Patent Application: US-85456201-A

Abstract:
the present invention related to two cdna clones , designated to pepdef and pepthi 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 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 . 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 . 2 ) contained an n - terminal secretory signal peptide that was cleaved after glycine at position 25 ( fig1 ). pepthi is a cys - rich polypeptide containing the consensus cys arrangement — c ( . . . ) c — x — x — x — c ( . . . ) g - x — c ( . . . ) c — x — c —. 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 dna 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 . 4 ) contained an n - terminal secretory signal peptide that was cleaved after alanine at position 27 ( fig1 ). pepdef is also a cys - rich polypeptide containing the consensus cys arrangement — c ( . . . ) c — x — x — x — c ( . . . ) g - x — c ( . . . ) c — x — c —. 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 . 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 . 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 . 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 . 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 ( fig4 ). 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 . 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 . 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 ). 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 . 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 ). 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 . 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 . 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 ). 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 ). 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 . 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 ( fig1 ). pepthi is a cys - rich polypeptide containing the consensus cys arrangement — c ( . . . ) c — x — x — x — c ( . . . ) g - x — c ( . . . ) c — x — c —. 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 ; fig1 ). 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 . 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 ( fig2 ). 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 . 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 ( fig2 ). 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 fig3 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 . 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 ( fig2 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 ( fig3 ). in compatible - unripe fruits , the accumulation of pepthi mrna was late , at 12 hai , and reached its maximum level at 72 hai . accumulation of pepdef mrna in the unripe fruit was very low ( fig3 ). as shown in fig2 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 . 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 ( fig4 ). 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 . 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 ( fig3 ). 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 ( fig2 ), 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 ( fig4 ) 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 ). 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 . 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 ( fig4 ). these data indicate that the pepthi gene is expressed via a ja - independent wound signal transduction pathway . 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 . 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 . 1 . altschul s f , madden t l , schäffer a a , zhang j , zhang z , miller w , lipman d j : gapped blast and psi - blast : a new generation of protein database search programs . nucleic acids res 25 : 3389 - 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