Patent Application: US-84837504-A

Abstract:
this invention relates to dual roles of the pepper esterase protein as a biocontrol agent that can elicit defense reactions in plant cells and as direct fungal inhibitor that can block fungal penetration into plant cells . the possible enzymatic actions of the pepest protein were elucidated in both fungus and plant to gain insight into the molecular mechanisms involved in the fungal resistance . exogenously treated pepest protein on the unripe pepper fruits decomposed the cuticles of the fruits and released glycerol and 9 - octadecenamide . the treatment resulted in the massive generation of hydrogen peroxide in the unripe pepper fruit and simultaneously elicited the expression of defense - related genes in both absence and presence of the fungus .

Description:
carboxylesterases are enzymes that catalyze the hydrolysis of compounds containing an ester bond . in plant - microbe interactions , a tobacco esterase and two arabidopsis lipase genes had been isolated in a hypersensitive reaction against phyto - pathogen . a pepper esterase gene ( pepest ) that is highly expressed during the incompatible interaction between the ripe fruit and c . gloeosporioides were cloned from pepper . the recombinant pepest protein expressed in e . coli ( fig1 a ) hydrolyzed p - nitrophenyl esters but did not show detestable level of lipase activity . since natural substrates for plant esterase are not identified yet , little is known about physiological role of the protein in resistance mechanism of the ripe pepper fruits . identification of compounds generated by pepest will help clarify the role of plant esterases in the defense system . so , the effect of pepest proteins by exogenous treatment was observed on the unripe pepper fruits in terms of resistance induction . in the present work , pepest protein was shown to sufficient to stimulate a complex defense response in pepper cells comprising the release of cutin monomer , h 2 o 2 generation , and defense - related gene activation . the results suggest that pepest gene product is involved in a defense mechanism against the fungal invasion in outer - epidermal cells of the ripe fruits . active oxygen species ( aos ) have been found to play a number of critical roles in defense responses during plant - pathogen interactions . they can function as messengers for activation of defense response genes beside the direct anti - microbial effect of aos . we used the unripe pepper fruit to correlate biochemically oriented elicitor experiments . a cutin monomer , 9 - octadecenamide , was released from the cuticle of the fruit when pepest protein was topically applied on the unripe pepper fruit ( fig2 ). in addition to cutin monomer solubilization , h 2 o 2 elicitation was also enhanced by the pepest protein . in the unripe fruits , h 2 o 2 generation was doubled at distinct time - points ( 6 h ) ( fig3 ). since it could function as activator of defense - related genes as suggested by liyama et al ( 1994 ), the expression of defense - related genes was determined in pepper after the treatment . several pepper pr genes were induced by exogenous application of pepest protein on the unripe fruits ( fig4 ). the expression patterns were similar to those by exogenous h 2 o 2 treatment . apparently , h 2 o 2 modulation can lead to enhanced disease resistance without significant negative effects . of particular interest in this respect is the observation that h 2 o 2 modulation is also activated by pepest treatment during the invasion of the fungus into pepper cells ( fig5 ). pr genes were locally induced in the infection site by adjacent application of pepest protein with fungal spore on the unripe fruits . taken together , these results suggest that the pepest gene as an esterase is involved in the defense response mechanism for the ripe fruit against pathogens . on the other hand , the viability of the fungus was severely impaired by pepest treatment even when the fungal hypha was still elongated ( fig6 ). ultimately , pepest protein could inhibit fungal development with mild fungicidal activity so that fungal colonization was completely blocked from the initial infection stage ( fig7 b ). to elucidate the important role of pepest protein in fungal morphogenesis , we analyzed the walls from germinated conidia of anthracnose fungus treated with pepest recombinant protein ( fig8 ). the surface of fungus was observed by using fitc - labeled concanavalin a ( con a ), which binds to the sugar residues on the cell wall ( fig9 a ). unlike normal fungus , the surface of the cell wall treated with pepest was perturbed when probed with cona - fitc . in the next to determine whether pepest hydrolyzes the cell walls , the structure of the walls was observed by using a scanning electron microscope ( fig9 b ). the surface of the fungal cell walls was covered with undefined mucilage materials in distilled water , but the skeletons of the cell wall structure were exposed by enzymatic action of pepest protein . the results strongly suggest that pepest protein can hydrolyze the materials covering the fungal walls . finally , a dithiane compound , 1 , 2 - dithiane - 4 , 5 - diol , was detected with glycerol in the solutions from pepest - treated fungal cell walls ( fig1 ). in conclusion , the pepper esterase ( pepest ) protects the susceptible unripe fruit against fungal infection by eliciting h 2 o 2 generation and subsequently inducing the expression of defense - related genes ( fig1 ). it was shown that the pepper esterase can hydrolyze the parts of plant cells as well as the surrounding cell layer of fungus . these results indicate that pepest protein can be used for disease control as an effective biocontrol agent ( fig1 ). application of pepest protein in pepper plants will benefit not only farmers by cutting their cultivation cost but also the environment by reducing the amount of pesticides used . monoconidial isolate kg13 of c . gloeosporioides was cultured on potato dextrose agar ( pda ) ( difco ) for 7 days in darkness at 28 ° c . conidia were harvested and suspended in sterile distilled water . ten microliters of spore suspension ( 5 × 10 5 spores / ml ) were used for drop inoculation in vitro and in vivo and amended with 10 μl ( final concentration of 1 μg ) of recombinant pepest and applied to cover glasses . as control , 1 μg of gst or sterile water was used . the cover glasses were incubated in humidified chamber at 25 ° c . in the dark . then , the spore suspension was stained with 0 . 1 % ( wt / vol ) cotton blue in lactophenol . spore germination and appressorium formation on the cover glass were observed under a light microscope ( olympus ). dosage - response experiments of recombinant pepest on fungal morphogenesis were performed . inoculation tests with spores amended with recombinant pepest per were performed on healthy unripe fruits as previously described ( oh et al ., 1998 ). the fruits were then placed in plastic boxes ( 25 × 16 × 6 cm ) containing wet paper towels to maintain 100 % relative humidity in darkness at 28 ° c . after incubation , the fruits were excised to 1 cm 2 at the drop - application site for the fungus . the samples were then frozen in liquid nitrogen . unripe mature - green pepper fruits were obtained from pepper plants under greenhouse conditions grown at gwangju , korea . 2 . solubilization of surface wax from pepper and fungus by pepest 10 μl of pepest protein ( 0 . 1 mg / ml ) was applied topically on the unripe pepper fruit . the spores were amended with 1 μg of pepest protein in 10 μl of distilled water . after 24 hrs of treatment , the soluble fraction was collected , lyophilized , and finally dissolved in chloroform . the wax extract was analyzed by gc - mass spectrometry ( hewlett packard ). 1 g of pepper fruits were frozen in liquid nitrogen and ground to powder in a mortar together with ice - cold 5 % tca and 45 mg of activated charcoal . the powder was centrifuged at 18000 g for 10 min at 0 ° c . and the supernatant was immediately filtered ( advantec 0 . 45 μm ) under pressure from a syringe to remove any traces of precipitated protein and activated charcoal . the filtrate was adjusted to ph 8 . 4 with nh 4 oh then refiltrate to remove the precipitate , which formed when extracts were neutralized . the amount of h 2 o 2 in the resulting extract was tested by pipetting 100 μl test solution into 100 μl of 0 . 5 mm luminol ( 3 - aminophthalhydrazide , sigma ) in 0 . 2m nh 4 oh ( ph 9 . 5 ). the test tube was placed in a measuring cuvette of aminco chem glow photometer for chemiluminescence ( cl ) emission initiated by injecting 100 μl of 1 mm k 3 fe ( cn ) 6 ( in 0 . 2m nh 4 oh ) into the mixture . the emitted photons were counted for 5 sec with a pulse integrator ( lumat lb9501 ). in addition , histochemical detection of h 2 o 2 was performed by an endogenous peroxidase - dependent staining procedure using dab ( 3 , 3 ′- diaminobenzidine ) as described by thordal - christensen et al . ( 1997 ). the unripe fruits treated with pepest protein were placed in a solution of 1 mg / ml dab for 8 hr . total rna was extracted by using trizol reagent ( gibco brl ) according to the manufacturer &# 39 ; s instructions . the equal amounts of total rna ( 10 μg per lane ) were separated on a 1 . 2 % denaturing agarose gels in the presence of formaldehyde . rna gel blotting , hybridization , and washing were conducted as described by the manufacturer of positively charged nylon membranes ( hybond n + ; amersham pharmacia biotech ). radiolabeled probes were prepared with [ α - 32 p ] dctp ( 3000 ci / mmol , perkinelmer ), using redi - prime ™ kit ( amersham pharmacia biotech ). membranes were washed twice with 2 × sspe , 0 . 1 % sds at 65 ° c . and once with 0 . 5 × sspe , 0 . 1 % sds at 65 ° c . probes for defense - related genes such as pr3 ( chitinase class ii ( cachi2 )), pr5 ( thaumatin - like protein ( tlp )), pr10 , pepthi , were used to examine their gene expression in pepper fruits by northern hybridization or rt - pcr . the primer pairs used for the pcr analysis were pr3 - 5 ′ ( atg gag ttc tct gta tca cca gtg g ) ( seq id no : 3 ), 3 ′ ( tcc gaa tgt cta aag tgg tac aag ) ( seq id no : 4 ), pr5 - 5 ′ ( atg ggc tat ttg aga tca tct ) ( seq id no : 5 ), 3 ′ ( tca cct ctc tgc aat caa tat ) ( seq id no : 6 ), pr10 - 5 ′ ( ctg aca agt cca cag cct cag ) ( seq id no : 7 ), 3 ′ ( gtt ctt tcc atg aca acc aat tg ) ( seq id no : 8 ), pepthi - 5 ′( ggg gga tcc aaa atg gct cgt tcc ) ( seq id no : 9 ), 3 ′( ctc ggt acc ctt tat tta att ttg tgt gac act ) ( seq id no : 10 ), respectively . a single colony of e . coli cells containing a recombinant pgex6p - 1 plasmid was picked to inoculate 5 ml of 2 × yta ( tryptone 16 g / l , yeast extract 10 g / l , nacl 5 g / l , ampicillin 100 mg / l ) medium and incubate for 12 – 15 hrs at 37 ° c . with vigorous shaking . dilute the culture 1 : 100 into fresh pre - warmed 2 × yta medium and grow at 37 ° c . with shaking until absorbance a 600 reaches 1 . 0 . then , 0 . 2 mm iptg at the final concentration was added to the culture and continued the incubation for additional 3 hrs 30 ° c . finally , the culture was centrifuged at 12 , 000 rpm for 10 minutes at 4 ° c . to sediment the cells . the supernatant was discarded . the cell pellet was completely suspended by adding 50 μl ice - cold 1 × pbs per ml of culture medium . suspended cells were disrupted using a sonicator on ice in short bursts . according to the manufacturer &# 39 ; s instructions , the lysate was bound to glutathione sepharose 4b and then the fusion protein was treated with 80 u of prescission protease in order to remove reduced gst from the pepest protein . protein content was determined by bradford method ( 1979 ). sds - page was performed on 10 % polyacrylamide gels . to partially separate pepest protein , the lysate was applied to 120 cm gel - filtration column ( sephadex g - 150 , sigma ). separated proteins in no . 9 to 26 fractions were mixed , lyophilized , and dissolved in 1 mm dithiothreitol ( dtt ) before use . fluorescein isothiocyanate ( fitc ) was freshly dissolved in dmso to 10 mg / ml and added to 1 mg / ml of purified pepest protein in 100 mm sodium bicarbonate ( ph 9 . 3 ) to final concentration of 1 mg / ml fitc . after incubation for 4 hrs in the dark at the room temperature , 1 m ethanolamine was added to inactivate the residual fitc . the solution was left in the dark for additional 2 hrs and applied for gel - filtration column chromatography using a 120 cm sephadex g - 50 ( sigma ) column to remove unconjugated dyes . separated samples are lyophilized . the conjugation between fitc and purified pepest was verified on sds - page gel with intense fluorescence of purified pepest under uv light . to observe the viability of the fungus , live / dead ® baclight ™ bacterial viability kit ( molecular probes ) was used according to the manufacturer &# 39 ; s instructions . live / dead consists of two reagents containing a mixture of fluorescent nucleic acid stains syto9 ( green ) and propidium iodide ( red ) in anhydrous dmso . pepper fruits pieces were prepared for scanning electron microscope ( sem ) after exogenous pepest protein treatment . small segments ( 5 mm long ) were vacuum - infiltrated in sodium phosphate buffer ( ph 7 . 0 ) containing 2 % sucrose , 3 % paraformaldehyde and 0 . 2 % glutaraldehyde on ice for 6 hrs . following this treatment , the material was slowly dehydrated in a graded ethanol series at room temperature . after the last change of ethanol , the samples were immediately submitted to critical point drying , gold coated , and observed with a scanning electron microscope ( hitachi ). fluorescein isothiocyanate - labeled cona ( sigma ) was used to examine the surface structure of the fungus after the treatment of pepest protein . germinated fungi were treated with 0 . 1 mg / ml fitc - cona in pbs and examined under a fluorescence microscope ( olympus ). the open reading frame of pepest cdna ( af122821 ) was inserted in - frame with the glutathion - s - transferase ( gst ) coding sequence in expression vector , pgex6p - 1 between ecori and xhoi sites . the gst - pepest fusion protein was expressed in e . coli and then purified . fig1 a shows that 63 . 5 kd gst - pepest fusion protein is cleaved with gst protein resulting a 36 . 5 kd pepest protein . to partially separate pepest protein , the lysate was applied to 120 cm gel - filtration column ( sephadex g - 150 , sigma ). in fig1 b , separated proteins in no . 9 to 26 fractions were separated on sds - page . and pepest protein was detected by western blotting in the same fraction ( fig1 c ). the cuticle is the outer protective layer , which covers the aerial part of higher plant . to penetrate into the underlying epidermis , fungus used an enzyme hydrolyzing plant cuticles . it has been reported that degradation products from the cuticle could activate defense reaction in the host plants . in the present experiment , pepest protein was topically applied on healthy unripe pepper fruit to clarify possible involvement of the degradation compound from cuticles . as the result , glycerol and 9 - octadecenamide was released from the cuticle of the fruit treated with pepest protein ( fig2 ). to demonstrate the effect of pepest protein on plant cells , generation of h 2 o 2 was examined as a parameter for defense and induction of defense related genes in the unripe fruit treated with pepest protein . in fig3 a , the results show that the application of distilled water on the unripe fruit as control did not affect hydrogen peroxide generation . however , dark brown precipitates were homogenously detected in the epidermal cells of unripe fruit treated with pepest protein for 6 hrs . to examine the time - course of differential accumulation of h 2 o 2 by pepest protein , the amount of hydrogen peroxide was measured during a day after the treatment . the accumulation pattern of h 2 o 2 associated with pepest treatment was quantitatively , but not qualitatively distinguishable from the application of distilled water as control ( fig3 b ). generation of h 2 o 2 was induced biphasically at 0 . 5 and 6 hrs after the treatment . generation of h 2 o 2 was significantly higher in the fruit treated with pepest protein than the control . to test whether pepest protein induces pr gene expression , rna blot analysis was performed on unripe fruits after treatment with pepest protein . also , we examined the level of pr gene transcripts in unripe fruits after treatment with hydrogen peroxide as h 2 o 2 is known to elicit resistance reaction in plants . pr genes such as pr3 , pr5 , pr10 and pepthi were induced in the fruits treated with hydrogen peroxide ( fig4 ). pr3 and pr5 were strongly induced from the early stage of incubation and then gradually decreased . in contrast , the transcripts of pr10 were strongly induced with time . upon pepest protein treatment , expression of pr genes showed similar patterns with those induced by hydrogen peroxide , but their level of expression slightly decreased , except for pepthi whose transcripts accumulated at 6 hrs after treatment . treatment of the unripe pepper with pepest protein led to the induction of defense related genes in the absence of the fungal pathogen , suggesting that pepest protein induced resistance protects pepper fruits from the fungal pathogen . to investigate whether the induction of pr genes occurs in infected fruits with anthracnose fungus , the expression pattern of pr genes was monitored in the treated fruits after inoculation of fungal spores . fig5 shows that the transcripts corresponding to pr5 and 10 genes accumulated only in the treated fruits at 72 hrs after inoculation . the conidia of c . gloeosporioides started to germinate at 1 hr after deposition on the surface of unripe pepper fruits or cover glass . one conidium produced a germination tube with swollen tip forming an appressorium . first apressorium developed at 6 hrs . however , normal development of the fungus was severely impaired by the application of pepest recombinant protein . fig7 a shows an abnormal development of the fungus treated with pepest protein on cover glass at 24 hrs . at low concentrations of pepest protein , the growing tip of hypha tended to elongate and sometimes produced new spores without developing appressorium . the growth of fungus was completely blocked at 0 . 5 mg / ml of pepest . viability of the fungus showing an abnormal growth pattern was examined by staining with live / dead ® baclight ™ bacterial viability kit based on two - color fluorescence assay of cell viability . the stains have different spectral characteristics and differ in their ability to penetrate fungal cell membranes . cells with intact membranes labeled with green fluorescence , whereas cells with damaged membranes stained fluorescent red . the results show that viability of the fungus was severely affected even at low concentration of pepest protein ( fig6 ). in addition , fungal morphology on the unripe pepper fruit was observed by using a scanning electron microscope ( hithachi ). the spores of c . gloeosporioides were inoculated on the unripe pepper fruits with pepest protein and then incubated for 24 hrs . fungal growth proceeded as usual with distilled water as control and germination tube with appressorium was observed ( fig7 a ). by treating with 1 μg of pepest protein , appressorium did not develop on the fungus , and the fungus showed severe abnormality at 5 μg of the protein . surface damage of the fungus after exogenous treatment with pepest protein to observe celullar localization of pepest protein , pepest protein was labeled with fitc as described above ( fig8 a ). gst protein was used as a control . the gst - fitc protein accumulated inside the fungal cells after 24 hrs of incubation . the growth and differentiation of the spores were not affected at all . however , pepest - fitc protein did attach to the outer surface of hypha ( fig8 b ). since pepest protein is a hydrolytic enzyme with esterase activity , the surface of the fungus treated with the protein was examined by both fluorescence and scanning electron microscopes . labeling of cell walls with cona - fitc resulted in an even fluorescence on the outside walls of the fungus germinated in distilled water as control . however , the labeled walls of the fungus treated with pepest protein appear severely altered . the pepest protein induced change in cell wall morphology indicates that pepest protein dissolves the materials deposited on the outside of the walls ( fig9 a ). to determine whether pepest protein hydrolyzes the cell wall , ultrastructure of the affected fungus surface was observed with a field - emission scanning electron microscope ( hitachi ). the surface of the fungal wall was covered with undefined mucilage materials in distilled water , but the skeletons of the wall structure was exposed after the treatment with pepest protein ( fig9 b ). decomposed materials from the treated fruits were analyzed using gc - mass spectrometry . two compounds were detected in the digested solution , glycerol and 1 , 2 - dithiane - 4 , 5 - diol ( fig1 ). by application of pepest protein in a dose - dependent manner , the fungal appressorium formation was significantly inhibited in the infected fruits with the anthracnose fungus . blocking of the appressorium development resulted in protection of the unripe fruit against the fungus ( fig1 ). the epidermal cells of pepper fruit remained healthy in the treated fruits at 72 hrs after inoculation with fungus while the fungus penetrated into the epidermal cells of untreated fruits . soluble proteins containing pepest protein were separated by size fractionation . fraction no . 9 to 26 containing the protein were mixed together . pepest protein accounted for 1 % of the separated protein fractions . the resulting protein mixture was lyophilized and dissolved in distilled water to 250 μg per milliliter of final concentration . then , dtt was added to the protein solution to 1 mm of final concentration , designated as formulakh1 . when the spores of anthracnose fungus were treated with formulakh1 , spore germination was completely inhibited as shown in fig1 . baudouin e , charpenteau m , roby d , marco y , ranjeva r , ranty b ( 1997 ) functional expression of a tobacco gene related to the serine hydrolase family . esterase activity towards short - 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