CN1224334A - Plant protection method - Google Patents

Abstract

A method of protecting a plant against a pathogen is described, the method comprising inducing expression of a plant defence gene by stimulating the jasmonate and/or ethylenc pathways. Also deseribed is a method of inducing expression of a plant defence gene, a composition which is capable of inducing expression of a plant defence gene and a method for screening compounds giving resistance-inducing activity. Preferably, the pathogen is a necrotrophic pathogen.

Description

Plant protection method

The present invention relates to a kind of method of protective plant opposing cause of disease.

More specifically, the present invention relates to a kind of new signal transduction pathway, it has caused and can avoid the protein expression that this cause of disease is encroached on by protective plant.

In particular, the present invention relates to the method for a kind of protective plant opposing cause of disease such as fungi and bacterium.

As everyone knows, be subjected to the plant of microorganism cause of disease infringement can induce the cause a disease defence Expression of Related Genes of associated protein (PR protein) of a group coding.These protein not only can be detected by cause of disease infringement position but also can detect in the blade that is infected, and they produce whole acquired resistance-be plant is invaded the resistance that performance strengthens once more to the microorganism cause of disease a kind of state.Know that also when microorganism pathogen infection blade caused the increase of endogenous salicylic acid level and induces PR protein subsequently, salicylic acid (SA) played an important role in the signal transduction pathway that causes the whole resistance that obtains.

The present invention relates to another kind of signal transduction pathway, activate this approach and show equally and induce cause of disease, particularly the resistance of microorganism cause of disease and this approach can have the purposes outside the salicylate pathway, or replace the purposes of salicylate pathway.

Previous work is verified induces resistance to late blight fungi Phytophthorainfestans (Cohen etc., 1993) at potato and tomato plant exogenous application jasmine acid esters (jasmonate) and methyl jasmine acid esters.Yet do not come the saying of protective plant opposing cause of disease by the expression of inducing plant defensin gene.

Treat two other plant/cause of disease systems with methyl jasmine acid esters, promptly barley/Erysiphe graminis (Kogel etc. 1995) or paddy rice/Magnaporthegisea (Schweizer etc., 1997) do not provide resistance.

Fully data shows the compound of the pathogeny evoked defense reaction of known activation salicylic acid dependent form, as salicylic acid itself, acetylsalicylic acid, 2,6-dichloro-isonicotinic acid (INA) and 1,2,3-diazosulfide-7-carbothioic acid S methyl esters can be induced the microorganism cause of disease to certain limit, comprises virus, the resistance of bacterium and fungi (White etc., 1979; Kessmann etc., 1994; Lawton etc., 1996; Friedrich etc.; Gorlach etc.).What is interesting is, 1,2,3-diazosulfide-7-carbothioic acid S methyl esters can not be protected infect (Lawton etc.) of the cause of disease Botrytis cinerea of tobacco plant opposing dead volume nutrition and Alternaria alternata.

Before identified three Arabidopsis genes already, i.e. PR-1, PR-2 (beta-1,3-glucanase) and PR-5 (permeating plain sample albumen (osmotin-like protein)), when being subjected to pathogen infection by collaborative and whole (Uknes etc., 1993 of inducing; Dempsey etc., 1993; Mauch-Mani and Slusarenko, 1994).These genes all can be induced by high level when exogenous application SA or INA, and seemingly a kind of synthetic compound (Ukens etc., 1992 of simulating the SA effect of INA; Cao etc., 1994).

The present invention's also find to encode inducing of gene of protective protein always do not depend on salicylate pathway, but may fully independently approach is relevant with one.

First of the present invention provides a kind of method of protective plant opposing cause of disease, and this method comprises by stimulating jasmine acid esters approach and/or ethene approach induced defence element (defensin) expression of gene.

Second portion of the present invention provides by use one or more ethene to plant, the jasmine acid esters is similar to the reagent of ethene or the effect of jasmine acid esters and produces the method that the reagent of oxidative stress (oxidative stress) comes the inducing plant phylaxin gene to express.

Third part of the present invention provides the composition of a kind of energy inducing plant phylaxin gene expression, comprises one or more jasmonic, jasmine acid esters, ethene, the reagent of analog vinyl or the effect of jasmine acid esters and the reagent that can produce oxidative stress.

The 4th part of the present invention provides the energy inducing plant compound that phylaxin gene is expressed, the compound that comprises one or more generation ethene, the signaling molecule that lipid is derived, salicylic acid, salicylic functional analog and active oxygen (reactiveoxygengenerating) compound of giving birth to.

The method that the 5th part of the present invention provides screening that the active compound of induction of resistance (induced defence element) is arranged, this method comprise using to some position of a plant species or plant and are considered to produce this class resistance and the inducing plant phylaxin gene is expressed or the compound of the gene of coordinate expression.

The 6th part of the present invention provide can inducing plant the phylaxin gene expression promoter, comprise by jasmonic or simulate the reagent and/or the ethene of its effect or simulate the zone that the reagent place of its effect induces.

The 7th part of the present invention provides a kind of promoter region, and this promoter region is included in the nucleotide sequence that provides among Figure 14, or with Figure 14 in the sequence of the basic homology of sequence that provides, or among its series of variation.

Preferably by stimulating jasmine acid esters approach and ethene approach to come the inducing plant phylaxin gene.

Preferred cause of disease is the cause of disease of dead volume nutrition (necrotrophic).

Preferred cause of disease is the microorganism cause of disease.

Preferred cause of disease is a fungi.

Preferably by using ethene, jasmonic or jasmine acid esters stimulate jasmine acid esters approach and/or ethene approach.Yet the reagent of using analog vinyl or jasmonic effect is also effective.And, but the expression of the reagent inducing plant defensin gene of the produced oxidizing force by using non-amount of herbicide seemingly.The example of this genoid comprises the biphenyl weed killer herbicide, if can produce the paraquat of superoxide anion or rose-red (rosebengal) that dibromide maybe can cause producing single oxygen species.

The stimulation of jasmine acid esters and/or ethene approach preferentially relates to signal transduction composition EIN2 and the COI1 of Arabidopsis.Yet the stimulation of these approach can relate to corresponding gene outcome in the other plant, and these gene outcomes are basically with coming from EIN2 and COI1.

Produce vinyl compound preferably from ethene, ethephon (ethephon) and 1-aminocyclopropane-1-carboxylic acid.

The signaling molecule that lipid is derived preferentially is selected from arachidonic acid and derivative thereof, linolenic acid and derivative thereof and jasmine acid esters and derivative thereof.

Activity is given birth to oxygen compound preferably from paraquat, dibromide, rose-red and tetrabromofluorescein.

Can use any plant species, foretell but particularly suitable plant comprises small gross, it is various that tobacco or Arabidopsis belong to.When using various that Arabidopsis belongs to, alexin can be the product of plant defense plain gene PDF1.2 (Figure 14), with the product of the sequence of PDF1.2 sequence or the basic homology of its series of variation.

As mentioned above, the also expression of inducing plant phylaxin gene of compound of simulation jasmine acid esters or ethene activity this means that this approach can be used to the compound of screening and activating endogenous defense mechanism.

Though in screening, can use whole plant, use the part of plant, particularly tissue may be more convenient as blade.

This detection can be undertaken by any suitable method, for example for relevant plant alexin, by using the antibody of anti-PDF1.1 or PDF1.2 gene outcome, or utilize reporter gene to detect as gus gene or the luciferase gene that is connected to the PDF1.2 promoter region.

An advantage of the inventive method is that this method does not relate to direct cytotoxicity or the potential harmful chemicals that influences viable microbial cells of use, but utilizes the chemicals of the defense mechanism that exists in the activated plant.

An advantage of the present composition is to utilize said composition to make the cause of disease of plant to some type, and for example the cause of disease to dead volume nutrition produces resistance.Perhaps, induce salicylic acid by use, the compound of jasmine acid esters and/or ethene approach, composition of the present invention can be used to protective plant opposing wide spectrum cause of disease.

A preferred embodiment of the present invention is that the plant of protection Arabidopsis kind is supported anti-fungal methods; this method comprises by the expression that stimulates jasmine acid esters and/or ethene approach to come the inducing plant phylaxin gene, wherein by handling the expression that blade comes the induced defence plain gene with 0.5 micromole's methyl jasmine acid esters in the atmosphere that contains 25ppm ethene.

The present invention more embodiment preferred is to resist the composition of the infringement inducing plant phylaxin gene expression of multiple cause of disease for protective plant, and wherein said composition comprises ethene, methyl jasmine acid esters and salicylic acid.In the method, salicylic acid dependent form defense pathway and jasmine acid esters and/or ethene approach all can be activated.

Term used in the present invention " its variant " refers to gene order is carried out any replacement, and variation is modified, and displacement lacks or adds one or more nucleotide and the product of the sequence that produces has disease-resistant former activity.This term also comprises basically the sequence with this gene order hybridization.It also comprises DNA and the DNA that is encoding to this gene order of small part with DNA hybridization of the present invention.This hybridization preferably occurs in low or high stringent condition or between.In general, it is among about 65 ℃ 3 * SSC that low stringent condition can be defined as environmental temperature, and high stringent condition is 0.1 about 65 ℃ * SSC.SSC is buffer solution 0.15MNaCI, the title of 0.015M trisodium citrate.3 * SSC is three times of SSC concentration etc.

Term " basic autoploidy " comprises in the gene order autoploidy of the nucleotide of necessity at least, so long as homologous sequence plays the phylaxin gene effect, promptly this gene outcome can produce the resistance to pathogenic.In general, autoploidy show as with gene order of the present invention have 60% or more nucleotide identical, more typical is 65%, preferred 70%, more preferably 75%, even more preferably 80% or 85%, and particularly preferably be 90%, 95%, 98% or 99% or higher autoploidy.

Term " microorganism " comprises bacterium, fungi and virus.

When microorganism was invaded, one of gene that is activated among the Arabidopsis was the plant defense plain gene, its expression do not rely on salicylic acid but need ethene and the jasmonic reaction path in functional component.Plant alexin is one of the long alkaline protein that is rich in cysteine of an about 5kDa family, relevant with the antimicrobial insect defensin of finding in the kind of various insects on its structure Plant such as (, 1995) Broekaert.The potent inhibitor that known many these plant alexins are conks, this shows that they work in host defense.Shown already that the expression of small gross fore-telling plant defense plain gene in rotaring gene tobacco plant provided the resistance to fungal pathogen Alternania Longipes (Terras etc., 1995).Pathogeny evoked plant defense plain gene is not to rely on unique gene that approach (being non-dependence salicylate pathway) activates by jasmine acid esters and/or ethene probably, but it can be used as a kind of indicator that detects this approach of activation.Yet any other gene that works in same approach can be used to same purpose.

As description more detailed among the following embodiment, coded plant alexinic two Arabidopsis genes have been found to be expression.These genes are found when the gene pool data of the sequence of the cDNA nucleotide sequence homology of searching and Rs-APE1 (a kind of known radish plant alexin), and gene pool number of registration Z27258 and T04323 are arranged, and be appointed as PDF1.1 and PDF1.2 respectively.Their sequence is presented in Figure 1A and 1B, and is marked as sequence number 1 and 2.In the cDNA storehouse of dry seeds mRNA (PDF1.1) or rice shoot mRNA (PDF1.2) preparation, these sequences are identified as expressed sequence tag.Described gene code contains a preceding protein of inferring a peptide signal and a maturation plant defensins function district, and they foretell protein with small gross has 98% and 92% identical.

By carrying out the expression pattern (Fig. 2) that RT-polymerase chain reaction (RT-PCR) is analyzed PDF1.1 and PDF1.2 to separating the RNA that handles from the DNA of different Arabidopsis organs enzyme.As the contrast of RT-PCR reaction, design a pair of primer with the sequence area of amplification, comprising the intron of one 99 base-pair corresponding to Arabidopsis ACTIN-1 gene (Nairn etc., gene magazine 1988).In the method, the pcr amplification product of genomic DNA can differentiate according to its size and the true RT-PCR product that obtains from RNA.As Fig. 2 finding, the RNA sample from whole analyzed tissues are obtained except that dry seeds, all produces the ACTIN-1 RT-PCR amplified production of desired size, and genomic DNA produces the above PCR product of an about 100bp.With the special primer of PDF1.1 has been shown that to the RT-PCR that carries out the RNA with siliqua and dry seeds is the product of template amplification.In any analyzed tissue of healthy plant, the special primer of PDF1.2 is not to producing the RT-PCR amplified production.Yet, when the blade RNA that is infected by Alternaria brassicicola MUCL2097 strain (a kind of not diffusion in time of this infringement of fungi that causes the downright bad infringement of brown) is carried out RT-PCR, detected the amplified production of expectation size.The pcr amplification product that obtains with genomic DNA is than more than the long 100bp of amplified production that obtains by the RT-PCR from the blade RNA that infects, thereby shows the intron in the special primer leap PDF-1.2 gene of PDF-1.2.This is not observing with the special primer of PDF1.1.

The result is: Arabidopsis contains two genes, the plant alexin of their coding height homologies but diverse expression pattern is arranged.PDF1.1 is expressed as dominance in seed, and is considered to and small gross is foretold Rs-AFP1 and Rs-AFP2 dna homolog, and PDF1.2 expresses in the blade that is subjected to the cause of disease infringement, and is considered to Rs-AFP3 and the RsAFP4 dna homolog (Terras etc., 1995) foretold with small gross.

PDF1.1 and PDF1.2 gene and their expression product are used in the further research, and these are studied below among the embodiment by more detailed description.

Two plant alexin expression of gene of label (EST) Sequence Identification by its expressed sequence the analysis showed that: they are differences to expressing, and have only one (PDF1.2) in these two genes to show the cause of disease inducibility.Though only identify plant alexin (EST) sequence of two homologies so far, the other plant phylaxin gene can exist and may express in Arabidopsis probably, because independently digestion with restriction enzyme Arabidopsis genomic DNA and the Southern marking analysis of using Rs-AFP2 cDNA to clone as hybridization probe demonstrate 3 to 5 bands (data are unlisted) that meet employed enzyme with four.At present, the expression pattern of other phylaxin genes of inferring still belongs to the unknown during causing a disease, and will need gene family is done careful detection.

In present work, we have found that plant alexin expression of gene (comprising PDF1.2 at least) is induced by the integral body of pathogen infection also, and this inducing followed a kind of reaction path that PR-protein is followed that obviously is different from.This is found based on several evidences.

At first, the Arabidopsis plant alexin is not subjected to inducing of outside SA of use or INA.On the contrary, with methyl jasmine acid esters or ethene or the compound paraquat of generation reactive oxygen species and the accumulation that rose-red processing but causes the plant alexin transcript.Secondly, the pathogeny evoked integral body of plant defense plain gene is expressed in the npr-1 Arabidopsis mutant strain and is not induced, and the SA of known this mutant strain is blocked (Cao etc., 1994) to PR-protein gene priming reaction approach.Its three, at the endogenous SA and the PR-1 of performance group moulding rising level, in the cprl Arabidopsis mutant strain of PR-2 and PR-5 transcript, the plant defense plain gene is not that (Bowling etc., 1994) are expressed on composing type ground.Its four, the mutant strain etr1-3 of Arabidopsis, the analytical proof of ein2 and coi1: as if inducing of plant alexin relates to from ethene and jasmine acid esters reaction path or its total composition in to the reaction of fungal infection.Therefore, these results show: two other antifungal proteins of separate class (respectively by PR-1 and plant alexin exemplified) all can be induced by the labeling process of uniqueness local and whole.

It is feature (Guzman and Ecker etc. that the ein2 mutant strain of Arabidopsis lacks the form reaction with when growth in the presence of ethene, 1990), this mutant strain is in fact at blade that cause of disease is handled be not subjected in the integral blade of cause of disease processing, and the pathogeny evoked property expression of plant defense plain gene all is blocked.On the contrary, part ethene non-sensitive type mutant strain (Chang etc., 1993), etr-3 mutant strain have the reaction of normal plant alexin in infected blade, but in infected integral blade, show as reduction but not the plant alexin gene expression that disappears.The incomplete inhibition of the plant alexin gene expression of this etr1-3 plant by pathogen infection most likely since the seepage of this special mutant allele cause.Known to handling the growth that suppresses root with methyl jasmine acid esters or corolla element (coronatine) (a kind of schizomycete toxin that plays the effect of jasmonic ester analogs), the coil mutant strain is than wild-type plant susceptibility low (Feys etc., 1994).This mutant strain handle in cause of disease with untreated integral blade in all show the pathogeny evoked plant alexin reaction of almost completely blocking-up.

Therefore, as if according to these analyses, EIN2 and COI1 are local and whole plant alexin is induced neededly, and that ETR1 shows as is only relevant with W-response.In these three genes, have only ETR1 to obtain identifying.ETR1 a kind of protein that is similar to bacterium bi-component histidine kinase receptor of encoding, and genetics and biochemistry evidence show that ETR1 is ethylene receptor (Schaller and Bleecker, 1995).In the ethylene reaction approach, gene outcome EIN2 acts on the downstream (Ecker, 1995) of ETR1.The feature of clear and definite COI1 not as yet so far, but think its relevant with the signal transduction that the jasmine acid esters excites (Feys etc., 1994).

What is interesting is, before had found that when infecting that the ein2 plant shows the chlorosis infringement (Ben etc., 1992) lower than wild type plant with strong strain Pseudomonas Syringae pv.Tomato.The mutant strain (before being called ein1-1) that carries the etr1-3 sudden change is similar with the wild type plant reaction.Show the disease symptoms that reduces though the ein2 plant is compared with the etr1-3 plant with wild type, P.Syringae pv.tomato bacterium breeds well equally in these three kinds of genotype.

When Fig. 9 is described in the cause of disease intrusion, cause a model of two independent approach of induced defence related gene.In this model, allergy is positioned on the bifurcation, because find the acd2 mutant strain (Greenberg etc. of the Arabidopsis of generation active anaphylaxis sample infringement, 1994) contain the plant alexin transcript degree and the PR-protein that increase and transcribe this level (Greenberg etc., 1994).The approach that causes the PR-protein gene to be expressed by salicylic acid relates to signal transduction component NPR1 and CPR1, and causes the approach of plant alexin gene expression to need EIN2 and COI1.As if also can have interchange to a certain degree between two kinds of defense reaction approach, and the inhibiting accumulation of SA makes jasmine acid esters approach and/or ethene approach, i.e. the activity of the non-dependence approach of SA strengthens.In this regard, acetylsalicylic acid shows the route of synthesis (Pena-Cprtes etc., 1993) that suppresses the jasmine acid esters, and SA is found the inducing action (Doares etc., 1995) of inhibition by the protease inhibitor of jasmonic generation.

By the non-dependence approach of above-described SA, can activate other defence related genes with plant alexin.First possible gene to be selected is Hel, and a kind of hevein sample (hevein-like) gene (PR-4), this gene are all induced (Potter etc., 1993) in local and integral body when virus infections.Hel is subjected to inducing by force of ethene, but only be subjected to SA a little less than induce, and PR-1, PR-2 and PR-5 are that non-ethene is derivable, but are strong SA derivable (Potter etc., 1993).Second kind of gene to be selected is thionin gene Thi2.1, and this gene is induced when fungal infection and when methyl jasmine acid esters is handled, but is not induced (Epple etc., 1995) when SA handles.The third possible gene to be selected is alkaline chitinase gene C HIT-B, and this gene is induced when ethene is handled the wild type plant, but is not induced (Chen and Bleecker, 1995) in ein2 or etr1-3 mutant strain.Tool is observed, and CHIT-B's induces and PR-1 inducing of PR2 and PR-5 relevant (Dempsey etc., 1993) in the blade of the virus infections of Arabidopsis.

But also relevant two evidences of cause of disease induced reaction approach existence independently in tobacco.First approach causes acid PR-protein gene such as PR-1, PR-2, and PR-3 (acid chitinase), PR-4 and PR-5 induce, and second approach causes the abduction delivering of alkaline beta-1,3-glucanase and alkaline chitinase gene.First group of gene activated by force by SA, and second group of gene is subjected to ethene to activate (Meins etc., 1991).What is interesting is, the rotaring gene tobacco plant of expressing a gene (G albumen suppressor) of coding Cholera Toxin A 1 subunit shows the constitutive expression of acid PR-protein gene, but the constitutive expression (Beffa etc., 1995) that does not have alkaline beta-1,3-glucanase and alkaline chitinase.When invading with tobacco mosaic virus (TMV), acid PR-protein is all induced (Ward etc., 1991 in local and integral body; Brederode etc. 1991).On the other hand, alkaline beta-1,3-glucanase and alkaline chitinase gene are also being induced by force by in the blade of inoculating, but at the evidence that has contradiction aspect its whole inducibility.Based on the RNA hybridization analysis, the plant that infects at TMV do not inoculate the transcript degree that obviously increases (Ward etc., 1991 that can not detect these genes in the blade; Brederode etc., 1991), and, in these blades, found the accumulation (Heitz etc., 1994) of corresponding proteins matter based on the Western hybridization analysis.

Can infer that in tobacco pathogeny evoked alkaline beta-1,3-glucanase and alkaline chitinase expression of gene are followed a kind of approach that is equivalent to cause the expression of inducing plant phylaxin gene in Arabidopsis.One significantly difference be that the abduction delivering at integral blade of plant alexin occurs on transcriptional level and the protein level.Therefore should consider that Arabidopsis is much smaller than tobacco, detect distance that W-response covers than short many of the latter at the former.Alkaline β-1 in the tobacco on the one hand, 3-dextranase and alkaline chitinase express and on the other hand between the plant alexin expression of gene among the Arabidopsis another tangible difference be that tobacco gene is the derivable (Brederode etc. of damage, 1991), and the plant defense plain gene among the Arabidopsis is quite different.

Our verified and a kind of pathogeny evoked protein of foretelling the anti-PDF1.2 analog antibody cross reaction that (Rs-AFP1) produce from small gross has a strong antifungal activity external.This protein is accumulated to very high level when fungal infection, promptly rise to 2% and 1% of gross protein respectively in the integral blade of inoculating and not being subject to processing.We had before shown to foretell from small gross with 25% the level that is about total soluble protein matter and induce the transgene tobacco of PDF1.2 analog that A.longipes is had higher resistance (Terras etc., 1995) than non-plant transformed.Based on these observations, plant alexin plays a role in host resistance probably.Present very strong evidence is that SA dependent form approach is important producing aspect some cause of disease resistance of (comprising P.syringaepv.tomato and Peronospora parasitica).In fact, the plant of once find expressing nagG concerning these cause of diseases with respect to more responsive (Delaney etc., 1994) the wild-type plant and, different with wild-type plant, they can not produce whole acquired resistance (Lawton etc., 1995).Evidence described herein shows that SA independent form approach (causing the activation of plant defense plain gene and possible other defence related genes) is also useful to resistance, although be to dissimilar cause of diseases.In fact, we have shown the ein2 that obviously is blocked and coil mutant strain are easier to be subjected to Botrytis cinerea than wild-type plant infection in SA independent form approach.

The present invention now will be only be further described by following embodiment and the diagram mode with embodiment, wherein:

Fig. 1 represents respectively expressed sequence tag Z27258 and the nucleotide sequence of T04323 and the amino acid sequence of deriving corresponding to PDF1.2 and PDF1.2.

(A) corresponding to the nucleotide sequence of the Z27258 of PDF1.1 and the amino acid sequence of deriving.Before be called At-AFP1 from the PDF1.1N petiolarea (Terras etc., 1993) experimentally determined by underscore.The nucleotide of 123 (T among the Z27258) is replaced by the G of appended sequence data in the position.

(B) corresponding to nucleotide sequence and the deduced amino acid of the T04323 of PDF1.2.

(C) amino acid sequence that will foretell the ripe Rs-AFP1 of seed and blade and Rs-AFP3 from small gross is arranged with the ripe functional areas contrast that ripe functional areas and the PDF1.2 of PDF1.1 derives respectively.

(A) and (B) corresponding in the position of the primer that is used in RT-PCR by the nucleotide sequence of underscore.

Fig. 2 is illustrated in the expression pattern of PDF1.1 and PDF1.2 among the Arabidopsis;

(A) use the right RT-PCR of PDF1.1 Auele Specific Primer to analyze.

(B) use the right RT-PCR of PDF1.2 Auele Specific Primer to analyze.

(C) use the right RT-PCR of ACTIN-1 Auele Specific Primer to analyze.

RT-PCR carries out at the total RNA of no DNA enzyme that is separated to from different Arabidopsis source.Root, stem, petal, open flower and siliqua from 7 age in week flowering plant collect.Blade and infected blade are collected from the plant in 4 ages in week.The blade that infects is cultivated with A.Brassicicola, collects after 3 days.

Fig. 3 represents to come the purifying and the sign of plant alexin of the Arabidopsis blade of self-infection;

(A) the alkaline protein component of the Arabidopsis blade (bottom) that the Arabidopsis blade (top) of separation health and A.brassicicola infect on C2/C18 silica gel reversed phase chromatography post.With linear gradient is nitro acetone this chromatographic column of wash-out in the trifluoracetic acid of 0.1% (v/v) of 0% to 50% (v/v).

(B) electrophoretic analysis contained protein in peak 1.(Phastgel High Density Pharmacia) goes up the protein of contained 200ng in the electrophoresis peak 1 and the Rs-AFP2 of 200ng purifying and also dyes at the SDS-PAGE gel.The size of molecular weight marker is that unit marks on the left side with the kilodalton.

(C) behind the SDS-PAGE electrophoresis, with the immune marking of anti-Rs-AFP1 antibody to peak 1 contained protein.In the load sample hole, add the purifying Rs-AFP of 200ng or the purifying peak 1 contained protein of 200ng.

(D) extracorporeal antifungal activity of A.brasssicicola (negative control, the left side) in the water, the Rs-AFP2 (centre) of 5 mcg/ml concentration of purifying or the peak of 5 mcg/ml purifying 1 contained protein (right side).22 ℃ of temperature baths are carried out little centrifugal after 24 hours.

Fig. 4 represents that plant alexin expresses in the Arabidopsis of fungal infection;

(A) PDF1.2 and 'beta '-tubulin (β-TUB) the RNA gel marking analysis of expression in the untreated integral blade (2 °) of blade (1 °) that cause of disease is handled and infected plant.All analyzed sample is the RNA of 4 micrograms.

(B) in the untreated integral blade (square frame) of blade (circle) that cause of disease is handled and infected plant plant alexin (PDF) content as the resisting of use antigen affinity purification-the Rs-AFP1 antiserum is determined at ELISA.Measured value is three independent means of measuring (± standard error).

Total RNA and protein are wherein collected with 5 * 10 from being separated to untreated whole Arsbidopsis blade that cause of disease is handled ⁵Back 0 hour of individual spore/milliliter inoculation 5 microlitre drops (5 in every blade), 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, the blade of 72 hours and 96 hours.

Fig. 5 represents among the Arabidopsis of chemical treatment and damage, the inducing of plant alexin;

(A) the RNA gel marking is analyzed in damaged or the (expression of β-TBU) of PDF1.2 and 'beta '-tubulin in the blade of specified chemical treatments.All analyzed samples are total RNA of 4 micrograms.

(B) content of plant alexin (PDF) is as being measured with the anti-Rs-AFP1 antiserum of antigen affinity purification by the ELISA method.Measured value is three independent mean values of measuring (± standard error).

Water with 5 microlitre drops (every blade 5), SA (5 mM), INA (1 mg/ml), paraquat (25 micromole), rose-red (20 mM), methyl jasmine acid esters (45 micromoles are in 0.1 (v/v) ethanol) or 0.1% (v/v) ethanol (0.1%EtOH) inoculation Arabidopsis blade.It is by carrying out in the cell that 20ppm concentration ethene is housed that plant is positioned over secluding air that ethene is handled.Check plant (in air) is cultivated in the same cell of no ethene.By causing damage in the method for blade upper cut with scalpel.The all 48 hour collections of leaf sample after handling beginning.This experiment repeats once with similar result.

Fig. 6 is illustrated among the wild type Arabidopsis (Col-0) and the inducing of plant alexin in the affected Arabidopsis mutant strain (npr1 and cpr1) in the SA-signal pathway.

This figure left part is represented the RNA gel marking analysis that PDF1.2 expresses.This sample is total RNA of 4 micrograms.

Plant alexin (PDF) content that the right side divides expression to use the anti-Rs-AFP1 antiserum of antigen affinity purification to be measured by the ELISA method.Measured value is three independent mean values of measuring (± standard error).

By on four lower floor's lotus throne leaves (rosette) blade (every blade 5) use 5 spore suspensions (5 * 10 ⁵Spore/milliliter) method inoculates A.brsssicicola (A.bras.) to the Arabidopsis plant.Check plant is with the water (H of 5 microlitre drops ₂O) handle with quadrat method.Inoculate after 3 days, collect blade (1 °) that cause of disease handled and plant and untreated blade equally.With described method (referring to: method) extract total RNA and protein.Twice of the experiment of repetition equifinality.

Fig. 7 is illustrated in the Arabidopsis wild type (col-0), is subjected in the Arabidopsis mutant strain (ein2 and etr1-3) that the ethylene reaction approach influences and the inducing of the plant alexin in a mutant strain (coil) that influenced by jasmine acid esters reaction path.

This figure left part is represented the RNA gel marking analysis that PDF1.2 expresses.This sample is total DNA of 4 microlitres.

Plant alexin (PDF) content that the right side divides expression to use the anti-Rs-AFP1 antiserum of antigen affinity purification to measure by the ELISA method.Measured value is three independent mean values of measuring (± standard error).

By on the rosette blade of four lower floors (every blade 5) use 5 microlitre drop spore suspensions (5 * 10 ⁵Spore/milliliter) method inoculates A.brassicicola (A.bras.) to the Arabidopsis plant.Check plant is with the water (H of 5 microlitre drops ₂O) handle with quadrat method.Inoculate after 3 days, collect blade (1 °) that cause of disease handled and plant and untreated blade equally.With described method (referring to: method) extract total RNA and protein.Twice of the experiment of repetition equifinality

Fig. 8 is illustrated in inducing of plant alexin in Arabidopsis wild type (col-0) and the Arabidopsis damage simulation mutant strain.

(A) the RNA gel marking of PDF1.2 expression is analyzed.All analyzed sample is total RNA of 4 micrograms.

(B) as passing through ELISA, plant alexin (PDF) content that the anti-Rs-AFP1 antiserum of use antigen affinity purification is measured.Measured value is three independent mean values of measuring (± standard error).

(C) total RNA separate from 5 ages in week the health that the acd2 plant is collected asymptomatic upper strata rosette blade (UH) and lower floor's rosette blade of the downright bad damage (LN) of performance, and healthy upper strata rosette blade (UH) and lower floor's rosette blade (LH) of the check plant (Col-0) of separation under similarity condition.This experiment repeats twice with equifinality.

Fig. 9 is two the independently approach that pass through that proposed, and promptly salicylic acid relies on the model that approach and jasmine acid esters and/or ethene rely on approach induced defence related gene.

But Figure 10 is illustrated in ethene and jasmine acid esters signal interaction during the PDF1.2 gene activation of the Arabidopsis plant that cause of disease invades three models of trans-substitution mutually.

When Figure 11 represents with A1ternaria brassicicola inoculation (sign of closing) or water simulation inoculation (sign of opening), Arabidopsis wild-type plant (Col-0, above) and the insensitive mutant strain of ethene (ein2, below) in the jasmonic level in each period.

Each data point is the mean value to two groups of two independent tests being done of every group of three plant.

When Figure 12 represents with Alternaria brassicicola inoculation (sign of closing) or water simulation inoculation (sign of opening), Arabidopsis wild-type plant (Col-0, above) and the insensitive mutant strain of jasmine acid esters (coil, below) in the ethylene production level in each period.The mean value of the data of Col-0 three independent experiments that to be each time points carry out with two plant species.The single experiment that the data of Coil are carried out with two plant species from each time point.

The inducing of plant alexin in Arabicopsis wild-type plant (Col-0) and the insensitive mutant strain of ethene (ein2) when Figure 13 is illustrated in and handles with the methyl jasmine acid esters (MeJA) in 0.1% ethanol (con) or 50 micromoles, 0.1% ethanol.

Figure 14 represents the nucleotide sequence of ArabidopsisPDF 1.2 genes.The nucleotide that encloses is represented first nucleotide of expressed sequence tag T04323.The amino acid of gene outcome is given under the corresponding codon in code area.Intron is represented with subscript character.

When Figure 15 represents with A.brassicicola (simulation or spore inoculating) or B.cinerea (simulation or leopard inoculation) inoculation, the β glucosiduronic acid enzymic activity of transgenosis pPDF1.2-GUS-tNOS Arabidopsis plant.Handle the blade (1 °) and the untreated blade of homophyletic (2 °) of collection and treatment in back 3 days.The result is represented as mean value ± standard error of four groups of two plant.

β glucosiduronic acid enzymic activity when Figure 16 represents with various chemical treatments in transgenosis pPDF 1.2-GUS-tNOSArabidopsis plant (A part) and the transgenosis pBg12-GUS-tNOS Arabidopsis plant (B part).

Figure 17 represents with β glucosiduronic acid enzymic activity in transgenosis pPDF1.2-GUS-tNOS tobacco (the also available Xanthi-nc) plant of tobacco mosaic virus or simulation inoculation.Virus or simulation inoculation be the blade under the blade that the tenderest full extension is opened just.These blades (1 °), the blade that the tenderest full extension is opened (2 °) and just in the blade on the blade that the tenderest full extension is opened (3 °) two days after handling (black stick), in 4 days (bright grey stick) or in 6 days (lead stick) collected respectively.

Figure 18 represents the β glucosiduronic acid enzymic activity in transgenosis pPDF1.2-GUS-tNOS tobacco (the also available Xanthi-nc) plant with various chemical treatments or damage.Handle the leaf sample of collection and treatment in back 48 hours.

When Figure 19 represents to contact 25ppm ethene, with 0.5 micromole's methyl jasmine acid esters (MeJA, in 0.1% ethanol), 333 micromole's ethephons, 25ppm ethene adds β glucosiduronic acid enzymic activity in the transgenosis pPDF1.2-GUS-tNOS Arabidopsis plant that 0.5 micromole's methyl jasmine acid esters handles, and the processing of suitable adjoining tree is: ingress of air and water and 0.1% Ethanol Treatment.

Figure 20 represents with after the fungal pathogen Botrytis cinerea inoculation, Arabidopsis wild type plant (Col-0, circle), the insensitive mutant strain of ethene (ein2, triangle) and the insensitive mutant strain of jasmine acid esters (coil, square) rotten.Data representedly contain carry out with 20 plant series of each plant lines four independently experiment (Col-0 and ein2) and three mean values of independently testing the standard deviation of (coil).

Figure 21 is illustrated in the Arabidopsis wild type plant (Col-0) and the ein2 of inoculation, in the blade of coil and npr1 mutant strain, and the lactophenol/trypan blue staining of the mycelial structure of fungi Peronospora parasitica pathovar Wela.The method biomaterial

Mutant strain npr1 (Cao etc., 1994) and cpr1 (Bowling etc., 1994) by doctor X.Dong provide (Duke University, Durham, NC, USA).Ethylene reaction mutant strain ein2 (Guzman and Ecker, 1990) and etrl-3 (Bleecker etc., 1988; Chang etc., 1993) from Arabidopsis living resources center, OH, USA obtains (number of registration is respectively CS3071 and CS3070) damage simulation mutant strain acd2 (Greenberg etc., 1994) provide (Massachusetts General Hospital by doctor F.Ausubel, Boston, USA).Jasmine acid esters reaction mutant strain coil (Feys etc., 1994) from the J.Turner acquisition (University of East Anglia, Norwich, UK).Because this mutant strain is recessive and causes that it is that the F2 that becomes of the seed growth by self-fertile COIl/coil semizygote plant is for the offspring (posteriori) in the plant that male sterile, coil mutant strain are accredited as.Therefore, F2 is carried out following different processing for colony, collected respectively and be that plant continue to grow up up to sowing from each individual blade.Those individualities that do not form siliqua have been accredited as the coil/coil genotype.Length according to the seedling root of external germination in 50 micromole's methyl jasmine acid esters is selected to the coil mutant strain that this disease detects in advance.Above listed whole mutant strain strains derive by Columbia (Col-0) ecotype.(MUCL 20297 for fungi A.brassicicola; Mycotheque UniversiteCatholique de Louvain, Louvain-1a-Neuve, adult Belgium) and the results of spore are carried out (Broekaert etc., 1990) by previous description.The plant strain growth condition, the application of chemicals and inoculation

The Arabidopsis seed is sowed on the mixing fertilizer that contains macronutrient complementary element (Asef.Didam, The Nehterlands) of plantation flower in culture dish.After planting, described seed at 4 ℃ by vernalization 2 days.(daytime, temperature was 20 ℃, and night, temperature was 18 ℃, at 100 μ Em in a growth greenhouse ^-2s ^-1Following 12 hours periodicity of illuminations of photon stream metric density) in cultivate after 5 days, be transplanted to contain that plantation that macronutrient replenishes mixes in the fertile flowerpot and with above-mentioned similarity condition under grow.Water by the pallet that loads flowerpot with running water.Reinforce the way damaged blade that terminal tweezers pulverize with having, or form otch with scalpel on branch, arteries and veins is complete in noting keeping.Paraquat (25 micromole), rose-red (20 mM), methyl jasmine acid esters (40 micromoles are in 0.1% (v/v) ethanol), 0.1% (v/v) ethanol, sodium salicylate (5 mM) and INA (1 mg/ml, 25% active component, but in wet powder, by Switzerland Basel, doctor H.Kessmann of Novartis provides) on blade, use 5 microlitre drops (5 in every blade) with the concentration that shows between the carriage.Methyl jasmine acid esters storage liquid is the ethanolic solution of 45 mMs.Ethene is handled and is undertaken by flowerpot being placed in air-locked translucent greenhouse, injects ethylene gas by a silica gel thin film in it.Check and verify ethylene concentration in the greenhouse with gas chromatography.The adjoining tree of ethene experiment is placed in the same greenhouse that does not have ethene.Spore suspension by using 5 microlitre drops is (in distilled water 5 * 10 ⁵Spore/milliliter) (5 in every blade) inoculation A.brassicicola on the blade of four lower floor's rosettes.Adjoining tree is handled equally with the distillation water droplet.The plant that has a drop of spore suspension and water is placed in the sowing pond that polystyrene covers by (if simultaneously handle different genotypic words) at random and keeps high humility 2 days to stimulate the infection of mycelia germlings.In 48 hours of spore suspension drop inoculation, the A.brassicicola of isolation and inoculation condition used herein produce limited yellow withered damage, and these damages can not further be spread.The RNA marking is analyzed

Phenol/LiCI method by (1996) such as Eggermont is from freezing liquid nitrogen and extract RNA in the tissue of-80 ℃ of storages.With every hole 4 microlitres the RNA sample is uploaded on formaldehyde-agar gel, and by 20 * SSC (Sambrook etc. 1989) capillary transfer method be imprinted onto on the positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany).Be to confirm the comparativity of RNA and the transfer of RNA, give and upload buffer solution and replenish 50 mcg/ml bromophenol blues, so that the RNA in the gel and marking naked eyes are as seen during ultraviolet irradiation.With the antisense RNA probes of digitoxin mark and marking prehybridization and hybridization and with foregoing immunochemistry fluorescence method colour developing (Terras etc., 1995).Dig RNA labelling kit with BoehringerMannheim is transcribed the probe that legal system is made the digitoxin mark by escape.With the SP6 RNA polymerase with by linearizing plasmid pZLI (BRL life Technologies, Gaithersburg, MD, USA) the synthetic PDF1.2 probe that contains Genbank number of registration T04323 expressed sequence tag of EcoR I.With T7 RNA polymerase and the linearizing plasmid pBluescript II SK (Stratagene that contains Genbank number of registration Z26191 expressed sequence tag of EcoR I, Lajolla, CA, USA) synthetic two plasmids of tubulin β-1 chain gene are from Arabidopsis living resources center (Columbus, 0H USA) obtains.Sample electrophoresis on the a-type double gel of analyzing with different probe also develops the color respectively.Reverse transcription PCR

(1 microgram is in 100 mM sodium acetates/5 mM magnesium sulfate, and pH5.0) the DNase I (Boehringer Manheim) of not having a RNase with 6 units continues 5 minutes 37, heats 5 minutes by 95 ℃ then, makes the DNA enzyme deactivation for total RNA.This RNA precipitates in molar sodium acetate and 66% (v/v) ethanol O.2 and spends the night, and collects in centrifugal 10 minutes by 10000xg, washes twice with 70% (v/v) ethanol and also is dissolved at last in the water of no RNA enzyme.With the birds myeloblastosis viral reverse transcriptase of 10 units (Pharmacia, Uppsala, Sweden) and total RNA of handling of 1 μ g DNA enzyme under 52 ℃, carried out reverse transcription reaction 60 minutes.Carry out reverse transcription reaction and be 15 mM cessation reactions to final concentration with homopolymer deoxyribosylthymine oligonucleotides (20-mer) by adding Na-EDTA.A (a thirtieth) reverse transcription reaction solution with the Taq polymerase of 2.5 units (Appligene, Pleasanton, CA, during USA) the 50 microlitre PCR that carry out according to people's such as Sambrook (1989) method react as template.30 circulations are carried out in described PCR reaction, at 55 ℃, use respectively PDF1.1 for 65 ℃ and 65 ℃, and the single-minded primer of PDF1.2 and ACTIN-1 is to increasing.The primer that is used for the PDF1.1 amplification is OWB260 (sense strand 5 '-GAGAGAAAGCTTGTTGTGCGAGAGGCCAAGTGGG-3 '); And OWB259 (antisense strand 5 '-GAGAGAGGATCCTGCAAGATCCATGTCGTGCTTTC-3 '), the primer that is used for the PDF1.2 amplification is: OWB240 (sense strand: 5 '-AATGAGCTCTCATGGCTAAGTTTGCTTCC-3 '); And OWB241 (antisense strand: 5 '-AATCCATGGAATACACACGATTTAGCACC-3 '); With the primer that is used for ACTIN-1 amplification be OWB270 (sense strand: 5 '-GGCGATGAAGCTCAATCCAAACG-3 '); And OWB271 (antisense strand: 5 '-GGTCACGACCAGCAAGATCAAGACG-3 ').Arabidopsis blade purifying plant alexin from the A.brassicicola infection

Distilled water (contrast) or A.brassicicola spore suspension (5 * 10 with 5 microlitre drops ⁵Spore/ml water) blade of inoculation Arabidopsis plant in 5 age in week, and be collected in after 3 days in the humidity sowing pond that transparent polystyrene lid arranged in inoculation.Prepare extract from the 20 blades gram water treatment or inoculation, and carry out purifying according to the method for (1995) such as previously described Terras fully.Carry out protein determination according to previous description (Terras etc., 1995) on the PhastGel High Density gel (Pharmacia) for preparing in advance, extracorporeal antifungal activity is measured and SDS-PAGE analyzes.Before SDS-PAGE analyzes, described according to (1992) such as Terras, with dithioerythritol and S-ethylization pyridine reduction protein example.Press (1995) such as Terras described, the protein that separates on 15% acrylamide SDS-PAGE gel carries out the immune marking.Elisa assay

From extracting buffer solution (10 mM sodium dihydrogen phosphates, 10 mM sodium hydrogen phosphates, 100 mM potassium chloride, 1.5% (w/v) crospovidone, pH7) the freezing blade separating substances protein in.In according to the resulting crude extract of Bradford (1976), use bovine serum albumin standard test protein concentration in vain.After thermal treatment (80 ℃, 10 minutes), in competitive ELISA, carry out the analysis of thermally-stabilised soluble protein fragment to crude extract.

(pH9.6) middle bag is continued 2 hours for 37 ℃ by ELISA microtiter plate (GreinerLabortechnik) for 10 mM sodium carbonate, 35 mM sodium bicarbonates to be cushioned liquid with 100ng/mlRsAFP2 at bag.Do not wrapping by the position with the cold fish glue from skin of 3% (w/v) (Sigma) (PBS) (37 ℃ 2 hours) sealing in phosphate buffered saline.50 times of the primary antibodies of dilution affinity purification and the diluted sample of while in the sample buffer of equal-volume (50 microlitre) are applied in the hole in containing 0.3% (w/v) gelatin PBS solution of 0.05% (v/v) Tween-20.This flat board was incubated 1 hour 37 ℃ of temperature.After washing for several times with the PBS that contains 0.1% (v/v) Tween20, the second antibody (with the goat anti-rabbit antibody of alkaline phosphatase coupling) that will contain 1000 times of the O.3% gel PBS liquid dilutions of 0.05% (v/v) Tween20 is filled in the dull and stereotyped hole (every hole 100 microlitres).Described flat board was incubated 1 hour 37 ℃ of temperature.With 1 mg/ml substrate buffer solution (20 mM sodium carbonate, 35 mM sodium bicarbonates, 5 mM magnesium chlorides, pH9.6) substrate 4-phosphoric acid nitrobenzene (the Janssen Chimica of concentration, Beerse, Belgium) after 37 ℃ of temperature are incubated 30-60 hour, the activity of the light absorption detection of alkaline phosphatase by measuring 450nm.With the protein example of 4 times of dilution series of triplicate preparation, and, detect plant alexin concentration with reference to Rs-AFP1 (Terras etc., 1992) the twice dilution series standard of purifying.

The sero-fast affinity purification of anti-Rs-AFP1 is undertaken by following method.By mixing and Affi-Gel10 matrix (Bio-Red Laboratories, Hercules, CA, USA) the 100 mM 3-N-morpholino propane sulfonic acid buffer solutions (pH7) of isopyknic 20 mg/ml purifying Rs-AFP1 preparation antigen affinity column.This mixed slurry spends the night under 4 ℃ of continuous gentle agitation.Behind the non-reactive site of 1 molar ethanolamine (pH8) the sealing matrix by adding 0.025 volume, with 10 mM Tris (pH7.5), 100 mM glycine (pH2.5), 10 mM Tris (pH8.8) and 100 mM triethylamines (pH11.5) order are washed this post, and saturated with 10 mM Tris (pH7.5).The anti-Rs-AFP1 antiserum of rabbit be diluted in the ImmunoPure Gentle antigen/antibody binding buffer liquid by twice (Pierce ChemicalCompany, Rockford, IL, USA) and for several times by this affinity column.After washing this post with the ImmunoPure Gentle antigen-antibody binding buffer liquid of 15 volumes, with the anti-Ps-AFP1 antibody of ImmunoPureGentle antigen-antibody elution buffer (Pierce Chemical Company) wash-out purifying.Monitor the wash-out of binding antibody by the light absorption of measuring 280nm.Elution fraction is by the PBS dialysed overnight.The amplification of Arabidopsis PDF1.2 gene promoter area, clone and dna sequence analysis

Regulate sequence based on the method for inverse PCR (this method detailed process referring to Gasch etc. 1992) 5 ' of the PDF1.2 gene that is used to increase.Corresponding to the Genbank number of registration of PDF1.2cDNA is the primer that expressed sequence tag (EST) dna sequence dna of T04323 is used to genomic 5 ' the genome flanking sequence of design amplification Arabidopsis.Total genomic dna is with people's such as Dellaporta method (Dellaporta), separate from Arabidopsis ecotype be Col-0 about 8 all ages plant 10 blades that restrain weight in wet bases.By be suspended in the CsCI solution and 45,000 rev/mins centrifugal, be further purified described DNA, the final densities of this CsCI solution is 1.55 mg/ml and the ethidium bromide that contains 0.75 mg/ml.From centrifuge tube, shift out the DNA of branch band then with syringe, use by the isoamyl alcohol layering and remove ethidium bromide, use precipitation with alcohol DNA then.Precipitated DNA is dissolved in the water, then with the restriction enzyme Sph1 of 10 units DNA1 hours of 37 ℃ of digestion 120ng in 40 microlitre reactant liquors.Known EST T04323 contains inner Sph1 recognition site (being positioned at 174 to 179 base places), therefore this enzyme is used to cut genomic fragment, described fragment contains preceding 178 bases of above-mentioned cDNA, swims over to any 5 ' sequence of first Sph1 recognition site in this genomic DNA on any intervening sequence and the est sequence.Described 65 ℃ of heat inactivations 10 minutes that are reflected at, simple centrifugal and with the DNA in the precipitation with alcohol suspension.Then in the standard 50 microlitre reactant liquors of the buffer solution (BoehringerMannheim) that the T4DNA ligase that uses 1 unit and this enzyme supplier provide 14 ℃ spend the night, connect the about 30ngDNA that is digested from body.Stopped coupled reaction in 10 minutes by 65 ℃ of heating, simple centrifugal and with the DNA in the precipitation with alcohol suspension.Add DNA sample that 10ng connects template then as 50 microlitre polymerase chain reactions (PCR), wherein contain 200 micromole dNTPs and each 1 micromolar primer 0WB257[5 ' GAGAGAGGATCCAACTTCTGTGCTTCCACCATTGC-3 ', BamH I recognition site is by underscore] and OWB256[5 '-GAGAGAAAGCTTGAAGCCAAGTGGGACATGGTCAGG-3 ', Hind III recognition site is by underscore].These primers correspond respectively to position 104-127 and the 133-156 in the EST T04323 sequence, but reversed in order, and be could increase under the situation that the body coupled reaction connects by the genomic fragment with terminal Sph1 recognition site at flanking sequence only.The PCR reactant liquor contains the TaqDNA polymerase (adding) of 1 unit and the reaction buffer that supplier (Appligene Inc.) provides when the first time, thermal cycle reaction reached 95 ℃.The PCR reaction is carried out according to following thermal cycle program.At first,, add the Taq polymerase during this period 95 ℃ of heating 4 minutes, then carry out 30 95 ℃ * 30 seconds, the circulation of 56 ℃ * 2 minutes (annealing temperature) and 72 ℃ * 1 minute, last circulation is 95 ℃ * 30 seconds, 56 ℃ * 2 minutes and 72 ℃ * 10 minutes.Add this reactant liquor sample be equivalent to 0.05 microlitre template as the PCR reaction second time, this reaction adds one and overlaps primer OWB255[5 '-GAGAGAGGATCCAGCAGCAAAGCGAACAAGAGCAGCG-3 ' BamH I recognition site by underscore except that not adding genomic DNA] other are all identical the replacement primer OWB257.Primer OWB255 is corresponding to 67-91 position in the T04323 sequence.Same thermal cycle program is carried out in this reaction, and different is that 56 ℃ of steps are enhanced 58 ℃.Obtain two dna fragmentations of about 1.3kb and 0.8kb.Separate big fragment from Ago-Gel, with the digestion of Hind III and BamH I and be connected among the pEMBL18+ that digests in advance with Hind III and BamH I.This clone is named as pJMiPCR-1t.Do the part order-checking with the M13 reverse primer to inserting fragment forward with dideoxy nucleotide termination reagent and M13.These partial sequences show that inserting fragment contains and be same as the sequence that those expections present from EST T04323, and identify the partial sequence that is used for the Sph1 restriction site flank that connects from body.Design and pJMiPCR-It insert the Oligonucleolide primers on the adjacent sequence in Sph1 site in the fragment then, and this sequence is contained the sequence at PDF1.2 gene 5 ' promoter region place by expection.Described primer is named as OWB273[5 '-AAGAAAGCTTATGCATGCATCGCCGCATCGATATCCC-3 ', and Hind III recognition site is by underscore].Described primer is used to and primer OWB276[5 '-GAGAGAAGCTTATTTTTATGTAAAATACACACGATTTAGCACC-3 ', Hind III recognition site is by underscore] PCR that is used in combination reaction, wherein contain sequence corresponding to position 292-326 in the EST T04323 sequence.5 ' the upstream sequence of PDR1.2 gene in case the inverse PCR reaction has been increased, these primers will increase and be positioned at the genomic DNA complete sequence of 326 5 ' the PDF1.2 genes of holding of T04323 sequence, and the Sph1 recognition site up to 5 ' upstream promoter district should be extended in described zone.Use the ecotypic 10ng complete genome of Arabidopsis group DNA as template, with above-mentioned these primers, 58 ℃ are carried out PCR for annealing temperature and react.Judge that as gel electrophoresis this PCR produces the single fragment that size is about 1.6kb.Downcut this Zone electophoresis band and use the digestion of Hind III from gel, be connected among the dephosphorylized pEMBL18+ of Hind III incision.This clone is named as pFAJ3085, and checks order fully with two chains of overlapping method to this insertion fragment with fluorescein-labeled pair of deoxidation reagent and Applied Biosystems 373A dna sequence analysis instrument.Make up PDF1.2 promotor-sub-construct of report and transform Arabidopsis and Tobacco

Use primer OWB272[5 '-GAGAGAGGATCCTGATGGAAGCAAACTTAGCCATG-3 ', BamH I recognition site is by underscore] and the part coding region sequence (1-1254 of fragment is inserted in the position at pFAJ3085) of 0WP273 amplification promoter fragment and PDF1.2 gene.This reaction uses the pFAJ3085 of 1ng to carry out as template and before using 56 ℃ of annealing temperatures.The fragment that obtains expecting is also used gel-purified.With Hind III and this fragment of BamH I double enzymolysis, be connected to then among the commercially available couple of vector plasmid pBI101.3 (Clontech Inc.).This carrier contains the T-DNA that can use Agrobacterium tumefaciens to be transferred to plant as medium.When in plant cell, expressing, an alternative marker gene that kalamycin resistance is provided is arranged on the T-DNA.In addition, one contains the polylinker of Hind III and BamH I recognition site and the UidA of Escherichia coli (coding beta-glucuronidase or GUS) cistron code area 5 ' next-door neighbour, and in this regional downstream adjacency is Agrobacteriumtumefacients (tNOS) rouge alkali synthetase gene.Being inserted into and causing a gene Fusion in the described zone by the PDF1.2 gene of OWB272 and OWB273 amplification by the Hind III/promotor of BamH I digestion, can expect that wherein beginning translation at the normal translation initiation of the expection position of PDF1.2 gene can cause producing and following N terminal sequence GUS fusion together: MAKFASIRIPGYGQSLM, wherein the underscore amino acid residue derives from the terminal PDF1.2 codon of seven N, standard (normal) character representation amino acids coding residue on the pBI101.3 carrier, and last italic M residue is the N terminal residue by the GUS enzyme of UidA cistron coding.The described carrier of inlaying the pPDF1.2-GUS-tNOS gene that contains is named as pFAJ3086.With OWB273 and commercially available primer [GUS Sequencing Primer, ClontechInc.5 '-TCACGGGTTGGGGTTTCTAC-3 '] the insertion fragment of amplification among the pFAJ 3086, and directly to the end sequence order-checking of PCR product to confirm that the PDF1.2 gene order is by correct combination.

Then, be to promote combination, contain the triparental mating that the E.coli HB101 bacterial strain of carrier pRK2013 carries out by use pFAJ3086 is transferred to Agrobacteriumtumefaciens strain LBA4404 (Ditta etc.).Transform the blade transplant of Nicotiana tabacum cv.Xanthi-nc and transform the environmental C24 of the Arabidopsis thaliana that uses the root explant with the A.tumefaciens strain that contains the pFAJ3086 carrier by blade spining disk method (leaf disc) (Horsch etc.).Disease detects

The disease detection of the last Botrytis cinerea of Arabidopsis is carried out as follows.(22 ℃ is 80uEm with the photon stream flux density to plant the Arabidopsis plant in the greenhouse on the potted plant mixing fertilizer ^-2s ^-1Periodicity of illumination 14 hours, relative moisture are 70%).In three weeks after planting, the blades that all launch with needle-punching method (every blade 3 pins) damage.Be suspended in Botrytis cinerea (10 in the strong potato glucose meat soup of false add (Difco) with 5 microlitre drops ⁵Spore/milliliter) suspension covers the injury.Postvaccinal plant is planted at random in the sowing pond of being stamped the transparent polystyrene lid and as top method and is cultivated, and different is that the photon stream flux density is lowered to 50 μ Em ^-2s ^-1Two days later, open lid, under similarity condition, continue to cultivate plant.When rotting fully, the stem that comprises the leaf that spray is most advanced and sophisticated and the tenderest thinks plant death.

It is as follows that Arabidopsis is subjected to Peronospora Parasitica INFECTION IN DETECTION.Screen the conidium of P.parasitica pathowar Wela by the infected blade of the environmental Weiningen of light sieve Arabidopsis in water.The conidium suspension is adjusted to 10 ^-5Spore/milliliter also is sprayed onto with the plant watering can on the plant in age all around.The plant that inoculated is planted in the sowing pond of transparent polystyrene lid and at 20 ℃ at random, and the photon stream metric density is 80 μ Em ^-2s ^-1Periodicity of illumination be to cultivate in 8 hours 7 days.Detect the progress of disease by dye blade with lactophenol/trypan blue staining method (Keogh etc.) to show the fungi structure.Embodiment 1

The plant alexin that antifungal activity arranged cause of disease stress the Arabidopsis blade in accumulate

For whether studying pathogeny evoked plant alexin biologically active among the Arabidopsis, we attempt to foretell the method protein purification of blade purifying Rs-AFP3 and Rs-AFP4 (Terras etc., 1995) by previous research and development from the small gross of fungal infection.In brief, this purification process comprises makes thick leaf protein matter extract cross the anion-exchange column of pH7.5, make unconjugated protein cross the cation exchange column of pH5.5, the protein of elution of bound under high ionic strength separates the protein of wash-out with the reverse chromatographic column of last mistake.Will from the reverse chromatography of the protein of not inoculating the Arabidopsis plant leaf with from the reverse chromatography of the blade of having inoculated A.brassicicola relatively, feasible only identifying at peak (Fig. 3 A peak 1) that the latter just occurs.SDS-PAGE analyzes protein in the component be presented at this peak and move (comigrating) (Fig. 3 B and 3C) altogether with Rs-AFP1 and show as a single 5kDa and be with.In the immune marking of SDS-PAGE preparing gel and with in the anti-Rs-AFP1 antiserum colour developing, this protein the same with Rs-AFP1 clearly visible (Fig. 3 C).And this protein component is proved to be the growth in vitro that suppresses A.brassicicola (Fig. 3 D) and Fusuarium culmorum (result does not show).Growth Inhibition is characterized by mycelial high branch and most advanced and sophisticated the expansion, as viewed at Rs-AFP1 (Fig. 3 D) and from other Brassicaseae, the corresponding plants alexin of PDF1.1 (being called At-AFP1 in the past) that comprises Arabidopsis seed (Terras etc., 1993) is observed.Embodiment 2 be subjected to cause of disease stress the whole inducing action of Arabidopsis plant alexin

We had shown before that local infection A.brassicicola can whole induce the plant defense plain gene (Terras etc. 1995) in the small gross fore-telling.For whether research also has a this situation in Arabidopsis, we are by ELISA (using the anti-Rs-AFP1 antiserum of antigen affinity purification) and analyze (use PDF1.2 is as alexinic probe) by the RNA gel marking and analyzed the Arabidopsis blade that is subjected to the A.brsssicicols infection and infected and inducing action plant alexin in the blade (integral blade) that is not subject to processing.On protein level, find that the following amount of plant alexin detection limit (0.05 microgram/milligram total soluble protein matter) during sampling before inoculating brings up to respectively up to 10 and 3 micrograms/milligram total soluble protein matter (Fig. 4 A) cause of disease processing and the untreated integral blade after inoculating 72 hours.Handle in cause of disease with untreated integral blade in the level of inoculation back alexin mRNA stable state all rise, and the amount of inoculating back 48 hours mRNA in two kinds of leaf samples all reaches maximum (Fig. 4 B).The inducing action of Arabidopsis plant alexin during embodiment 3 chemical treatments

Studied as of late be subjected to cause of disease stress the time derivative gene at least may part be subjected to salicylic acid (SA) or 2, the inducing of 6-dichloro-isonicotinic acid (INA), the latter is a kind of synthetic compound (Ward etc., 1991) of simulating the salicylic acid effect.In Arabidopsis, shown PR-1 already, PR-2 and PR-3 are subjected to that SA and INA handle induces (Uknes etc.) by force.Yet no matter pass through the analysis of the RNA marking still by the detection of ELISA, SA or INA all can not observe the inducing action (Fig. 5) of plant alexin gene expression when handling.On the contrary, the application of ethene or methyl jasmine acid esters obviously increases the plant alexin expression of gene.For example in air, cultivate (contrast of ethene) or use the increase that does not detect plant alexin gene expression in 0.1% (v/v) ethanol (the solution contrast of methyl jasmine acid esters) sample in suitable control treatment.(PR-1, PR-2 PR-3) had before demonstrated and ethene processing irrelevant (Lawton etc., 1994) PR protein gene among the Arabidopsis.Integrate, these data show that plant alexin gene and PR-protein gene induced by on the same group chemicals not.

Chemicals paraquat and rose-red also tested its influence to plant alexin gene expression.With paraquat or known activating oxide superoxide anion and single oxygen molecule (Bowler etc., 1992 of causing respectively of rose-red processing plant; Green and Fluhr, 1995), and therefore cause partial pressure of oxygen (oxidative stress).Plant alexin protein and mRNA are by paraquat or rose-red induce by force (Fig. 5).

The damage of Arabidopsis blade does not cause the plant alexin mRNA (Fig. 5) of the level that can detect when processing detected in back 48 hours.Because temporarily opened (Berger etc., 1995 in the time that known many damage induced genes are lacked relatively after processing; Warner etc., 1993), we also confirm after damage 3,6,9,12 by analysis of the RNA marking and ELISA, 24 and 48 hours, and the plant alexin expression of gene.Yet,,, all can not detect the plant alexin expression of gene no matter damage still is to press from both sides broken blade with tweezers to cause (result does not show) by making otch with scalpel at the time point of any analysis.The integral body of plant alexin is induced jasmine acid esters and/or the ethene dependence approach followed among the embodiment 4Arabidopsis

Because SA and INA are can not inducing plant alexinic synthetic, we have further studied the effect of SA in the plant alexin after fungal infection is induced.Therefore, we have detected in known SA signal pathway by plant alexin expression of gene in the Arabidopsis plant of genetic modification.The Npr mutant strain shows the SA signal pathway of blocking-up, because it can not express PR protein gene (Cao etc., 1994) when SA processing or pathogen infection.On the contrary, cpr mutant strain even SA and the just constantly rising of PR protein level without any infection signal the time.

Water simulation inoculation or inoculate the plant of affected different Arabidopsis strains in the SA signal pathway with the A.brasssicicola spore suspension, and after 72 hours collection and treatment and untreated blade.Analyze and ELISA detection plant alexin expression of gene by the RNA marking.When inoculating wild type (Col-0) plant with A.brassicicola, compare with simulation inoculation plant respective vanes, the plant alexin expression of gene is all induced (Fig. 6) in cause of disease processing and untreated integral blade.Npr1 mutant strain and cpr1 mutant strain plant defense plain gene are all induced equally when being subjected to A.brassicicola and infecting, and do not observe constitutive expression in the cpr1 plant of simulation inoculation.The alexinic approach of embodiment 5 inducing plants needs the composition in ethene and the jasmine acid esters reaction path

As implied above, after using exogenous ethene or methyl jasmine acid esters, the plant alexin accumulation is arranged in the Arabidopsis blade.For understanding fully this effect of two plant species hormone in plant alexin is induced, we have studied in the insensitive mutant strain ein2 of ethene and etr1-3 (Guzman and Ecker, 1990; Chang etc., 1993) and corolla is plain and the middle plant alexin expression of gene of methyl jasmine acid esters non-sensitive type mutant strain coil (Feys etc., 1994).

The influence of inducing the fungal infection that obviously is subjected to ethene non-sensitive type or jasmine acid esters non-sensitive type mutant strain of plant alexin.As if the expression of plant alexin almost is blocked fully in ein2 and coil plant.Handled by cause of disease with the postvaccinal ein2 of A.brassicicola and coil plant and untreated integral blade in level in the wild type plant sample respective vanes handled than similar cause of disease of plant alexin level hang down 30 times at least.The blocking-up of pathogeny evoked plant alexin expression of gene may occur in transcriptional level in ein2 and coil plant, because do not detect plant alexin mRNA in the plant leaf that cause of disease is handled.In the etr1-3 plant, the plant alexin expression of gene is enhanced the level of finding in the wild type blade that is similar to the cause of disease processing in the blade that cause of disease is handled.Yet in the untreated intact leaf of etr1-3 plant, the accumulation of plant alexin is added to the level than low 3 times of the level in the wild type plant integral blade.Described inducing action is consistent in three are independently tested, but induces the degree difference.Embodiment 6 plant alexin in damage simulation mutant strain acd2 is induced by composing type

The formed damage of allergy (Greenberg etc. 1994) that the spontaneous generation of Arabidopsis acd2 mutant strain produces when being similar to and standing strong bacterial invasion by the wild type plant.Because before being displayed in the asymptomatic and downright bad blade all accumulation, this mutant strain had high-caliber PR protein to transcribe this, (Greenberg etc., 1994), and thinking is worth plant alexin expression of gene in the detection acd2 plant.Collect healthy asymptomatic upper strata rosette blade and the downright bad lower floor's rosette blade of performance respectively from the acd2 plant in 5 ages in week, and collect healthy the upper and lower rosette blade from wild type (Col-0) plant that is grown under the similarity condition.According to the detection of being undertaken by ELISA, the acd2 plant has accumulated very high-caliber plant alexin, estimates to constitute respectively asymptomatic blade and 5% and 10% (Fig. 8) that total soluble protein matter in the downright bad blade that damages is arranged.Level in analysis demonstration of the RNA marking and the wild type plant compares, and the plant alexin transcript degree is obviously improved (Fig. 8 A) in the downright bad and asymptomatic blade of acd2.Embodiment 7 ethene and jasmine acid esters are by Equal-position Signal pathway activation PDF1.2 gene

Interact for a certain reason and influence this expression of gene by ethylene reaction the get involved observation prompting ethene of pathogeny evoked expression of mutant strain (coil) blocking-up PDF1.2 and the jasmine acid esters of mutant strain (ein2) and jasmine acid esters reaction of getting involved.In concept, can dream up three ethene and the interactional model of jasmine acid esters (Figure 10).

The ethene that first model prompting cause of disease identification causes increasing generates, and causes stimulating the generation of jasmine acid esters and PDF1.2 subsequently to activate thus.Second model is identical with first, and the grade between the signal of different is ethene and jasmine acid esters reverses.At last, the 3rd model hypothesis ethene and jasmine acid esters do not work in a sequential manner, but work by the equipotential approach, and wherein when cause of disease was discerned, both all needed to be activated to induce the PDF1.2 gene.

Before shown with Alternaria brassicicola inoculation Arabidopsis wild type plant (Col-0) and caused jasmine acid esters level rising (Penninckx etc., 1996) in the blade.First model prophesy rising of this jasmine acid esters level when pathogen infection can not occur among the ethene non-sensitive type mutant strain ein2, yet according to model 2 and 3, the ein2 sudden change can not influence the jasmine acid esters approach that cause of disease stimulates.For testing the prophesy that these are helped mutually, infect ein2 and wild type plant (Col-0) with cause of disease A.brassicicola, thereafter in different time monitoring jasmine acid esters level.As shown in figure 11, jasmine acid esters level begins after 24 hours to rise and more suddenly rises after inoculation and to reach peak value in about 72 hours from inoculating beginning in back 48 hours in inoculation in the wild type plant of fungi inoculation.In the coil mutant strain, the nucleus formation that cause of disease stimulates clearly is not cancelled fully, and even with respect to wild type effect more obvious.Therefore, these results think contradiction with the prophesy that produces according to model 1.

Be further to screen model 2 and 3, measured by wild type plant and ciol mutant strain the ethene nucleus formation when inoculation reacts to A.brassicicola.Model 2 prophesies are when cause of disease is invaded, and the ability that the coil mutant strain stimulates ethene to generate will be blocked, and ethylene reaction is not lowered in the coil mutant strain and model 3 promptings are compared with the wild type plant.Making ethene generation level with A.brassicicola inoculation wild type plant is that the about twice in the plant is inoculated in simulation, reaches peak level (Figure 12) in back 60 hours in inoculation.The similar double growth of ethene generation level is also observed (Figure 12) in the coil mutant plant that A.brassicicols handles.With respect to the wild type plant, ethene generation level on average exceeds twice approximately in the coil mutant strain of inoculation and simulation inoculation.The ethene nucleus formation that stimulates in view of cause of disease in the coil mutant strain obviously is not eliminated, and the gained conclusion is that model 2 is unreliable.Therefore the model 3 that proposes equipotential ethene and jasmine acid esters signal pathway is and the corresponding to unique model of whole observations.

Another mode that confirms the reliability of model 1 is to handle wild type plant and ein2 mutant strain and detect the plant alexin level by ELISA two days later in processing subsequently with methyl jasmine acid esters.Handling the wild type plant with 50 micromole's methyl jasmine acid esters makes the plant alexin level than at least 100 times of the plant alexin height in the wild type plant of solvent processing.On the contrary, the plant defense cellulose content of the ein2 mutant strain of handling with the methyl jasmine acid esters plant of handling do not rise (Figure 13) with respect to solvent.This observation contradicts with model 1, and this model prophesy jasmine acid esters induces the accumulation of PDF1.2 not to be subjected to the influence of ein2 sudden change.

Handle the fact and the model 3 basic noncontradictories that can activate the PDF1.2 gene separately with methyl jasmine acid esters, cause promoting that the generation of ethene is firmly to establish (Crapski and Saniewski, 1992) because handle plant by external application methyl jasmine acid esters.The plant of handling separately non-sterile soil growth with ethene causes promoting the accumulation (referring to embodiment 3) of plant alexin, this also obviously and model 3 inconsistent.Yet, when (25ppm) handles the aseptic plant that is grown on the agar medium separately with ethene, do not observe derivative PDF1.2 accumulation (result is not shown).Handle aseptic plant with methyl jasmine acid esters and still induce the PDF1.2 accumulation, most likely owing to stimulate ethene to generate simultaneously.Ethene stimulates PDF1.2 accumulate in non-sterile plant but does not have plant change that the observation of this accumulation can contact with microorganism in the soil by hypothesis in aseptic plant they obtain explanation to the reactivity of ethene processing.

It should be noted that when considering that model 3 is most probable model, it more may be interactional model between best illustration ethene and the jasmine acid esters that further experiment can demonstrate one of other models.The exploitation of the write down marker gene of jasmine acid esters and/or ethene dependent form alexin approach in embodiment 8Arabidopsis and the tobacco

By PCR method, use primer (gene pool number of registration: T04323) clone ArabidopsisPDF1.2 gene promoter based on the PDF1.2cDNA sequence.This method produces the clone of the genomic DNA fragment of 1616bp, and its sequence is displayed on Figure 14.The sequence of this genomic fragment is positioned at 1202-1296, and 1388-1616 matches just with the PDF1.2cDNA sequence that comes from position 1-326.Be considered to representing the 91bp intron that is positioned at coding PDF1.2 signal peptide inside being arranged in the interruption of 1297-1387 with respect to this genome sequence pairing of described cDNA sequence.Synonym (consensus) sequence observed in sequence and other plant gene around the intron montage position of prophesy is consistent.Importantly, this genome sequence transcribing of PDF1.2 gene outcome of containing the cDNA5 ' sequence of 1201bp and 1232bp expection opened the beginning upstream.Then contrived experiment detect the PDF1.2 genetic transcription open the beginning password 5 ' dna sequence dna whether may contain the promotor of being with regulating element, the inducing action that local and whole expression of this gene that described regulating element decision is pathogeny evoked and jasmine acid esters and other chemicals stimulate, described inducing action will be induced the accumulation of PDF1.2 gene outcome.For this purpose, to comprise that the promoter element of inferring is connected to the code area (coding beta-glucuronidase or GUS) of Escherichia coli UidA gene together with the preceding 1254bp of the genomic fragment in the zone of 5 ' untranslated leader and part coding seven PDF1.2 codons as the corresponding circle of sensation of translation, and then snap on the Agrobacterium tumefaciens rouge alkali synthetase terminator (tNOS).The pPDF1.2-GUS-tNOS expression cassette that is produced is transferred in the plant conversion carrier, transforms by basis (based) root conversion method and enters the environmental C24 of Arabidopsis thaliana.

The pPDF1.2-GUS-tNOS transgenosis to jasmonic handle the ability react at first six independently the offspring of T0 transgenosis ArRbidopsis strain be detected.The seed that makes each strain is at the Skoog agar medium that contains 1% sucrose and 50 micromole's kanamycin or aseptic germination on the same medium that 10 micromole's jasmonics replenish arranged.The back 10 days results seedling that germinate are cooked substrate with 4 methyl umbrella shape acyl group (umbelliferyl) glucosiduronic acids and measure their β glucosiduronic acid enzymic activity according to the method (1987) of Jefforson.β glucosiduronic acid enzymic activity is standardized into every sample total protein content, as using bovine serum albumin(BSA) determined as standard by the method (1976) of Bradford.

As shown in table 1, all tested lines show that β glucosiduronic acid enzymic activity is significantly increased (from 9 to 25 times of scopes) with respect to the seedling that is not subject to processing in the seedling that the jasmine acid esters is handled.The increase of the β glucosiduronic acid enzymic activity that the strongest relative jasmine acid esters of strain 19 performance is induced, this strain is further cultivated to obtain T3 that transgenosis isozygotys for seed by self-pollination.This strain is used to during subsequently all analyze.Table 1 lack or have germinate in 10 micromole's jasmonics after, be loaded with pPDF1.2-GUS-tN0S genetically modified six independently 10 day age of Arabidopsis strain β glucosiduronic acid enzymic activity in the seedling

Transgenic strain	β glucosiduronic acid enzymic activity		Multiple is induced
				Contrast	JA handles
	????3 ????4 ????5 ????6 ????7 ????19	?115 ?113 ?296 ?149 ?21 ?8				????2895 ????978 ????2676 ????2166 ????203 ????202	????25.2 ????8.7 ????9.0 ????14.5 ????9.7 ????25.2

* when 37 ℃ of per minutes and every milligram of soluble protein, 4-methyl 7 hydroxycoumarins of representing with pmoles that discharge from 4-methyl umbrella shape acyl group (umbelliferyl) glucosiduronic acid

Detect demonstration by the RNA gel marking and ELISA, during with fungal pathogen Alernoariabrassicicola inoculation Arabidopsis blade, the PDF1.2 gene is induced by integral body.By in inoculation in 48 hours after handling transfer-gen plant and do not inoculate measure in the integral blade β glucosiduronic acid enzymic activity assessed to inoculation fungal pathogen Alternaria brassicicola and Botrytis cinerea aitiogenic around the expression of pDF1.2-GUS-tNOS reporter gene in the age transgenosis Arabidopsis plant.By applying 5 microlitre drop conidal spore suspensions (5 * 10 ⁵Spore/ml water) (4 on every leaf) inoculation A.brassicicola to lower floor's rosette blade.By on the acupuncture wound of lower floor's rosette blade, covering 5 * 10 of 5 microlitre drops ⁵Spore/strong potato glucose the meat soup of milliliter false add conidium 1 spore suspension (Difco) inoculation B.cinerea (4 wounds of every leaf, 1 of every wound).As shown in figure 15, the blade of being inoculated with A.brassicicols or B.cinerea inoculation back and all obviously improve with respect to β glucosiduronic acid enzymic activity in the blade of simulation inoculation for the integral blade of being inoculated.This shows that the pPDF1.2-GUS-tNOS reporter gene is the suitable mark of the whole pathogeny evoked gene expression of Arabidopsis.

With the Arabidopsis pPDF1.2-GUS-tNOS transfer-gen plant in age around the various chemical treatments to study the ability that it induces described reporter gene to express.The chemicals that uses in these tests is jasmonic (JA, 500 micromoles are in 0.1% ethanol), methyl jasmine acid esters (MeJA, 50 micromoles are in 0.1% ethanol), salicylic acid (SA, the 5 mM aqueous solution), 2,6-dichloro-isonicotinic acid (INA is in the 1 mg/ml water), 1,2,3-diazosulfide-7-carbothioic acid S methyl esters (BTH, 300 micromole's aqueous solution), paraquat (PQ, 25 micromole's aqueous solution).On the blade that plant is launched fully, use described chemical solutions, drop in upper strata blade surface (5 on every leaf) with 5 microlitre drops.One group of plant separately is also processed by contact ethene (25ppm) in the sealing greenhouse.With chemicals or suitable control treatment after 48 hours, from the plant collection blade handled and measure β glucosiduronic acid enzymic activity.

As shown in figure 16, use JA, MeJA, or paraquat handles and to have induced the pPDF1.2-GUS-tNOS reporter gene, and use SA, and INA, BTH or ethene then do not act on.These find further certainly based on the PDF1.2 gene RNA gel marking in the reaction of the processing of chemicals (ethene handle except) to be analyzed and the ELISA detection.Handle the PDF1.2 level (referring to embodiment 3) of the plant that increases non-sterile soil growth though find ethene, this gas hormone does not start the accumulation (referring to embodiment 7) of the PDF1.2 of aseptic plant.Therefore, as if non-sterile condition of culture increases the reactivity of Arabidopsis plant to ethene.The reactivity of this increase may be enough to activate endogenous PDF1, and 2 genes are inlayed reporter but can not activate pPDF1.2-GUS-tNOS.

For confirming SA, whether INA and BTH are used under the condition of inducing the pathogeny evoked gene of SA-dependent form, also comprise Arabidopsis β-1 carrying, inlaying of β glucuronidase code area under the control of 3-dextranase PR-2 type gene (being called Bg12) promotor carried out above-mentioned test (Bowling etc. on the genetically modified transfer-gen plant, 1994, the 6th phase of plant cell magazine 1485-1857 page or leaf).This transgenosis is abbreviated as pBg12-GUS-tNOS below.As shown in Figure 3, handle transgenosis pBg12-GUS-tNOS Arabidopsis plant with SA and its functional analogue INA and BTH and really can cause the β glucosiduronic acid enzymic activity that rises appreciably, and JA, MeJA, ethene or paraquat are all without any influence.Before shown SA, INA and BTH induce the pathogeny evoked gene of SA dependent form among the Arabidopsis, as PR-1, and PR-2 and PR-5 (Uknes etc., 1992).

In a word, these data show that the pPDF1.2-GUS-tNOS reporter gene does not have W-response to pathogen infection when having SA to get involved, thereby show that it is suitable a write down mark of the pathogeny evoked gene activation of SA independent form, and may also be suitable the write down mark of the SA independent form resistance of inducing.

In order to test the mark whether the pPDF1.2-GUS-tNOS promotor can be used as the pathogeny evoked gene expression of SA independent form in the plant beyond the Arabidopsis, also the pPDF1.2-GUS-tNOS gene is introduced among the tobacco cultivation kind Xanthi-nc by the blade rotating disk conversion method of Agrobacterium mediation.Select the transgenosis homozygote of T2 for plant.Proper behind the blade under the blade of the tenderest expansion fully with the tobacco mosaic virus inoculation, whole pathogeny evoked expression in the transgenic strain of detection plant in 8 age in week.Tobacco cultivation kind Xanthi-nc has resistance and resists virus by producing allergy tobacco mosaic virus, therefore prevents that virus is diffused into beyond the damage.Collect the tenderest blade that launches fully at postvaccinal 2,4 and 6 days respectively and be positioned at the tenderest blade that launches fully on the blade, and measure β glucosiduronic acid enzymic activity.By at 50 mM sodium phosphate buffers (pH7), grind by the blade of the pre-tobacco cultivation kind Hicks that infects of tobacco mosaic virus by per 10 milliliters of buffer solutions, 1 gram tissue.10 times of this suspension be diluted in the buffer solution and grind by carborundum powder after be applied on the tobacco leaf.Adjoining tree is only with buffer solution and powder simulation inoculation.As shown in figure 17, β glucosiduronic acid enzymic activity obviously increases in the blade integral blade back 4 days with taking from inoculation to take from back two days being inoculated of inoculation.In addition, by salicylic acid (SA, the 5 mM aqueous solution), methyl jasmine acid esters (MeJA, 50 mMs, 0.1% ethanolic solution), the inducibility of damage check reporter gene is handled and passed through to paraquat (PQ, 25 micromole's aqueous solution).According to 8 age in week plant the blade that launches fully on four drops, 100 microlitres use chemicals.By twisting blade into pieces with 2 centimetres interval test is done in damage with the tweezers that the sawtooth end is arranged.In processed blade and suitable contrast, measure the content of handling back 48 hours β glucuronidase.

As shown in figure 18, methyl jasmine acid esters and paraquat all cause 35 times the growth of being higher than of β glucuronidase.On the contrary, damage and salicylic acid are handled and are only caused 2 active multiplications long.Confirm that by the RNA gel marking analysis of in the tobacco plant of handling with the same manner, carrying out the activation of tobacco PR-1 shows that (situation that is similar to Arabidopsis) this gene only induced by salicylic acid, and not by methyl jasmine acid esters, (not providing) induced in paraquat or damage.

These results show that the PDF1.2 promotor is activated in the non-dependence approach of SA of the tobacco plant of section (a kind of the Solanaceae of belonging to), and when cause of disease was invaded, it can all induced by integral body in the plant.Therefore, the pPDF1.2-GUS-tNOS reporter gene can be used to different plants and reaches screening and induce the compound of SA independent form defense pathway, the purpose of microorganism or physical damnification processing.Embodiment 9 is by ethene and jasmine acid esters co-induction plant alexin promotor

Our relevant jasmine acid esters and ethene can have activating this gene synergism with combining of ethene by discovery (referring to embodiment 7) the prompting jasmine acid esters of equipotential pathway activation PDF1.2 gene.For testing this hypothesis, ethene and methyl jasmine acid esters are combined with the dosage that its every kind compound can not activate the pPDF1.2-GUS-tNOS gene in the transgenosis Arabidopsis report plant separately.As shown in figure 19, in the atmosphere that contains 25ppm ethene, handle the Arabidopsis blade with 0.5 micromole's methyl jasmine acid esters drop and cause very high β glucosiduronic acid enzymic activity, and each independent processing does not cause the β glucosiduronic acid enzyme level of increase.When in conjunction with 333 micromolar ethephons (a kind of compound that produces ethene when being sprayed onto on the plant) and 0.5 micromole's methyl jasmine acid esters in conjunction with the time detect tangible but low-level synergy (Figure 19).In this test, ethephon is utilized with the speed than low 10 times of the speed that just is usually used in the activated plant aging course.

These find that prompting can cause the cause of disease of more effective stimulation natural resistance opposing Special Category with the mixture process plant of activating the compound of ethylene reaction approach and jasmine acid esters reaction path respectively.Embodiment 10 jasmine acid esters and/or ethene rely on approach and play an important role in Arabidopsis resists the defence of some cause of disease

For test is subjected to the disease neurological susceptibility of the Arabidopsis that jasmine acid esters and/or ethene dependent form defense pathway influence, develop a kind of biological detection that is used for sac fungi dead volume nutrition fungal pathogen Botrytis cinerea (epigamous=Botrytinia fuckeliana).This detection comprises with Botrytis cinerea spore suspension covering acupuncture wound, detects by the death of inoculation plant then.Figure 20 shows with a series of wild type Arabidopsis plant (Col-0), the number of dead plant in the test that insensitive mutant strain of ethene (ein2) and the insensitive mutant strain of jasmine acid esters (coil) are formed.On average, all in the ein2 mutant strains of inoculation 41% and the coil mutant strains of all inoculations in 86% dead after 16 days, in contrast, have only 1% wild type plant death (Figure 20).After rotting, ein2 and coil plant have a large amount of conidiums to form.The mutant strain that preliminary test shows is influenced by salicylic acid dependent form defense reaction, npr1 infect the resistance that has as the Col-0 wild type to Botrytis cinerea.These results point out that jasmine acid esters and/or ethene dependent form defense reaction approach are to setting up the importance that the Arabidopsis plant is resisted the resistance of grey mold fungi Botrytis cinerea.

Also use oomycetes biotroph (biotrophic) fungal pathogen Peronosporaparasitica pathovar Wela to carry out wild type (Col-0) and mutant strain npr1, the disease biological of ein2 and coil detects.The fungi structure that detects in the blade by the method for using lactic acid phenol/trypan blue staining detects the infection that this fungi is caused.These experiments show Col-0, and ein2 and coil do not support any cause of disease growth that records, and the npr1 mutant strain is then formed the hypha,hyphae severe infections (Figure 21) of egg spore in a large number.

These data show that jasmine acid esters described herein and/or ethene dependent form defense pathway are successfully to defend Botrytis cinerea but not Peronospora parasitica infects a crucial decisive factor of tendency in a word, and concerning salicylic acid dependent form defense pathway then antithesis.Therefore, in plant as if at least two kinds effectively the complete dissimilar cause of diseases of opposing unique defense pathway in action.Importantly, this finds that prompting when not only inducing jasmine acid esters and/or ethene dependent form defense pathway but also inducing the mixture of compound of salicylic acid dependent form defense pathway, will realize a spectrum induction resistance.

According to inference the viewed disease sensitivity phenotype who has improved of the ethene insensitive and insensitive Arabidopsis mutant strain of jasmine acid esters, if when some cause of disease of host defense, jasmine acid esters and/or the pathogeny evoked defense reaction of ethene dependent form play a major role, and then can expect can cause the susceptibility of some cause of disease is reduced with the compound treatment plant of activating this reaction path.

At least to B.cinerea, we have shown that at least the Arabidopisis plant is by activating jasmine acid esters and/or ethene dependent form gene but not salicylic acid dependent form gene can be set up resistance, can explain thus this cause of disease, 1,2, the phylactic shortage (Lawton etc.) that the 3-diazosulfide-7-carbothioic acid S methyl esters is provided.

Nearly all crop plants to certain limit but not one, two kind of pathogenic microorganism be responsive.Therefore the chemicals that importantly is used for disease control provides resistance to wide as far as possible cause of disease spectrum.We have described the unverified defense pathway that is called jasmine acid esters and/or ethene dependent form defense pathway in this paper forward part, and described defense pathway not only is different from the salicylic acid dependent form defense pathway furtherd investigate but also substantially to controlled special cause of disease provides resistance by activating salicylic acid dependent form defense pathway in addition.Can infer by with blending constituent (cocktails) with not only activated salicylic acid dependent form defense pathway but also activated the jasmine acid esters and/or the compound treatment plant of ethene defense pathway can obtain wider disease control spectrum from these observations.

Concerning the personnel that are familiar with this professional domain, apparent other method of modifying of the present invention do not depart from the scope of the present invention.Screening for example of the present invention also can be used to plant is carried out chemistry, biology, and the screening that microorganism or physical treatment are done is to cause the resistance to salicylic acid independent form damage by disease and insect.List of references

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Claims (25)

1. the method for protective plant opposing cause of disease, this method comprises by stimulating the expression of jasmine acid esters and/or ethene approach inducing plant phylaxin gene.

2. the process of claim 1 wherein that plant alexin expression of gene or a kind of expression of coordinating expressing gene are stimulated jasmine acid esters and ethene approach to induce.

3. claim 1 or 2 method, wherein cause of disease is a dead volume nutrition cause of disease.

4. one of any method of claim 1 to 3, wherein cause of disease is the microorganism cause of disease.

5. one of any method in the aforementioned claim, wherein cause of disease is a fungal pathogen.

6. one of any method in the aforementioned claim, the material that wherein said approach is applied in one or more stimulates, described material is an ethene, jasmine acid esters or jasmonic sample compound, the reagent of analog vinyl effect, reagent, the reagent of the analog vinyl effect of non-amount of herbicide, the reagent of the simulation jasmine acid esters effect of non-amount of herbicide and the reagent that causes oxidative stress of the effect of simulation jasmonic.

7. one of any method of aforementioned claim, wherein the stimulation of jasmine acid esters and/or ethene approach relates to signal transduction composition EIN2 and the COI1 of Arabidopsis.

8. by using one or more ethene to plant, jasmine acid esters, the method for the reagent of analog vinyl or the effect of jasmine acid esters and the reagent inducing plant phylaxin gene expression that causes oxidative stress.

9. the method for claim 7 or claim 8, the reagent that wherein can cause oxidizing force are the rose-red or tetrabromofluoresceins that produces bis-phenol weed killer herbicide such as the paraquat or the dibromide of superoxide anion or cause producing single oxygen species.

10. can inducing plant the phylaxin gene composition of expressing, comprise one or more jasmonic, jasmine acid esters, ethene, the reagent of analog vinyl or the effect of jasmine acid esters and cause the reagent of oxidative stress.

11. can inducing plant the phylaxin gene composition of expressing, comprise one or more generation vinyl compound, the signaling molecule that lipid is derived, salicylic acid, salicylic functional analogue and produce the compound of active oxygen.

12. the composition of claim 11, the compound that wherein produces ethene is selected from ethene, ethephon and 1-aminocyclopropane-1-carboxylic acid.

13. the composition of claim 11 or claim 12, wherein the lipid signaling molecule of deriving is selected from arachidonic acid and derivative thereof, linolenic acid and derivative thereof and jasmine acid esters and derivative thereof.

14. any composition of claim 11 to 13, wherein the compound of active oxygen generation is selected from paraquat, dibromide, rose-red and tetrabromofluorescein.

15. screening is used for the method for the compound of reactance power activity, this method comprises to the position of plant or plant uses the expression that the compound of this species resistance is provided and detects the gene of plant defense plain gene or coordinate expression under a cloud.

16. the method for claim 15, wherein said cause of disease are dead volume nutrition cause of diseases.

17. the method for claim 15 or claim 16, wherein said cause of disease are the microorganism cause of diseases.

The method that one of 18. claim 15 to 17 is any, wherein said cause of disease is a fungi.

19. one of any method of claim 15 to 18, wherein said plant is a radish, tobacco or Arabidopsis.

20. the method for claim 19, wherein said plant is Arabidopsis, and described alexin is the product (Figure 14) of plant defense plain gene PDF1.2, and a kind of and PDF1.2 have sequence or its series of variation of basic autoploidy.

The method that one of 21. claim 15 to 20 is any, wherein said compound is used to blade.

22. the method that one of claim 15 to 21 is any wherein detects by the antibody that uses PDF1.1 or PDF1.2 gene outcome.

23. can inducing plant the phylaxin gene expression promoter, comprise by jasmonic or simulate the reagent and/or the ethene of its effect or simulate the zone that the reagent place of its effect induces.

24. be comprised in the promoter region in the shown nucleotide sequence of Figure 14, or the sequence of basic autoploidy or its series of variation arranged with the shown nucleotide sequence of Figure 14.

25. basically as one of any diagram of front reference described method, composition and promotor.

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