). However, JA could not significantly induce the PepThi mRNA in both ripe and unripe fruits. The expression level of PepDef mRNA was not changed in both ripe and unripe fruits by SA. Interestingly, the expression of PepDef mRNA by JA increased in the unripe fruit, but decreased slightly in the ripe fruit. Taken together, these results suggest that the PepThi and PepDef genes are expressed via different signal transduction pathways during ripening.  
         [0021]    The PepDef and PepThi genes can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepDef and PepThi genes of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.  
       EXAMPLES  
       [0022]    Fungal Inoculum and Plant Material  
         [0023]    Monoconidial isolate KG13 of  C. gloeosporioides  was cultured on potato dextrose agar (Difco, USA) for 5 days in darkness at 27° C. Sterile distilled water was added and conidia were harvested through four layers of cheesecloth to remove mycelial debris. Ten μl at 5×10 5  conidia/ml of  C. gloeosporioides  was used for the inoculation of both unripe and ripe pepper fruit as described (Oh et al., 1998).  
         [0024]    Both ripe-red and unripe-mature-green fruits of pepper cv. Nokkwang were grown and harvested under green-house conditions. For wound treatments, five healthy ripe and unripe fruits were deeply scratched by a knife and incubated under relative humidity of 100% at 27° C. in the dark. Ten μl of SA (0.5 and 5 mM) and JA (4 and 40 μM) was applied to both ripe and unripe sets of five fruits. After incubation under the condition described above, the fruits were excised to 1 cm 2  at the application site and frozen in liquid nitrogen. Leaf, root, and stem samples were harvested from 3-week-old plants and handled as described above for fungal inoculation and wounding.  
         [0025]    mRNA Differential Display  
         [0026]    Total RNA was extracted from healthy and infected ripe and unripe fruits using RNeasy Plant kit (Qiagen, Germany) according to the manufacturer&#39;s instruction. We used total RNA as template for the reverse transcriptase reaction and performed differential display with [α 33 P]dATP instead of [α 35 S]dATP (Liang and Pardee, 1992). Anchored primers and random-arbitrary primers were purchased from Operon Technologies (Alameda, Calif., USA). PCR-amplified cDNA fragments were separated on denaturing 5% polyacrylamide gels in Tris-borate buffer. cDNAs were recovered from the gel, amplified by PCR, and cloned into pGEM-T easy vector (Promega, USA) as described (Oh et al., 1995).  
         [0027]    Construction and Screening of cDNA Library  
         [0028]    Poly(A) +  mRNA was purified from total RNA of unripe fruits at 24 and 48 h after inoculation with  C. gloeosporioides  using Oligotex mRNA Kit (Qiagen, Germany). The cDNA library (2.5×10 5  plaque-forming unit with the mean insert size of 1.2 kb) was constructed in the cloning vector XZAPII (Stratagene, Germany) according to the manufacturer&#39;s instruction.  
         [0029]    A partial cDNA, designated pddThi, from the differential display was used as a probe to screen the  C. gloeosporioides -induced pepper cDNA library. After three rounds of plaque hybridization, positive plaques were purified. The pBluescript SK phagemid containing cDNAs was excised in vivo from the ZAP Express vector using the ExAssit helper phage.  
         [0030]    DNA Sequencing and Homology Search  
         [0031]    The cDNA sequencing was performed with an ALFexpress automated DNA sequencer (Pharmacia, Sweden). Analysis of nucleotide and amino acid sequences was performed using the DNASIS sequence analysis software for Windows, version 2.1 (Hitachi, Japan). The multiple sequence alignment was produced with the Clustal W program. For a homology search, cDNA sequence was compared to the NCBI non-redundant databases using the BLAST electronic mail server (Altschul et al., 1997).  
         [0032]    RNA Blot and Hybridization  
         [0033]    Total RNA (10 μg/lane) from each plant tissue used in this study was separated on 1.2% denaturing agarose gels in the presence of formaldehyde. RNA gel-blotting, hybridization and washing were conducted as described by the manufacturer of the positively charged nylon membrane employed (Hybond N + ; Amersham, UK). Radiolabeled probes were prepared with [α 32 P]dCTP (Amersham) using a random primer-labeling kit (Boehringer Mannheim, Germany).  
         [0034]    Cloning and Characterization of Thionin-Like cDNAs  
         [0035]    [0035] C. gloeosporioides  showed the incompatible interaction with ripe-red fruits of pepper and the compatible interaction with unripe-mature-green fruits (Oh et al., 1998). We isolated several cDNAs induced from the ripe fruit, but not from the unripe fruit by the fungal infection using mRNA differential display. By nucleotide sequence analysis of cDNAs, two cDNA fragments were identified to be thionin homologs. One cDNA was full length and was similar to j1-1 cDNA that encodes a fruit specific defensin (Meyer et al., 1996). We named the defensin as PepDef ( pep per  def ensin). Another cDNA fragment, designated pddThi, showed homology to γ-thionin from tobacco (Gu et al., 1992). In preliminary RNA gel blot analysis, the two mRNAs accumulated to high levels in the incompatible interaction. A full-length cDNA clone of pddThi was isolated from a cDNA library prepared from pepper fruits 24 and 48 h after inoculation with the fungus. The full-length clone was designated pPepThi ( pep per  thi onin) and sequenced.  
         [0036]    The pPepThi cDNA is 506 bp in length with 9 bp of 5′-untranslated region and 245 bp of 3′-untranslated region including the poly(A) tail (GenBank AF112443). The pPepThi clone represented a full-length cDNA of the 0.5 kb transcript identified by RNA gel blot analysis. The cDNA contained one open reading frame encoding a polypeptide of 9.5 kDa with 84 amino acids. The deduced amino acid sequence of PepThi contained an N-terminal secretory signal peptide that was cleaved after glycine at position 25 (FIG. 1). PepThi is a Cys-rich polypeptide containing the consensus Cys arrangement —C( . . . )C—X—X—X—C( . . . )G-X—C( . . . )C—X—C—.  
         [0037]    A sequence alignment showed that the PepThi shared significant homology (identity and similarity: 50% and 64%, respectively) to a flower-specific y-thionin from tobacco (Gu et al., 1992) and to several other γ-thionins from Nicotiana species and tomato (Milligan and Gasser, 1995; FIG. 1). PepThi protein showed 29% identity for the whole coding region to a pepper defensin protein PepDef. PepThi did not have nucleotide sequence homology to thionins and was different from other γ-thionins. Thus, we assigned PepThi as a thionin-like protein.  
         [0038]    Expression Pattern and Induction by Fungal Infection and Wounding  
         [0039]    To examine the PepThi gene expression in various organs and its inducibility by fungal inoculation and wounding, RNA gel blot analysis was performed using total RNAs prepared from fruits, leaves, stems, and roots of pepper plants at 24 h after treatments. The expression of Peplhi gene was observed in ripe fruits, leaves, stems, and roots (FIG. 2). The basal and non-induced level of PepThi gene was higher in the leaves and roots than in the fruits and stems. In the fruits, the PepThi mRNA was highly induced by fungal infection and wounding. Also, the accumulation of the PepThi mRNA increased in the stems with fungal infection and wounding. However, the level of PepThi mRNA was not significantly changed in the leaves and roots by the treatments.  
         [0040]    We hybridized the PepDef cDNA to the same blot that was used for the hybridization of PepThi cDNA. The basal level of PepDef gene was very high in the ripe fruit, and undetectably low in the unripe fruit (FIG. 2). The PepDef mRNA was not detected in leaves, stems, and roots even after the treatments. PepDef protein is wound-inducible in the unripe fruit at 3 days after treatment (Meyer et al., 1996). However, the accumulation of PepDef mRNA was not significantly induced in the unripe fruit by fungal infection and wounding for 24 h or 48 h. Interestingly, the level of PepDef mRNA was reduced in the ripe fruit by fungal infection and wounding. These phenomena were also observed in the ripe fruit by fungal infection and JA treatment (see FIGS. 3 and 4). These results suggest that PepThi and PepDef genes are developmentally and organ-specifically regulated, and the induction by fungal infection and wounding is also subject to developmental regulation.  
         [0041]    Differential Induction by Fungal Infection During Fruit Ripening  
         [0042]    In our previous study for fungal morphogenesis on the surface of fruits, conidial germination, initial and mature infection hypha were observed at 2, 12, and 24 h after inoculations (HAIs), respectively (Oh et al. 1998). The initial anthracnose symptoms were detected only on the unripe fruit at 2 days after inoculation, resulting in typical sunken necrosis within 5 days after inoculation. To examine the time course of the induction of PepThi or PepDef mRNAs in response to the fungal infection, RNA gel blot analysis was performed with the ripe and unripe fruits at 0, 3, 6, 12, 24, 48, and 72 HAI using PepThi and j1-1 cDNAs as probes. The uninoculated incompatible-ripe fruit contained a basal level of PepThi mRNA (FIGS. 2 and 3). However, the expression of PepThi was rapidly induced in the ripe fruit upon fungal infection and reached a maximum at 48 and 72 HAIs (FIG. 3). In compatible-unripe fruits, the accumulation of PepThi mRNA was late, at 12 HAI, and reached its maximum level at 72 HAI.  
         [0043]    Accumulation of PepDef mRNA in the unripe fruit was very low (FIG. 3). As shown in FIG. 2, PepDef expression was suppressed by fungal infection in the ripe fruit. The transcript levels dropped until 48 HAI, and had begun to increase again 72 HAI. Since PepDef gene was highly expressed in the ripe fruit and PepThi gene was induced in the ripe fruit by the fungal infection, these genes may be involved in the defense mechanism during fruit ripening against the phytopathogen.  
         [0044]    Induction and Suppression During Fruit Ripening by JA and SA  
         [0045]    To identify the inducers of PepThi and PepDef gene expression from fruits, RNA gel blot analysis was performed with the unripe and ripe fruits treated with exogenous JA and SA for 24 h. The PepThi mRNA was highly accumulated in the unripe fruit compared to in the ripe fruit by SA at 5 mM (FIG. 4). However, JA could not significantly induce the PepThi mRNA in both ripe and unripe fruits. The expression level of PepDef mRNA was not changed in both ripe and unripe fruits by SA. Interestingly, the expression of PepDef mRNA by JA increased in the unripe fruit, but decreased slightly in the ripe fruit. Taken together, these results suggest that the PepThi and PepDef genes are expressed via different signal transduction pathways during ripening.  
         [0046]    Discussion  
         [0047]    Fungal-inducible thionin genes were identified in several plant/fungus interactions, such as in  Arabidopsis/Fusarium oxysporum  f.sp.  matthiolae  (Epple et al., 1995), barley/ Stagonospora nodorum  (Titarenko et al., 1993; Stevens et al., 1996), and barley/the mildew fungus (Boyd et al., 1994; Bohlmann et al., 1998). Relevant to these findings, the accumulation of barley leaf thionin in papillae and in the cell wall surrounding the infection peg was higher in the incompatible interaction than that in the compatible one (Ebrahim-Nesbat et al., 1989, 1993). Similar phenomena have been reported for many other plant and pathogen interactions. The induction of PepThi mRNA was observed to be faster in the incompatible interaction of ripe pepper fruits with the fungus (FIG. 3).  
         [0048]    The PepThi gene was induced during the early conidial germination of the fungus, before infection hyphae formation (Oh et al., 1998) and even before appressorium formation (Kim et al., 1999). These results suggest that signaling compounds released/produced during fungal germination result in the expression of PepThi gene in the epidermal cells of the incompatible-ripe fruit. Since the PepThi gene is expressed in various organs of pepper plants and its expression level is enhanced by fungal inoculation and wounding (FIG. 2), PepThi thionin-like protein could play a role in conferring systemic protection for the plants against both biotic and abiotic stresses. Also, the induction of PepThi gene in the unripe fruit by SA (FIG. 4) is consistent with a systemic protection role. SA plays an important role in the signal transduction pathway leading to the systemic acquired resistance (Gaffney et al., 1993).  
         [0049]    The expression of the PepDef gene is regulated during fruit ripening. Similarly, several defensins and thionins are specifically expressed in reproductive organs, such as flowers in tobacco (Gu et al., 1992) and Arabidopsis (Epple et al., 1995), pistils in petunia (Karunanandaa et al., 1994), and seeds in radish (Terras et al., 1995). These findings suggest that both defensins and thionins are possibly involved in the defense mechanism for protecting the reproductive organ against pathogens or wounds. Further, thionins and other Cys-rich proteins exhibit synergistically enhanced antifungal activity (Terras et al., 1993). Therefore, the concerted expression of both PepDef and PepThi genes during ripening could confer disease resistance in the ripe fruit during the early fungal infection process.  
         [0050]    The responses to exogenous JA and SA treatments in pepper during fruit ripening are different for both PepDef and PepThi genes. JA as a chemical elicitor induces thionin genes in Arabidopsis (Epple et al., 1995; Vignutelli et al., 1998) and barley (Andresen et al., 1992), and defensin genes in Arabidopsis (Penninckx et al., 1996), in addition to other wound inducible genes (Hildmann et al., 1992; Reinbothe et al., 1994). SA also induces a thionin gene in barley leaf (Kogel et al., 1995) as well as PR proteins (Ward et al., 1991; Uknes et al., 1992). A JA-independent wound induction pathway that shows opposite regulation to the JA-dependent one was identified in Arabidopsis (Rojo et al., 1998). In the present study, the PepThi gene is strongly inducible in the unripe fruit by SA and wounding, but not by JA (FIG. 4). These data indicate that the PepThi gene is expressed via a JA-independent wound signal transduction pathway.  
         [0051]    Since the PepDef gene is induced in the unripe fruit by JA, it is probably regulated via the octadecanoid pathway (Peña-Cortés et al., 1995; Bergey et al., 1996). The slightly suppression of the PepDef gene in the ripe fruit by JA and wounding is puzzling, since both JA in the unripe fruit result in the induction of PepDef RNA. The possible explanation is that JA may elicit other signals that are able to activate genes in response to JA. These additional signals may result in the inhibition of PepDef expression in the ripe fruit.  
         [0052]    This present study shows that a defensin and a thionin-like protein that may have defensive roles are deployed via different signal transduction pathways and may protect pepper fruits against the anthracnose fungus.  
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         [0071]    19. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J: Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261: 754-756 (1993).  
         [0072]    20. Garcia-Olmedo F, Molina A, Segura A, Moreno M: The defensive role of nonspecific lipid-transfer proteins in plants. Trend Microbiol 3: 72-74 (1995).  
         [0073]    21. Goodman R N, Novacky A J: The Hypersensitive Reaction in Plants to Pathogens. A Resistance Phenomenon. APS Press, St. Paul, Minn., USA (1994).  
         [0074]    22. Gu Q, Kawarta E F, Mores M-J, Wu H-M, Cheung A Y: A flower specific cDNA encoding a novel thionin in tobacco. Mol Gen Genet 234: 89-96 (1992).  
         [0075]    23. Hildmann T, Ebneth M, Peña-Cortés H, Sanches-Serrano J J, Willmitzer L, Prat S: General roles of abscisic acid and jasmonic acids in gene activation as a result of mechanical wounding. Plant Cell 4: 1157-1170 (1992).  
         [0076]    24. Karunanandaa B, Singh A, Kao T: Characterization of a predominantly pistil-expressed gene encoding a γ-thionin-like protein of Petunia inflata. Plant Mol Biol 26: 459-464 (1994).  
         [0077]    25. Kim W G, Cho E K, Lee E J: Two strains of  Colletotrichum gloeosporioides  Penz. causing anthracnose on pepper fruits. Korean J Plant Pathol 2: 107-113 (1986).  
         [0078]    26. Kim K D, Oh B J, Yang J: Differential interactions of a  Colletotrichum gloeosporioides  isolate with green and red pepper fruits. Phytoparasitica 27: 97-106 (1999).  
         [0079]    27. Kogel K-H, Ortel B, Jarosch B, Atzom R, Schiffer R, Wastemack C: Resistance in barley against the powdery mildew fungus ( Erysiphe graminis  f.sp.  hordei ) is not associated with enhanced levels of endogenous jasmonates. Eur J Plant Pathol 101: 319-332 (1995).  
         [0080]    28. Liang P, Pardee A B: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971 (1992).  
         [0081]    29. Linthrost H J M: Pathogenesis-related proteins of plants. Crit Rev Plant Sci 10: 123-150 (1991).  
         [0082]    30. Lotan T, Ori N, Fluhr R: Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell 1: 881-887 (1989).  
         [0083]    31. Manandhar J B, Hartman G L, Wang T C: Conidial germination and appressorial formation of  Colletotrichum capsici  and  C. gloeosporioides  isolates from pepper. Plant Dis 79: 361-366 (1995).  
         [0084]    32. Meyer B, Houlné G, Pozueta-Romero J, Schantz M-L, Schantz R: Fruit-specific expression of a defensin-type gene family in bell pepper. Upregulation during ripening and upon wounding. Plant Physiol 112: 615-622 (1996).  
         [0085]    33. Milligan S B, Gasser C S: Nature and regulation of pistil-expressed gene in tomato. Plant Mol Biol 28: 691-711 (1995).  
         [0086]    34. Oh B J, Balint D E, Giovannoni J J: A modified procedure for PCR-based differential display and demonstration of use in plants for isolation of gene related to fruit ripening. Plant Mol Biol rep 13: 70-81 (1995).  
         [0087]    35. Oh B J, Kim K D, Kim Y S: A microscopic characterization of the infection of green and red pepper fruits by an isolate of  Colletotrichum gloeosporioides.  J Phytopathol 146: 301-303 (1998).  
         [0088]    36. Peña-Cortés H; Fisahn J, Willmitzer L: Signals involves in wound-induced proteinase inhibitor II gene expression in tomato and potato plants. Proc Natl Acad Sci USA 92: 4106-4113 (1995).  
         [0089]    37. Penninckx I A, Eggermont K, Terras F R, Thomma B P, De Samblanx G W, Buchala A, Metraux J P, Manners J M, Broekaert W F: Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8: 2309-2323 (1996).  
         [0090]    38. Ponstein A S, Bres-Vloemans S A, Sela-Buurlage M B, van den Elzen P J M, Melchers L S, Comelissen B J C: A novel pathogen- and wound-inducible tobacco ( Nicotiana tabacum ) protein with antifungal activity. Plant Physiol 104: 109-118 (1994).  
         [0091]    39. Prusky D, Plumbley R A, Kobiler I: The relationship between the antifungal diene levels and fungal inhibition during quiescent infections of  Colletotrichum gloeosporioides  in unripe avocado fruits. Plant Pathol 40: 45-52 (1991).  
         [0092]    40. Reinbothe S, Mollenhauer B, Reinbothe C: JIP and RIPs: the regulation of plant gene expression by jasmonates in response to environmental cues and pathogens. Plant Cell 6: 1197-1209 (1994).  
         [0093]    41. Rojo E, Titarenko E, León J, Berger S, Vancanneyt G, Sanchez-Serrano J J: Reversal protein phosphorylation regulates jasmonic acid-dependent and—independent wound signal transduction pathways in  Arabidopsis thaliana. Plant J  13: 153-165 (1998).  
         [0094]    42. Salzman R A, Tikhonova I, Bordelon B P, Hasegawa P M, Bressan R A: Coordinate accumulation of antifungal proteins and hexoses constitutes a developmentally controlled defense response during fruit ripening in grape. Plant Physiol 117: 465-472 (1998).  
         [0095]    43. Stevens C, Titarenko E, Hargreaves J A, Gurr S J: Defense-related gene activation during an incompatible interaction between  Stagonospora  ( Septoria )  nodorum  and barey ( Hordeum vulgare  L.) coleoptile cells. Plant Mol Biol 31: 741-749 (1996).  
         [0096]    44. Swinbume T R: Post-Harvest Pathology of Fruits and Vegetables. Academic Press, NY, USA (1983).  
         [0097]    45. Terras F R G, Egermont K, Kovaleva V, Raikhel N V, Osborn R W, Kester A, Rees S B, Torrekens S, Van Leuven F, Vanderleyden J, Cammue B P A, Broekaert W F: Small cystein-rich antifungal proteins from radish: their role in host defense. Plant Cell 7: 573-588 (1995).  
         [0098]    46. Terras F R G, Schoofs H M E, Thevissen K, Osborn R W, Vanderleyden J, Cammue B P A, Broekaert W F: Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitors. Plant Physiol 103: 1311-1319 (1993).  
         [0099]    47. Titarenko E, Hargreaves J, Keon J, Gurr S J: Defense-related gene expression in barley coleoptile cells following infection by  Septoria nodorum.  In Mechanisms of Plant Defense responses, Fritig B and Legrand M (eds), pp. 308-311. Kluwer Academic Publisher, Dordrecht (1993).  
         [0100]    48. Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J: Acquired resistance in Arabidopsis. Plant Cell 4: 645-656 (1992).  
         [0101]    49. Van Etten H D, Mattews D E, Mattews P S: Phytoalexin detoxification: Importance for pathogenicity and practical implications. Annu Rev Phytopathol 27:143-164 (1989).  
         [0102]    50. Vignutelli A, Wasternack C, Apel K, Bohlmann H: Systemic and local induction of an Arabidopsis thionin gene by wounding and pathogens. Plant J 14: 285-295 (1998).  
         [0103]    51. Ward E R, Uknes S J, Williams S C, Dincher S S, Wiederhold D L, Alexander D C, Ahl-Goy P, Metraux J-P, Ryals J A: Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3: 1085-1094 (1991).   
     
       
       
         1 
         
           
             
4 
 
           
           
             
               1685 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               Arabidopsis thaliano  
             
             
               CDS  
                57..1511  
                /note= “amino acid transporter”
 
             
              1 

CTTAAAACAT TTATTTTATC TTCTTCTTGT TCTCTCTTTC TCTTTCTCTC ATCACT         56 

ATG AAG AGT TTC AAC ACA GAA GGA CAC AAC CAC TCC ACG GCG GAA TCC      104 
Met Lys Ser Phe Asn Thr Glu Gly His Asn His Ser Thr Ala Glu Ser 
  1               5                  10                  15 

GGC GAT GCC TAC ACC GTG TCG GAC CCG ACA AAG AAC GTC GAT GAA GAT      152 
Gly Asp Ala Tyr Thr Val Ser Asp Pro Thr Lys Asn Val Asp Glu Asp 
             20                  25                  30 

GGT CGA GAG AAG CGT ACC GGG ACG TGG CTT ACG GCG AGT GCG CAT ATT      200 
Gly Arg Glu Lys Arg Thr Gly Thr Trp Leu Thr Ala Ser Ala His Ile 
         35                  40                  45 

ATC ACG GCG GTG ATA GGC TCC GGA GTG TTG TCT TTA GCA TGG GCT ATA      248 
Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile 
     50                  55                  60 

GCT CAG CTT GGT TGG ATC GCA GGG ACA TCG ATC TTA CTC ATT TTC TCG      296 
Ala Gln Leu Gly Trp Ile Ala Gly Thr Ser Ile Leu Leu Ile Phe Ser 
 65                  70                  75                  80 

TTC ATT ACT TAC TTC ACC TCC ACC ATG CTT GCC GAT TGC TAC CGT GCG      344 
Phe Ile Thr Tyr Phe Thr Ser Thr Met Leu Ala Asp Cys Tyr Arg Ala 
                 85                  90                  95 

CCG GAT CCC GTC ACC GGA AAA CGG AAT TAC ACT TAC ATG GAC GTT GTT      392 
Pro Asp Pro Val Thr Gly Lys Arg Asn Tyr Thr Tyr Met Asp Val Val 
            100                 105                 110 

CGA TCT TAC CTC GGT GGT AGG AAA GTG CAG CTC TGT GGA GTG GCA CAA      440 
Arg Ser Tyr Leu Gly Gly Arg Lys Val Gln Leu Cys Gly Val Ala Gln 
        115                 120                 125 

TAT GGG AAT CTG ATT GGG GTC ACT GTT GGT TAC ACC ATC ACT GCT TCT      488 
Tyr Gly Asn Leu Ile Gly Val Thr Val Gly Tyr Thr Ile Thr Ala Ser 
    130                 135                 140 

ATT AGT TTG GTA GCG GTA GGG AAA TCG AAC TGC TTC CAC GAT AAA GGG      536 
Ile Ser Leu Val Ala Val Gly Lys Ser Asn Cys Phe His Asp Lys Gly 
145                 150                 155                 160 

CAC ACT GCG GAT TGT ACT ATA TCG AAT TAT CCG TAT ATG GCG GTT TTT      584 
His Thr Ala Asp Cys Thr Ile Ser Asn Tyr Pro Tyr Met Ala Val Phe 
                165                 170                 175 

GGT ATC ATT CAA GTT ATT CTT AGC CAG ATC CCA AAT TTC CAC AAG CTC      632 
Gly Ile Ile Gln Val Ile Leu Ser Gln Ile Pro Asn Phe His Lys Leu 
            180                 185                 190 

TCT TTT CTT TCC ATT ATG GCC GCA GTC ATG TCC TTT ACT TAT GCA ACT      680 
Ser Phe Leu Ser Ile Met Ala Ala Val Met Ser Phe Thr Tyr Ala Thr 
        195                 200                 205 

ATT GGA ATC GGT CTA GCC ATC GCA ACC GTC GCA GGT GGG AAA GTG GGT      728 
Ile Gly Ile Gly Leu Ala Ile Ala Thr Val Ala Gly Gly Lys Val Gly 
    210                 215                 220 

AAG ACG AGT ATG ACG GGC ACA GCG GTT GGA GTA GAT GTA ACC GCA GCT      776 
Lys Thr Ser Met Thr Gly Thr Ala Val Gly Val Asp Val Thr Ala Ala 
225                 230                 235                 240 

CAA AAG ATA TGG AGA TCG TTT CAA GCG GTT GGG GAC ATA GCG TTC GCC      824 
Gln Lys Ile Trp Arg Ser Phe Gln Ala Val Gly Asp Ile Ala Phe Ala 
                245                 250                 255 

TAT GCT TAT GCC ACG GTT CTC ATC GAG ATT CAG GAT ACA CTA AGA TCT      872 
Tyr Ala Tyr Ala Thr Val Leu Ile Glu Ile Gln Asp Thr Leu Arg Ser 
            260                 265                 270 

AGC CCA GCT GAG AAC AAA GCC ATG AAA AGA GCA AGT CTT GTG GGA GTA      920 
Ser Pro Ala Glu Asn Lys Ala Met Lys Arg Ala Ser Leu Val Gly Val 
        275                 280                 285 

TCA ACC ACC ACT TTT TTC TAC ATC TTA TGT GGA TGC ATC GGC TAT GCT      968 
Ser Thr Thr Thr Phe Phe Tyr Ile Leu Cys Gly Cys Ile Gly Tyr Ala 
    290                 295                 300 

GCA TTT GGA AAC AAT GCC CCT GGA GAT TTC CTC ACA GAT TTC GGG TTT     1016 
Ala Phe Gly Asn Asn Ala Pro Gly Asp Phe Leu Thr Asp Phe Gly Phe 
305                 310                 315                 320 

TTC GAG CCC TTT TGG CTC ATT GAC TTT GCA AAC GCT TGC ATC GCT GTC     1064 
Phe Glu Pro Phe Trp Leu Ile Asp Phe Ala Asn Ala Cys Ile Ala Val 
                325                 330                 335 

CAC CTT ATT GGT GCC TAT CAG GTG TTC GCG CAG CCG ATA TTC CAG TTT     1112 
His Leu Ile Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Gln Phe 
            340                 345                 350 

GTT GAG AAA AAA TGC AAC AGA AAC TAT CCA GAC AAC AAG TTC ATC ACT     1160 
Val Glu Lys Lys Cys Asn Arg Asn Tyr Pro Asp Asn Lys Phe Ile Thr 
        355                 360                 365 

TCT GAA TAT TCA GTA AAC GTA CCT TTC CTT GGA AAA TTC AAC ATT AGC     1208 
Ser Glu Tyr Ser Val Asn Val Pro Phe Leu Gly Lys Phe Asn Ile Ser 
    370                 375                 380 

CTC TTC AGA TTG GTG TGG AGG ACA GCT TAT GTG GTT ATA ACC ACT GTT     1256 
Leu Phe Arg Leu Val Trp Arg Thr Ala Tyr Val Val Ile Thr Thr Val 
385                 390                 395                 400 

GTA GCT ATG ATA TTC CCT TTC TTC AAC GCG ATC TTA GGT CTT ATC GGA     1304 
Val Ala Met Ile Phe Pro Phe Phe Asn Ala Ile Leu Gly Leu Ile Gly 
                405                 410                 415 

GCA GCT TCC TTC TGG CCT TTA ACG GTT TAT TTC CCT GTG GAG ATG CAC     1352 
Ala Ala Ser Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met His 
            420                 425                 430 

ATT GCA CAA ACC AAG ATT AAG AAG TAC TCT GCT AGA TGG ATT GCG CTG     1400 
Ile Ala Gln Thr Lys Ile Lys Lys Tyr Ser Ala Arg Trp Ile Ala Leu 
        435                 440                 445 

AAA ACG ATG TGC TAT GTT TGC TTG ATC GTC TCG CTC TTA GCT GCA GCC     1448 
Lys Thr Met Cys Tyr Val Cys Leu Ile Val Ser Leu Leu Ala Ala Ala 
    450                 455                 460 

GGA TCC ATC GCA GGA CTT ATA AGT AGT GTC AAA ACC TAC AAG CCC TTC     1496 
Gly Ser Ile Ala Gly Leu Ile Ser Ser Val Lys Thr Tyr Lys Pro Phe 
465                 470                 475                 480 

CGG ACT ATG CAT GAG TGAGTTTGAG ATCCTCAAGA GAGTCAAAAA TATATGTAGT     1551 
Arg Thr Met His Glu 
                485 

AGTTTGGTCT TTCTGTTAAA CTATCTGGTG TCTAAATCCA ATGAGAATGC TTTATTGC     1611 

AAACTTCATG AATCTCTCTG TATCTACATC TTTCAATCTA ATACATATGA GCTCTTCC     1671 

AAAAAAAAAA AAAA                                                     1685 

 
           
           
             
               485 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              2 

Met Lys Ser Phe Asn Thr Glu Gly His Asn His Ser Thr Ala Glu Ser 
  1               5                  10                  15 

Gly Asp Ala Tyr Thr Val Ser Asp Pro Thr Lys Asn Val Asp Glu Asp 
             20                  25                  30 

Gly Arg Glu Lys Arg Thr Gly Thr Trp Leu Thr Ala Ser Ala His Ile 
         35                  40                  45 

Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile 
     50                  55                  60 

Ala Gln Leu Gly Trp Ile Ala Gly Thr Ser Ile Leu Leu Ile Phe Ser 
 65                  70                  75                  80 

Phe Ile Thr Tyr Phe Thr Ser Thr Met Leu Ala Asp Cys Tyr Arg Ala 
                 85                  90                  95 

Pro Asp Pro Val Thr Gly Lys Arg Asn Tyr Thr Tyr Met Asp Val Val 
            100                 105                 110 

Arg Ser Tyr Leu Gly Gly Arg Lys Val Gln Leu Cys Gly Val Ala Gln 
        115                 120                 125 

Tyr Gly Asn Leu Ile Gly Val Thr Val Gly Tyr Thr Ile Thr Ala Ser 
    130                 135                 140 

Ile Ser Leu Val Ala Val Gly Lys Ser Asn Cys Phe His Asp Lys Gly 
145                 150                 155                 160 

His Thr Ala Asp Cys Thr Ile Ser Asn Tyr Pro Tyr Met Ala Val Phe 
                165                 170                 175 

Gly Ile Ile Gln Val Ile Leu Ser Gln Ile Pro Asn Phe His Lys Leu 
            180                 185                 190 

Ser Phe Leu Ser Ile Met Ala Ala Val Met Ser Phe Thr Tyr Ala Thr 
        195                 200                 205 

Ile Gly Ile Gly Leu Ala Ile Ala Thr Val Ala Gly Gly Lys Val Gly 
    210                 215                 220 

Lys Thr Ser Met Thr Gly Thr Ala Val Gly Val Asp Val Thr Ala Ala 
225                 230                 235                 240 

Gln Lys Ile Trp Arg Ser Phe Gln Ala Val Gly Asp Ile Ala Phe Ala 
                245                 250                 255 

Tyr Ala Tyr Ala Thr Val Leu Ile Glu Ile Gln Asp Thr Leu Arg Ser 
            260                 265                 270 

Ser Pro Ala Glu Asn Lys Ala Met Lys Arg Ala Ser Leu Val Gly Val 
        275                 280                 285 

Ser Thr Thr Thr Phe Phe Tyr Ile Leu Cys Gly Cys Ile Gly Tyr Ala 
    290                 295                 300 

Ala Phe Gly Asn Asn Ala Pro Gly Asp Phe Leu Thr Asp Phe Gly Phe 
305                 310                 315                 320 

Phe Glu Pro Phe Trp Leu Ile Asp Phe Ala Asn Ala Cys Ile Ala Val 
                325                 330                 335 

His Leu Ile Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Gln Phe 
            340                 345                 350 

Val Glu Lys Lys Cys Asn Arg Asn Tyr Pro Asp Asn Lys Phe Ile Thr 
        355                 360                 365 

Ser Glu Tyr Ser Val Asn Val Pro Phe Leu Gly Lys Phe Asn Ile Ser 
    370                 375                 380 

Leu Phe Arg Leu Val Trp Arg Thr Ala Tyr Val Val Ile Thr Thr Val 
385                 390                 395                 400 

Val Ala Met Ile Phe Pro Phe Phe Asn Ala Ile Leu Gly Leu Ile Gly 
                405                 410                 415 

Ala Ala Ser Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met His 
            420                 425                 430 

Ile Ala Gln Thr Lys Ile Lys Lys Tyr Ser Ala Arg Trp Ile Ala Leu 
        435                 440                 445 

Lys Thr Met Cys Tyr Val Cys Leu Ile Val Ser Leu Leu Ala Ala Ala 
    450                 455                 460 

Gly Ser Ile Ala Gly Leu Ile Ser Ser Val Lys Thr Tyr Lys Pro Phe 
465                 470                 475                 480 

Arg Thr Met His Glu 
                485 

 
           
           
             
               1740 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               Arabidopsis thaliana  
             
             
               CDS  
                80..1558  
                /product= “amino acid transporter”
 
             
              3 

CTATTTTATA ATTCCTCTTC TTTTTGTTCA TAGCTTTGTA ATTATAGTCT TATTTCTCTT     60 

TAAGGCTCAA TAAGAGGAG ATG GGT GAA ACC GCT GCC GCC AAT AAC CAC CGT     112 
                     Met Gly Glu Thr Ala Ala Ala Asn Asn His Arg 
                       1               5                  10 

CAC CAC CAC CAT CAC GGC CAC CAG GTC TTT GAC GTG GCC AGC CAC GAT      160 
His His His His His Gly His Gln Val Phe Asp Val Ala Ser His Asp 
             15                  20                  25 

TTC GTC CCT CCA CAA CCG GCT TTT AAA TGC TTC GAT GAT GAT GGC CGC      208 
Phe Val Pro Pro Gln Pro Ala Phe Lys Cys Phe Asp Asp Asp Gly Arg 
         30                  35                  40 

CTC AAA AGA ACT GGG ACT GTT TGG ACC GCG AGC GCT CAT ATA ATA ACT      256 
Leu Lys Arg Thr Gly Thr Val Trp Thr Ala Ser Ala His Ile Ile Thr 
     45                  50                  55 

GCG GTT ATC GGA TCC GGC GTT TTG TCA TTG GCG TGG GCG ATT GCA CAG      304 
Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln 
 60                  65                  70                  75 

CTC GGA TGG ATC GCT GGC CCT GCT GTG ATG CTA TTG TTC TCT CTT GTT      352 
Leu Gly Trp Ile Ala Gly Pro Ala Val Met Leu Leu Phe Ser Leu Val 
                 80                  85                  90 

ACT CTT TAC TCC TCC ACA CTT CTT AGC GAC TGC TAC AGA ACC GGC GAT      400 
Thr Leu Tyr Ser Ser Thr Leu Leu Ser Asp Cys Tyr Arg Thr Gly Asp 
             95                 100                 105 

GCA GTG TCT GGC AAG AGA AAC TAC ACT TAC ATG GAT GCC GTT CGA TCA      448 
Ala Val Ser Gly Lys Arg Asn Tyr Thr Tyr Met Asp Ala Val Arg Ser 
        110                 115                 120 

ATT CTC GGT GGG TTC AAG TTC AAG ATT TGT GGG TTG ATT CAA TAC TTG      496 
Ile Leu Gly Gly Phe Lys Phe Lys Ile Cys Gly Leu Ile Gln Tyr Leu 
    125                 130                 135 

AAT CTC TTT GGT ATC GCA ATT GGA TAC ACG ATA GCA GCT TCC ATA AGC      544 
Asn Leu Phe Gly Ile Ala Ile Gly Tyr Thr Ile Ala Ala Ser Ile Ser 
140                 145                 150                 155 

ATG ATG GCG ATC AAG AGA TCC AAC TGC TTC CAC AAG AGT GGA GGA AAA      592 
Met Met Ala Ile Lys Arg Ser Asn Cys Phe His Lys Ser Gly Gly Lys 
                160                 165                 170 

GAC CCA TGT CAC ATG TCC AGT AAT CCT TAC ATG ATC GTA TTT GGT GTG      640 
Asp Pro Cys His Met Ser Ser Asn Pro Tyr Met Ile Val Phe Gly Val 
            175                 180                 185 

GCA GAG ATC TTG CTC TCT CAG GTT CCT GAT TTC GAT CAG ATT TGG TGG      688 
Ala Glu Ile Leu Leu Ser Gln Val Pro Asp Phe Asp Gln Ile Trp Trp 
        190                 195                 200 

ATC TCC ATT GTT GCA GCT GTT ATG TCC TTC ACT TAC TCT GCC ATT GGT      736 
Ile Ser Ile Val Ala Ala Val Met Ser Phe Thr Tyr Ser Ala Ile Gly 
    205                 210                 215 

CTA GCT CTT GGA ATC GTT CAA GTT GCA GCG AAT GGA GTT TTC AAA GGA      784 
Leu Ala Leu Gly Ile Val Gln Val Ala Ala Asn Gly Val Phe Lys Gly 
220                 225                 230                 235 

AGT CTC ACT GGA ATA AGC ATC GGA ACA GTG ACT CAA ACA CAG AAG ATA      832 
Ser Leu Thr Gly Ile Ser Ile Gly Thr Val Thr Gln Thr Gln Lys Ile 
                240                 245                 250 

TGG AGA ACC TTC CAA GCA CTT GGA GAC ATT GCC TTT GCG TAC TCA TAC      880 
Trp Arg Thr Phe Gln Ala Leu Gly Asp Ile Ala Phe Ala Tyr Ser Tyr 
            255                 260                 265 

TCT GTT GTC CTA ATC GAG ATT CAG GAT ACT GTA AGA TCC CCA CCG GCG      928 
Ser Val Val Leu Ile Glu Ile Gln Asp Thr Val Arg Ser Pro Pro Ala 
        270                 275                 280 

GAA TCG AAA ACG ATG AAG AAA GCA ACA AAA ATC AGT ATT GCC GTC ACA      976 
Glu Ser Lys Thr Met Lys Lys Ala Thr Lys Ile Ser Ile Ala Val Thr 
    285                 290                 295 

ACT ATC TTC TAC ATG CTA TGT GGC TCA ATG GGT TAT GCC GCT TTT GGA     1024 
Thr Ile Phe Tyr Met Leu Cys Gly Ser Met Gly Tyr Ala Ala Phe Gly 
300                 305                 310                 315 

GAT GCA GCA CCG GGA AAC CTC CTC ACC GGT TTT GGA TTC TAC AAC CCG     1072 
Asp Ala Ala Pro Gly Asn Leu Leu Thr Gly Phe Gly Phe Tyr Asn Pro 
                320                 325                 330 

TTT TGG CTC CTT GAC ATA GCT AAC GCC GCC ATT GTT GTC CAC CTC GTT     1120 
Phe Trp Leu Leu Asp Ile Ala Asn Ala Ala Ile Val Val His Leu Val 
            335                 340                 345 

GGA GCT TAC CAA GTC TTT GCT CAG CCC ATC TTT GCC TTT ATT GAA AAA     1168 
Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Ala Phe Ile Glu Lys 
        350                 355                 360 

TCA GTC GCA GAG AGA TAT CCA GAC AAT GAC TTC CTC AGC AAG GAA TTT     1216 
Ser Val Ala Glu Arg Tyr Pro Asp Asn Asp Phe Leu Ser Lys Glu Phe 
    365                 370                 375 

GAA ATC AGA ATC CCC GGA TTT AAG TCT CCT TAC AAA GTA AAC GTT TTC     1264 
Glu Ile Arg Ile Pro Gly Phe Lys Ser Pro Tyr Lys Val Asn Val Phe 
380                 385                 390                 395 

AGG ATG GTT TAC AGG AGT GGC TTT GTC GTT ACA ACC ACC GTG ATA TCG     1312 
Arg Met Val Tyr Arg Ser Gly Phe Val Val Thr Thr Thr Val Ile Ser 
                400                 405                 410 

ATG CTG ATG CCG TTT TTT AAC GAC GTG GTC GGG ATC TTA GGG GCG TTA     1360 
Met Leu Met Pro Phe Phe Asn Asp Val Val Gly Ile Leu Gly Ala Leu 
            415                 420                 425 

GGG TTT TGG CCC TTG ACG GTT TAT TTT CCG GTG GAG ATG TAT ATT AAG     1408 
Gly Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met Tyr Ile Lys 
        430                 435                 440 

CAG AGG AAG GTT GAG AAA TGG AGC ACG AGA TGG GTG TGT TTA CAG ATG     1456 
Gln Arg Lys Val Glu Lys Trp Ser Thr Arg Trp Val Cys Leu Gln Met 
    445                 450                 455 

CTT AGT GTT GCT TGT CTT GTG ATC TCG GTG GTC GCC GGG GTT GGA TCA     1504 
Leu Ser Val Ala Cys Leu Val Ile Ser Val Val Ala Gly Val Gly Ser 
460                 465                 470                 475 

ATC GCC GGA GTG ATG CTT GAT CTT AAG GTC TAT AAG CCA TTC AAG TCT     1552 
Ile Ala Gly Val Met Leu Asp Leu Lys Val Tyr Lys Pro Phe Lys Ser 
                480                 485                 490 

ACA TAT TGATGATTAT GGACCATGAA CAACAGAGAG AGTTGGTGTG TAAAGTTTAC      1608 
Thr Tyr 
CATTTCAAAG AAAACTCCAA AAATGTGTAT ATTGTATGTT GTTCTCATTT CGTATGGT     1668 

CATCTTTGTA ATAAAATTTA AAACTTATGT TATAAATTAT AAAAAAAAAA AAAAAAAA     1728 

AAAAAAAAAA AA                                                       1740 

 
           
           
             
               493 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              4 

Met Gly Glu Thr Ala Ala Ala Asn Asn His Arg His His His His His 
  1               5                  10                  15 

Gly His Gln Val Phe Asp Val Ala Ser His Asp Phe Val Pro Pro Gln 
             20                  25                  30 

Pro Ala Phe Lys Cys Phe Asp Asp Asp Gly Arg Leu Lys Arg Thr Gly 
         35                  40                  45 

Thr Val Trp Thr Ala Ser Ala His Ile Ile Thr Ala Val Ile Gly Ser 
     50                  55                  60 

Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln Leu Gly Trp Ile Ala 
 65                  70                  75                  80 

Gly Pro Ala Val Met Leu Leu Phe Ser Leu Val Thr Leu Tyr Ser Ser 
                 85                  90                  95 

Thr Leu Leu Ser Asp Cys Tyr Arg Thr Gly Asp Ala Val Ser Gly Lys 
            100                 105                 110 

Arg Asn Tyr Thr Tyr Met Asp Ala Val Arg Ser Ile Leu Gly Gly Phe 
        115                 120                 125 

Lys Phe Lys Ile Cys Gly Leu Ile Gln Tyr Leu Asn Leu Phe Gly Ile 
    130                 135                 140 

Ala Ile Gly Tyr Thr Ile Ala Ala Ser Ile Ser Met Met Ala Ile Lys 
145                 150                 155                 160 

Arg Ser Asn Cys Phe His Lys Ser Gly Gly Lys Asp Pro Cys His Met 
                165                 170                 175 

Ser Ser Asn Pro Tyr Met Ile Val Phe Gly Val Ala Glu Ile Leu Leu 
            180                 185                 190 

Ser Gln Val Pro Asp Phe Asp Gln Ile Trp Trp Ile Ser Ile Val Ala 
        195                 200                 205 

Ala Val Met Ser Phe Thr Tyr Ser Ala Ile Gly Leu Ala Leu Gly Ile 
    210                 215                 220 

Val Gln Val Ala Ala Asn Gly Val Phe Lys Gly Ser Leu Thr Gly Ile 
225                 230                 235                 240 

Ser Ile Gly Thr Val Thr Gln Thr Gln Lys Ile Trp Arg Thr Phe Gln 
                245                 250                 255 

Ala Leu Gly Asp Ile Ala Phe Ala Tyr Ser Tyr Ser Val Val Leu Ile 
            260                 265                 270 

Glu Ile Gln Asp Thr Val Arg Ser Pro Pro Ala Glu Ser Lys Thr Met 
        275                 280                 285 

Lys Lys Ala Thr Lys Ile Ser Ile Ala Val Thr Thr Ile Phe Tyr Met 
    290                 295                 300 

Leu Cys Gly Ser Met Gly Tyr Ala Ala Phe Gly Asp Ala Ala Pro Gly 
305                 310                 315                 320 

Asn Leu Leu Thr Gly Phe Gly Phe Tyr Asn Pro Phe Trp Leu Leu Asp 
                325                 330                 335 

Ile Ala Asn Ala Ala Ile Val Val His Leu Val Gly Ala Tyr Gln Val 
            340                 345                 350 

Phe Ala Gln Pro Ile Phe Ala Phe Ile Glu Lys Ser Val Ala Glu Arg 
        355                 360                 365 

Tyr Pro Asp Asn Asp Phe Leu Ser Lys Glu Phe Glu Ile Arg Ile Pro 
    370                 375                 380 

Gly Phe Lys Ser Pro Tyr Lys Val Asn Val Phe Arg Met Val Tyr Arg 
385                 390                 395                 400 

Ser Gly Phe Val Val Thr Thr Thr Val Ile Ser Met Leu Met Pro Phe 
                405                 410                 415 

Phe Asn Asp Val Val Gly Ile Leu Gly Ala Leu Gly Phe Trp Pro Leu 
            420                 425                 430 

Thr Val Tyr Phe Pro Val Glu Met Tyr Ile Lys Gln Arg Lys Val Glu 
        435                 440                 445 

Lys Trp Ser Thr Arg Trp Val Cys Leu Gln Met Leu Ser Val Ala Cys 
    450                 455                 460 

Leu Val Ile Ser Val Val Ala Gly Val Gly Ser Ile Ala Gly Val Met 
465                 470                 475                 480 

Leu Asp Leu Lys Val Tyr Lys Pro Phe Lys Ser Thr Tyr 
                485                 490

Technology Classification (CPC): 2