WO2004099415A1

WO2004099415A1 - A new stress-resistance transcription factor (capf1) gene, its protein and a transfected plant by the gene

Info

Publication number: WO2004099415A1
Application number: PCT/KR2003/001937
Authority: WO
Inventors: Doil Choi; So-Young Yi; Young Hee Joung; Sanghyeob Lee; Jeong-Mee Park
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2003-05-07
Filing date: 2003-09-23
Publication date: 2004-11-18
Also published as: KR20040096006A; AU2003264962A1; KR100577142B1

Abstract

The present invention relates to the EREBP/AP2-type hot pepper(Capsicum annuum) transcription factor, CaPF1, induced by plant pathogen and environmental stresses. More particularly, the present invention relates to a DNA encoding or an expression vector for plant transformation capable of providing plants with stress resistance due to the above DNA contained, a plant cell transfected with the expression vector the stress resistant plants or its seeds tranformed with the above expression vector, and the method for preparing stress resistant plants, in which the above DNAs are introduced into plants. According to the present invention, useful plants, which have resistance to various biotic or biotic stresses, can be bred by simple and systemic method.

Description

A NEW STRESS-RESISTANCE TRANSCRIPTION FACTOR (CAPFl) GENE, ITS PROTEIN AND A TRANSFECTED PLANT BY THE GENE

FIELD OF THE INVENTION

The present invention relates to CaPFl (Capsicum annuum Pathogen and

Freezing tolerance related protein 1) which is the EREBP/AP2-type hot pepper(Capsicum annuum) transcription factor, induced by plant pathogen and environmental stresses, the gene encoding the transcription factor and the stress resistance plant transfected by the gene.

BACKGROUND OF THE INVENTION

During their life cycle, plants have to deal with various environmental stress conditions. Biotic and abiotic stress factors cause adverse effects on the growth and productivity of crops. To adjust the changes from environment, plants trigger active defense responses via a number of signal transduction pathways. Among which, what is most intriguing is to induce the expression of various defense-related genes.

Particularly, alterations in the expression of transcription factors has a major influence on plants under stress. There are a variety of transcription factor families, containing a distinct type of DNA binding domain in Arabidopsis. The transcription factors include EREBP/AP2, bZIP, Myp, WRKY and proteins having zinc finger domain, and researches have been being conducted to find out that their expression is inducing under what kind of stress conditions (Rushton et al., 1998; Shinozaki and Yamaguchi-Shinozaki, 2000). For example, Tomato overexpressing Pti4 which is the tomato ethylene- response-factor (ERF), displayed increased resistance to the fungal pathogen, Erysiphe orontii, and increased tolerance to the bacterial pathogen of plants, Pseudomonas syringae pv. tomato. Pti4 is presumed to function as a transcriptional activator to regulate the expression of GCC box-containing genes (Gu et al., 2002; Wu et al., 2002).

In another case, overexpression of CBP1/DREBP1 and DERBP1A which is EREBP/AP2 type transcription factors of Arabidopsis, resulted in enhanced tolerance to environmental stresses, such as drought, salt with high concentration, and freezing (Jaglo-Ottosen et al., 1998). These two transcription factors interact with the CRT/DRE box which is cold-responsive promoter sequence, and regulate the expression of target genes (Kasuga et al., 1999).

Tsil, which is one of the tobacco, ERFs binds both to the GCC box and CRT/DRE box. It was reported that Tsil overexpressed tobacco improved tolerance to salt with high concentration and pathogens. However, only pathogen defense-related genes were constitutively expressed in transformants, and it is unclear whether high salinity-related genes were constitutively expressed in Tsil overexpressed tobacco (Park et al., 2001).

Common regulatory components, including phytohormones, participate in separate signaling pathways. Salicylic acid (SA), ethylene (ET) and jasmonic acid (JA) possibly act as secondary signals in pathogen resistance signal transduction process after pathogen attack, and induce the expression of many defense-related genes (Yang et al., 1997).

Drought and high salinity production cause geneation of a large quanity of abscisic acid(ABA). Exogenously treated ABA regulates the expression of genes responding to dehydration and cold stress. These findings suggest that differences in expression patterns of genes participating in biotic and abiotic- stress stem from transcription factors being different from each other and phytohormones regulating differently each other. Recent studies provide evidences for sharing signaling mechanism responding to biotic and abiotic stress. For the first evidence, the gene expression profiles observed during an incompatible interaction between plant and pathogenic fungus is similar to those induced and expressed from wounding (Durrant et al., 2000). For the second evidence, experiments with cDNA microarrays reveal that genes expressed by inoculation of fungal pathogen are similar to genes expressed after treating cold/drought stress (Schenk et al., 2000; Seki et al., 2001).

For the third evidence, another global gene expression approach using microarrays with 402 Arabidopsis transcription factors revealed a clear overlap of genes expressed in response to stress treatment which is different from each other. There was also significant overlap with the genes expressed during senescence (Chen et al., 2002).

However, despite accumulating data that resistance mechanisms of plant responding to stresses, different from each other overlap, the molecular biologic mechanisms underlying this smdies are yet unknown. Thus, a thorough knowledge at the molecular level, related to biotic and abiotic stress defense mechanism of plants, or to interaction between biotic and abiotic stresses is essential to understand how plants activate the correct responses to various environmental stress factors. DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a novel transcription factor, which give a plant stress resistance and the gene for the factor. Another object of the present invention is to provide a plant stress resistance transformed with the gene for the novel transcription factor, which gives a plant stress resistance.

To achieve the above objects, the present invention is based on the fact that a novel gene, CaPFl (Capsicum annuum Pathogen and Freezing tolerance related protein 1) encoding putative EREBP/AP2 type transcription factor protein was isolated from hot pepper cDNA library(refer to http://plant.pdrc.re.kr ) and characterized.

Moreover, the present invention provides a DNA encoding stress- resistance inducible transcription factor having more than 80% of homology to the amino acid sequence of SEQ ID NO: 2.

In the present invention, the DNA is preferably CaPFl gene having the base sequence of SEQ ID NO: 1, or a DNA capable of hybridizing with the above gene under strict condition.

In the present invention, the stress can be biotic stress such as viral, bacterial, fungal pathogen or abiotic stress such as wound, cold, chemicals.

Furthermore, the present invention provides a plant stress resistant inducible transcription factor protein, which is encoded by the above each DNA. The above transcription factor proteins have EREBP/AP2 domain, and are preferably CaPFl protein having the amino acid sequence of SEQ ID NO: 2. In addition, the present invention provides expression vectors containing the above each DNA, plant cells transformed with the above expression vector, stress resistant plants transformed with the above expression vector or the seeds. The present invention also provides a method for preparing stress resistant plants, in which the above various DNAs or expression vectors are introduced into plants and the method of regulating stress resistance of plants in which the expression of the above various stress resistance inducible transcription factor protein is changed.

Hereinafter, the present invention will be described in detail.

According to the present invention, it has been found that transcripts of CaPFl genes are induced from leaves treated with ethephon or jasmonic acid (JA) as well as tissues infected by host-incompatible and nonhost-incompatible pathogens. Over-expression of CaPFl shows resistance to bacterial pathogens and constitutively induces expression of PR- or environmental stress defense-related genes. Therefore, plants transformed with CaPFl showed strong resistance to bacterial pathogen infection or freezing stress.

It is presumed that CaPFl gene is involved in the signal transduction pathways of biotic or abiotic stress defense response because EREBP/AP2 type of CaPFl transcription factor is preferentially induced by pathogen infection, ethephon treatment or freezing treatment.

Now, various properties of CaPFl will be explained in detail hereinafter. (1) CaPFl expression is induced upon hypersensitive response in hot pepper.

To select genes showing hypersensitive response-specific expression, CaPFl was selected by performing differential display using mRNA of hot pepper inoculated with Xanthomonas axonopodis pv. glycines.

RNA gel blot analysis was performed to verify that CaPFl expression was induced both under the non-host incompatible (interaction between the hot pepper and the Xanthomonas axonopodis pv. glycines), or host-incompatible (interaction between the hot pepper and the Xanthomonas campestris pv. vesicatoria) conditions. The analysis revealed CaPFl gene expression was induced specifically at the site induced of hypersensitive response induced by bacteria in a hot pepper.

(2) CaPFl overexpression induces activation of defense related genes in tobacco and Arabidopsis

To analyze the CaPFl function in stress defense mechanism, CaPFl overexpressing Arabidopsis and tobacco were prepared. Defense related genes were commonly activated in both different plant species. PR2, PDF1.2 and GST 1 (Arabidopsis, tobacco) genes as biotic stress related defense genes, were expressed constitutively, and COR47, COR6.6 and COR78 (Arabidopsis), as freezing stress-related genes were expressed constitutively.

(3) CaPFl overexpressions in tobacco and Arabidopsis induce the resistance to bacterial pathogen In the present invention, the function of CaPFl in defense mechanism has been found by CaPFl overexpression in tobacco and Arabidopsis.

Resistance to Pseudomonas syringae pv. tomato DC3000 was increased in CaPFl overexpressed Arabidopsis, and resistance to Pseudomonas syringae pv. tabaci was increased in CaPFl overexpressed tobacco. It is presumed that such phenomena are caused by the increased expression of PR- or defense- related genes induced by the CaPFl overexpression. From these observations, it can be concluded that the CaPFl gene plays important role in the communication between the pathways generating resistance to bacteria.

(4) CaPFl overexpression in Arabidopsis induces the resistance to freezing stress

For the freezing tolerance test, CaPFl transgenic Arabidopsis and control

Arabidopsis plants were cultured at 25°C for 3 weeks. Plants were transferred to

-5°C for 24 hr and freezed plants were numbered. Control Arabidopsis plants died over 90% and CaPFl transgenic plants survived over 70%. The above observation showed that the tolerance to freezing was increased in the CaPFl overexpressed Arabidopsis

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an electrophoretic picture of RNA gel blotting analysis showing CaPFl expression according to disease resistance in the hot pepper tissue inoculated with pathogenic bacteria.

FIG. IB is an RNA gel blotting picture showing the CaPFl expression specific to tissue in the hot pepper.

FIG. 2 is an electrophoretic picture showing the result of DNA gel blotting analysis for total DNAs of hot peppers treated with Dral, EcoRI, Hindlll, or Xbal using CaPFl cDNA as a probe.

FIG. 3 is an RNA gel blotting picture showing the CaPFl expression induced by biotic stress.

FIG. 4 is an RNA gel blotting picture showing the CaPFl expression induced by treatment of phytohormone or environmental stress.

FIG. 5 a picture showing a specific interaction between CaPFl and GCC box or CRT/DRE box, which are the promoter sequences related plant stress resistance.

FIG. 6 is a blotting picture showing constitutive expression of defense related genes by overexpression of CaPF 1 in Arabidopsis.

FIG. 7A, FIG. 7B and FIG. 7C represent a blotting picture, an individual picture and a chart respectively showing that defense related genes were constitutively expressed and resistance to plant pathogen bacteria was increased, in CaPFl overexpressed tobbaco. FIG. 8 A and FIG. 8B represent respectively a picture and a chart showing that the resistance to freezing stress and plant pathogenic bacteria were increased in CaPFl overexpressing Arabidopsis.

DETAILED DESCTIPTION OF THE INVENTION The present invention will be described in more detail with the following examples and Examples, which are intended to illustrate the examples of the present invention, and therefore they should not be considered to restrict the scope of the present invention.

Example 1 : Isolation/Characterization of the CaPFl gene To examine the regulation mechanism in a plant stress resistant response, CaPFl gene was isolated from hot pepper plants and characterized.

Eight-week-old hot pepper plants (Capsicum annunm cv. Bugang) grown in a growth chamber under a regimen of 16 hr light and 8 hr darkness at 27±2^°C was used as a model system in this example.

(1) 8 week-old hot peppers were syringe-infiltrated with Xanthomonas axonopodis pv. Glycines 8ra; Xag 8ra culture suspension(lX10⁸cfu/ml) and 1 mM MgCl₂ as a control. Then, cDNA library induced by bacterial disease was constructed by isolating mRNA from each individual at each time. Clones having full sequence was isolated using CaPFl DNA fragment selected from mRNA differential display, as a probe

Said CaPFl gene cDNA sequence was analyzed with conventional method (SEQ ID NO: 1). As a result of analyzing, it was revealed that the CaPFl gene consists of nucleotide sequence of 1.4kb. It was also revealed that CaPFl gene contains a single ORF(open reading frame) encoding a putative amino acid sequence(SEQ ID NO: 2) that consists of 369 amino acid residue and having a calculated molecular mass of 41 kDa.

The protein structure analysis based on putative amino acid sequence showed that CaPFl protein contains two domains, EREBP/AP2 DNA binding domain composed of 57 amino acid sequences and a domain which short basic residues and acidic residues signal-transducing to nucleus, are gathered. The gene, which is most homologous in amino acid sequence among gene groups having EREBP/AP2 domain, is AS30 of Arabidopsis which is known to be a cadmium-induced protein. (2) RNA gel blotting was performed according to the conventional method to observe the CaPFl expression at each fixed time according to the method previously described in (1) (FIG.1A). As shown in FIG. 1A, the blotting analysis revealed that the expression of CaPFl gene and PR-4 which is, the resistance related gene were amplified as time goes by.

(3) The transcriptional specificity of CaPFl transcript was examined in several tissues. 18 hours after inoculation, total RNAs were isolated from 8 tissues of roots, stems, leaves, flowers and fruits tissues etc of infected hot pepper plants, for RNA gel blotting analysis, according to the conventional method (Choi et al., 1996). Total RNA 20μg from each sample was fractionated by formaldehyde containing agarose gel electrophoresis method (Sambrook et al., 2001) and transferred onto a nylon membrane (Armersham, USA) for blotting. The fractioned RNAs were blotted and the blotted RNAs were hybridized with full- length CaPFl cDNA labeled with ³²P.

As shown in FIG. IB, transcripts corresponding to CaPFl gene were abundantly found in flowers, stems and germinating seeds. However, almost no transcript was found in leaf-tissues without environmental stress treatment. Generally, EREBP/AP2 type transcription factors were known to be expressed abundantly in roots but the CaPFl expressing amount in roots was very small.

It can be suggested from the above fact that the CaPFl gene was tissue specifically expressed. (4) The copy number of CaPFl in a hot pepper genome was examined by genome DNA gel blot analysis

According to the known method (Lee et al., 2002), genome DNAs were isolated from hot pepper leaves. 20μg genome DNA was treated with enough EcoRI, Hindlll, Xbal and Dral. Treated genome DNA was isolated by 0.7% agarose gel electrophoresis and denatured. Then the DNA was blotted onto Nylon membrane (Amersham, USA). Southern blotting was performed according to the conventional method (Church and Gilbert 1984) using full-length CaPFl cDNA labeled with ³²P-dCTP and gene specific sequence of 3 'end-fragment as probes (FIG.2). D, E, H and X represent experimental groups treated with Dral, EcoRI, Hindlll and Xbal in FIG. 2.

As shown in FIG. 2, 5 bands from the analysis using full-length cDNA and one band from the analysis using gene specific sequence of 3 'end- fragment, were observed. Such results show that several EREBP/AP2 type genes and only one CaPFl gene are present in a hot pepper genome.

Example 2 : Analysis of CaPFl gene expression by biotic stresses

In the aforementioned example 1, the induction of CaPFl gene expression was observed in a hot pepper when a soybean was inoculated with pathogen X. axonopodis 8ra causing hypersensitive response (HR) by inducing non-cyclic resistance in a hot pepper and bacterial pustule in a soybean. To reconfirm such HR specific expression, the HR was induced by inoculating Xanthomonas campestris pv. vesicatoria race 3(Xcv race3) with the concentration of lX10⁸cfu/ml into leaves of ECW (bs2/bs2) which is, hypersensitive pepper cultivar and leaves of ECW-20R (BS2/BS2), resistant pepper cultivar. Such pathogen expresses avrBS2 genes. After inoculating, total RNA was isolated at each various time and then CaPFl gene expression was analyzed through Northern blotting (FIG.3).

As shown in FIG.3, although no visual response was observed in ECW (hypersensitive plant cultivar) 36 hours after inoculating, CaPFl genes and PR-4 genes were abundantly transcribed within 24 hours, and HR was also induced in ECW-20R (hot pepper cultivar with resistance).

Example 3: Analysis of CaPFl Expression by Various Abiotic Stresses Induction of the CaPFl expression in plants was examined under abiotic stress (FIG. 4).

Ethylene (ET), Salicylic acid (SA), Jasmonic acid (JA), etc. were reported as hormones playing important roles in signal transduction mechanism related plant defense. Induction of CaPFl genes expression was examined after said hormones were sprayed in a hot pepper. CaPFl gene expression was induced 30 minutes after Ethylene (ET) or jasmonic acid (JA) treatment.

However, Salicylic acid (SA) treatment did not induce the expression (FIG.4A).

Ethephon represents ethylene releasing compound and MJ represents methyl jasmonate in FIG.4A.

Induction of CaPFl gene expression was examined by performing treatment of drying (mannitol (0.4 M) treating), low temperature(4°C) and salt with high concentration(0.4 M) which are representative environmental stresses.

Although all treated stresses induced the expression, especially cold stress treatment induced the rapid and strong expression (FIG.4B). Example 4: Analysis of whether a specific interaction exists between CaPFl transcription factor and promoter sequence GCC box or CRT/DRE box

The promoter sequence specifically interacting with EREBP/AP2 type transcription factors has been reported already. It was examined whether the

CaPFl protein as transcription factor interacts specifically with GCC box

(Stokinger et al., 1997) or CRT/DRE box (Butter and Singh, 1997) as a promoter sequence of defense related gene.

SEQ ID NO: 3 (GCC box): ATAAGAGCCGCCACTAAAAT SEQ ID NO: 4 (CRT/DRE box): ATTTCATGGCCGACCTGCTTTT

According to the known protein preparing method (New England Biolabs Version 3.03, 'Protein fusion and purification system'manual), CaPFl gene was fused with maltose-binding protein (MBP) gene (MBP:CaPFl) to be expressed in E. coli, and was purely isolated to be used in gel retardation assay. The base sequence of GCC box and CRT/DRE box were artificially synthesized and used (FIG.5).

The result of said experiment showed that CaPFl protein interacted commonly with GCC box and CRT/DRE box as shown in FIG. 5. Such a characteristic is regarded as CaPFl 's own property, where the known EREBP/AP2 type protein interacts with GCC box or CRT/DRE box.

Example 5: CaPFl overexpression effect 1 in Arabidopsis and tobacco plants - Constitutive expression of defense related and cold related genes The analysis was performed to examine the relation between CaPFl 004/099415

overexpression, and defense related gene(PR gene) and cold responsive gene(COR gene).

After CaPFl cDNA was cloned to pMBPl vector having 35S promoter (modified from pBI121 (Clontech Co. USA)) and overexpressed in Arabidopsis, RNA was isolated from the transgenic second-generation plants to be used in RNA gel blots. The probe was chosen from PR genes having GCC box in promoter, such as PR2 (Uknes et al., 1992), PDF1.2 (Penninckx et al., 1996), etc. and COR genes having CRT/DRE box in promoter, such as COR47 (X90959), COR6.6 (X55053), COR78 (RD 29A: Park et al., 2001), etc. The above COR6.6 and COR47 probes were cloned by PCR using primer set consisting of SEQ ID NO: 5/6 and SEQ ID NO: 7/8 respectively. The primer set for PCR synthesis of COR78/RD29A, PDF1.2 and PR2 probe, were used as described in the references (Park et al., 2001; Penninckx et al., 1996; Uknes et al., 1992). SEQ ID NO: 5 : 5 '-AGTATATCGGATGCGGCAGT-3 '

SEQ ID NO: 6: 5'-CAAACGTAGTACATCTAAAGGGAGAA-3'

SEQ ID NO: 7: 5'-CGACGAGAAAGCAGAGGATT-3'

SEQ ID NO: 8: 5'-ATGTCCCACTCCCACATCAT-3'

All said gene probes were constitutively expressed without environmental stress treatment in transgenic arabidopsis. The defense related genes (PR2, 3, 4, 5 gene) were constitutively expressed without environmental stress treatment in trangenic tobacco which belongs to the Solanaceous plant family the same as a hot pepper. The same results were shown in Arabidopsis (FIG. 6). In FIG. 6, control is empty vector-transformed negative control, and line 3, 8 and 22 represent the plant having relatively improved resistance to disease and cold stress among the CaPFl transgenic second generations (that is, CaPFl overexpressed Arabidopsis).

Such a result showed that the same interaction as between specific promoter sequences from example 4 (GCC box, CRT/DRE box) and CaPFl protein, occurred in vivo.

Example 6: CaPFl overexpression effect 2 in Arabidopsis and tobacco plants -Increase of tolerance to plant pathogenic bactrial infection and cold treatment CaPFl overexpressed transgenic plants constitutively expressing various defense related genes were examined whether tolerance to stresses was increased in the plants compared to normal plants.

(1) Increase of resistance to pathogenic bacteria of tobacco The pepper bacterial spot pathogen, Xanthomonas campestris pv. vesicatoria race 3 (Xcv race3) was inoculated into tobacco plants and the result was analyzed(FIG.7A, FIG.7B and FIG.7C). In FIG.7A, C represents the control (non-transgenic plant), the others represent transgenic clones

Compared to the non-transgenic plant (control: C), transcription of CaPFl and resistance related genes (PR2, 3, 4, 5 gene) was increased in the transgenic plant transformed with CaPFl gene according to the present invention(FIG.7A), and visual disease symptoms were hardly observed (FIG.7B). The number of bacterial pathogens was very small in the leaves of transgenic plant inoculated leaves at different times after infection compared to control (FIG.7C). For example, a reduction of over one hundred fold in the number of live bacteria was detected after inoculation in tobacco plant tissues, compared to non-transgenic plants (empty vector-transformed control plants).

The above fact suggests that tobacco plants have tolerance to bacterial diseases by overexpression of CaPFl transcription factor.

(2) Increase of resistance to cold stress and pathogenic bacteria in Arabidopsis

CaPFl overexpressed Arabidopsis was left alone at -5 °C for 24 hours and the number of survived plants were measured. The measurement showed over 90% of non-transgenic plants were frozen to death, and over 70% of transgenic plants survived (FIG.8A). Col-0 represents control (non-transgenic plant) in FIG. 8A.

Moreover DC3000 which is tomato pathogenic bacteria was inoculated into Arabidopsis plant as pathogenic stress and then the result was analyzed (FIG. 8B). As shown in FIG.8B, visual disease symptoms were hardly observed in the transgenic plant transformed with CaPFl gene according to the present invention compared to non-transgenic plant (control: C). Moreover, number of bacterial pathogens was very small in the leaves of transgenic plant at different times after infection compared to control. For example, a reduction of ten to one hundredfold in the number of bacteria was detected after inoculation in tissues of transgenic Arabidopsis, compared to empty vector-transformed control plants.

The above fact suggests that Arabidopsis plants have tolerance to bacterial diseases by overexpression of CaPFl transcription factor. INDUSTRIAL APPLICABILITY

According to the present invention, useful plants, which have resistance to both various plant pathogenic bacteria and cold stress, can be bred by simple and systemic method. The novel gene for transcription factor of the present invention, which gives a plant stress resistance, and the protein expressed from the gene are derived from hot peppers which is a crop that have been used for a long time. Therefore, transformed plants and agriculmral products which are safer can be obtained using the present invention. Furthermore, the present invention provides the resistant mechanism of plants to environmental stresses. Therefore, it can be used as a basis for the studies in the related field and development of technologies.

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Claims

WHAT WE CLAIMED IS:

1. A DNA encoding stress-resistance inducible transcription factor (CaPFl) having more than 80% of homology with amino acid sequence of SEQ ID NO:2.

2. The DNA according to claim 1, wherein said DNA has the base sequence of SEQ ID NO: 1.

3. A DNA capable of hybridizing with the DNA of claim 2 in a strict condition.

4. The DNA according to claim 1, wherein said stress is viral, bacterial, fungal pathogen or abiotic stress.

5. A plant stress resistant inducible transcription factor protein which is encoded by the DNA of any one of claims 1 to 4.

6. The protein according to claim 5, which has an EREBP/AP2 domain.

7. The protein according to claim 5, wherein said transcription factor protein is the CaPFl having amino acid sequence of SEQ ID NO: 2.

8. An expression vector having the DNA of any one of claims 1 to 4.

9. A plant cell transformed with the expression vector of claim 8.

10. A stress resistant plant or its seed transformed with the expression vector of claim 8.

11. A method for producing a stress resistant plant which comprises introducing the DNA of any one of claims 1 to 4 into a plant.

12. A method for controlling stress resistance of a plant which comprises changing expression of the stress resistant inducible transcription factor protein of claim 5.