KR100343975B1 - TRANSGENIC PLANTS WITH DIVERGENT SCaM4 OR SCaM5 GENE TO ACHIEVE MULTIPLE DISEASE RESISTANCE - Google Patents

Abstract

PURPOSE: A gene transferred plant which shows multi-diseases resistance to a wide range of pathogenic organisms of plant by being transferred to heterogeneous genes of soybean calmodulin(SCaM) such as SCaM4 or SCaM5 and plant cell thereof are provided. CONSTITUTION: A gene transferred plant, cells or offspring thereof express genes encoding SCaM4, which is deposited on KCTC(Korean Collection for Type Cultures) as a deposit number of 0543BP and has a sequence number of 1, or soybean calmodulin 5(SCaM5), which is deposited on KCTC as a deposit number of 0544BP and has a sequence number of 3. The multi-diseases resistance of higher plant life regarding one or more of the pathogenic organisms of plant is improved by transferring the gene transferred plant using two SCaM4 or SCaM5 gene vectors into the genes encoding SCaM4 or SCaM5.

Description

TRANSGENIC PLANTS WITH DIVERGENT SCaM4 OR SCaM5 GENE TO ACHIEVE MULTIPLE DISEASE RESISTANCE}

The present invention relates to transgenic plants and plant cells transformed with the calmodulin isoform genes of soybean, SCaM4 or SCaM5, which show multi-disease resistance to a wide range of plant pathogens. The present invention also provides an expression vector having the genes and a host cell into which the genes in the expression vector are introduced to make a pathogen resistant plant. Transgenic plants expressing heterologous SCaM4 or SCaM5 show increased resistance to fungi, bacteria and viruses that normally invade the plant.

More and more pathogens are constantly attacking plants. Very large economic losses occur when pathogens attack higher plants that have insufficient natural defense mechanisms or fail to respond and defend against damage caused by pathogens. Therefore, it is very important to control various plant pathogens in agriculture, and extensive efforts have been focused on controlling pathogenic diseases of crops. However, breeding programs for screening crops that are more resistant to pathogens have rarely succeeded. Moreover, if preformed plant defense mechanisms are inadequate, plants do not detect pathogens, or an active defense response is ineffective, pathogens invade successfully and disease develops accordingly.

Plant resistance to invading pathogens occurs with the development of complex arrays of defensive reactions. If the pathogen has a specific avirulence (avr) gene and the plant host is homogeneous, local lesions are formed at the site of the pathogen's infection, resulting in inhibition of the growth of the pathogen. Response (hypersensitive response). Therefore, plants expressing a specific resistance (R) gene have specific resistance to pathogens expressing the corresponding avr gene. In addition to hypersensitivity reactions, secondary defense reactions can be initiated that allow uninfected parts of the plant to resist a variety of toxic pathogens (called systemic acquired resistance (SAR)). Interactions between plants and pathogens include a series of defenses, including rapid increases in oxidative reactions, transient increases in Ca ²⁺ , accumulation of salicylic acid, synthesis of high-level onset proteins, biosynthesis of phytoalexin, and activation of protective genes. Induce signaling. The results so far indicate that Ca ²⁺ signals are involved in certain plant defense reactions. Influx of Ca ²⁺ ions is one of the first events to occur in cells attacked by pathogens and appears to be essential for the activation of defense reactions such as phytoalexin biosynthesis, induction of defense-related genes and hypersensitivity cell death. However, no direct evidence is available regarding the involvement of CaM in plant disease resistant responses.

Various approaches have been used to control toxic fungi, bacteria, viruses and nematodes. One approach is to use naturally occurring bacteria that have the ability to inhibit or interfere with fungi or nematodes. Another approach is to multiply resistance, which focuses primarily on the manipulation of negative resistance genes that make a small quantitative contribution to the overall resistance of the plant. However, sometimes plants do not recognize pathogens and thus do not cause plant defense mechanisms by induction. Moreover, the protection provided by these approaches is much more limited and far less pronounced than the protection afforded by proper organizational acquired resistance.

The transgenic plant of the present invention is characterized by the level of specific differentiated SCaM4 or SCaM5 protein (0.3-0.5 μg per mg of total soluble leaf protein) at levels similar to those of SCaM1, SCaM2 and SCaM3, which are highly conserved CaM variants in wild type plants Have It has been found that the transgenic plants provided by the present invention exhibit long lasting and widespread resistance, similar to systemic acquired resistance to invasion of various pathogens, ie fungal, viral and bacterial pathogens. This is the first in vivo evidence of the central role of SCaM4 and SCaM5 for a wide range of pathogens, including viruses, bacteria and fungi, as well as for functional differences between CaM isoforms.

The present invention provides higher plant cells that are transformed with the SCaM4 or SCaM5 gene and exhibit enhanced multi-resistance against pathogenic attack by one or more plant pathogens. In addition, the present invention provides an expression vector having SCaM4 or SCaM5 capable of transforming cells of higher plants.

1 is a schematic diagram of a plant expression vector pLES9804 having an exogenous transgene encoding SCaM4 protein,

2 is a schematic diagram of a plant expression vector pLES9805 having an exogenous transgene encoding SCaM5 protein,

Figure 3 shows the rapid induction of SCaM4 and SCaM 5 during plant defense reaction.

(A) is the effect of a fungal elicitor on the expression of calmodulin (SCaM) genes in soybean. Soybean cell suspension culture (hereinafter referred to as 'SB-P') was treated with a fungal derivative prepared from Fusarium solani , and the total RNA was isolated according to the indicated time, SCaM1, SCaM4, SCaM5 , phenylalanine ammonia-lyase (PAL) and β-tubulin mRNAs were analyzed.

(B) is the change in SCaM protein level according to the fungal derivative treatment. SB-P cells were treated as described in (A), and SCaM1 / by immunoblotanalysis using anti-SCaM4 antibody or anti-SCaM1 antibody, respectively. The relative protein levels of SCaM4 / 5 versus 2/3 were examined. The relative protein levels of SCaM isoforms compared to band intensities and regions of known amounts of standard SCaM1 or SCaM4 proteins were calculated by scanning densitometry.

(C) is the effect of various protective signal molecules on the expression of the SCaM genes. SB-P cells were treated for 1 hour as indicated in the line above and mRNA levels of SCaM genes were examined by Northern blot analysis. In the figure, (dH ₂ O) is a control group using water, (Psg) is Pseudomonas syringae pv. Glycinea having avrC gene, and (FE) is a phytoptora paracytica variant Nicotiane ( fungal derivatives prepared from phytophthora paracitica var.nicotianae ), (FE + CHX) are fungal derivatives + 1 μg / ml cycloheximide, (FE + BAPTA) are fungal derivatives + 5 mM 1,2- Bis- (o-aminophenoxy) -ethane-N, N, N ', N'-tetraacetic acid [1,2-bis- (o-aminophenoxy) -ethane-N, N, N', N'-tetraacetic acid)], (Ca ²⁺ + A23187) is 25 μM Ca ²⁺ ionopore A23187 + 5 mM calcium chloride (CaCl ₂ ), (H ₂ O ₂ ) is 2 mM hydrogen peroxide, (G-GO) Glucose and glucose oxidase system, (X-XO) are xanthine and xanthine oxidase system, (SA) is 2mM salicylic acid, (JA) is 10 μM jasmonic acid, (ABA) is 100 μM abscisic acid.

Figure 4 is a phenotype of transgenic tobacco plants continuously expressing either SCaM4 or SCaM5.

(A) is developmentally controlled formation of necrotic sites, such as spontaneous disease lesions of transgenic plants. The entire morphology of the 8-week representative transgenic SCaM4 plant (right), where the lesions appear, was found only in older leaves near the bottom of the plant. The left side shows normal wild type plants of similar age.

(B) Magnification of spontaneous lesions such as spots formed on the leaves of 10 week old SCaM5 transgenic plants (right). For comparison, normal leaves of similar age are shown on the left.

(C and D) are the accumulation of UV-stimulating fluorescent material in the cell walls of spontaneously formed lesions. Cortical fluorescence after microscopic examination of spontaneous lesions under specific interference comparison and staining nuclei of leaf tissues with 4 ', 6-diaimidino-2-phenylindole (epifluorecence) (D) optics.

5 is a continuous expression of PR protein genes in transgenic SCaM4 and SCaM5 tobacco plants.

(A) immunoblots showing enhanced SCaM4 and SCaM5 protein levels in transgenic plants. SCaM protein levels were analyzed for total soluble protein (50 μg) of three independent transgenic plants using either anti-SCaM4 or anti-SCaM5 antibodies.

(B) continuous expression of PR protein genes in transgenic plants. Wild type (WT), control transgenic plant (CT) with empty vector without SCaM gene and three representative independent transgenic plants expressing SCaM4 (S4TG) or three representative independent transgenic plants expressing SCaM5 ( Total RNA of S5TG) was isolated to examine mRNA levels of tobacco SAR genes (31). # Represents the number of transgenic plant systems.

(C) Expression of PR protein gene in transgenic SCaM4 and SCaM5 plants without lesions.

(D) SA-dependent PR protein gene expression. The effects of PR protein gene expression of SCaM4 or SCaM5 continuously expressed in wild type plants or nahG transgenic plants were investigated by RNA gel blot analysis using PR1a and Pr5 probes. Data shown are results from at least 10 representative independent transgenic plants.

6 shows enhanced disease resistance of transgenic tobacco plants expressing SCaM4 or SCaM5 continuously.

(A) are disease responses to the phytofungal pathogen, the phytophthora parasitea strain nicotiane. Signs of disease were found in the plants 7 days after inoculation. Representative results are shown for transgenic plants expressing wild type (WT) and SCaM4 (S5TG) or transgenic plants expressing SCaM5 (S5TG).

(B and C) are fluorescence micrographs of leaves infected with the toxic phytophthora parasitea strain Nicotiane. Infected cells were cleared, stained with aniline blue and irradiated with UV light fluorescence microscopy. Fungal mycelium clearly spreads in the leaves (B) of wild-type plants, but in the leaves (C) of transgenic plants, the fungal hyphae do not grow as detectable and UV-in cells surrounding the sites where fungi have penetrated. Stimulating fluorescent material has accumulated. Rulers represent 100 μm.

(D) In planta bacterial growth. The leaves of mature wild type (WT), transgenic SCaM-4 plants (S4TG) and transgenic SCaM-5 plants (S5TG) were inoculated with Pseedomonas syringae pv. Tabacci at 105 cfu / ml for 5 days. The bacterial growth in the plant was observed. Data points represent the method of two measurements of five independent plant systems.

(E) shows enhanced resistance of SCaM4 and SCaM5 transgenic plants to TMV, a nontoxic pathogen. The second or third fully unfolded young leaves from the top of the plants were gently rubbed with carborundum, inoculated with 2 μg of TMV per leaf, and the development of HR lesions was observed for at least 5 days. Data shown is the number of HR lesions formed on this plant. In all of these pathogen tests, control transgenic plants transformed with empty vectors without the SCaM gene showed essentially similar results to wild type plants (data not shown).

The present invention relates to transgenic plants and plant cells that have been transformed with soy calmodulin isoform 4 or 5 (SCaM4 or SCaM5) and exhibit very enhanced resistance to a wide range of plant pathogens. The present invention also provides a host cell into which the genes in the expression vector are introduced to make the expression vector having the genes and pathogen resistant plants. Transgenic plants expressing heterologous SCaM show increased resistance to fungi, bacteria and viruses that normally invade the plant.

In one more preferred embodiment of the invention, cells of tobacco plants are transformed with the differentiated SCaM4 or SCaM5 genes. Transformed tobacco plants exhibit enhanced multi-disease resistance against various pathogen attacks (fungi, viruses and bacteria). Increased multi-disease resistance will increase crop yields and reduce the amount of fungicides used to control corruption, enabling more competitive agricultural sales.

The soybean-derived SCaM4 and SCaM5 genes are novel and differentiated calmodulin isoforms with the unique ability to activate calmodulin-dependent enzymes. SCaM5 shows about 78% identity with the nucleotide sequence of SCaM4.

The transgenic plant according to the present invention relates to a plant or plant material having at least one or more genetically recombinant recombinant nucleic acid expression cassettes encoding SCaM4 or SCaM5. More preferably, the transgenic tobacco plant of the present invention has at least one of SCaM4 or SCaM5 or both.

As used herein, 'overexpressed' SCaM is a protein produced in an amount greater than that produced intracellularly. For example, overexpression can be achieved by combining a transgene with an appropriate constitutive promoter to ensure that the transgene is continuously expressed. The transgene can be linked to a powerful inducible promoter in order to be overexpressed only when needed selectively. Appropriate levels of overexpression include those in which the transgene expresses at least 10 times more than the naturally occurring level of a particularly preferred intracellular transgene. Sustained promoters suitable for use in the practice of the present invention include cauliflower mosaic virus 35S (CaMV35S) promoters known in the art and widely available (US Pat. No. 5,097,925) and the like.

SCaM4 and SCaM5 described herein are encoded by recombinant transgene molecules. The term 'transgene' as used herein refers to DNA or RNA molecules. The transgene used herein encodes a biologically active amino acid sequence, a protein.

Expression cassettes generally contain a transgene encoding an SCaM4 or SCaM5 protein. The expression cassette refers to a structural gene for synthesizing a desired protein, that is, a DNA molecule capable of directing transcription and translation of cDNA. The expression vector consists of at least one transgene encoding a desired protein, at least one promoter coupled to enable the transgene to operate, and a transcription terminator sequence. Thus, the fragment encoding the protein is transcribed into a transcript capable of providing the desired protein by translation under the control of the promoter site. The location and orientation of the appropriate reading frame of the various fragments of the expression cassette are within the knowledge of those of ordinary skill in the art.

As used herein, the term 'plasmid' or 'vector' refers to an annular double-stranded DNA loop that does not bind to a chromosome. Plasmids have DNA that enables them to express the DNA sequences contained therein, and those sequences are effectively associated with other sequences, that is, promoter sequences, that can affect their expression. More preferred vectors for producing the transgenic tobacco plants of the present invention are the plasmids pLES9804 and pLES9805 described in the Examples section below.

The term 'multi-disease resistance', when used in the context of comparing the resistance level between the transgenic plants of the present invention and other plants, despite the attack of the pathogen compared to the non-transgenic plant or a single genetically transgenic plant And the ability of the transgenic plant of the present invention to maintain the desired phenotype. The level of resistance can be determined by comparing the physical properties of the plant according to the invention with the physical properties of the non-transgenic plants that are exposed or not exposed to infection of the pathogen.

Methods applicable to culturing such cells with genes encoding heterologous proteins as well as methods for introducing the constructs used herein into appropriate host cells are generally known in the art. According to the invention, the vector is introduced into the host cell by Agrobacterium tumefaciens EHA101. In addition to plant transformation vectors derived from Agrobacterium, high-speed ballistic penetration methods can be used to insert the DNA constructs of the present invention into plant cells. Another method of introduction is the fusion of protoplasts with other protoplasts. DNA can also be introduced into plant cells by electroporation.

In one embodiment of the invention, two expression cassettes with transgenes encoding SCaM4 or SCaM5, respectively, are prepared as described in the Examples. The expression cassette is combined into one expression vector to form a DNA construct comprising two individual genes encoding either SCaM4 or SCaM5. The vector is inserted into Agrobacterium tumefaciens cells with a degraded Ti plasmid.

After transformation, transformed plant cells or plants comprising the DNA construct can be identified using a selectable marker. The cells can be cultured in growth media with appropriate antibiotics to select the transformed plant cells.

After selecting the transformed cells, the expression of the desired heterologous gene can be confirmed. MRNA encoded by the inserted DNA by Northern blot hybridization can be detected simply. In addition, the DNA sequence inserted by Southern blot hybridization can be confirmed. Once the presence of the desired transgene is confirmed, the entire plant can be regenerated. Any plant that can be cultured from cultured cells or tissues can be transformed by the present invention.

Genetically transgenic plants of the present invention can be produced by sexually crossing two separate transgenic plants having an expression cassette having at least one transgene with a cross-pollination method. Depending on the species being bred, one of many standard breeding techniques may be used.

Plants to be used in the practice of the present invention include both monocotyledons and dicotyledons. Typical monocotyledonous plants to be used in the practice of the present invention include rice, wheat, corn, barley and other grains, and typical dicotyledonous plants include tobacco, tomatoes, potatoes, soybeans, and the like.

The invention will be explained in more detail by the following non-limiting examples.

<Result>

SCaM4 is 1,429 bp in length, has a 5 'untranslated region of 664 bp, a protein coding sequence of 450 bp and a 3' untranslated region of 315 bp. The protein encoding site consists of 149 amino acids. ATTAAA, a polyadenylation signal, appeared in the 3 'untranslated region.

Nucleotide sequences were compared with CaMs of other plants, SCaM-1, 2, 3 and bovine CaM, and showed more than 70% sequence homology with their protein encoding sites. However, since the 5 'and 3' untranslated regions of SCaM-4 do not show significant homology with other SCaMs, this cDNA clone may be thought to be derived from other gene transcripts in the soybean genome. In the case of the actin gene, the nucleotide sequence differs only 11-15% between plant actin and non-plant actin, and only 6-9% within the actin family members of soybeans. SCaM-4 differs greatly by more than 30% compared to SCaM-1, 2 and 3.

Comparing the estimated amino acid sequences of CaM and bovine CaM and SCaM-4 of higher plants, it can be seen that SCaM-4 has more than 80% sequence identity with the estimated amino acid sequence. If not, the plant CaMs are at least 95% identical to each other. Interestingly, the estimated amino acid sequences of SCaM-4 are very different. SCaM-4 is 32 amino acids substituted for SCaM-1,2,3, 26 amino acids for potato CaM and 28 amino acids for bovine CaM.

A notable substitution is the substitution of phenylalanine (Phe) with tyrosine (Tyr) in the third Ca ²⁺ -binding domain. In bovine CaM, tyrosine (Tyr) residues were thought to have been phosphorylated to function to react with target proteins in the absence of Ca ²⁺ . The second notable substitution claim 1 Ca ²⁺ - lysine (Lys) in the positively charged binding domain is replaced with glutamic acid (Glu) negatively charged, claim 2 Ca ²⁺ - the aspartic acid-proline (Pro) in the binding domain Substituted with (Asp). Ca ²⁺ - binding domain was substituted for Ca ²⁺ - indicated that the binding affinity and structural changes are affected. Another notable substitution is the substitution of glycine ⁴¹ (Gly ⁴¹ ) with aspartic acid (Asp) and serine ⁸¹ (Ser ⁸¹ ) with alanine (Ala). This residue is necessary for helix bending sites to fix two lobes on a wide variety of basic amphoteric α-helices at various targets. Glycine ⁴¹ (Gly ⁴¹⁾ would have been highly conserved in evolution, because of the smaller size to be a sharp bending. Larger and charged aspartic acid (Asp) will interfere with this warpage. In addition, substitution of valine-145 (Val-145) with three short, unbranched methionines (Met) in the hydrophobic moieties can provide individual contact for activation of specific targets.

SCaM-5 is composed of 68bp 5 'untranslated region, 450bp open reading frame and 397bp 3' untranslated region. The protein encoding site consists of 149 amino acids. No polyadenylation signal was found.

Nucleotide sequences of CaMs of other plants, SCaM-1, -2, -3, -4 and bovine CaM show more than 70% sequence homology within their protein encoding sites. However, the 5 'and 3' untranslated regions of SCaM-5 do not show significant homology with other SCaMs, suggesting that this cDNA clone is derived from other genes in the soybean genome. SCaM-5 has an interesting sequence motif of ATTTA in the 3 'untranslated region. This motif is known as the mRNA destabilizing motif of eukaryotic cells recognized by RNAase or other cellular factors.

SCaM-4 exhibits at least 80% sequence homology when compared to the higher amino acid CaM and bovine CaM and the estimated amino acid sequence of SCaM-5. Interestingly, the speculative amino acid sequences of SCaM-5 are very different from other SCaMs and plant CaMs. SCaM-5 is the most differentiated SCaM isoform with 18 amino acid substitutions in SCaM-4. Such substitutions include the replacement of five aspartic acids (Asp) with glutamic acid (Glu) as found in SCaM-1 and -2. One notable substitution of the amino acid residues at SCaM-4 and -5 is the tyrosine ⁹⁹ (Tyr ⁹⁹ ) residue found in the third Ca ²⁺ -binding domain. Tyrosine ⁹⁹ (Tyr ⁹⁹ ) residues in the third Ca ²⁺ -binding domain were found only in animal CaM and not yet in plant CaM sequences. Tyr ⁹⁹ is considered as a target site for phosphorylation by src kinases (src kinase) and the insulin receptor kinase (insulin receptor kinase). In addition, the serine ⁸¹ (Ser ⁸¹ ) residues conserved in all identified CaM sequences are substituted with alanine (Ala) or glutamic acid (Glu) at SCaM-4 and -5, respectively. In vitro (in vitro) serine ⁸¹ (Ser ⁸¹⁾ residue is observed to be phosphorylated by casein kinase II (casein kinase II). As is clearly identified from the sequence comparison, SCaM-5 can be classified into a novel type of CaM group together with SCaM-4.

Treatment of soy cell suspension cultures with non-specific fungal derivatives prepared from Fusarium Solani resulted in mRNAs encoded by SCaM4 and SCaM5 , whose sequences are the two different SCaM genes that differ from other CaM genes and belong to different CaMs . Significantly increased more than 10 times (FIG. 3A). Their mRNA levels peaked at 1 hour and slowly decreased to 12 hours to basal level. The basic expression levels of these two SCaM genes in untreated cells were low compared to the basic expression levels of the highly conserved SCaM genes SCaM1, SCaM2 and SCaM3 . The expression of these three conserved SCaM genes was not activated by derivatization as compared to SCaM4 and SCaM5 (FIG. 3A). Consistent with changes in SCaM mRNA levels, protein levels of SCaM4 / 5 but not SCaM1 / 2/3 also increased within 30 minutes after treatment (FIG. 3B). After 2 hours, the SCaM4 / 5 protein level was about 0.5 µg / mg of their highest water-soluble protein and gradually decreased after 12 hours. In contrast, SCaM1 / 2/3 protein levels were not significantly changed by fungal derivative treatment. Interestingly, the induction of SCaM4 and SCaM5 gene expression occurred earlier than the induction of the phenylalanine ammonia-lyase gene, which began to increase mRNA levels 3 hours after treatment (see FIG. 3A). Derivatives made from the Phytophthora parasitica varieties Nicotiane ( Phytophthora parasitica var. Nicotianae ) have a similar effect on these CaM gene expression (see Figure 3C).

Next, we examined the effects of various potential derivatives of plant defense-related genes to learn more about the molecular signals involved in the induction of SCaM4 and SCaM5 gene expression. Pseudomonas syringa e pv. Glycinea ( Psg), which has a phytoptopara parasitica derivative and avrC , a bacterial pathogen, effectively induced SCaM4 and SCaM5 gene expression. Cycloheximide, a protein synthesis inhibitor, did not block the induction of SCaM4 and SCaM5 by phytophthora paracitica derivatives. Conversely, Ca ²⁺ chelator, 1,2-bis- ( o -aminophenoxy) -ethane-N, N, N ', N'-tetraacetic acid [1,2-bis- ( o -aminophenoxy)- ethane-N, N, N ', N'-tetraacetic acid; BAPTA] did not induce such SCaM4 and SCaM5 , whereas A23187, a Ca ²⁺ -ion transporter, alone alone induced sufficient expression of SCaM4 and SCaM5 as effectively as fungal derivatives (FIG. 3C). However, application of 1-5 mM exogenous salicylic acid (SA) or 1-10 mM hydrogen peroxide (H ₂ O ₂ ) did not induce SCaM4 and SCaM5 gene expression over 24 hours (data not shown). ). Hydrogen peroxide production systems (glucose and glucose oxidase) and peroxide production systems (xanthine and xanthine oxidase) also did not induce the expression of the SCaM4 and SCaM5 genes. Two unrelated signal molecules, jasmonic acid (JA) and abscisic acid (ABA), also failed to induce SCaM4 and SCaM5 . These results suggest that transcriptional activation of SCaM4 and SCaM5 genes by induction is a very rapid process that does not require protein synthesis and occurs prior to the synthesis and accumulation of reactive oxygen species (ROS) and SA, or other disease-resistant signaling pathways. Indicates that it belongs. Their activation was also specific; Signaling compounds associated with other stress responses such as wounds (JA) and deficiency (ABA) did not induce SCaM4 and SCaM5 . However, their induction seems to be mediated by an increase in intracellular Ca ²⁺ concentrations.

It was constructed continuity of cauliflower transgenic tobacco plants that continuously express the SCaM4 SCaM5 or under the control of a mosaic virus 35S promoter in order to investigate the biological function of a temporary differentiation in plant defense reactions SCaM conformers body. Transgenic plants expressing SCaM4 or SCaM5 were selected by Northern blot analysis and confirmed by immunoblot analysis. Interestingly, transgenic SCaM4 and SCaM5 plants sometimes spontaneously produced necrotic spots such as disease in their leaves (Figures 4A, 4B). These lesions initially appeared in the oldest mature leaves, and the top three or four young leaves did not appear. These results suggest that spontaneous lesion formation is genetically regulated in transgenic plants. Control transgenic plants transformed with the untransformed wild type grown under the same conditions and an empty vector without SCaM4 and SCaM5 did not show this indication. To determine whether these lesions were lesions similar to HR, they were examined by fluorescence microscopy. Lesions similar to HR accumulate fluorescent substances in the cell walls that can be easily identified by irradiation with UV light, whereas necrotic areas caused by mechanical wounds or freezing and thawing do not have such fluorescence. The leaves of transgenic plants accumulate vivid UV-stimulating fluorescent material in their lesions, suggesting that these necrotic lesions are similar to HR-like lesions (Figure 4C, 4D).

Sustained expression of these differentiated CaM variants, SCaM4 and SCaM5, did not affect the levels of highly conserved intracellular tobacco CaM recognized by anti-SCaM1 antibodies (FIG. 5A). Anti-SCaM1 antibodies recognized the same highly conserved CaM variants, SCaM1, SCaM2 and SCaM3 proteins, but did not recognize the various SCaM4 and SCaM5 proteins. Anti-SCaM4 antibodies cross reacted with SCaM5 but not with highly conserved SCaM isoforms (16, data not shown). The amount of SCaM4 and SCaM5 in these transgenic plants is estimated to be 0.3-0.5 µg per mg of total soluble leaf protein, which is similar to the highest level of SCaM4 / 5 protein induced by fungal derivatives in soybean cells (Fig. 3B). In addition, the levels of SCaM4 and SCaM5 in transgenic plants were not higher than the protein levels of the highly conserved CaM variants, SCaM1, SCaM2 and SCaM3. Thus, the phenotype of these transgenic plants is not due to the high overexpression of SCaM4 or SCaM5, which may cause abnormal cellular responses, but rather the level of these differentiated CaM isoforms similar to those after activation of these genes by pathogen attack. It seems to be because.

Spontaneous lesions that form mutations sometimes indicate increased expression of genes encoding pathogenesis-related (PR) proteins and increased resistance to pathogens. Interestingly, in the absence of pathogens, transgenic SCaM4 and SCaM5 plants expressed high levels of all nine SAR marker genes in tobacco normally activated during SAR development (FIG. 5B). Wild-type plants grown under the same conditions and plants transformed with empty vectors from controls without SCaM4 and SCaM5 did not express these genes. SAR-associated genes were continuously expressed during the growth and development of SCaM4 and SCaM5 transgenic plants and clearly expressed in the leaves of aseptically grown young seedlings with no developing lesions (FIG. 5C). Therefore, the expression of PR genes in transgenic SCaM4 and SCaM5 plants was independent of pathogenesis, suggesting that PR gene expression was not the result of cell death in transgenic plants.

SA is known as a natural signal molecule for the activation of certain plant defense reactions. Application of exogenous SA to tobacco leaves results in a similar response to pathogen-induced SAR, such as PR protein synthesis. In addition, the levels of intracellular SAs in many mock mutations are substantially higher than those of wild-type plants without pathogen attack. In addition, transgenic plants that continuously express the bacterial nahG gene encoding SA degrading enzymes lack the ability to induce SAR. Therefore, we investigated whether disease resistance in SCaM4 or SCaM5 transgenic plants is due to an increase in intracellular SA levels. Surprisingly, SA levels in SCaM4 and SCaM5 transgenic plants were comparable to those of wild type (Table 1).

In Table 1 below the levels of SA (μg / g) were determined from the 4-5th middle leaves from above of the mature plants as described. Data were averaged twice from five independent transgenic plant systems. Bound SA levels were below detection limits in uninfected plants. Intracellular SA levels in plants infected with tobacco mosaic virus were measured 10 days after inoculation.

Intracellular Salicylic Acid (SA) Levels in Transgenic SCaM-4 and SCaM-5 Plants Plants Uninfected TMV infection Liberated SA Liberated SA Combined SA Wild type 0.042 ± 0.005 2.8 ± 0.5 2.2 ± 0.5 Transgenic SCaM-4 0.031 ± 0.010 4.1 ± 1.0 2.4 ± 0.8 Transgenic SCaM-5 0.035 ± 0.005 2.5 ± 0.5 1.6 ± 0.5

In addition, infection of Tobacco mosaic virus (TMV) resulted in similar SA levels in transgenic plants and wild-type plants, so that expression of SCaM4 and SCaM5 in transgenic plants did not affect intracellular SA accumulation. Not shown. In line with this observation, the presence of the nahG gene and the expression did not inhibit the PR gene is constantly expressed in SCaM4 SCaM5 and transgenic plants (FIG. 5D). These results strongly indicate that SA is not involved in plant disease resistance responses mediated by SCaM4 or SCaM5.

Finally, we evaluated whether transgenic SCaM4 and SCaM plants have changed resistance to pathogens. We inoculated transgenic plants and wild plants with Phytophthora parasitica var.nicitianae (the pathogen of black shank disease), an oomycete fungal pathogen. . On day 5 after inoculation of phytophthora parasitica, signs of disease began to appear in wild-type plants but not in transgenic plants. On day 7 after inoculation, wild-type plants died of disease only 8 days after vaccination due to severe disease signs of withering leaves and rotting stems. However, transgenic plants survived healthy without detectable disease symptoms (FIG. 6A). The leaves of wild-type plants infected with fungi were found to have little callose deposited and fungal hyphae spread uninterrupted (FIG. 6B). In contrast, inoculated transgenic plants lacked the growth of such fungal hyphae and instead showed bright fluorescence associated with the deposition of autofluorescein and the deposition of carloose in the cells surrounding the fungal infiltration (FIG. 6C). This is a characteristic of HR. Interestingly, the phytophthora parasitika strain Nicotiane is a malignant pathogen for untransformed Xanthi-nc tobacco cultivars of the parent but does not cause HR in normal conditions. The transgenic plants also exhibited increased resistance to the malignant bacterial pathogen Pseudomonas syringae pv. Tabacci (Pst). They successfully blocked the development of disease symptoms, and growth in transgenic plants of Pst was delayed more than 10-fold compared to growth in wild-type plants (FIG. 6D). The transgenic SCaM4 and SCaM5 plants also showed increased resistance to TMV, a malignant viral pathogen. In transgenic plants, TMV-induced HR lesions developed about 12 hours earlier than wild-type plants. There were also about 5 times fewer and smaller TMV-induced lesions in transgenic plants (FIG. 6E), which is a two feature associated with enhanced resistance to TMV.

In the present invention, the main role of CaM, a major Ca ²⁺ signal transducer in plant defense signals, is to be demonstrated. As a result, it is known that the differentiated CaM isoforms act as signal receptors and transporters for pathogenic Ca ²⁺ signals. Can be. Differentiated CaM isoforms resemble immediate early genes, such as fos or jun, in animal systems in which external stimuli activate their expression to induce cellular responses. Thus differentiated CaM isoforms are representative of novel inducible components of plant defense reactions.

Transgenic plants consistently expressing these differentiated CaM dimorphs had a phenotype similar to that of spontaneous mock lesion mutations; But there are several notable differences. The first concerns the causal relationship between cell death and PR gene expression. PR gene expression is closely linked to apoptosis in lsd and acd mutations, but is independent of apoptosis in SCaM4 and SCaM5 transgenic plants. The second major difference is the SA dependency. SA levels in most mock mutations are substantially higher than SA levels in normal plants. Conversely, in SCaM4 and SCaM5 transgenic plants, disease resistance was activated without simultaneously increasing intracellular SA levels. In addition, the removal of SA from these transgenic plants by co-expression of the nahG gene did not block the continuous expression of the PR gene. This observation strongly indicates that the differentiated isoforms activate the plant disease resistance response through the SA-independent pathway (s).

The present invention provides the first in vivo evidence for functional differences between plant CaM isoforms. Only differentiated CaM isoforms can be induced by pathogens to cause a protective response in transgenic plants, while other highly conserved CaM isoforms such as SCaM1 and SCaM2 did not have this property (data not shown). Since differentiated CaM isoforms do not activate NAD kinase, the Ca ²⁺ / CaM pathway in ROS production is thought to be mediated by highly conserved CaM isoforms. These observations indicate that the highly conserved CaM isoforms mediate ROS increase while the differentiated CaM isoforms activate predetermined cell death and protective gene expression, which contributes to the cooperative role of CaM isoforms in plant defense responses against pathogens. Support the model.

Transgenic plants that consistently express several different transgenes have been shown to have mock-type phenotypes and altered disease resistance. These genes include the opsin gene of Halobacterium , the mutant ubiquitine ubR48 gene, the cholera toxin subunit A1 gene, and the yeast invertase. These transgenic plants increase the level of intracellular SAs, indicating that their altered disease resistance response is the result of SA accumulation that can be thought of as belonging to metabolic stress induced by these transgenes. Therefore, it is not clear whether these genes are true components of the plant defense pathway (s). Therefore, it can be seen that the differentiated CaM isoforms are one of the first 'natural' pathogen-inducible components of plant defense signals that are constantly expressed and lead to increased disease resistance. The present invention not only enhances our understanding of the path (s) leading to plant disease resistance, but also presents new opportunities for genetically engineered plants that are resistant to a wide range of pathogens.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the invention and do not limit the scope of the invention.

The procedures of the DNA recombination technique described below are those known in the art and commonly applied. These techniques and various other techniques are generally done according to the molecular cloning-experimental manual (Sambrook et al., 1989). All general citations are provided throughout this document. All information contained herein is incorporated by reference.

Example 1--Isolation of Calmodulin cDNAs from Soybeans

Isolation of SCaM-4 cDNA Clones from Soybeans

To isolate the SCaM cDNA clone, Stratagene's λZAP soybean cDNA library consisting of the polytops of the four-day seedlings and the poly (A) ⁺ RNA of the semi-growths was used. Library screening was done using Amersham's ECL gene detection system. The genomic calmodulin clone of rice, cam-2 , was used as probe DNA. About 35,000 phages were applied to E. coli XL1-Blue and transferred to Amersham's Highbond N ⁺ (Hybond N ⁺ ) nylon membrane. The membranes to which phages were transferred were denatured by soaking in 0.5 M sodium hydroxide (NaOH) solution and 1.5 M sodium chloride (NaCl) solution for 5 minutes. The membrane was then neutralized by dipping twice in 0.5 M tris hydrochloric acid solution (Tris-HCl) (pH 8.0) and 1.5 M aqueous sodium chloride solution at room temperature twice. The membrane was immersed in 0.4N aqueous sodium hydroxide solution for 2 minutes and alkali fixed. The membrane was pretreated for hybridization at 37 ° C. for 2 hours with hybridization buffer containing 0.5 M sodium chloride and 5% blocking agent. The membrane was then hybridized with probe DNA labeled horseradish peroxidase for 6 hours at 37 ° C. with gentle shaking of the membrane. After hybridization, 6M urea, 0.4-fold SSC, 0.5% SDS was washed twice at 37 ° C. for 20 minutes and twice at room temperature for 2 minutes with 2 times SSC. The membrane was then detected with detection reagents A and B for 1 minute and then exposed to Kodak X-OMAT AR film for 10 minutes. Positively hybridized plaques were further purified with the next round of plaque hybridization.

To obtain cDNA inserts, purified positive plaques were excised in vivo with pBluescript-SK (−) with helper phage R408. 200 μl of XL1-Blue host cells (OD ₆₀₀ = 1.0) 200 μl of λZAP phage stock containing about 1 × 10 ⁶ pfu / ml and 1 μl of R408 helper phage (1 × 10 ⁶ pfu / Ml). After incubating the mixture for 15 minutes at 37 ° C., 5 ml of 2-fold YT medium (16 g of Bacto tryptone per liter, 10 g of Bacto Yeast Extract and 10 g of sodium chloride) was added. After incubation at 37 ° C. for 3 hours with shaking, the mixture was heated at 70 ° C. for 20 minutes to inactivate helper phage and kill bacteria. Next, the mixture was centrifuged at 4000 g for 10 minutes and the supernatant was transferred to a sterile test tube and stored at 4 ° C. To obtain phagemid excised from this stock, 200 μl of XL1-blue host cell (OD ₆₀₀ = 1.0) and 200 μl of phagestock were combined and then incubated at 37 ° C. for 15 minutes. 50 μl of the mixture was applied to LB / ampicillin plates and incubated at 37 ° C. for 12 hours. Ampicillin resistant colonies with the obtained phagemid were observed.

Applications Open Biosystems Inc. stand 373A automated DNA bases (DNA sequencer) using the Applied Biosystems Inc. (Applied Biosystem) of the Taq Dye Primer Cycling sequencing kit has (Taq Dye Primer Cycling Sequencing Kit) for double-stranded plasmid DNA A sequencing reaction was performed. Hitachi DNASIS on IBM PCs or GCG packages (Genetics Computer Group, University of Wisconsin) on DEC Station 3300 / Ultrix system Analyzed.

ΛZAP cDNA libraries were screened using cam-2 , a genomic clone of rice as a probe, to isolate cDNA encoding soybean CaM. SCaM-1, 2 and 3 were isolated from 62 positive clones among 3.5 × 10 ⁴ clones to confirm the sequence in advance. 15 clones with very low intensity were randomly selected and screened 2-3 times. Partial nucleotide sequences and restriction enzyme mapping of these clones were used to select one clone with a very long cDNA size and a different restriction map pattern compared to other SCaMs.

The finally selected clone was a complete cDNA and was named SCaM-4 (SEQ ID NO: 1). There are two Hind III enzyme sites and one EcoR I enzyme site in the internal sequence. Therefore, the Hind III fragment, two EcoR I- Hind III fragments were subcloned for pBluscript SK (-), and the EcoR I- Xho I fragment was self-ligation on pBluscript SK (-). Then, the sequences of the fragments were confirmed in both directions. The long Hind III fragment was identified by a deletion sequencing method using ExoIII enzyme.

Isolation of SCaM-5 cDNA Clones from Soybeans

To isolate SCaM cDNA clones, Stratagene's λZAP soybean cDNA library consisting of the polytops of the four-day seedlings and the poly (A) ⁺ RNAs of the semi-growths were used. Libraries were screened using the ECL gene detection system from Amersham. The genomic calmodulin clone of rice, cam-2 , was used as probe DNA. About 50,000 phages were applied to E. coli XL1-Blue and transferred to Amersham's Highbond N ⁺ (Hybond N ⁺ ) nylon membrane. The membranes to which phages were transferred were denatured by soaking in 0.5 M sodium hydroxide (NaOH) solution and 1.5 M sodium chloride (NaCl) solution for 7 minutes. Subsequently, the membrane was immersed in a 0.5 M tris hydrochloric acid solution (Tris-HCl) (pH 7.5) and a 1.5 M aqueous sodium chloride solution at room temperature for 7 minutes to neutralize. The membrane was then pretreated for hybridization with hybridization buffer containing 0.5 M sodium chloride and 5% blocking agent at 37 ° C. for 2 hours. The membrane was then hybridized with probe DNA labeled horseradish peroxidase for 12 hours at 37 ° C. with gentle shaking of the membrane. After hybridization, 6M urea, 0.4 times SSC, 0.5% SDS was washed twice at 37 ° C. for 20 minutes and twice at 37 ° C. for 2 minutes with SSC. Positively hybridized plaques were visualized using detection reagents A and B for 1 minute and exposed to Kodak X-OMAT AR film for 10 minutes. Positively hybridized plaques were further purified with the next round of plaque hybridization. To obtain cDNA inserts, purified positive plaques were excised in vivo with pBluescript-SK (−) with helper phage R408. 200 μl of XL1-Blue host cells (OD ₆₀₀ = 1.0) were mixed with 200 μl of λZAP phage stock containing about 1 × 10 ⁶ pfu / ml and 1 μl of helper phage R408. The mixture was incubated at 37 ° C. for 15 minutes and then 5 ml of 2-fold YT medium was added. After incubation at 37 ° C. for 3 hours with shaking, the mixture was heated at 70 ° C. for 20 minutes to inactivate helper phage and kill bacteria. Next, the mixture was centrifuged at 4000 g for 10 minutes and the supernatant was transferred to a sterile test tube and stored at 4 ° C. To obtain phagemids excised from the stock, 200 μl of XL1-blue host cells (OD ₆₀₀ = 1.0) were combined with 100 μl of phagestock and incubated at 37 ° C. for 20 minutes. 50 μl of the mixture was applied to LB / ampicillin plates and incubated at 37 ° C. for 15 hours. Alkaline lysis minipreps were used to determine whether ampicillin resistant colonies had appropriate phagemids.

Nucleotide sequence of the application of Biosystems Inc. 373A using the open standing automated DNA bases have applied Biosystems, Inc. Taq dye primer cycling sequencing kit (Taq Dye Primer Cycling Sequencing Kit) double-stranded plasmid DNA as specified by the manufacturer The reaction was carried out. Nucleotide sequences were analyzed using Hitachi's DNASIS on an IBM PC or using the GCG package (Genetics Computer Group, University of Wisconsin) in a DEC Station 3300 / Ultrix system.

ΛZAP cDNA libraries were screened using cam-2 , a genomic clone of rice as a probe, to isolate cDNA encoding soybean CaM. SCaM-1, -2, -3 and -4 were isolated from 62 positive clones among 3.5 x 10 ⁴ clones to confirm the sequence in advance. However, the fifth clone cut off the 5 'region. To obtain a complete clone, the library was rescreened with specific probes made from 3 ′ untranslated sites. By analyzing partial nucleotide sequences and restriction enzyme mapping of four clones, one clone with the same nucleotide sequence as the truncated clone was selected. The selected clone was shown to be complete cDNA. This clone was named SCaM-5. There is one EcoR I enzyme site and one Sac I enzyme site in the internal sequence.

Example 2--Configuration of Expression Vectors

Composition of the plasmid pLES9804

For overexpression of SCaM-4 (SEQ ID NO: 1) in tobacco plants, SCaM-4 was administered under the control of the Cauliflower Mosaic Virus 35S subunit (CaMV35S) promoter site and the Agrobacterium nopaline synthase terminator. The branch consists of two-component vectors. The pGA643 binary vector was digested with Hpa I. SCaM-4 cDNA was digested with Rsa I. The 854 bp DNA fragment digested with RsaI from SCaM-4 cDNA was directly cloned into pGA643 vector digested with HpaI to give pLES9804. The vector in Agrobacterium tumefaciens EHA101 / pLES9804 has been deposited with Accession No. 0545BP to the Korean Collection for Type Culture (KCTC) in accordance with the Budapest Treaty.

Composition of the plasmid pLES9805

For overexpression of SCaM-54 (SEQ ID NO: 3) in tobacco plants, SCaM-5 was administered under the control of the Cauliflower Mosaic Virus 35S subunit (CaMV35S) promoter site and the Agrobacterium nopaline synthase terminator. The branch consists of two-component vectors. pGA643 binary vector was digested with Hpa I. SCaM-5 cDNA was digested with EcoR I. The sock end of the DNA fragment (562bp) of the SCaM-5 cDNA is converted to a blunt end with the appropriate Klenow fragment and linked to a T4 DNA ligase to give a pLES9805 binary vector. Cloned into. The vector in Agrobacterium tumefaciens EHA101 / pLES9805 has been deposited with accession number 0546BP to KCTC (Korean Collection for Type Culture) in accordance with the Budapest Treaty.

Example 3-Generation of Genetically Converted Tobacco Plants

Transgenic tobacco plants expressing SCaM-4 were produced by leaf disc transformation mediated by Agrobacterium. Tobacco leaf plaques were made from 8-week-old young tobacco plants and soaked for 5 minutes in a solution containing MS salts, 3% sucrose, 2 mg / L NAA, 0.5 mg / L BA and 0.05% MES. Agrobacterium tumefaciens EHA101 with pLES9804 composition was inoculated into leaf bed for 2 days. After washing with sterile MS basal medium, leaf beds were selected as medium [0.5 mg / l BA, MS salts, 3% sucrose, 100 mg / l kanamycin, 250 mg / l. Sudopen and 0.8% agar]. A month later, the regenerated shoots were transferred to a rooting medium containing MS salts, 3% sucrose, 100 mg / l kanamycin, 250 mg / l pseudofen and 0.8% agar. . Rooted plants were transferred to a bedbed mixed vermiculite, perlite and peat moss in a 1: 1: 1 ratio and grown at 25 ° C. with a 16-hour low in the growth chamber. 32 regenerated plants grew. 13 of them expressed SCaM-4 transcripts and SCaM-4 protein.

Transgenic tobacco plants expressing SCaM-5 were produced by leaf bed transformation mediated by Agrobacterium. Tobacco leaf beds were made from young tobacco plants 8 weeks old and soaked for 5 minutes in a solution containing MS salts, 3% sucrose, 2 mg / L NAA, 0.5 mg / L BA and 0.05% MES. Agrobacterium tumefaciens with pLES9805 composition was inoculated into leaf bed for 2 days. After washing with sterile MS basal medium, leaf beds were selected as medium [0.5 mg / l BA, MS salts, 3% sucrose, 100 mg / l kanamycin, 250 mg / l. Sudopen and 0.8% agar]. A month later, the regenerated shoots were transferred to a rooting medium containing MS salts, 3% sucrose, 100 mg / l kanamycin, 250 mg / l pseudofen and 0.8% agar. . Rooted plants were transferred to a bedbed mixed vermiculite, perlite and peat moss in a 1: 1: 1 ratio and grown at 25 ° C. with a 16-hour low in the growth chamber. 32 Plants grew in the soil. Among them, 19 independent transgenic plants expressed SCaM-5 transcripts and proteins.

R1 progeny of transgenic plants expressing high levels of SCaM4 or SCaM5 were used in the experiments, maintaining daytime temperatures of 25 ° C, night temperature of 20 ° C, photoperiod of 16 hours and 65% relative humidity. .

Example 4 --- Analysis of SCaM Expression in Transgenic Plants

Immunblotting-polyclonal antibodies against two SCaM isoforms, SCaM-1 and SCaM-4, with 10 mg of each purified SCaM protein subcutaneously immunized with goats with Freund's complete adjuvant (Immunoblotting-Polyclonal antibodies) was prepared. 1 mg of protein was continuously injected at 3 week intervals with Freund's adjuvant. 50 μg of total water soluble protein isolated from transgenic plant systems independent of wild type plants was electrophoresed on 13.5% SDS polyacrylamide sels. Proteins were transferred to PVDF membranes from Millipore and incubated with anti-SCaM-1 or anti-SCaM-4 antibodies. After incubation with ICN biomedical (horseradish peroxidant-bound anti-chlorine IgG) protein bands were detected using the ECL system. The amount of SCaM-4 or SCaM-5 protein in transgenic tobacco plants is estimated to be 0.3-0.5 μg / mg of total soluble leaf protein, respectively.

Northern RNA hybridization was performed by Northern blot hybridization from a control transgenic plant with an empty vector without a wild type, an SCaM-4 or SCaM-5 gene, and three representative independent transgenic plants expressing either SCaM-4 or SCaM-5. Separated. 10 [mu] g of the isolated total RNA was isolated and denatured on 1.5% agarose formaldehyde gels. Ethidium Bromide was included to demonstrate the same loading of RNA. Transferred to Gene Screen Membranes and hybridized with ³² P labeled gene specific probes in Church's buffer for 16 hours at 65 ° C. SCaM-4 specific probes were obtained by purification of SCaM-4 or SCaM-5 cDNA digested with SCaM-4 or EcoR I or Xho I on a gel. After incubation, the filters were washed twice with 20 times with 2x SSC, twice with 20x with 1x SSC and once with 20x with 0.5x SSC, then 20 minutes with 0.5x SSC, 0.1% SDS at 60 ° C. Wash once. The filters were exposed to X-ray film for 1 hour at −80 ° C. for 6 days.

Example 5-Analysis of Disease Resistance of Genetically Converted Plants

The transgenic tobacco plants of the present invention described above were analyzed for fungal, bacterial and viral infections. The results are shown in FIG.

TMV treated-tobacco plants [ Nicotiana tabacum cv. Xanthi nc (NN) / SCaM-4 or -5 transgenic] were grown at 25 ° C. in a growth chamber programmed with a 16 hour photoperiod. 7-8 week old tobacco plants were inoculated with carborundum and TMV (U1 strain, 50 mM phosphate buffer solution at 2 μg / ml or pH 7.0, as indicated, with rubbing on leaves fully swollen or with buffer solution only). After infection, the plants were washed by spraying sterilized water to remove carborundum from the leaves The plants maintained the plants at 25 ° C. through infection in the growth chambers and observed the development of HR lesions for more than 5 days. Plants transfected with empty vectors without the SCaM-4 or SCaM-5 gene were simultaneously tested as controls.

Results of fungal treatment

Suspended phytophthora parasitiica strain Nicotiane mycelium in sterilized water and added 1 g of dried tobacco leaves per liter [V8 agar plate [200 ml of V8 juice per liter, 3.0 g of calcium carbonate (CaCO ₃ ) and 18 g of agar (pH 6.15)] was incubated at 25 ° C. under continuous light. The mycelia were propagated for 2-4 weeks, then cut into small rectangles of about 8 mm 2 and attached to agar medium (tobacco plants less than 4 weeks). They were transferred to a culture chamber in a 16 hour photoperiod at 25 ° C. The development of HR lesions was observed for at least 5 days. Plants transfected with the empty vector were simultaneously tested as controls.

Bacteria treatment

Pseudomonas Syringe Variant Tabashi (ACTC 11528) was grown overnight at 28 ° C. in King's B medium with 50 μg / ml riampicin, washed twice, resuspended, and 10 mM magnesium chloride Dilute with (MgCl ₂ ). Small leaves of healthy leaves were inoculated by infiltration of a suspension of 10 ⁵ bacterial cells per ml using a needleless 1 ml hypodermic syringe. One side of each leaf was inoculated into four sites with about 20 μl of bacterial suspension per wound, and the other side was vaccinated with 20 μl of 10 mM magnesium chloride. Leaf punchers 6 mm in diameter were made with a puncher. The leaves were immersed in 10 mM magnesium chloride to extract bacteria, and serial dilutions were applied to King's B medium containing 50 μg of rifampicin per mL and 10 μg of Malidixic acid per mL. Colonies were counted 48 hours after incubation at 28 ° C. Bacterial growth in plants was observed for at least 5 days. The development of HR lesions was also observed for more than 5 days. Plants transfected with the empty vector were simultaneously tested as controls.

Transgenic tobacco plants transformed with pLES9804 or pLES9805, which exhibit multiple disease resistance to a wide range of pathogens, have been deposited in the Korean Collection for Type Cultures (KCTC) under the Budapest Treaty (KCTC Accession Nos. 0543BP and 0544BP, respectively). .

The heterologous SCaM genes of the present invention as described above are one of the components of the plant defense signal that is continuously expressed in the plant to induce disease resistance to the pathogens of the plant, the transgenic plant expressing the heterologous SCaM is normally Shows increased resistance to fungi, bacteria and viruses that invade plants.

Claims (9)

Transgenic plant cells transformed with genes encoding soy calmodulin 4 (SCaM4) or soy calmodulin 5 (SCaM5) (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) and express SCaM4 or SCaM5 and the progeny of the cells . The transgenic plant cell of claim 1, wherein the transgenic plant cell is a tobacco plant and a dicot species. A transgenic plant transformed with a gene encoding SCaM4 or SCaM5 (KCTC Accession No. 0543BP and KCTC Accession No. 0544BP) and expressing SCaM4 or SCaM5, its cells or the offspring of the plant. The transgenic plant of claim 3, wherein the transgenic plant is a dicot species. Transforming said plant with a gene encoding SCaM4 or SCaM5 to enhance multi-disease resistance of higher plants against attack of one or more plant pathogens. 6. A method according to claim 5, wherein the plant is a dicot species. The method of claim 5, wherein the cells of the plant are transformed using two SCaM4 or SCaM5 gene vectors. 8. The bicomponent SCaM4 or SCaM5 gene vector according to claim 7 comprising a cDNA nucleotide sequence encoding SCaM4 or SCaM5 and regulatory elements for expression of SCaM4 or SCaM5 in plant cells. KCTC Accession No. 0545BP and KCTC Accession No. 0566BP). The method of claim 8, wherein the regulatory components are 35S promoter, translation enhancer sequence and nopaline synthase termination signal of cauliflower mosaic virus. Vector selected from the group consisting of.