JP2011041527A - Plasmid, ralstonia solanacearum, method of monitoring, method of evaluation, and method of screening - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plasmid reinforcing the expression of a labeling material in Ralstonia solanacearum, the Rastonia solanacearum reinforcing the expression of the labeling material, a method for monitoring various pathogenic bacteria including the Rastonia solanacearum, a method for evaluating sensitivity or resistance to the various pathogenic bacteria including the Rastonia solanacearum, and a method for screening test substances effective to the various pathogenic bacteria including the Rastonia solanacearum. <P>SOLUTION: This plasmid has a replication module of the genome of a phage ϕRSS1 having an infective capacity to the Rastonia solanacearum and an expression cassette of the labeling substance. The expression cassette has a phage ϕRSB2-originated MCP (major coat protein) promotor region, a gene encoding the labeling material, and a phage ϕRSB2-originated MCP terminator region. The method of monitoring is performed by culturing a plant so as to elongate its root and/or stem along an agar medium surface made inclined, and observing the labeling substance. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a plasmid having an expression cassette for a gene encoding a labeling substance, a bacterial wilt fungus transformed with the plasmid, a method for monitoring pathogenic bacteria such as bacterial wilt fungus by observing the labeling substance, and the monitoring The present invention relates to a method for evaluating the susceptibility or resistance of a test plant to a pathogen such as bacterial wilt disease, and a method for screening a test substance effective against the pathogen such as bacterial wilt using the monitoring method. .

  Bacterial wilt (blight blight) is a disease that causes serious agricultural damage by infecting and killing more than 200 kinds of plants including solanaceous plants. If the disease progresses rapidly, it may die with the bluish color remaining. It is characterized by producing milky white mucus (bacterial mucus) when cutting the stalk of the ground part of a plant with bacterial wilt and soaking it in water.

The bacterial wilt fungus (scientific name Ralstonia solanacearum ) invades the plant body from the wound of the root of the plant, and then grows in the vascular bundle. And an extracellular polysaccharide is produced in large quantities. As a result, the water flow through the vascular bundle deteriorates, so that the infected plant wilts and eventually dies. The bacterial wilt fungus is weak to dryness and can easily be killed by acid or alkali. However, it has the property to survive stably for many years in water, and even if it is sterilized with chemicals, it survives deep underground. Is generated. For this reason, it is known as a very difficult disease to control.

  At present, for the control of bacterial wilt disease, a classic rotation method or soil treatment with methyl bromide or chloropicrin is performed. However, the use of existing pesticides has a great impact on the environment and the human body. Therefore, alternative means for effectively controlling bacterial wilt disease have been developed. Patent Document 1 describes three types of bacteriophages (bacteriophage type 1, bacteriophage type 2, and bacteriophage type 3) that are lytic to bacterial wilt. These bacteriophages show lytic properties against 6 bacterial wilt strains (C-319, M4S, Ps29, Ps65, Ps72, Ps74) having different pathogenicity, epidemiological characteristics, and physiological / biochemical properties. In addition, a control agent for bacterial wilt fungus, a method for controlling bacterial wilt fungus, a soil conditioner and a soil improvement method using these as active ingredients are also described.

  Patent Document 2 describes a bacteriophage exhibiting a broader lysis spectrum than each bacteriophage described in Patent Document 1, a control agent for bacterial wilt disease using the bacteriophage, and the like. This bacteriophage shows not only the 6 bacterial wilt fungi described above, but also 9 strains of MAFF106603, MAFF106611, MAFF21270, MAFF21271, MAFF21272, MAFF301556, MAFF301358, MAFF730138 and MAFF730139. Non-patent document 1 and Non-patent document 2 include four types of bacteriophages that specifically infect bacterial wilt, bacteriophage φRSS1 (hereinafter referred to as phage φRSS1), bacteriophage φRSM1 (hereinafter referred to as phage φRSM1), bacteriophage. The phage φRSL1 and bacteriophage φRSA1 have been described. Of these, two phages, phage φRSS1 and phage φRSM1, have the property of inhibiting growth without lysing bacterial wilt.

  On the other hand, in order to elucidate the infection mechanism and pathogenesis mechanism of bacterial wilt fungus, a method for elucidating the ecology of bacterial wilt fungus using bioluminescence has been developed. Conventionally, plasmids used for labeling bacterial wilt (for example, plasmids derived from pKT240) can be replicated and maintained in bacterial wilt. However, under conditions where there is no selective pressure due to a drug (for example, kanamycin), it quickly drops off from bacterial wilt. For this reason, such plasmids are not suitable for long-term stable labeling in plants or soil where drug selection pressure cannot be applied.

  In order to label bacterial wilt bacteria, there is a method of inserting a target gene into the genome using a transposon or the like. When this method is used, the target gene is stably retained even without drug selection pressure. However, when a gene is inserted into the genome using a transposon or the like, there is a possibility that the expression pattern of the gene may change due to rearrangement of the genome of the host bacterial wilt disease or position effect due to insertion. When using the method of inserting into a genome, since these side effects must be evaluated, a complicated procedure is required.

  Patent Document 3 and Non-Patent Document 3 describe a plasmid containing the specific sequence of phage φRSS1 described above. The plasmids described in these references contain a phage φRSS1 genomic replication module and are easily incorporated into bacterial wilt. In addition, when this plasmid is used, the plasmid can be stably replicated and maintained in bacterial wilt even under conditions where there is no drug selection. Therefore, various useful genes can be easily introduced into bacterial wilt. Furthermore, when the plasmid is no longer needed, it can be removed without changing the genome information of bacterial wilt.

  These documents include, for example, a plasmid pRSS11 (hereinafter referred to as pRSS11) in which a kanamycin resistance cassette is connected between plasmids composed of a replication module derived from phage φRSS1, or a kanamycin resistance cassette and Green Fluorescent Protein uv (hereinafter referred to as GFPuv). The plasmid pRSS12 into which the gene has been inserted (hereinafter referred to as pRSS12) is described. By using pRSS12, even if there is no selective pressure by kanamycin, it can be stably retained in bacterial wilt. Furthermore, the location of bacterial wilt can be easily labeled by expressing a labeling substance such as GFPuv protein.

  As a method for confirming the location of bacterial wilt fungus, inoculating a fluorescently labeled bacterial wilt fungus in a plant (tomato) cultivated in soil, and after a certain time, at a position away from the seeds of the species. There is a method of preparing a section and observing the section with a fluorescent stereomicroscope. From this observation result, it is possible to observe the upward or downward movement of bacterial wilt in the plant and the movement in the vascular bundle.

  When using the monitoring method of bacterial wilt fungus by observing a section of a plant cultivated in such a soil, when monitoring over time, the plant cultivated in the soil is periodically excavated, and the stem is A method of cutting, observing the cross section, and then replanting is also conceivable. However, in such a method, cutting and replanting have a great influence on the plant, and it is difficult to say that the bacterial wilt fungus is monitored in a natural environment. Therefore, when testing the pathogenicity of bacterial wilt fungus against a certain plant, in the conventional method, the plant to be tested is first cultivated in soil for about one month. Thereafter, these stems or roots are inoculated with the test bacteria, and cultivation is continued. Finally, the appearance of disease symptoms (discoloration of leaves and stems, wilting, etc.) in these strains is indexed and statistically processed. For this reason, it takes about 2 to 4 weeks for observation / determination (Non-Patent Document 4). Thus, when monitoring bacterial wilt fungi for plants cultivated in soil, a period of one month or longer is required. Therefore, the development of a simple and short-time monitoring method is desired.

JP 2005-278513 A JP 2007-252351 A JP 2009-55881 A

Takashi Yamada, Takeru Kawasaki, Shoko Nagata, Akiko Fujiwara, Shoji Usami and Makoto Fujie.New bacteriophages that infect the phytopathogen Ralstonia solanacearum. Microbology, 153, 2630-2639 (2007) Takeru Kawasaki, Shoko Nagata, Akiko Fujiwara, Hideki Satsuma, Makoto Fujie, Shoji Usami, and Takashi Yamada.Genomic Characterization of Filamentous Integrative Bacteriophages, φRSS1 and φRSM1, That Infect Ralstonia solanacearum.J. Bacteriol, 189, 5792-5802 (2007) Kawasaki, T., Satsuma, H, Fujie, M, Shoji, U., and Yamada, T. Monitoring of Phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage φRSS1. J. Biosci. Bioeng., 104, 451-456 (2007) Mitsuo Hotta and Kenichi Tsuchiya, Microbial Genetic Resource Manual (12) -Ralstonia solanacearum-, National Institute for Agrobiological Resources (2002)

  Patent Document 3 describes that when a plasmid into which a gene capable of expressing a labeling substance is inserted (specifically, pRSS12) is prepared, both a promoter and an operator region can be incorporated. Examples of such promoter and operator regions include various commonly used ones such as lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like.

  However, for example, when luminescence of green fluorescent protein (GFP) in a plant infected with bacterial wilt disease transformed by pRSS12 is observed, autofluorescence derived from the chloroplast of the plant is also observed. Therefore, it is necessary to more clearly label bacterial wilt bacteria in the plant body, and it is desired to enhance the fluorescence intensity by GFP.

  The present invention has been made in view of the above circumstances, and a plasmid capable of enhancing the expression of a labeling substance such as GFP and enhancing the fluorescence intensity due to, for example, GFP when transformed with bacterial wilt. Bacterial wilt fungus with increased concentration of labeling substance in the transformed fungus, method for monitoring bacterial wilt fungus using the bacterial wilt fungus, method for evaluating sensitivity or resistance to bacterial wilt fungus, and effective against bacterial wilt fungus Screening method for a test substance, and further, a plasmid capable of transforming a pathogenic bacterium that infects a plant and having a property of producing a labeling substance when infected with the pathogenic bacterium, the pathogenic bacterium using the transformed pathogenic bacterium Monitoring method, evaluation method for susceptibility or resistance to the pathogen, and screening method for test substances effective against the pathogen An object of the present invention is to provide a.

  The plasmid according to the first aspect of the present invention is a plasmid containing a replication module of the genome of phage φRSS1 having the ability to infect bacterial wilt, and is described in (a) SEQ ID NO: 2 derived from phage φRSB2 A base sequence identical to the sequence of the MCP promoter region and a base sequence in which one or several bases are deleted, substituted, added or inserted, or a combination thereof, and the sequence is mutated, A promoter region having a base sequence selected from base sequences having equivalent actions, (b) a gene encoding a labeling substance downstream of the promoter region, and (c) a gene encoding downstream of the labeling substance, The same nucleotide sequence as the sequence of the MCP terminator region described in SEQ ID NO: 3 derived from phage φRSB2, and A base sequence selected from a base sequence having one or several bases deleted, substituted, added, inserted, or a combination thereof, and having a mutation equivalent to the MCP terminator. And a terminator region having an expression cassette.

  In the first aspect, preferably, the replication module has the same base sequence as the base sequence from the 1st to 1720th and 5800 to 6135th of the phage φRSS1 genome described in SEQ ID NO: 1. 1 or several bases deleted, substituted, added to the base sequence from the 1st to 1720th positions and the 5800 to 6135th positions in the genome of the phage φRSS1 described in SEQ ID NO: 1 A nucleotide sequence in which the sequence is mutated by insertion or a combination thereof, and is equivalent to the nucleotide sequence from 1st to 1720th and 5800 to 6135th of the phage φRSS1 genome described in SEQ ID NO: 1 A nucleotide sequence having the following action.

  Preferably, the plasmid further has a gene encoding a selection marker.

  Further preferably, the gene encoding the selection marker is a kanamycin resistance gene.

  Preferably, the replication module has a site cleaved by a restriction enzyme, and the expression cassette is incorporated into the site. More preferably, the site cleaved by the restriction enzyme is an EcoRI site.

  Preferably, the labeling substance is a chemiluminescent protein or a fluorescent protein.

  Further preferably, the labeling substance is a fluorescent protein.

  Preferably, the labeling substance is green fluorescent protein (GFP).

  More preferably, the labeling substance is green fluorescent protein mut2 (GFPmut2).

  The bacterial wilt fungus according to the second aspect of the present invention is transformed with the plasmid according to the first aspect of the present invention.

  The method for monitoring bacterial wilt fungus according to the third aspect of the present invention is the growth, reduction or behavior of bacterial wilt fungus by observing the labeling substance expressed in the bacterial wilt fungus according to the second aspect of the present invention. It is characterized by observing.

  In the third aspect described above, preferably, the plant is infected with the bacterial wilt fungus according to the second aspect of the present invention, and the labeling substance expressed in the bacterial wilt fungus in the plant body is observed, whereby blue in the plant body is observed. It is characterized by observing the growth, decrease or behavior of the bacterial wilt.

  Further preferably, the observation of the growth, decrease or behavior of bacterial wilt fungus in the plant body is performed by observing the labeling substance in a transverse section of the plant infected with bacterial wilt fungus.

  Preferably, the observation of the growth, reduction or behavior of bacterial wilt fungus in the plant body is performed by roots and / or along the surface of the agar medium inclined at an inclination angle with respect to the horizontal plane. It is characterized by cultivating the stem to grow and observing the labeling substance produced in the plant cultured on the surface of the agar medium.

  Further preferably, the inclination angle is 15 to 75 degrees with respect to a horizontal plane.

  Preferably, the labeling substance is observed over time, and the growth, decrease or behavior of bacterial wilt fungus is observed in real time.

  The method for evaluating bacterial wilt fungus according to the fourth aspect of the present invention uses the monitoring method according to the third aspect of the present invention, and uses the growth, decrease or behavior of the bacterial wilt fungus as an indicator. It is characterized by evaluating susceptibility or resistance to a disease fungus.

  A screening method for a test substance effective against bacterial wilt fungus according to the fifth aspect of the present invention provides a test substance to a plant infected or diseased with bacterial wilt fungus according to the second aspect of the present invention. A test substance effective against bacterial wilt disease is screened by observing using the method for monitoring bacterial wilt disease according to the third aspect of the invention.

  The monitoring method according to the sixth aspect of the present invention has a property of producing a labeling substance by introducing a foreign gene containing a gene encoding a labeling substance into a pathogenic bacterium that infects a plant. In order to observe the growth, decrease, or behavior, the plant infected with the pathogen is cultured so that the roots and / or stems extend along the surface of the agar medium inclined at an inclined angle with respect to the horizontal plane, and cultured on the surface of the agar medium. It is characterized by observing the labeling substance produced in the plant body.

  In the sixth aspect, preferably, the inclination angle is 15 to 75 degrees with respect to a horizontal plane. Further preferably, the inclination angle is approximately 45 degrees with respect to a horizontal plane.

  Preferably, the labeling substance is observed over time, and the growth, decrease or behavior of pathogenic bacteria is observed in real time.

  The evaluation method according to the seventh aspect of the present invention uses the monitoring method according to the sixth aspect of the present invention, and uses the growth, decrease or behavior of the pathogenic bacteria as an index, and the susceptibility or resistance to the pathogenic bacteria by plant varieties. It is characterized by evaluating.

  The screening method according to the eighth aspect of the present invention has a property of producing a labeling substance by introducing a foreign gene containing a gene encoding a labeling substance into a pathogenic bacterium that infects a plant. A test substance effective for the pathogen is screened by giving a test substance to the plant and observing it using the monitoring method according to the sixth aspect of the present invention.

  Since the plasmid according to the first aspect of the present invention has an expression cassette in which a gene encoding a labeling substance is sandwiched between a specific promoter region and a terminator region, the labeling substance is transformed when bacterial wilt fungus is transformed. Expression can be enhanced. According to the bacterial wilt fungus according to the second aspect of the present invention, a bacterial wilt fungus with enhanced expression of the labeling substance can be obtained. Moreover, since the plasmid in the bacterial wilt fungus has the property of being retained in the bacterial wilt fungus for a long time even under conditions where there is no drug selection pressure, it is also suitable for use in the monitoring mentioned below. . According to the method of monitoring bacterial wilt disease according to the third aspect of the present invention, since the expression of the labeling substance of bacterial wilt fungus is enhanced, the growth or migration of bacterial wilt fungus is clearly and easily monitored. Can do. According to the evaluation method for bacterial wilt fungus according to the fourth aspect of the present invention, since the expression of the labeling substance of bacterial wilt fungus is enhanced, the sensitivity or resistance to bacterial wilt fungus in plant varieties is clearly and easily evaluated. can do. According to the screening method for a test substance effective against bacterial wilt fungus according to the fifth aspect of the present invention, since the expression of the labeling substance of bacterial wilt fungus is enhanced, the test effective against bacterial wilt fungus Substances can be screened clearly and conveniently.

  In the monitoring method according to the sixth aspect of the present invention, the growth or migration of pathogenic bacteria that infect plants can be observed very simply. In the evaluation method according to the seventh aspect of the present invention, since the monitoring method is applied, the susceptibility or resistance to pathogenic bacteria in plant varieties can be evaluated very simply. Also in the screening method according to the eighth aspect of the present invention, since the monitoring method is applied, a test substance effective against pathogenic bacteria can be clearly and easily screened.

It is a schematic diagram which shows the structure of pRSS12. It is a schematic diagram which shows the specific structure of pRSS1S. It is a schematic diagram which shows the structure of pRSS1S-GFPuv. It is a schematic diagram which shows the structure of pRSS1S-GFPmut2. It is quantitative comparison data of the fluorescence intensity of pRSS12 and pRSS1S-GFPuv which concern on Example 2. FIG. It is quantitative comparison data of the fluorescence intensity of pRSS1S-GFPuv and pRSS1S-GFPmut2 according to Example 3. It is the photograph replaced with drawing by the fluorescence stereomicroscope of the cross section of the stem infected with the bacterial wilt fungus transformed using pRSS12 which concerns on the reference example 2. FIG. It is the photograph replaced with drawing by the fluorescence stereomicroscope of the cross section of the stem which infected the bacterial wilt fungus transformed using pRSS12 which concerns on the reference example 3. FIG. It is a photograph replaced with drawing which shows a mode that the bacterial wilt fungus transformed using pRSS12 which concerns on Example 4 was infected to a tomato, and it culture | cultivated on the surface of an agar medium. It is a photograph replaced with drawing which shows a mode that the bacterial wilt fungus transformed using pRSS1S-GFPmut2 which concerns on Example 5 was infected to a tomato, it culture | cultivated on the surface of an agar medium, and observed in the bright field with the stereomicroscope. It is a photograph replacing a drawing which shows a state where a bacterial wilt fungus transformed with pRSS1S-GFPmut2 according to Example 5 was infected with tomato, cultured on the surface of an agar medium, and observed with a fluorescent stereomicroscope. It is the photograph replaced with drawing by the fluorescence stereomicroscope which shows a mode that the bacterial wilt fungus transformed using pRSS12 which concerns on Example 6 was infected with tomato. It is a figure which shows the penetration | invasion rate of the bacterial wilt disease by the tomato kind | variety based on Example 6. FIG. In the plasmid which concerns on Embodiment 1, it is a schematic diagram which shows the general structure of the plasmid which has the gene which codes a selection marker.

  First, the present inventors constructed a plasmid pRSS1S (hereinafter, pRSS1S), which is a kind of a basic plasmid before introducing a gene encoding a labeling substance. FIG. 2 is a schematic diagram showing a specific structure of pRSS1S. The upper diagram shows the structure of the genomic DNA of phage φRSS1. The lower diagram shows the structure of pRSS1S. pRSS1S is derived from phage φRSS1 as is pRSS12. Similarly to pRSS12, pRSS1S has a replication module constructed by removing the structural module, assembly, and secretion module from the DNA of phage φRSS1. However, unlike pRSS12, the replication module possessed by pRSS1S consists of the 1st to 1720th base sequence and the 5800 to 6661th base sequence of the genomic DNA sequence of phage φRSS1.

  The replication module has the same nucleotide sequence as the 1st to 1720th and 5800 to 6135th base sequences of the phage φRSS1 genome described in SEQ ID NO: 1, or to SEQ ID NO: 1. One or several bases are deleted, substituted, added or inserted, or a combination thereof is mutated from the 1st to 1720th and 5800 to 6135th base sequences of the described phage φRSS1 genome. Including a base sequence having an action equivalent to that of the 1st to 1720th and 5800 to 6135th base sequences of the phage φRSS1 genome described in SEQ ID NO: 1 Anything is acceptable.

  In addition, when a target region of this genomic DNA is amplified by the PCR method, a sequence that can be cleaved by a specific restriction enzyme (specific restriction enzyme cleavage site) is added to one primer to specifically restrict the amplified DNA fragment. It is desirable to add an enzyme cleavage site. In pRSS1S, an EcoRI site is added as a specific restriction enzyme cleavage site to the amplified fragment of about 2.6 kbp. In addition to the EcoRI site, specific restriction enzyme cleavage sites used for such purposes include sites corresponding to restriction enzymes such as BamHI, HindIII, PstI or NotI.

By ligating a gene encoding a selectable marker (selectable marker cassette) to this amplified product, a basic one of the preferred form of the plasmid of the present invention is constructed. In pRSS1S, a pRSS1S of about 4.0 kbp is constructed by ligating a kanamycin resistance cassette (Km cassette).

  Next, the present inventors searched for a novel promoter in order to more strongly express a gene encoding a labeling substance such as a GFP gene in bacterial wilt. In many bacteriophages infected with bacterial wilt, we investigated promoters that express the genes and searched for those that are constantly highly expressed. In particular, a search was performed for the promoter of bacteriophage φRSB2 (hereinafter, phage φRSB2). Phage φRSB2 is a novel bacteriophage that the inventors have isolated with bacterial wilt in Hiroshima Prefecture. The phage φRSB2 has been confirmed to be a bacteriophage similar to the phage φRSB1, which is an already known phage isolated by the present inventors.

  Examination of the promoter of each gene of phage φRSB2 MCP (major coat protein), primese, DNA polymerase or tail protein revealed that expression of the target gene can be enhanced by using the nucleotide sequence of the promoter region of the phage φRSB2 MCP gene. It was. Furthermore, it has been found that the expression level is further enhanced by adding the base sequence of the terminator region of the MCP gene downstream of the target gene. The base sequence of the MCP promoter region of phage φRSB2 consists of the base sequence shown in SEQ ID NO: 2, and the base sequence of the terminator region of MCP of phage φRSB2 consists of the base sequence shown in SEQ ID NO: 3.

  First, a cassette for expressing the GFP gene was constructed using the promoter region and terminator region of the MCP gene of phage φRSB2. As the GFP gene, GFPuv (a gene used in Examples according to Patent Document 3) or GFPmut2 was used. FIG. 3 is a schematic diagram of the structure of plasmid pRSS1S-GFPuv (hereinafter, pRSS1S-GFPuv). pRSS1S-GFPuv is constructed by inserting an expression cassette consisting of the promoter region of the MCP gene, the GFPuv gene and the terminator region of the MCP gene at the position of the EcoRI site of pRSS1S shown in FIG.

  FIG. 4 is a schematic diagram of the structure of plasmid pRSS1S-GFPmut2 (hereinafter, pRSS1S-GFPmut2). pRSS1S-GFPmut2 is constructed by inserting an expression cassette consisting of the promoter region of the MCP gene, the GFPmut2 gene, and the terminator region of the MCP gene at the position of the EcoRI site of pRSS1S shown in FIG. For details of GFPmut2, see FACS-optimized mutants of the green fluorescent protein (GFP), Brendan P. Cormack, Raphael H. Valdivia and Stanley Falkow, Gene, 173 (1996) 33-38.

  By transforming bacterial wilt fungi using the plasmids of the present invention, such as pRSS1S-GFPuv, pRSS1S-GFPmut2, etc., the present inventors expressed GFPuv or GFPmut2 in bacterial wilt fungi, and compared their fluorescence intensities. An experiment was conducted. As a result, it was confirmed that the use of the MCP promoter region and MCP terminator region of phage φRSB2 was effective in enhancing the fluorescence intensity. Usually, in observations performed with a fluorescent stereomicroscope, excitation is often performed with blue light. Thus, comparing the results of fluorescence intensity with pRSS1S-GFPuv and pRSS1S-GFPmut2, it was found that using GFPmut2 for fluorescence labeling was more effective for enhancing fluorescence intensity than with GFPuv for observation by blue light excitation.

  In addition, the present inventors have developed a specific method for monitoring bacterial wilt disease using the plasmids of the present invention, such as pRSS1S-GFPuv and pRSS1S-GFPmut2. As described above, when a bacterial wilt fungus is transformed using these plasmids, the bacterial wilt fungus can be fluorescently labeled with the expressed GFPuv or GFPmut2. Therefore, it was found that bacterial wilt fungi in plants can be monitored by inoculating plants infected with bacterial wilt fungi such as tomatoes with bacterial wilt fungus transformed with these plasmids. Specifically, the present inventors conducted a monitoring experiment for bacterial wilt using two methods.

  In the first specific method, a fluorescently labeled bacterial wilt fungus is inoculated into a plant (tomato) cultivated in soil, and after a certain period of time, a section at a distance from the species is prepared from the plant inoculation site. . The prepared section was observed with a fluorescent stereomicroscope. From this observation result, it was possible to observe the upward or downward movement of bacterial wilt in the plant and the movement in the vascular bundle.

When using the monitoring method of bacterial wilt fungus by observing a section of a plant cultivated in such a soil, when monitoring over time, the plant cultivated in the soil is periodically excavated, and the stem is A method of cutting, observing the cross section, and then replanting is also conceivable. However, in such a method, cutting and replanting have a great influence on the plant, and it is difficult to say that the bacterial wilt fungus is monitored in a natural environment. Therefore, when testing the pathogenicity of bacterial wilt fungus against a certain plant, in the conventional method, the plant to be tested is first cultivated in soil for about one month. Thereafter, these stems or roots are inoculated with the test bacteria, and cultivation is continued. Finally, the appearance of disease symptoms (discoloration of leaves and stems, wilting, etc.) in these strains is indexed and statistically processed. For this reason, it takes about 2 to 4 weeks for observation and determination. (See Mitsuo Hotta and Kenichi Tsuchiya, Microbial Genetic Resource Manual (12) -Ralstonia solanacearum- , National Institute of Agrobiological Resources (2002)). Thus, when monitoring bacterial wilt fungi for plants cultivated in soil, a period of one month or longer is required.

  Accordingly, the present inventors have developed a more convenient method for monitoring bacterial wilt disease as a second specific method. In this monitoring method, a target plant is sown on the surface of an agar medium inclined at 15 to 75 degrees, preferably 30 to 60 degrees, more preferably 40 to 50 degrees, and even more preferably about 45 degrees with respect to a horizontal plane. And cultivated to extend along this slope. Thereafter, about 7 days after sowing, the target plant is inoculated with bacterial wilt fungus transformed with the plasmid of the present invention. Then, without producing a stem section or the like, it is possible to observe the bacterial bacterial infection site in the plant body with a fluorescent stereomicroscope while maintaining the cultivation state. For this reason, the time-lapse monitoring of the bacterial wilt fungus in the plant body can be performed more easily and early in the cultivation while suppressing the influence on the plant to be observed.

In addition, the present inventors performed this monitoring using bacterial wilt that was transformed with pRSS12 or pRSS1S-GFPmut2, but monitoring is possible regardless of which is used. It was confirmed. Among them, stronger fluorescence intensity could be obtained by using pRSS1S-GFPmut2.

From this result, infect this second specific monitoring method and plants such as Fusarium molds such as Fusarium oxysporum , blast fungus ( Magnaporthe grisea ) or soil pathogenic bacteria ( Erwinia carotovora ) It was found that even when a foreign gene containing a gene encoding a labeling substance was introduced into the pathogen and combined with the property of producing the labeling substance, the growth or migration of the pathogen could be observed very easily. . Since it has such effects, if this monitoring technique is applied, it becomes possible to clearly and easily screen test substances effective against specific pathogens and to evaluate sensitivity or resistance to specific pathogens in plant varieties. I also understood that.

  That is, the present inventors studied a method for evaluating sensitivity or resistance to bacterial wilt using this monitoring method. First, bacterial wilt fungus transformed with pRSS12 was inoculated on the surface of an agar medium to various tomato varieties with known susceptibility and resistance to bacterial wilt. As a result, it was found that the invasion state of the bacterial wilt fungus varies depending on the sensitivity or resistance of the cultivar to the bacterial wilt fungus. Details are shown in the Examples, and it was found that the rate of infestation of bacterial wilt bacteria to the lateral roots varies greatly depending on either sensitivity or resistance, so the above conclusion was reached.

  Hereinafter, the plasmid, bacterial wilt fungus, bacterial wilt fungus monitoring method, susceptibility to bacterial wilt fungus evaluation method, etc., test substance effective screening method against bacterial wilt fungus, etc. This will be described in detail. In this specification, the term “comprising” (or “having”) includes the meaning of “consisting of”, that is, “consisting of”. In the present specification, “inoculation” and “infection” have the same meaning.

(Plasmid)
Embodiment 1 of the present invention relates to a plasmid having an expression cassette for a gene encoding a labeling substance. That is, the plasmid according to the first embodiment is a plasmid containing a replication module of the phage φRSS1 genome having infectivity against bacterial wilt, and comprises an MCP promoter region derived from the phage φRSB2, and the promoter region It has an expression cassette including a gene encoding a labeling substance downstream and an MCP terminator region derived from phage φRSB2 downstream of the gene encoding the labeling substance. Specific examples include pRSS1S-GFPuv or pRSS1S-GFPmut2 as shown in FIG. 3 or FIG.

  The meaning of the term “replication module” is as described above. The “phage φRSS1 genomic replication module” is preferably the 1st to 1720th sequence of the base sequence described in SEQ ID NO: 1 in the sequence listing, and any of 5800 to 6135th to 6661th sequence. This refers to a sequence (preferably 5800 to 6662th sequence). However, not only the same base sequence as the base sequence, but also a base sequence in which one or several bases are deleted, substituted, added or inserted, or a combination thereof is mutated in the base sequence It doesn't matter. That is, a person skilled in the art can appropriately change the sequence within the range that can maintain the affinity for the above-described base sequence, specifically the affinity for bacterial wilt.

  The homology in the sequence in which such a mutation has occurred includes homology of 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. More specific examples of the range of homology include 60% to 99%, 65% to 99%, 70% to 99%, 75% to 99%, 80% to 99%, 85% to 99%, 90% % -99%, 60% -95%, 65% -95%, 70% -95%, 75% -95%, 80% -95%, 85% -95%, 90% -95%, etc. Can be mentioned.

  This homology is similarly applied to homology in a sequence in which a mutation occurs in the base sequence described below.

  In the plasmid according to the first embodiment, the “MCP promoter region derived from phage φRSB2” preferably refers to a promoter sequence having the same base sequence as described in SEQ ID NO: 2. However, not only a base sequence identical to the base sequence but also a promoter comprising a base sequence in which one or several bases are deleted, substituted, added or inserted, or a combination thereof is mutated in the base sequence It does not matter if it is an area. That is, a person skilled in the art can appropriately change the sequence within a range that functions as a promoter region that enhances the expression of a gene encoding a labeling substance. The sequence of the promoter region also includes a ribosome binding sequence (an SD sequence or a sequence considered to be an SD sequence).

  The same applies to the “MCP terminator region derived from phage φRSB2”, preferably a terminator sequence having the same base sequence as described in SEQ ID NO: 3. However, not only the same base sequence as the base sequence but also a terminator comprising a base sequence in which one or several bases are deleted, substituted, added or inserted, or a combination thereof is mutated in the base sequence. It does not matter if it is an area. That is, those skilled in the art can appropriately change the sequence within a range that functions as a terminator region that enhances the expression of the gene encoding the labeling substance.

  The “plasmid” according to the first embodiment is used in a general sense that it is an extrachromosomal genetic element that can autonomously replicate independently of the host chromosome and can be stably inherited. Is. Further, as described above, the plasmid according to the first embodiment has an expression cassette including a phage φRSS1 genome replication module, a gene encoding a labeling substance, and the like. That is, it may consist only of the phage φRSS1 genome replication module and the expression cassette, or may be incorporated into various known plasmids, for example, plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19 derived from E. coli. Also included.

  Furthermore, as described above, since the plasmid according to the first embodiment has a gene encoding a labeling substance, the gene according to the labeling substance can be introduced into a host cell (green wilt fungus). The “gene encoding the labeling substance” is not particularly limited as long as it can function as a label, but is a chemiluminescent protein such as a luciferase gene or a gene encoding green fluorescent protein (hereinafter referred to as GFP), or / A gene encoding a fluorescent protein is preferred. A general gene includes a gene encoding GFP. Furthermore, in the case of GFP, GFPmut2 is most preferable.

  In addition to the “phage φRSS1 genome replication module” and the “expression cassette containing a gene encoding a labeling substance” in the plasmid according to the first embodiment, various “genes encoding a selection marker”, etc. Can also be included. As the “gene encoding the selectable marker”, those commonly used can be used in a conventional manner. For example, antibiotic resistance genes such as tetracycline, ampicillin, neomycin or kanamycin are exemplified. FIG. 14 shows a schematic diagram of the general structure of the plasmid according to the first embodiment having such a structure. In FIG. 14, X means a labeling substance X.

  In the plasmid according to the first embodiment, the “expression cassette” has both the MCP promoter region and the MCP terminator region. However, as another form of the plasmid according to the first embodiment, it may have only the MCP promoter region. In addition, as shown in FIG. 3 or FIG. 4, the “expression cassette” is preferably the 1st to 1720th sequence of the base sequence described in SEQ ID NO: 1 and the 5800th to 6135th to the 6662th sequence. It is inserted at the position of the EcoRI site of pRSS1S having a “phage φRSS1 genomic replication module” consisting of a sequence (preferably 5800 to 6662th sequence) and a “gene encoding a selection marker”. In the specific example of the plasmid according to the first embodiment shown in FIG. 3 or FIG. 4, the “gene encoding the selection marker” is the kanamycin resistance gene (Km cassette), and the “labeling substance is encoded in the“ expression cassette ”. “Gene” is described as a gene (GFPuv or GFPmut2) encoding a green fluorescent protein.

  Thus, in the plasmid according to the first embodiment, “MCP promoter region derived from phage φRSB2” and “MCP terminator region derived from phage φRSB2” are inserted. As described above, the MCP promoter region is located upstream of the target gene (“gene encoding the labeling substance”) (preferably the MCP promoter region and the MCP terminator region, and the MCP promoter region is located upstream of the target gene and the When the MCP terminator region is inserted downstream of the target gene, the gene expression can be enhanced. Therefore, when the plasmid according to Embodiment 1 is introduced into bacterial wilt and transformed, the expression of the “gene encoding the labeling substance” can be enhanced, and the bacterial wilt is labeled more clearly. Can do.

  Furthermore, since the plasmid according to the first embodiment contains a genome replication module of phage φRSS1, it is easily incorporated into bacterial wilt fungi. In addition, bacterial wilt bacteria can stably replicate and retain the plasmid even under conditions where there is no drug selection (see Patent Document 3 for the stability of the plasmid according to the present embodiment in bacterial wilt fungus). . When the plasmid is no longer needed, it can be removed without changing the genome information of bacterial wilt.

  DNA fragments related to “replication module”, “MCP promoter region derived from phage φRSB2”, “MCP terminator region derived from phage φRSB2”, “gene encoding labeling substance” and “gene encoding selection marker” Can be prepared by using a general PCR (Polymerase Chain Reaction) technique, which is known to those skilled in the art, as shown in the Examples described later. Insertion of “gene encoding labeling substance”, insertion of “gene encoding selection marker”, insertion of “MCP promoter region derived from phage φRSB2”, insertion of “MCP terminator region derived from phage φRSB2”, etc. Can also be performed using standard recombinant DNA techniques (see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). For example, it can be performed by a known method using a restriction enzyme and DNA ligase.

  For the DNA fragments related to “MCP promoter region derived from phage φRSB2” and “MCP terminator region derived from phage φRSB2”, DNAs having the nucleotide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3 are known to those skilled in the art. And can be obtained by synthesis. For example, it is possible to synthesize DNA having a desired base sequence by outsourcing to Nippon Gene Co., Ltd. The target expression cassette can be obtained by ligating the thus synthesized MCP promoter region and MCP terminator region with a gene encoding a labeling substance. In addition, a primer for amplifying a gene encoding a labeling substance including the base sequence of the MCP promoter region (forward) and a primer for amplifying a gene encoding the labeling substance including the base sequence of the MCP terminator region ( The target expression cassette can also be obtained by amplifying a DNA fragment by PCR using a DNA containing the base sequence of the gene encoding the labeling substance as a template using (reverse).

(Green wilt fungus)
The second embodiment of the present invention relates to bacterial wilt disease transformed with the plasmid according to the first embodiment.

  As a method for introducing the plasmid according to Embodiment 1 into bacterial wilt, conventionally known methods can be used. For example, transformation can be performed by an electroporation method. The plasmid according to Embodiment 1 has a very strong affinity for bacterial wilt and can be introduced into bacterial wilt relatively easily.

  The bacterial wilt fungus into which the plasmid has been introduced can be selected by a conventional method using a selection marker or the like. As the selective medium, a nutrient medium for cultivating bacterial wilt can be used. By culturing the selected transformed cell (transformed bacterial wilt fungus) in a nutrient medium, the introduced gene can be expressed in the transformed cell. It is preferable that the nutrient medium contains a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of bacterial wilt (transformant).

  Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean cake, potato extract and the like. In addition, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be included as desired. . As a preferable nutrient medium of the transformed bacterial wilt fungus according to the second embodiment, a CPG medium can be mentioned.

  According to the bacterial wilt fungus according to the second embodiment, the bacterial wilt fungus that is transformed with the plasmid according to the first embodiment and has enhanced luminescence or fluorescence intensity by the labeling substance can be obtained. Moreover, since the plasmid in bacterial wilt disease has the property of being retained in bacterial wilt disease for a long time even in the absence of drug selection pressure, it is used for monitoring bacterial wilt disease referred to below. Is also suitable.

(Monitoring method for bacterial wilt disease)
Embodiment 3 of the present invention relates to a method for monitoring bacterial wilt disease. In the present specification, “monitoring of bacterial wilt fungus” refers to observing the growth, decrease or behavior of bacterial wilt fungus by observing the labeling substance. Observation of growth or reduction of bacterial wilt fungus also includes measuring the amount of growth or reduction of bacterial wilt fungus in soil or plants. In addition, in the observation of the behavior of bacterial wilt disease in the soil or plant body, the observation of the location of bacterial wilt fungus in the soil or plant body, It also includes the meaning of detection.

  The method for monitoring bacterial wilt fungus according to the third embodiment includes observing the growth, reduction or behavior of bacterial wilt fungus according to the second embodiment with the labeling substance expressed in the bacterial wilt fungus. by. Observation of growth, decrease or behavior of bacterial wilt fungus refers to observation of bacterial wilt fungus under various places and conditions such as in soil, in plants, or on a nutrient medium.

  Since the bacterial wilt fungus according to Embodiment 2 has enhanced light emission or fluorescence intensity due to the labeling substance, the labeling substance can be clearly and easily observed. Thereby, a bacterial wilt fungus can be monitored. As described above, the labeling substance is not particularly limited as long as it can function as a label. For example, a chemiluminescent protein such as a luciferase gene or a gene encoding green fluorescent protein, and / or a fluorescent protein is encoded. The gene is preferred. For example, green fluorescent protein (GFP), preferably GFPmut2.

  In observing the labeling substance, a conventionally known method suitable for observing each labeling substance can be used. As an observation instrument, it can observe using a stereomicroscope, an optical microscope, a dark field microscope, a fluorescence microscope, an electron microscope, or a fluorescence stereomicroscope, for example. That is, any means capable of visualizing bacterial wilt bacteria labeled with a labeling substance may be used. When green fluorescent protein (GFP) is used as the labeling substance, it can be preferably observed using a fluorescent stereomicroscope. In this specification, the case where the term “observation” is used includes the case where an observation instrument suitable for observing the labeling substance is used.

  In the method for monitoring bacterial wilt disease according to the third embodiment, preferably, the plant is infected with the bacterial wilt fungus according to the second embodiment, and the labeling substance expressed in the bacterial wilt fungus in the plant body is observed. Observe the growth, decrease or behavior of bacterial wilt in plants.

In the case of infecting a plant with bacterial wilt, it can be performed using a conventionally known method. For example, the stalk or root of a plant can be perforated with a needle coated with a bacterial wilt culture solution. Alternatively, for example, it can also be carried out by placing a bacterial wilt culture solution in the soil in which plants are planted. In this case, the growth, decrease or behavior of bacterial wilt fungus in the infected plant is observed. Furthermore, as described in the Examples, an infection method newly developed by the present inventors, such as applying a culture solution of bacterial wilt fungus that has been transformed by cutting the root tips of the plant seedlings, may be used. (See Mitsuo Hotta and Kenichi Tsuchiya, Microbial Genetic Resource Manual (12) -Ralstonia solanacearum- , National Institute of Agrobiological Resources (2002), etc.).

  When a plant is infected, for example, the labeling substance is observed by observing the labeling substance in a cross section of the plant. Here, the “cross section of a plant” is a section in which the roots, leaves, or stems of a plant are cut by a cutting means such as a razor, and the state of a path through which bacterial wilt fungi such as vascular bundles of the plant pass can be observed. Say.

  Alternatively, preferably, in order to observe the growth, decrease or behavior of bacterial wilt fungus in the plant body, the plant infected with bacterial wilt fungus is cultured so as to extend along the surface of the agar medium inclined at an angle to the horizontal plane. , By observing the labeling substance in the plant cultured on the surface of the agar medium. Hereinafter, such a gradient agar medium will be described in detail.

  As a component of the agar medium, a conventionally known component may be included for growing plants. For example, agar and hyponex powder (http://vegetea.naro.affrc.go.jp/kinou/shukakugoseiri/page021.html) as shown in detail in the examples and for plant culture such as Murashige & Skoog (MS) According to the composition of mediums known in the art such as medium, B5 medium, N6 medium, AA medium (see Biochemical Experimental Method 41, Introduction to Plant Cell Engineering, edited by Atsushi Komazaki and Kouji Nomura, Academic Publishing Center) p17-39) In addition, an agar medium composed of those containing various necessary components can be used. The content of agar components such as agar in this agar medium is 1 to 2 w / v%, preferably 1.3 to 1.7 w / v%, more preferably about 1.5 w / v%. The inclination angle of the surface of the agar medium is not particularly limited as long as the plant can grow so that the roots and / or stems extend along the surface of the agar medium, but preferably 15 to 75 degrees with respect to the horizontal plane. Is 30 to 60 degrees, more preferably 40 to 50 degrees, and still more preferably about 45 degrees. That is, the root or / and stem of the plant grows so as to extend along the surface of the agar medium, and the labeling substance in the plant has an inclination angle that can be directly observed with a fluorescent stereomicroscope without digging out the plant. means. At this time, the inclination angle may be either when a petri dish having an agar medium formed horizontally is cultivated on an inclined surface, or when the agar medium itself in the petri dish is inclined and placed on a horizontal surface for cultivation.

  Further, preferably, the labeling substance is observed over time, and the growth, decrease or behavior of bacterial wilt fungus is observed in real time. “Observation in real time over time” means to observe the growth, decrease or behavior of bacterial wilt disease every time a certain time elapses.

  Among the methods for monitoring bacterial wilt fungus according to the third embodiment, according to the method using a gradient agar medium, it is possible to more easily observe the growth, reduction or behavior of bacterial wilt fungus labeled with a labeling substance. it can. For example, by incorporating the labeled bacterial wilt fungus into a plant, the location of the bacterial wilt fungus in the plant can be observed, for example, directly by a fluorescent stereomicroscope. It can be used for drug research. Furthermore, by observing the labeled bacterial wilt fungus in real time, the mechanism of bacterial wilt fungus infection in the plant can be elucidated.

  According to the method for monitoring bacterial wilt disease according to the third embodiment, the bacterial wilt fungus in soil can be dispersed by spraying the bacterial wilt fungus labeled with a labeling substance into the soil, in addition to observation in the plant body. The amount, and even the state of growth can be observed. This observation can be used to study the mechanism of bacterial wilt infection from soil to plants. In addition, the method of cultivating plants infected with bacterial wilt on a slanted agar medium is suitable for research of bacterial wilt fungus at the laboratory level. Compared with, experiments (monitoring) can be performed in a short period of time. Furthermore, since the bacterial wilt fungus according to Embodiment 2 is used and the luminescence intensity by the labeling substance is enhanced, the bacterial wilt fungus can be clearly and easily monitored.

(Evaluation method for bacterial wilt disease)
Embodiment 4 of the present invention relates to an evaluation method for bacterial wilt disease. The evaluation method for bacterial wilt fungus according to the fourth embodiment uses the monitoring method according to the third embodiment and uses the growth, decrease or behavior of the bacterial wilt fungus as an index, or the susceptibility to bacterial wilt fungus by plant varieties, or Assess tolerance. Preferably, as described above, the growth, decrease or movement of bacterial wilt fungus in the plant body as an indicator is observed along the surface of the agar medium in which the plant infected with bacterial wilt fungus is inclined with respect to the horizontal plane. The culture is preferably performed by elongating and observing the labeling substance in the plant cultured on the surface of the agar medium.

  As shown in detail in the Examples, it has been found that plant varieties having sensitivity or resistance to bacterial wilt disease have greatly different growth or migration when infected with bacterial wilt disease. In particular, bacterial wilt fungi invaded from the main root to the lateral root in a short period of time in sensitive varieties, but did not invade into the lateral root in a short period of time in resistant varieties. Furthermore, it was found that sensitive varieties invade the entire plant including veins, and depending on the cultivar, leakage of green fluorescence from bacterial wilt fungus was observed from the root tip of the lateral root. Therefore, in the evaluation method for bacterial wilt fungus according to the fourth embodiment, the growth, decrease or movement of bacterial wilt fungus is used as an index, and the plant is used against bacterial wilt fungus by using the monitoring method according to the third embodiment. Whether it is sensitive or resistant.

  For example, when many intrusions into the lateral root are observed as in the experimental results of the ponderosa and large-scale longevity shown in the examples, it can be said that the plant has sensitivity to bacterial wilt. On the other hand, if the invasion into the lateral root is not observed as much as the B-barrier experimental results, it can be said that the plant has resistance to bacterial wilt. Therefore, preferably, in the evaluation method according to the fourth embodiment, the growth, reduction, or behavior of the bacterial wilt fungus as an index is the invasion (ratio) of the bacterial wilt fungus according to the second embodiment into the side roots of the plant. In this way, the sensitivity or resistance to bacterial wilt is evaluated.

  According to the method for evaluating bacterial wilt fungus according to the fourth embodiment, it is possible to determine whether each plant variety is sensitive or resistant to bacterial wilt fungus. Therefore, it can be used for the development of plant varieties resistant to bacterial wilt and can be used to reduce damage to bacterial wilt. Moreover, since the bacterial wilt fungus according to Embodiment 2 is used and the luminescence or fluorescence intensity by the labeling substance is enhanced, the sensitivity or resistance to bacterial wilt fungus can be clearly and easily evaluated.

(Screening method of test substance effective against bacterial wilt disease)
Embodiment 5 of the present invention relates to a screening method for a test substance effective against bacterial wilt. The screening method for a test substance effective against bacterial wilt fungus according to the fifth embodiment provides a test substance to a plant infected and diseased with bacterial wilt fungus according to the second embodiment, and relates to the third embodiment. The test substance effective against bacterial wilt is screened by observing using the bacterial wilt fungus monitoring method. Preferably, in the method for monitoring bacterial wilt disease according to Embodiment 3, a monitoring method using a gradient agar medium is used.

  The “test substance” given is an organic compound of various molecular sizes (nucleic acid, peptide, protein, lipid (simple lipid or complex lipid (such as phosphoglyceride, sphingolipid, glycosylglyceride or cerebroside)), prostaglandin, isoprenoid, terpene or Steroids))) or inorganic compounds can be used. The “test substance” may be derived from a natural product or synthesized. In the latter case, for example, an efficient screening system can be constructed using a combinatorial synthesis technique. A cell extract or a culture supernatant may be used as a test substance.

  The “test substance effective against bacterial wilt fungus” means, for example, the “test subject” in the case of a plant variety judged to be sensitive to bacterial wilt fungus in the evaluation method for bacterial wilt fungus according to Embodiment 4. “Substance” refers to a substance that can suppress the spread of bacterial wilt fungus in the body (side roots and veins) of the plant variety. Moreover, even if it is a plant variety judged to be resistant to bacterial wilt, it is applicable if it is a substance that can further suppress the spread of bacterial rot in the plant body.

  When confirming the effectiveness of the “test substance” with respect to a plant planted in the soil, it can be confirmed, for example, by mixing the “test substance” with the soil around the plant. In addition, when the effectiveness of the “test substance” is confirmed for the plant on the surface of the agar medium as described above, for example, it can be confirmed by including the “test substance” in the components of the agar medium. .

  According to the screening method for a test substance effective against bacterial wilt disease according to the fifth embodiment, a test substance effective against bacterial wilt disease (fungus) can be found. By using this discovered test substance, it is possible to reduce the damage caused by bacterial wilt. Moreover, since the bacterial wilt fungus according to Embodiment 2 is used and the intensity of light emission and / or fluorescence by the labeling substance is enhanced, screening of test substances effective against the bacterial wilt fungus can be performed clearly and easily. It can be carried out.

(Monitoring method, evaluation method and screening method)
Embodiment 6 of the present invention relates to a monitoring method, an evaluation method, and a screening method using a gradient agar medium.

  The monitoring method according to Embodiment 6 of the present invention has a property of producing a labeling substance by introducing a foreign gene containing a gene encoding a labeling substance into a pathogenic bacterium that infects the plant, In order to observe the growth, decrease, or behavior, the plant infected with the pathogen is cultured so that the roots and / or stems extend along the surface of the agar medium inclined at an inclined angle with respect to the horizontal plane, and cultured on the surface of the agar medium. It is characterized by observing the labeling substance produced in the plant body.

  Further, as an application of this monitoring method, there are an evaluation method for evaluating the susceptibility or resistance to pathogenic bacteria by plant varieties, and a method for screening a test substance effective against the pathogenic bacteria.

  A preferred embodiment of the monitoring method according to the sixth embodiment of the present invention and the evaluation method and the screening method to which the monitoring method is applied is that the pathogen used includes the transformed bacterial wilt of the present invention. The above-mentioned monitoring method for bacterial wilt disease and its application, except that the pathogen has the property of producing a labeling substance by introducing a foreign gene including a gene encoding the labeling substance into the pathogen This is substantially the same as the preferred embodiment of the evaluation method and screening method described above.

Here, the term “foreign gene including a gene encoding a labeling substance” to be introduced into a pathogenic bacterium is one that can be easily introduced into a pathogenic bacterium and that can exhibit the effect of producing a labeling substance within the introduced pathogenic bacterium. There is no particular limitation, and such a method for preparing a foreign gene and a method for introducing it into a pathogenic bacterium can be used without any particular limitation in methods generally used in this field. If a preferable example is shown, what has a "replication module" derived from a bacteriophage corresponding to a pathogenic bacterium and a "gene encoding a labeling substance" as components, such as the plasmid of the present invention, specifically, For example, pRSS12, pRSS1S-GFPuv mentioned above, pRSS1S-GFPmut2, etc. are mentioned.

Moreover, examples of the “pathogenic fungus that infects plants” to which the monitoring method according to the sixth embodiment of the present invention and the evaluation method and screening method applying the monitoring method are applicable include Fusarium molds such as Fusarium oxysporum , Examples include pathogenic bacteria well known in this field, such as pathogenic bacteria ( Magnaporthe grisea ) and soil pathogenic bacteria ( Erwinia carotovora ).

In addition, in Embodiments 3 to 6 of the present invention, the “plant” includes at least 33 kinds of 200 or more plants that are reported to be infected with bacterial wilt fungi such as tomato, eggplant or tobacco (Mitsuo Horita, In Kenichi Tsuchiya, Microbial Genetic Resource Manual (12) -Ralstonia solanacearum- , Agricultural Bioresource Research Institute (2002), etc.), in Embodiment 6 of the present invention, Fusarium genus molds and rice blasts described above are further used. This includes plants that are reported to be infected with pathogenic bacteria, soil pathogenic bacteria, and the like.

  As a component of the agar medium, a conventionally known component may be included for growing plants. For example, agar and hyponex powder (http://vegetea.naro.affrc.go.jp/kinou/shukakugoseiri/page021.html) as shown in detail in the examples and for plant culture such as Murashige & Skoog (MS) According to the composition of mediums known per se, such as medium, B5 medium, N6 medium, AA medium (see Biochemical Experimental Method 41, Introduction to Plant Cell Engineering, edited by Atsushi Komazaki and Kouji Nomura, Academic Publishing Center, p. 17-39) In addition, an agar medium composed of those containing various necessary components can be used. The content of agar components such as agar in this agar medium is 1 to 2 w / v%, preferably 1.3 to 1.7 w / v%, more preferably about 1.5 w / v%. The inclination angle of the surface of the agar medium is not particularly limited as long as the plant can grow so that the roots and / or stems extend along the surface of the agar medium, but preferably 15 to 75 degrees with respect to the horizontal plane. Is 30 to 60 degrees, more preferably 40 to 50 degrees, and still more preferably about 45 degrees. That is, the root or / and stem of the plant grows so as to extend along the surface of the agar medium, and the labeling substance in the plant has an inclination angle that can be directly observed with a fluorescent stereomicroscope without digging out the plant. means. At this time, the inclination angle may be either when a petri dish having an agar medium formed horizontally is cultivated on an inclined surface, or when the agar medium itself in the petri dish is inclined and placed on a horizontal surface for cultivation.

  In the monitoring method, the evaluation method, and the screening method according to the sixth embodiment, pRSS12 invented by some of the present inventors can be used, and this structure is also described for reference.

FIG. 1 is a schematic diagram showing a specific structure of the pRSS 12. That is, FIG. 1 shows the structure of pRSS12 described in Patent Document 3 and the phage φRSS1 genomic DNA that is the origin thereof. The upper diagram in FIG. 1 shows the structure of the genomic DNA of phage φRSS1. The lower diagram of FIG. 1 shows the structure of pRSS12. The entire base sequence of phage φRSS1 is registered in DDBJ as accession number AB259124. The entire sequence consisting of 6662 bases of the genomic DNA of phage φRSS1 with accession number AB259124 is shown in SEQ ID NO: 1 in the sequence listing.

  In general, the genome of an Ff-like phage such as phage φRSS1 includes a plurality of structures called “modules” in which genes having related functions are grouped. For example, a “replication module” contains genes gII, gV, and gX involved in rolling circle DNA replication. The genomes of Ff-like phages have three types in common: a “replication module”, a “structural module”, and an “assembly and secretion module”. These modules are arranged on the genome in the above order, and this arrangement is also maintained in the phage φRSS1. As used herein, the term “replication module” means a module containing genes gII, gV, and gX involved in rolling circle DNA replication, or from the genomic structure of an Ff-like phage, It means the part except “module” and “assembly and secretion module”.

  To create pRSS12, first, a plasmid consisting of about 1/3 (2248 bases) of the phage φRSS1 DNA is constructed by removing the structural module, assembly and secretion module (ORF4 to ORF11) from the phage φRSS1 DNA. To do. In the portion consisting of 2248 bases, ORF1 to ORF3 and IG of the DNA of phage φRSS1 are encoded. This sequence consists of the 1st to 1720th base sequence and the 6135th to 6662th base sequence of the genomic DNA sequence of phage φRSS1.

  As shown in the lower diagram of FIG. 1, pRSS12 is resistant to GFP-Km (GFP gene (GFPuv gene) and kanamycin between positions 1720 and 1721 of about one-third of DNA (2248 bases) of phage φRSS1. About 4.7 kbp plasmid containing the cassette bound). As a precaution, pRSS12 uses the lac promoter derived from E. coli for the expression of the GFP gene (Patent Document 3). For detailed preparation methods and the like of pRSS12, refer to Patent Document 3 and Non-Patent Documents 1 to 3.

The bacteriophages (phage φRSS1 and phage φRSB2) used in this example and reference examples are those isolated from bacterial wilt ( Ralstonia solanacearum ). Phage φRSS1 was propagated using bacterial wilt strain C319. In addition, the strain C319 is a strain of race 1 isolated from tobacco, and is a strain distributed from San-in Construction Industry Co., Ltd. (Izumo City, Shimane Prefecture). The phage φRSB2 is a novel bacteriophage hosted by bacterial wilt fungi isolated from Hiroshima Prefecture by the present inventors.

  The bacterial wilt fungus strain MAFF106611 used in this Example and Reference Example is described in the Microbial Genetic Resource Utilization Manual (12) published by the Ministry of Agriculture, Forestry and Fisheries Bioresource Research Institute, Mitsuo Hotta and Kenichi Tsuchiya, March 2002. It is a strain and can be sold from the Agricultural Bioresource Genebank, Ministry of Agriculture, Forestry and Fisheries. For details of each phage isolation method and each strain, see Patent Documents 1 to 3 and Non-Patent Documents 1 to 3 described in Background Art.

  Various bacterial wilt strains were stirred at 200 to 300 rpm at 28 ° C. and cultured in CPG medium. A CPG medium containing 0.1% casamino acid, 1% peptone and 0.5% glucose was used. Each phage was propagated and purified by a single plaque isolation method. When the OD600 of the bacterial cell culture reached 0.1 units, 0.01 to 0.05 moi (the relative number of phages relative to the number of bacteria) was added. Thereafter, the cells are cultured for 16 to 18 hours, and cells are removed by centrifugation. After passing through a 0.45 μm filter, CsCl (9.4 g / 20 mL) is added to the phage suspension, and ultracentrifugation is performed. Performed further purification. Purified phage was stored at 4 ° C. until used.

  In addition, observation with a fluorescence stereomicroscope is performed using an MF16F fluorescence stereomicroscope (Leica Microsystems, Heidelberg, Germany) using GFP2 and GFP3 filters and / or an Olympus BH2 fluorescence microscope (Olympus, Tokyo, Japan), and a CCD camera ( Keyence VB-6010; Osaka, Japan).

  Hereinafter, Examples and Reference Examples relating to a method for preparing pRSS12, pRSS1S-GFPuv and pRSS1S-GFPmut2, bacterial wilt fungus transformed with these plasmids, and a monitoring method using these bacterial wilt fungi will be described. . First, the preparation method of pRSS12 and the transformation to bacterial wilt are described.

(Reference Example 1)
In order to prepare pRSS12, first, a DNA fragment of about 1/3 length (about 2248 bases) lacking the structural protein and the morphogenic module part from the genomic sequence of phage φRSS1 was amplified by PCR (Polymerase Chain Reaction) method. did. The entire sequence of phage φRSS1 is registered in DDBJ as accession number AB259124. The entire genomic DNA sequence of phage φRSS1 is shown in SEQ ID NO: 1. As shown in FIG. 1, the primer includes a forward primer 12-F1 (SEQ ID NO: 4, 5′-cggaattctatccggagtaacgaaaag-3 ′) having a base sequence corresponding to the 3 ′ terminal portion of ORF11 in the phage φRSS1 genome, and Reverse primer 12-R1 (SEQ ID NO: 5, 5′-atggaattctccttgagatggaggttgag-3 ′) having a base sequence corresponding to the 5 ′ end portion of ORF4 was used. An EcoRI site is added to each of these primers. This PCR was performed using MY-Cycler (manufactured by Bio-Rad) under standard conditions. As a template, the phage φRSS1 RF-DNA (1 ng) was used for 25 cycles. The obtained DNA fragment was purified according to a conventional method.

  Further, a kanamycin resistance cassette was excised from the plasmid pUC4-KIXX (Amersham Biosciences) with EcoRI. This kanamycin resistance cassette fragment was inserted into the EcoRI site of pGFPuv (Takara Bio Inc.) containing the GFP gene to construct pGFPuv-Km in which the kanamycin resistance cassette and the GFP gene (GFPuv) were bound. The orientation of each gene was confirmed by a restriction enzyme mapping method.

  Furthermore, using the constructed pGFPuv-Km as a template, a DNA sequence fragment (hereinafter referred to as GFPuv-Km) related to the binding portion of the kanamycin resistance cassette and GFPuv was amplified by PCR. As the primer, forward primer 12-F2 (SEQ ID NO: 6, 5'-gagcgccgaattcgcaaaccgcctctcc-3 ') and reverse primer 12-R2 (SEQ ID NO: 5, 5'-ttgacaccagacaagttggtaatggtag-3') were used. As shown in the lower diagram in FIG. 1, pRSS12 was constructed by binding this PCR product (GFPuv-Km) to the amplified fragment derived from the above-described phage φRSS1. The pRSS12 thus constructed becomes a plasmid having the 1st to 1720th nucleotide sequence of the phage φRSS genome, the 6135th to 6662th nucleotide sequence of the same genome, the kanamycin resistance cassette, and the GFPuv gene.

  About introduction | transduction to the bacterial wilt fungus which concerns on obtained pRSS12 according to description of a manufacturer's instruction | indication using Gene Pulser Xcell (made by Biorad), it is 2 mm cuvette at 2.5 kV, and bacterial wilt fungus MAFF106611 strain Were introduced by electroporation. The resulting transformants were selected on CPG medium containing 15 μg / mL kanamycin.

Example 1
Examples relating to methods for preparing pRSS1S-GFPuv and pRSS1S-GFPmut2 will be described. First, in order to prepare pRSS1S to be the basis of these plasmids, in the same manner as in the case of pRSS12 described above, the length of about 1/3 in which the structural protein and the morphogenic module portion are missing from the genomic sequence of phage φRSS1 ( A DNA fragment of 2583 bases) was amplified by the PCR method. In addition, compared with the case of pRSS12, the sequence of the forward primer is slightly different. As shown in the upper diagram of FIG. 2, the primers include forward primer 1S-R (SEQ ID NO: 8, 5′-agacatcgacattcggcacatcgc-3 ′) having a base sequence corresponding to the vicinity of ORF11 of phage φRSS1; and Reverse primer 1SL (SEQ ID NO: 9, 5′-atggaattctccttgagatggaggttgag-3 ′) having a base sequence corresponding to the 5 ′ end portion of ORF4 was used. Note that an EcoRI site is introduced into the 1S-L primer.

  This PCR was performed under standard conditions using MY-Cycler (Bio-Rad). As a template, the phage φRSS1 RF-DNA (1 ng) was used for 25 cycles. The obtained DNA fragment was purified according to a conventional method. A fragment of the kanamycin resistance cassette was bound to this DNA fragment to construct pRSS1S as shown in the lower diagram of FIG. The kanamycin resistance cassette was obtained by excising from plasmid pUC4-KIXX (manufactured by Amersham Biosciences) with EcoRI, blunting and phosphorylation. The pRSS1S constructed in this way becomes a plasmid having the base sequence from 1 to 1720 of the phage φRSS genome, the base sequence from 5800 to 6662 of the same genome, and the kanamycin resistance cassette (Km cassette).

  Next, an expression cassette consisting of a phage φRSB2 MCP (major coat protein) gene promoter (SEQ ID NO: 2), a GFP (GFPuv or GFPmut2) gene, and an MCP gene terminator (SEQ ID NO: 3) was prepared. Phage φRSB2 is a novel bacteriophage hosted by bacterial wilt, and the inventor isolated from Hiroshima Prefecture. First, the promoter sequence of the MCP gene was amplified by PCR using the prepared phage φRSB2 DNA as a template. At that time, MCP-F-72 (SEQ ID NO: 10, 5′-cccgaattctggaacgccgcgtcgctgcctcgtaag-3 ′) is used as the forward primer, and MCP-R-74 (SEQ ID NO: 11, 5′-tgtggttctccttgaaaagtgatcagggac-3 ′) is used as the reverse primer. It was. An EcoRI site is introduced into the forward primer MCP-F-72. PCR was performed under standard conditions using MY-Cycler (manufactured by Bio-Rad) in 25 cycles.

  Similarly, the terminator sequence of the MCP gene was amplified by PCR using the phage φRSB2 DNA as a template. At that time, Term-F-77 (SEQ ID NO: 12, 5′-tggccgtcctccaacccaagcata-3 ′) was used as the forward primer, and Term-R-R1-71 (SEQ ID NO: 13, 5′-cctgaattctccctgctgggtccccttggggtggtgc-3 ′) was used as the reverse primer. Was used. An EcoRI site is introduced in the reverse primer Term-R-Rl-71. The conditions for PCR are the same as for PCR of promoter sequences.

  Next, an GFP (GFPuv or GFPmut2) gene was ligated between the amplified MCP promoter and MCP terminator, and cut at the EcoRI site to prepare an expression cassette. As for GFPuv and GFPmut2, pMRP9-1 (Davies, DG et al. The involvement of cell-to-cell signals in the development of a bacterial) is a plasmid containing a gene encoding pGFPuv (Takara Bio Inc.) and GFPmut2. biofilm. Science 280, 295-298 (1998).). DNA fragments of GFPuv and GFPmut2 were amplified by PCR using pGFPuv and pMRP9-1 as a template and a primer for amplifying the coding region of each GFP, respectively. Further, ligation of these DNA sequence fragments (MCP promoter, GFPuv or GFPmut2, and MCP terminator) was ligated by T4 DNA ligase using Ligation High (manufactured by Toyobo Co., Ltd.). The orientation of GFPuv or GFPmut2, MCP promoter region, and MCP terminator region was confirmed by PCR.

  The GFPuv or GFPmut2 expression cassette thus prepared was introduced into the EcoRI site of pRSS1S described above. The ligation method is the same as described above. When the GFPuv expression cassette is introduced, as shown in FIG. 3, the 1st to 1720th nucleotide sequence of the phage φRSS genome, the 5800 to 6661th nucleotide sequence of the genome, MCP promoter region, GFPuv gene, MCP terminator region, And pRSS1S-GFPuv with a kanamycin resistance cassette. On the other hand, when the GFPmut2 expression cassette was introduced, as shown in FIG. 4, the 1st to 1720th nucleotide sequence of the phage φRSS genome, the 5800 to 6662th nucleotide sequence of the same genome, the MCP promoter region, the GFPmut2 gene, and the MCP terminator PRSS1S-GFPmut2 with the region and kanamycin resistance cassette.

  For introduction into bacterial wilt fungus related to the obtained pRSS1S-GFPuv or pRSS1S-GFPmut2, using Gene Pulser Xcell (manufactured by Bio-Rad Laboratories, Inc.) at 2.5 kV according to the manufacturer's instructions. A 2 mm cuvette was introduced into the bacterial wilt fungus MAFF106611 by electroporation. The resulting transformants were selected on CPG medium containing 15 μg / mL kanamycin.

(Example 2)
Hereinafter, Examples relating to quantitative comparison of fluorescence emission in bacterial wilt disease transformed with pRSS12 and pRSS1S-GFPuv will be described.

As described in Reference Example 1 and Example 1, the prepared pRSS12 and pRSS1S-GFPuv were introduced into bacterial wilt fungus MAFF106611 by electroporation. Next, the strain containing each plasmid was cultured in a CPG medium until the logarithmic growth phase, and was suspended in distilled water after centrifugation. The density | concentration of the microbe was adjusted so that it might become OD600 = 0.5 (1.5 * 10 < ⁸ > cells / ml). A dilution series of the suspension was prepared, and the fluorescence intensity of the bacterial suspension was measured using a multiplate reader (Infinit M200, Tecan). 200 μl of the suspension was used for measurement, and excited with 485 nm blue light, and 518 nm green fluorescence was measured and quantified.

FIG. 5 is quantitative comparison data of fluorescence intensity between pRSS12 and pRSS1S-GFPuv. That is, FIG. 5 shows the fluorescence intensity (relative value) of the suspension of bacteria containing pRSS12 or pRSS1S-GFPuv at each cell concentration (cell / ml). In addition, the fluorescence intensity of the suspension with a cell concentration of 1.5 × 10 ⁸ cells / ml of bacterial wilt disease transformed with pRSS12 was set to 1. As shown in FIG. 5, the suspension of bacterial wilt fungus transformed with pRSS1S-GFPuv at a cell concentration of 1.5 × 10 ⁸ cells / ml is about 1.6 times that of pRSS12. The fluorescence intensity is enhanced.

  As shown in FIG. 1 and FIG. 3, pRSS12 and pRSS1S-GFPuv differ in whether an MCP promoter and an MCP terminator are incorporated. In pRSS12, the lac promoter derived from E. coli is used for the expression of the GFP gene. The gene used as a fluorescent label in any plasmid is GFPuv. From the above results, it was found that when the bacterial wilt disease was fluorescently labeled, the use of the MCP promoter and MCP terminator of phage φRSB2 was effective in enhancing the fluorescence intensity. That is, it was found that detection of bacterial wilt and monitoring in plants can be observed more clearly and easily by using a plasmid incorporating the promoter and terminator sequences.

(Example 3)
Hereinafter, Examples relating to quantitative comparison of fluorescence emission in bacterial wilt disease transformed with pRSS1S-GFPuv and pRSS1S-GFPmut2 will be described.

As described in Example 1, the prepared pRSS1S-GFPuv and pRSS1S-GFPmut2 were introduced into bacterial wilt fungus MAFF106611 by electroporation. Next, in the same manner as in Example 2, the strain containing each plasmid was cultured in a CPG medium until the logarithmic growth phase, and was suspended in distilled water after centrifugation. The density | concentration of the microbe was adjusted so that it might become OD600 = 0.5 (1.5 * 10 < ⁸ > cells / ml). A dilution series of the suspension was prepared, and the fluorescence intensity of the bacterial suspension was measured using a multiplate reader. 200 μl of the suspension was used for measurement, and excited with 485 nm blue light, and 518 nm green fluorescence was measured and quantified.

FIG. 6 is quantitative comparison data of fluorescence intensity between pRSS1S-GFPuv and pRSS1S-GFPmut2. That is, FIG. 6 shows the fluorescence intensity (relative value) of a bacterial suspension containing ppRSS1S-GFPuv or pRSS1S-GFPmut2 at each cell concentration (cell / ml). In addition, the fluorescence intensity of the suspension with a cell concentration of 1.5 × 10 ⁸ cells / ml of bacterial wilt disease transformed with pRSS1S-GFPuv was set to 1. As shown in FIG. 6, the suspension of bacterial wilt bacteria transformed with pRSS1S-GFPmut2 at a cell concentration of 1.5 × 10 ⁸ cells / ml is 10 times or more compared to pRSS1S-GFPuv. The fluorescence intensity is enhanced.

  As shown in FIGS. 3 and 4, pRSS1S-GFPuv and pRSS1S-GFPmut2 are different in that the gene used as a fluorescent label is GFPuv or GFPmut2. Both the MCP promoter and MCP terminator are incorporated. From the above results, when labeling bacterial wilt bacteria with a fluorescent label, the fluorescence intensity could be further enhanced by using GFPmut2.

(Reference Example 2)
Hereinafter, a reference example in which bacterial wilt fungus transformed with pRSS12 is inoculated on a stem of tomato and the bacterial wilt fungus is monitored by a cross section of the plant will be described.

After preparing pRSS12 by the method described in Reference Example 1, bacterial wilt fungus MAFF106611 strain was transformed. The resulting bacterial wilt of the bacterial wilt is inoculated into the stem of a tomato plant (variety: Large Fushou, Takii Seed Co., Ltd.) that has been cultivated in soil for 4 weeks after sowing and attached 4-6 leaves. Bacterial fungi were monitored. The seedlings of tomato used for inoculation were sown at 25 ± 3 degrees in soil containing peat moss and vermiculite. Bacterial inoculation was performed by piercing the stem with a needle coated with bacterial wilt fungus (Mitsuo Hotta, Kenichi Tsuchiya, Microbial Genetic Resource Manual (12) -Ralstonia solanacearum- bacterial wilt fungus, National Institute of Agrobiological Resources) (2002), pages 17-21). The bacterial wilt culture solution was cultivated in a CPG medium and diluted with distilled water so that the concentration of the bacterium was 1.0 × 10 ⁸ cells / ml.

  120 hours after inoculation, a cross section of a stem was prepared and observed with a fluorescent stereomicroscope. FIG. 7 is a photograph replacing a drawing by a fluorescent stereomicroscope of a cross section of a stem infected with bacterial wilt disease transformed with pRSS12 according to Reference Example 2. Cross sections of the stems were made with a razor at 10 mm intervals from the injection point. A in FIG. 7 is a section 20 mm above the infection site. B in FIG. 7 is a section 10 mm above the infected site. C of FIG. 7 is a section of an infection site (perforation site, injection point). D in FIG. 7 is a section 10 mm above the infection site. E in FIG. 7 is a section 20 mm below the infection site. F in FIG. 7 is a section of a control not inoculated with bacterial wilt. In FIG. 7, fluorescence (GFPuv) derived from bacterial wilt is detected in green. On the other hand, autofluorescence derived from plant chloroplasts is detected in red. The scale in the figure is 1 mm.

  The moving distance and moving speed of bacterial wilt fungus during the observation period according to the present example are determined from the distance between the infected site and the section where the farthest fluorescence (GFPuv) is observed. Further, as shown in FIG. 7, fluorescence (GFPuv) is observed in a section 20 mm away from the infected site, but fluorescence (GFPuv) is not observed in a section 20 mm below the infected site.

  As shown in A to E of FIG. 7, when the bacterial wilt fungus moves the tomato stem, it was found that the upward movement speed is faster than the downward movement speed. Specifically, it was found that the upward moving speed was 0.4 to 0.8 mm / h, and the downward moving speed was 0.1 to 0.2 mm / h. Thus, by inoculating (infecting) a plant stem with bacterial wilt fungus transformed with pRSS12 and observing the fluorescence (GFPuv) of a section of the plant stem, It was found that observation (ie monitoring) such as follow-up or proliferation can be performed. In Reference Example 2, bacterial wilt disease transformed with pRSS12 is used, but it is suggested that such monitoring can be performed with pRSS1S-GFPuv and pRSS1S-GFPmut2 in the same manner. In particular, when the results of Examples 2 and 3 are considered, it is suggested that monitoring can be performed more clearly and simply by using pRSS1S-GFPuv or pRSS1S-GFPmut2, preferably pRSS1S-GFPmut2.

(Reference Example 3)
It is considered that the movement of water through the xylem conduit influences the difference in the movement speed of bacterial wilt bacteria above and below the infected site described in Reference Example 2. Therefore, the present inventors conducted an experiment regarding further monitoring of bacterial wilt. Hereinafter, a more detailed reference example for inoculating a bacterial wilt fungus transformed with pRSS12 onto a stem of tomato and monitoring the bacterial wilt fungus with a cross section of the plant will be described.

After preparing pRSS12 by the method described in Reference Example 1, bacterial wilt fungus MAFF106611 strain was transformed. The obtained bacterial wilt of bacterial wilt is inoculated by perforation to the stems of tomatoes (variety: Large Fushou, Takii Seed Co., Ltd.) cultivated in soil for 4 weeks after sowing (Mitsuo Hotta and Kenichi Tsuchiya, Microbial genetics Resource manual (12) -bacterial wilt fungus Ralstonia solanacearum- , National Institute of Agrobiological Resources (2002), pages 17 to 21) Cross sections were prepared 4 to 6 days later and bacterial wilt fungus was monitored. FIG. 8 is a photograph replacing a drawing by a fluorescent stereomicroscope of a cross section of a stem infected with bacterial wilt fungus transformed with pRSS12 according to Reference Example 3. Please refer to the photo of the property submission for the colors described below.

  FIG. 8A shows a schematic diagram of an infection experiment according to Reference Example 3. In FIG. 8A, the yellow line schematically shows the vascular bundle in the tomato stem. The arrow indicates the position where the bacterial wilt of tomato stem was infected with bacterial wilt fungus by the perforation method. B or D in FIG. 8 is a photograph taken by a fluorescent stereomicroscope of a cross section of a stem below a pedicle branch point indicated by a broken line B or D of A. C of FIG. 8 is a photograph taken by a fluorescent stereomicroscope of a cross section of a stem at a striatum branch point indicated by a broken line C of A. FIG. E in FIG. 8 is a photograph taken by a fluorescent stereomicroscope of the cross section of the stem at the striatum branch point indicated by the broken line E in A. The scale in the figure is 1 mm.

  As shown in FIGS. 8B to 8E, a vascular bundle infected with bacterial wilt is observed as green fluorescence (indicated by an arrowhead). Autofluorescence derived from plant chloroplasts is detected in red. As shown in FIG. 8C, when the vascular bundle infected with bacterial wilt was connected to the petiole, infection was also observed in the petiole. In addition, fluorescence (GFPuv) is not observed in vascular bundles other than those connected to the petiole. On the other hand, as shown in FIG. 8E, the fluorescence (GFPuv) is not observed in the petiole unless the vascular bundle connected to the petiole is infected.

  From the above results, it was found that bacterial wilt bacteria move in the plant mainly in the longitudinal direction along the vascular bundle. In addition, it was confirmed that movement between vascular bundles was limited, that is, bacterial wilt fungi were not transferred between a plurality of vascular bundles constituting a plant stem in a short period of time. For example, as shown in FIG. 8C, when a vascular bundle infected with bacterial wilt enters the petiole, bacterial wilt is not observed in other vascular bundles.

  Based on the experimental results of Reference Example 3 as described above, not only the observation of fluorescence (GFPuv) in the section of the plant stem as described in Reference Example 2, but also examination and consideration of the continuity of the vascular bundle in the plant body. By observing cross sections of various parts such as plant stems, leaves, and roots, it is possible to observe (ie, monitor) bacterial wilt fungus detection, migration, tracking, or growth in more detail. Was suggested. In Reference Example 3, bacterial wilt disease transformed with pRSS12 is used, but it is suggested that such monitoring can be performed with pRSS1S-GFPuv and pRSS1S-GFPmut2 in the same manner. In particular, when the results of Examples 2 and 3 are considered, it is suggested that monitoring can be performed more clearly and simply by using pRSS1S-GFPuv or pRSS1S-GFPmut2, preferably pRSS1S-GFPmut2.

Example 4
Hereinafter, Examples relating to the case where a bacterial wilt fungus transformed with pRSS12 is infected with a tomato germinated on the surface of an agar medium will be described.

  Tomato (variety: large Fushou, Takii Seed Co., Ltd.) seeds were surface sterilized, germinated on the surface of an agar medium in a square petri dish, and cultured aseptically. The components of the agar medium contained 0.15 w / v% Hyponex powder (Hyponex Japan Co., Ltd.), 0.5 w / v% sucrose and 1.5 w / v% agar, and were adjusted to pH 5.8. The square petri dish in culture was cultivated for 7 to 21 days in a 28-degree greenhouse at a light-dark cycle of 16 hours light period and 8 hours dark period (manufactured by Sanyo). During cultivation, the agar medium was cultivated at an angle of 45 degrees, and the tomato roots were cultivated so as to extend straight along the agar medium.

After preparing pRSS12 by the method described in Example 1, bacterial wilt fungus MAFF106611 strain was transformed. The obtained bacterial wilt of bacterial wilt was infected with tomatoes 7 days after seeding on the surface of the agar medium. As a method of infection, a new method for infecting bacterial wilt fungi by cutting the root tips of tomato seedlings and applying the transformed bacterial wilt fungus culture medium (2 μl, 1 × 10 ⁸ cells / ml) was newly developed. did. By this method, bacterial wilt disease was infected and observed with a fluorescence stereomicroscope.

  FIG. 9 is a photograph instead of a drawing showing a state where bacterial wilt fungus transformed with pRSS12 according to Example 4 was infected with tomato and cultured on the surface of the agar medium. FIG. 9A is a photograph of tomato seedling before infection 7 days after sowing. In the photographs taken with the fluorescent stereomicroscopes B to F in FIG. 9 described below, fluorescence derived from bacterial wilt is detected in green, and autofluorescence derived from the chloroplast of the plant is detected in red.

  FIG. 9B is a photograph taken with a fluorescent stereomicroscope of the root 12 hours after infection. Growth of bacterial wilt is observed as green fluorescence (GFPuv). C of FIG. 9 is a photograph taken by a fluorescence stereomicroscope at the boundary between the root and the hypocotyl 24 hours after the infection. FIG. 9D is a photograph taken with a fluorescent stereomicroscope of the hypocotyl 48 hours after infection. It can be seen that bacterial wilt fungus has invaded the hypocotyl. FIG. 9E is a photograph taken with a fluorescent stereomicroscope of the lateral root 48 hours after infection. It can be seen that the bacterial wilt fungus has started invading the lateral roots. F in FIG. 9 is a photograph taken with a fluorescent stereomicroscope of the seedling root and hypocotyl infected with bacterial wilt bacteria not containing pRSS12, and is a control. Green fluorescence due to GFPuv is not observed. In addition, as for the scale in FIG. 9, A is 2 cm and B thru | or F are 2 mm.

  From the above results, by infecting a plant cultivated with an agar medium by such a method with bacterial wilt fungus transformed by pRSS12, observation of bacterial wilt fungus detection, migration, tracking, or growth (ie, Monitoring) can be performed more easily over time. That is, it is not necessary to prepare a stem section as shown in the reference example described above, and it is not necessary to cut the plant every time it is observed. In addition, since the roots do not enter the agar medium, the resolution of observation with a fluorescent stereomicroscope can be improved. Further, since the cultivation is performed on the surface of the agar medium, the observation can be performed in a short period of time. In addition, although bacterial wilt disease transformed with pRSS12 is used in Example 4, it is suggested that such monitoring can be performed with pRSS1S-GFPuv and pRSS1S-GFPmut2 in the same manner. In particular, considering the results of Example 2 and Example 3, it is suggested that clearer and easier monitoring can be performed. A similar example related to pRSS1S-GFPmut2 is shown in Example 5 below.

(Example 5)
Hereinafter, Examples relating to a case where a tomato is infected with bacterial wilt fungus transformed with pRSS1S-GFPmut2 and cultured on the surface of an agar medium will be described.

  After preparing pRSS1S-GFPmut2 by the method described in Example 1, bacterial wilt fungus MAFF106611 strain was transformed. The obtained bacterial wilt of bacterial wilt was infected with tomatoes 7 days after seeding on the surface of the agar medium. The details of the components of the agar medium, the tomato cultivation method, the bacterial wilt infection method and the like are the same as in Example 4.

  FIG. 10 is a photograph replacing a drawing showing a state where bacterial wilt fungus transformed with pRSS1S-GFPmut2 according to Example 5 was infected with tomato, cultured on the surface of an agar medium, and observed in a bright field with a stereomicroscope. is there. FIG. 11 is a photograph replacing a drawing which shows a state where bacterial wilt fungus transformed with pRSS1S-GFPmut2 according to Example 5 was infected with tomato, cultured on the surface of an agar medium, and observed with a fluorescent stereomicroscope. The scale A in FIGS. 10 and 11 is 3 mm. In FIG. 11, fluorescence derived from bacterial wilt is detected in green, and autofluorescence derived from the chloroplast of the plant is detected in red.

  In Example 5, the bacterial wilt disease was monitored over time at 12 hours, 18 hours, 24 hours and 48 hours after infection. As in the case of pRSS12 described in Example 4, as shown in FIG. 11, growth to the roots of bacterial wilt fungus was observed 12 hours after infection. Invasion into the lateral root was observed 18 and 24 hours after infection, and fluorescence labeling with GFPmut2 appeared more strongly. Forty-eight hours after infection, the fluorescent label also appears stronger on the hypocotyl and stem.

  From the above results, in the same manner as in Example 4, by infecting a plant cultivated with an agar medium by such a method with bacterial wilt fungus transformed with pRSS1S-GFPmut2, detection, movement, It can be seen that observation (ie monitoring) such as follow-up or proliferation can be performed more easily over time. That is, it is not necessary to prepare a stem section as shown in the reference example described above, and it is not necessary to cut the plant every time it is observed. In addition, since the roots do not enter the agar medium, the resolution of observation with a fluorescent stereomicroscope can be improved. Further, since the cultivation is performed on the surface of the agar medium, the observation can be performed in a short period of time.

  In addition, when comparing the lateral root 48 hours after infection shown in E of FIG. 9 with the lateral root 48 hours after infection shown in FIG. 11, the infection conditions and culture conditions are the same, but pRSS1S shown in FIG. -Stronger fluorescence intensity is obtained when transformed with GFPmut2. That is, it was found that bacterial wilt bacteria can be monitored more easily and clearly than when pRSS12 is used. This is consistent with the result according to Example 3.

(Example 6)
Hereinafter, Examples relating to evaluation of susceptibility or resistance to bacterial wilt fungi according to the variety of tomatoes cultivated on the surface of the agar medium using the bacterial wilt fungus transformed with pRSS12 will be described.

  After preparing pRSS12 by the method described in Reference Example 1, bacterial wilt fungus MAFF106611 strain was transformed. The resulting bacterial wilt of bacterial wilt was infected with tomato for 1 or 3 weeks after seeding on the surface of the agar medium. The details of the components of the agar medium, the tomato cultivation method, the bacterial wilt infection method and the like are the same as in Example 4. As the tomato, varieties of Ponderosa, Large Fuju, and B-Barrier (Takii Seed Co., Ltd.) were used. Among them, Ponderosa and large-scale longevity are varieties sensitive to bacterial wilt, and B-barrier is a cultivar resistant to bacterial wilt.

  FIG. 12 is a photograph replacing a drawing with a fluorescent stereomicroscope showing a state in which tomato was infected with bacterial wilt fungus transformed with pRSS12 according to Example 6 and cultured on the surface of the agar medium. Fluorescence derived from bacterial wilt (GFPuv) is detected in green, and autofluorescence derived from the chloroplast of the plant is detected in red.

  FIG. 12A shows the state of seedling after 96 hours after infection when infected with a ponderosa (susceptible variety) one week after sowing. Already invasion into the veins was observed. FIG. 12B shows the state of the root tip after 96 hours after infection when infected with a ponderosa one week after sowing. It was observed that bacterial wilt was leaking from the root tip of the side root. Although not shown in the photograph, when the bacterial wilt fungus was infected one week after sowing, the root tip, lateral root, and hypocotyl were confirmed by fluorescence (GFPuv) in all cultivars.

  C of FIG. 12 shows the state of the root after 120 hours have passed after infecting the ponderosa 3 weeks after sowing. The growth of bacterial wilt disease was confirmed by green fluorescence (GFPuv), and invasion into the lateral roots also occurred. D of FIG. 12 shows the state of the hypocotyl after 120 hours have passed after infecting bacterial wilt in Ponderosa 3 weeks after sowing. The growth of bacterial wilt was confirmed by green fluorescence (GFPuv). E of FIG. 12 shows the state of the root after 120 hours have elapsed after infecting B-barrier (resistant varieties) 3 weeks after sowing with bacterial wilt. The growth of bacterial wilt on the main root was confirmed by fluorescence (GFPuv). There was no intrusion into the lateral root. FIG. 12F shows the state of the hypocotyl after 120 hours have elapsed after infecting B-barrier with the bacterial wilt 3 weeks after sowing. Little fluorescence (GFPuv) was observed in the hypocotyl. Note that the scale in the photograph is 2 mm.

  From the above results, it was not possible to determine susceptibility or resistance to bacterial wilt when infecting plants for 1 week after sowing, and susceptibility to bacterial wilt when infecting plants for 3 weeks after sowing. It was also found that the invasion rate of bacteria varies depending on the resistance. When infected 3 weeks after sowing, green fluorescence derived from bacterial wilt fungus is observed in all susceptible varieties, including leaf veins, and leakage of bacterial wilt fungus is observed from the root tip of lateral root depending on the cultivar. I found out. On the other hand, in the resistant varieties, the growth of the fungus was mainly limited to the main root, and no invasion into the side root was observed. Therefore, in order to investigate in detail the invasion rate of bacteria due to susceptibility or resistance to bacterial wilt, further experiments on the invasion of bacterial wilt into the lateral root were performed.

  As in the case of FIG. 12, first, bacterial wilt fungi were inoculated at the root tips of tomatoes (Ponderosa, large Fuju and B-barrier) grown on the surface of the agar medium for 3 weeks. Thereafter, the ingress of bacterial wilt into the lateral roots of each variety was observed and monitored over time. FIG. 13 is a table showing the invasion rate of bacterial wilt fungus by tomato variety according to Example 6. Each cultivar was observed 72 hours, 90 hours and 108 hours after inoculation with bacterial wilt (Time after infection). The observation was performed using 12 lateral roots for Ponderosa, 16 for Oogata-fukujyu, and 14 for B-barrier.

  As a result, as shown in FIG. 13, at 72 hours after the infection, invasion of bacteria into the lateral roots was observed at a high rate in Ponderosa and large-scale longevity which are varieties sensitive to bacterial wilt. On the other hand, in the B-barrier which is a variety resistant to bacterial wilt, the rate of invasion of the bacteria was low. Similar results were observed 90 hours after infection and 108 hours after infection.

  From the results according to Example 6, observation (ie, monitoring) of bacterial wilt fungus can be detected, transferred, followed, or propagated, and the monitoring result can be used as an index to evaluate the susceptibility or resistance to bacterial wilt fungus by plant varieties. Was suggested. Specifically, it was found that the sensitivity or resistance to bacterial wilt fungus can be examined by examining the invasion rate of bacterial wilt fungus into the lateral root at about 72 hours after infection. In Example 6, bacterial wilt disease transformed with pRSS12 is used, but it is suggested that an evaluation method using such monitoring can be performed with pRSS1S-GFPuv and pRSS1S-GFPmut2 in the same manner. Is done. In particular, considering the results of Examples 2 and 3, pRSS1S-GFPmut2 makes it easier to evaluate sensitivity or resistance because monitoring can be performed in a clearer and simpler manner and in a shorter time than conventionally performed methods. It is suggested that it can be done.

  Furthermore, the above-mentioned result has the property of producing a labeling substance by introducing a foreign gene including a gene encoding a labeling substance into a pathogen that infects a plant, including the transformed bacterial wilt of the present invention. If the `` the pathogenic fungus '' is used, the monitoring method for bacterial wilt disease described above and the monitoring method for various pathogenic bacteria according to the evaluation method and screening method applying the same, and the evaluation method and screening method applying the same are implemented. It shows you can.

  According to the present invention, a plasmid capable of enhancing the intensity of luminescence or fluorescence by a labeling substance when transformed with bacterial wilt, bacterial wilt transformed with the plasmid, bacterial wilt by observing the labeling substance Monitoring method, a method for evaluating susceptibility or resistance to bacterial wilt using the monitoring method, and a method for screening a test substance effective against bacterial wilt using the monitoring method are provided.

  In the method for monitoring bacterial wilt fungus transformed with the plasmid, the growth, decrease or behavior of bacterial wilt fungus in the soil or plant body can be clearly and easily observed by enhancing the expression of the labeling substance. it can. Moreover, the plasmid in the bacterial wilt fungus is suitable for use in such a monitoring method because it has the property of being retained in the bacterial wilt fungus for a long time even under conditions where there is no drug selection pressure. Such observations can be useful in research on bacterial wilt, such as drug research or bacterial mechanism for bacterial wilt. Among such methods for monitoring bacterial wilt, especially the method for culturing and monitoring a plant on the surface of an agar medium is suitable for research of bacterial wilt on a laboratory level. Monitoring can be performed in a short period of time compared to the case of monitoring with inoculation with blight fungus. In addition, the method for evaluating the sensitivity or resistance to bacterial wilt can be used for the development of varieties resistant to bacterial wilt. Furthermore, screening for test substances can also lead to a reduction in damage caused by bacterial wilt.

  Furthermore, if the monitoring method of the present invention and the evaluation method and screening method applying the monitoring method are used, the pathogenic bacterium that infects the plant contains a gene encoding a marker substance. A monitoring method for various pathogens using the “pathogen having the property of introducing a foreign gene and producing a labeling substance”, and an evaluation method and a screening method using the same can be implemented.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

  The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (21)

  A plasmid comprising a phage φRSS1 genomic replication module capable of infecting bacterial wilt, comprising (a) the same nucleotide sequence as the sequence of the MCP promoter region described in SEQ ID NO: 2 derived from phage φRSB2, and A base sequence selected from a base sequence having one or several bases deleted, substituted, added, inserted, or a combination thereof, and having a mutation equivalent to the MCP promoter. A promoter region comprising: (b) a gene encoding a labeling substance downstream of the promoter region; and (c) a MCP according to SEQ ID NO: 3 derived from phage φRSB2 downstream of the gene encoding the labeling substance. The same nucleotide sequence as that of the terminator region and one or several bases deleted from or A terminator region having a base sequence selected from a base sequence having a sequence equivalent to that of an MCP terminator, the sequence of which has been mutated by addition or insertion or a combination thereof. Characteristic plasmid.   The replication module has the same base sequence as the 1st to 1720th and 5800 to 6135th base sequences of the phage φRSS1 genome set forth in SEQ ID NO: 1, or the sequence set forth in SEQ ID NO: 1. From 1 to 1720 and from 5800 to 6135 in the genome of phage φRSS1 from 1 to 6662, one or several bases are deleted, substituted, added or inserted, or combinations thereof, and the sequence is mutated. A generated base sequence comprising a base sequence having an action equivalent to that of the 1st to 1720th and 5800 to 6135th base sequences of the phage φRSS1 genome described in SEQ ID NO: 1 The plasmid according to claim 1, wherein   The plasmid according to claim 1 or 2, further comprising a gene encoding a selection marker.   The plasmid according to claim 3, wherein the gene encoding the selection marker is a kanamycin resistance gene.   The plasmid according to any one of claims 1 to 4, wherein the replication module has a site cleaved by a restriction enzyme, and the expression cassette is incorporated into the site.   The plasmid according to claim 5, wherein the site cleaved by the restriction enzyme is an EcoRI site.   The plasmid according to any one of claims 1 to 6, wherein the labeling substance is a chemiluminescent protein or a fluorescent protein.   The plasmid according to any one of claims 1 to 7, wherein the labeling substance is a fluorescent protein.   The plasmid according to any one of claims 1 to 8, wherein the labeling substance is green fluorescent protein (GFP).   The plasmid according to any one of claims 1 to 9, wherein the labeling substance is green fluorescent protein mut2 (GFPmut2).   A bacterial wilt that is transformed with the plasmid according to any one of claims 1 to 10.   A method for monitoring the growth, decrease or behavior of bacterial wilt fungi by observing the labeling substance expressed in the bacterial wilt fungus according to claim 11.   Observing the growth, reduction or behavior of bacterial wilt fungus in the plant body by infecting the plant with the bacterial wilt fungus according to claim 11 and observing the labeling substance expressed in the bacterial wilt fungus in the plant body. The monitoring method according to claim 12, which is characterized by:   14. The monitoring according to claim 13, wherein the observation of the growth, decrease or behavior of bacterial wilt fungus in the plant body is performed by observing the labeling substance in a cross section of a plant infected with bacterial wilt fungus. Method.   The monitoring method according to any one of claims 13 to 14 is used to evaluate the susceptibility or resistance to bacterial wilt fungi by plant varieties using as an index the growth, decrease or behavior of the bacterial wilt fungus. Evaluation method.   A test substance is given to a plant infected or diseased with the bacterial wilt fungus according to claim 11 and observed using the monitoring method according to any one of claims 13 to 14, thereby And screening for effective test substances.   It shall have the property of introducing a foreign gene including a gene encoding a labeling substance into a pathogen that infects the plant to produce the labeling substance, and the growth, decrease, or behavior of the pathogen in the plant body is observed, and the pathogen is infected. The cultured plant is cultured so that roots and / or stems extend along the surface of the agar medium inclined at an inclination angle with respect to the horizontal plane, and the labeling substance produced in the plant cultured on the surface of the agar medium is observed. A monitoring method characterized by being performed.   The monitoring method according to claim 17, wherein the inclination angle is 15 to 75 degrees with respect to a horizontal plane.   The monitoring method according to any one of claims 17 to 18, wherein the labeling substance is observed over time, and the growth, decrease or behavior of pathogenic bacteria is observed in real time.   Evaluation using the monitoring method according to any one of claims 17 to 19, wherein the susceptibility to or resistance to the pathogen by a plant variety is evaluated using the growth, decrease or behavior of the pathogen as an index. Method.   18. A test substance is given to a plant infected with or affected by a pathogen that has a property of introducing a foreign gene including a gene encoding a label into a pathogen that infects the plant, A screening method comprising screening a test substance effective against the pathogen by observing using the monitoring method according to any one of 19 items.