Patent Application: US-49930200-A

Abstract:
the present invention related to a cdna clone , designated to pepcyp 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 .

Description:
the present invention has identified a cdna clone , designated to pepcyp , from the incompatible interaction between pepper and the pepper anthracnose fungus colletotrichum gloeosporioides using mrna differential display and cdna library screening . the 1781 bp full - length sequence of pepcyp gene ( fig1 ) contains one open reading frame of 1506 bp from the first translation start ( atg ) at nucleotide position 4 to a translational stop ( tga ) at position 1509 ( genbank af122821 ). the nucleotide sequences of picc6 encode a polypeptide of 502 amino acids with a calculated molecular mass of 56 . 8 kda . a putative polyadenylation site was identified at 22 bp downstream of the stop codon . the amino acid sequence of this cdna is highly homologous to the genes encoding cytochrome p450s found in plants . therefore , the picc6 clone was designated pepcyp for pepper cytochrome p450 . the pepcyp protein contains a hydrophobic membrane anchor region in the n terminal region ( amino acid residues 1 to 27 ) ( bozak et al . 1990 ) ( fig2 ). a heme - binding domain ( residues 435 to 440 ), pfgxgxrxcxg , ( seq id no : 3 ) is located in the c terminal region of the polypeptide ( frey et al . 1995 ). sequence identity showed that the highest level was 59 % with a potato cytochrome p450 protein ( cyps . ch ) from a solanum chacoense line rich in glycoalkaloids ( hutvágner et al . 1997 ) ( fig2 ). sequence identity was 52 % and 48 % with cyp71d8 and cyp71d9 from soybean treated with an elicitor , respectively ( schopfer and ebel 1998 ). the identities with other cyp71 subfamilies were 46 % with avocado cyp71a1 ( bozak et al . 1990 ), 41 % with catmint cyp71a5 ( clark et al . 1997 ), and 40 % with arabidopsis cyp71b6 ( mizutani et al . 1998 ). the minimum identity of amino acid sequence required to assign a cytochrome p450 within the same family should be higher than 40 % ( nebert et al . 1991 ). thus , the pepper gene belongs to the cyp71 family . in the tobacco and phytopathogenic bacterium pseudomonas solanacearum interaction , the first cytochrome p450 gene hsr515 of tobacco that was expressed during the hypersensitive reaction was isolated ( czernic et al . 1996 ). the hsr515 protein shared 36 % identity with the pepcyp . we examined whether the expression of pepcyp gene was fruit - specific by fungal infection or inducible by other treatments . rna gel blot analysis was performed with total rnas prepared from fruits , leaves , stems , and roots of the pepper plants at 24 h after fungal inoculation or wounding . the expression of pepcyp gene was observed only in fruits , but not in leaves , stems , and roots after treatments ( fig3 a ). interestingly , the pepcyp mrna was induced in both ripe and unripe fruits by fungal infection , but wounding caused the induction of this mrna only in the ripe fruit . we further examined whether the wound - inducible pepcyp expression is inducible by aba or ja treatments . rna gel blot analysis was performed with total rnas prepared from the application sites of both ripe and unripe fruits drop - applied with aba or ja for 24 h . pepcyp mrna highly accumulated only in the ripe fruit treated with ja at 40 μm ( fig3 b ). however , aba did not affect the expression of pepcyp in both ripe and unripe fruits . to test whether a high concentration of ja is able to induce the expression of pepcyp in the unripe fruit , ja was applied to the unripe fruit at 100 , 400 , and 1000 μm . no induction of pepcyp expression was observed in the unripe fruit treated with ja ( data not shown ). we examined whether the induction of time - course of pepcyp mrna by c . gloeosporioides inoculation correlated with fungal morphogenesis and symptom development . rna gel blot analysis was performed with both unripe and ripe fruits at 0 , 3 , 6 , 12 , 24 , 48 , and 72 hais . the pepcyp mrna was not detected in both ripe and unripe fruits with water inoculation without fungal spores as a control . however , the accumulation of pepcyp mrna was detected in both ripe and unripe fruits from 12 hai ( fig4 ). in the unripe fruit , the expression of pepcyp gene is transient and peaks at 24 hai before rapidly declining to barely detectable levels at 48 and 72 hai . in contrast , in the ripe fruit , the expression level remains elevated . thus , the results show that the pepcyp gene is inducible by fungal infection and is differentially expressed in compatible and incompatible interactions . to examine the localization and accumulation of pepcyp mrna during early infection , we performed in situ hybridization using a gene - specific antisense or sense rna probe of pddicc6 ( fig1 ) with sections . the transverse - sections were prepared from the infection sites of both ripe and unripe fruits at 24 and 72 hais , respectively . the transcript of pepcyp was not detectable in uninoculated unripe ( fig5 a ) and ripe fruits ( fig5 d ) hybridized with anti - sense or sense rna probe ( data not shown ). in unripe fruit , fungus with infection hypha started to invade outer epidermal cells at 24 hai ( fig5 b ) ( oh et al . 1998 ). the accumulation of pepcyp mrna at 24 hai was localized only in the epidermal cells that were highly vacuolated , but not in the cortical cell layers ( fig5 b ). when the fungus colonized the outer epidermal cells at 72 hal the induction - level of transcripts was very low or undetectable ( fig5 c ). in ripe fruit , fungal invasion was rarely observed at 24 hai ( fig5 e ), and even at 72 hai ( fig5 f ). this result shows that fungal invasion and colonization are inhibited in incompatible - ripe fruit during early infection . the accumulation of the transcripts in the epidermal cells at 24 hai was sustained up to 72 hai . these results suggest that the expression of the pepcyp gene is localized to the epidermal cell layers of the ripe fruit during incompatible interaction . the pepcyp gene 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 pepcyp gene 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 . monoconidial isolate kg13 of c . gloeosporioides was cultured on potato dextrose agar ( difco , detroit , mich .) for 5 days in darkness at 28 ° c . sterile distilled water was added and conidia were harvested through four layers of cheesecloth to remove mycelial debris . ten μl of 5 × 10 5 conidium / ml of c . gloeospioides was used for drop - inoculation on both ripe and unripe pepper fruits as described ( oh et al . 1998 ). both ripe - red and unripe - mature - green fruits of pepper cv nokkwang were grown and harvested under greenhouse conditions . for wound treatments , five healthy ripe and unripe fruits were deeply scratched with a knife and incubated at 100 % relative humidity at 27 ° c . in the dark . ten μl of aba at 4 and 40 μm , or ja at 4 and 40 μm were drop - applied to both ripe and unripe sets of five fruits , respectively . after incubation under the conditions described above , the fruits were excised to 1 cm 2 at the drop - application site for the fungus , aba or ja , and at the wounding site . the samples were then 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 . total rna was extracted from healthy or infected ripe and unripe fruits using the rneasy plant kit ( qiagen , hilden , germany ) according to the manufacturer &# 39 ; s instructions . 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 ( operon , alameda , calif .). pcr - amplified cdna fragments were separated on denaturing 5 % polyacrylamide gels in tris - borate buffer . cdnas were recovered from the get , amplified by pcr , and cloned into pgem - t easy vector ( promega , madison , wis .) as described ( oh et al . 1995 ). poly ( a ) + mrna was purified from total rna of unripe - green fruits at 24 and 48 h after inoculation with c . gloeosporioides using the oligotex mrna kit ( qiagen ). the cdna library ( 2 . 5 × 10 5 plaque - forming unit with a mean insert size of 1 . 2 kb ) was constructed in the cloning vector λzapii ( stratagene , heidelburg , germany ) according to the manufacturer &# 39 ; s instructions . a partial cdna , designated pddicc6 , from the differential display analysis was used as a probe to screen the c . gloeosprioides - 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 . cdna sequencing was performed with an alfexpress automated dna sequencer ( amersham pharmacia biotech , buckinghamshire , uk ). analysis of nucleotide and amino acid sequences was performed using the dnasis sequence analysis software for windows , version 2 . 1 ( hitachi , san bruno , calif .). 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 ). 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 pharmacia biotech ). radiolabeled probes were prepared with [ α - 32 p ] dctp ( amersham pharmacia biotech ) using a random primer - labeling kit ( boehringer , mannheim , germany ). pepper fruits were fixed in 1 % glutaraldehyde / 2 % paraformaldehyde in 100 mm sodium phosphate buffer ph 7 . 0 , dehydrated in ethanol and embedded in paraffin . tissues were transverse - sectioned at 10 μm in thickness and stained with dapi ( 10 μg / ml ) to examine the infection hypha of the fungus in pepper fruits ( russell et al . 1975 ). pddicc6 was used to prepare gene - specific dig - labeled antisense rna probes using t7 rna polymerase or sense rna probes using sp6 rna polymerase . hybridization steps were performed according to the manufacture &# 39 ; s recommendation ( boehringer , mannheim , germany ). to isolate genes differentially induced from the ripe fruit but not from the unripe fruit in response to the fungal infection , we used mrna differential display ( liang and pardee 1992 ). differential display was performed with total rnas prepared from both unripe and ripe fruits at 24 and 48 h after fungal inoculation . the cdnas amplified from the ripe fruit were excised from the gel , re - amplified , and cloned . rna gel blot analysis with these clones was performed to confirm their differential expression . a cdna clone , named pddicc6 for the incompatible capsicum annuum / colletotrichum gloeosporioides interaction , hybridized to a transcript of 1 . 8 kb which accumulated to high levels in the incompatible interaction ( data not shown ). in order to isolate the full - length cdna clone , the insert of pddicc6 was used as a probe for plaque hybridization using a cdna library prepared from mrna extracted from the unripe fruit at 24 and 48 h after inoculation with the fungus . a clone containing the longest insert from cdna library screening was designated picc6 , isolated and sequenced . the 3 ′ region of picc6 clone contained the nucleotide sequence of pddicc6 as expected . sequence analysis and characterization of pepcyp cdna the 1781 bp full - length sequence ( fig1 ) contains one open reading frame of 1506 bp from the first translation start ( atg ) at nucleotide position 4 to a translational stop ( tga ) at position 1509 ( genbank af122821 ). the nucleotide sequences of picc6 encode a polypeptide of 502 amino acids with a calculated molecular mass of 56 . 8 kda . a putative polyadenylation site was identified at 22 bp downstream of the stop codon . the amino acid sequence of this cdna is highly homologous to the genes encoding cytochrome p450s found in plants . therefore , the picc6 clone was designated pepcyp for pepper cytochrome p450 . the pepcyp protein contains a hydrophobic membrane anchor region in the n terminal region ( amino acid residues 1 to 27 ) ( bozak et al . 1990 ) ( fig2 ). a heme - binding domain ( residues 435 to 440 ), pfgxgxrxcxg , is located in the c terminal region of the polypeptide ( frey et al . 1995 ). sequence identity showed that the highest level was 59 % with a potato cytochrome p450 protein ( cyps . ch ) from a solanum chacoense line rich in glycoalkaloids ( hutvágner et al . 1997 ) ( fig2 ). sequence identity was 52 % and 48 % with cyp71d8 and cyp71d9 from soybean treated with an elicitor , respectively ( schopfer and ebel 1998 ). the identities with other cyp71 subfamilies were 46 % with avocado cyp71a1 ( bozak et al . 1990 ), 41 % with catmint cyp71a5 ( clark et al . 1997 ), and 40 % with arabidopsis cyp71b6 ( mizutani et al . 1998 ). the minimum identity of amino acid sequence required to assign a cytochrome p450 within the same family should be higher than 40 % ( nebert et al . 1991 ). thus , the pepper gene belongs to the cyp71 family . in the tobacco and phytopathogenic bacterium pseudomonas solanacearum interaction , the first cytochrome p450 gene hsr515 of tobacco that was expressed during the hypersensitive reaction was isolated ( czernic et al . 1996 ). the hsr515 protein shared 36 % identity with the pepcyp . fruit - specific induction of pepcyp gene by fungal inoculation : up - regulation during ripening , and upon wounding and jasmonic acid treatments c . gloeosporioides causes anthracnose diseases on the fruit of various plant species ( daykin 1984 ; dodd et al . 1991 ; kim et al . 1986 , manandhar et al . 1995 , prusky et al . 1991 ). thus , we examined whether the expression of pepcyp gene was fruit - specific by fungal infection or inducible by other treatments . rna gel blot analysis was performed with total rnas prepared from fruits , leaves , stems , and roots of the pepper plants at 24 h after fungal inoculation or wounding . the expression of pepcyp gene was observed only in fruits , but not in leaves , stems , and roots after treatments ( fig3 a ). interestingly , the pepcyp mrna was induced in both ripe and unripe fruits by fungal infection , but wounding caused the induction of this mrna only in the ripe fruit . jasmonic acid ( ja ) is a plant hormone with roles in mechanical wounding responses ( creelman et al . 1992 ; creelman and mullet 1997 ). aba is hypothesized to be a key component in wound - signaling cascade leading to the activation of a defense gene ( pena - cortés et al . 1996 ; wasternack and partheir 1997 ). thus , we further examined whether the wound - inducible pepcyp expression is inducible by aba or ja treatments . rna gel blot analysis was performed with total rnas prepared from the application sites of both ripe and unripe fruits drop - applied with aba or ja for 24 h . pepcyp mrna highly accumulated only in the ripe fruit treated with ja at 40 μm ( fig3 b ). however , aba did not affect the expression of pepcyp in both ripe and unripe fruits . to test whether a high concentration of ja is able to induce the expression of pepcyp in the unripe fruit , ja was applied to the unripe fruit at 100 , 400 , and 1000 μm no induction of pepcyp expression was observed in the unripe fruit treated with ja ( data not shown ). in our previous studies ( kim et al . 1999 ; oh et al . 1998 ), higher levels of the appressorium and infection hypha formations were observed on the unripe fruit than on the ripe fruit at 12 h and 24 h after inoculation ( hai ), respectively . initial anthracnose symptoms were detected only on the unripe fruit after 48 hal , and typical sunken necrosis occurred within 120 hai . thus , we examined whether the induction of time - course of pepcyp mrna by c . gloeosporioides inoculation correlated with fungal morphogenesis and symptom development . rna gel blot analysis was performed with both unripe and ripe fruits at 0 , 3 , 6 , 12 , 24 , 48 , and 72 hais . the pepcyp mrna was not detected in both ripe and unripe fruits with water inoculation without fungal spores as a control . however , the accumulation of pepcyp mrna was detected in both ripe and unripe fruits from 12 hai ( fig4 ). in the unripe fruit , the expression of pepcyp gene is transient and peaks at 24 hai before rapidly declining to barely detectable levels at 48 and 72 hai . in contrast , in the ripe fruit , the expression level remains elevated . thus , the results show that the pepcyp gene is inducible by fungal infection and is differentially expressed in compatible and incompatible interactions . a cdna for the pr - 2 gene from nicotiana glutinosa was hybridized to the same blots to serve as a molecular marker for the activation of plant defense responses . in the unripe fruit , a basal level of pr - 2mrna was not detected , but the accumulation of pr - 2mrna was detected at 12 hai ( fig4 ). and a biphasic accumulation of pr - 2 mrna was observed at 12 and 72 hais . in contrast , in the ripe fruit , a basal level of pr - 2 mrna was detected . the expression of pr - 2 gene was rapidly induced in the ripe fruit at 3 ha and reached a maximum at 48 hai . to examine the localization and accumulation of pepcyp mrna during early infection , we performed in situ hybridization using a gene - specific antisense or sense rna probe of pddicc6 ( fig1 ) with sections . the transverse - sections were prepared from the infection sites of both ripe and unripe fruits at 24 and 72 hais , respectively . the transcript of pepcyp was not detectable in uninoculated unripe ( fig5 a ) and ripe fruits ( fig5 d ) hybridized with anti - sense or sense rna probe ( data not shown ). in unripe fruit , fungus with infection hypha started to invade outer epidermal cells at 24 hai ( fig5 b ) ( oh et al . 1998 ). the accumulation of pepcyp mrna at 24 hai was localized only in the epidermal cells that were highly vacuolated , but not in the cortical cell layers ( fig5 b ). when the fungus colonized the outer epidermal cells at 72 hai , the induction - level of transcripts was very low or undetectable ( fig5 c ). in ripe fruit , fungal invasion was rarely observed at 24 hai ( fig5 e ), and even at 72 hai ( fig5 f ). this result shows that fungal invasion and colonization are inhibited in incompatible - ripe fruit during early infection . the accumulation of the transcripts in the epidermal cells at 24 hai was sustained up to 72 hai . these results suggest that the expression of the pepcyp gene is localized to the epidermal cell layers of the ripe fruit during incompatible interaction . as a first step to investigate the molecular mechanisms involved in the incompatible interaction between the ripe fruit of pepper and c . gloeosporioides , several cdnas were isolated that were differentially expressed in the ripe fruit by fungal infection , but not in the unripe fruit . in this study with one of these cdnas , we showed the characterization of the pepcyp gene that encodes a protein homologous to plant cytochrome p450 ( bozak et al . 1990 ; frey et al . 1995 ). cytochrome p450s in plants are membrane - bound proteins involved in several metabolic pathways related to the defense mechanisms ( maule and ride 1983 ; kessmann et al . 1990 ). some genes encoding these proteins are induced by wounding ( batard et al . 1997 ; frank et al . 1996 ). in a plant - phytopathogenic bacterium interaction , the tobacco cytochrome p450 gene , hsr515 , was isolated during hypersensitive reaction ( czernic et al . 1996 ). in this study of a fungal - plant interaction , a pepper cytochrome p450 gene , pepcyp , was differentially expressed in compatible and incompatible interactions . transcript levels of the two interactions were very different with maintenance of elevated levels in the incompatible interaction and a very substantial reduction in the compatible interaction . together with the hsr515gene in a bacterial - plant interaction , the isolation of pepcyp in the pepper and fungus interaction suggests a new role for cytochrome p450s in plant - pathogen interactions . sequence comparison showed that pepcyp protein shared highest homology to the cyps . ch from a solanum chacoense line rich in glycoalkaloids ( hutvágner et al . 1997 ) as well as cyp71d8 and cyp71d9 from soybean treated with an elicitor ( schopfer and ebel 1998 ). a possible role of cyps . ch was suggested to be involved in the synthesis of stress - inducible metabolites . cyp71d8 and cyp71d9 may have a variety of functional roles in terpenoid metabolism ( christoffersen et al . 1995 ). the antimicrobial sesquiterpenoid phytoalexin , capsidol ( chavez - moctezuma and lozoya - gloria 1996 ; watson and brooks 1984 ), was synthesized in pepper challenged with fungus ( ward 1976 ) and an abiotic elicitor , uv light ( back et al . 1998 ). therefore , these data raise the possibility that pepcyp functions in the pepper plant &# 39 ; s defense against fungal infection . the expression of pepcyp gene in the pepper fruit in response to fungal inoculation and wounding ( fig3 and 4 ) supports a possible role of pepcyp involved in the plant &# 39 ; s defense mechanism . the first cytochrome p450 , cyp71a , in plants was identified during avocado fruit ripening ( bozak et al . 1990 ). in this study , a basal level of pepcyp mrna was not detected in ripe or unripe fruits or other various organs of pepper . however , the induction of pepcyp was detected only in fruit after fungal inoculation ( fig3 a ). in addition , the expression of pepcyp was induced only in ripe fruit by wounding and ja treatment ( fig3 a and b ). thus , these results suggest that pepcyp is developmentally and fruit - specifically regulated , and the induction is upregulated during fruit ripening in response to wounding and ja . ja is reported to have roles in mechanical wounding responses ( creelman et al . 1992 ; creelman and mullet 1997 ) and in activating genes for plant disease resistance ( johnson et al . 1989 ; xu et al . 1994 ; reinbothe et al . 1994 ). however , the role of ja during the fruit ripening has not been well studied , in contrast to ethylene ( theologis 1992 ). a few cases that methyl ja triggers the ripening process of climacteric fruits including tomato and apple with ethylene production were reported ( czapski and saniewski 1992 ; saniewski et al . 1987a , 1987b ). however , the role of ja in nonclimacteric fruits such as pepper , grape and strawberry has not been reported . fruit ripening represents a genetically synchronized developmental process unique to plants . generally , ripe fruit is accompanied by an increased susceptibility to pathogen infection ( prusky et al . 1991 ; swinburn 1983 ). as one of the reproductive organs of the plants , the fruit must be protected from pathogens or abiotic stresses . pr proteins and several antifungal proteins that are responsible for the protection against pathogens during fruit ripening have been identified ( fils - lycaon et al . 1996 ; meyer et al . 1996 ; salzman et al . 1998 ; tattersall et al . 1997 ). in the present study , the expression of pepcyp gene was detected only in the ripe fruit after fungal inoculation or wounding . we propose that the pepcyp gene is involved in the defense mechanism for the ripe fruit in order to maintain fruit integrity and to protect seed maturation against biotic and abiotic stresses . initial and mature infection hypha of c . gloeosporioides developed on pepper fruits at 12 and 24 hais , respectively ( oh et al . 1998 ). pepcyp mrna in the fruit started to accumulate from 12 hai and increased at 24 hai ( fig4 ). thus , it is likely that pepcyp gene expression occurs when the fungus directly invades the fruit by infection hypha . in microscopic and in situ hybridization observations , although cells didn &# 39 ; t directly contact with the fungus , the induction of pepcyp transcript was detected throughout the epidermal cell layers . this result suggests that transcripts are induced by plant - derived defense signals generated after the fungus invasion . on the other hand , the accumulation of pr - 2 mrna in the ripe fruit at 3 hai when the fungus germinates suggests that this gene is induced early in the incompatible interaction by fungal elicitors rather than plant - derived signals . the induction of pepcyp and pr - 2 mrnas was observed to be higher and faster , respectively , in the incompatible interaction than in the compatible interaction . these similar phenomena have been reported for many other plant - pathogen interactions ( ebrahim - nesbat et al . 1989 ; 1993 ). thus , higher and faster expression of many defense genes including pepcyp and pr - 2 may confer disease resistance for the ripe fruit against fungal infection . in summary , the present study showed that active fungal invasion and colonization processes are suppressed in the incompatible - interacting ripe fruit . notably , pepcyp mrna accumulated to higher levels in the ripe fruit in response to the fungal infection . the transcript is mainly localized in the epidermal cell layers of the pepper fruit after the fungal inoculation . we suggest that the pepcyp gene product plays a critical role in the plant &# 39 ; s defense mechanism against the fungal invasion and colonization of the epidermal cells of the fruit in the incompatible interaction . it remains to be elucidated how the cytochrome p450 protein provides an effective defense against the fungal infection in pepper . adikaram , n . k . b ., brown , a . e ., and swinburne , t . r . 1983 . observations on infection of capsicum annuum fruit by glomerella cingulata and colletotrichum capsici . trans . brit . mycol . soc . 80 : 395401 . altschul , s . f ., madden , t . l ., schäffer , a . a ., zhang , j ., zhang , z ., miller , w , and lipman , d . j . 1997 . gapped blast and psi - blast : a new generation of protein database search programs . nucleic acids res . 25 : 3389 - 3402 . back , k ., he , s ., kim , k . u ., and shin , d . h . 1998 . cloning and bacterial expression of sesquiterpene cyclase , a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in uv - challenged leaves of capsicum annuum . plant cell physiol . 39 : 899 - 904 . bailey , j . a ., o &# 39 ; connell , 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