JPH07203989A - Method and apparatus for screening of pathogenicity of pathogenic microorganism and method and apparatus for screening of blight resistance of plant - Google Patents

Abstract

PURPOSE:To provide a process and apparatus capable of quickly, easily, objectively and accurately screening microorganisms by pathogenicity. CONSTITUTION:This screening apparatus is provided with a specimen positioning means 80 to position each of the 1st specimen and the 2nd specimen prepared by dividing a specimen of the plant to be examined into two parts, an inoculation means 50 to inoculate a specimen with the 1st pathogenic microorganism containing a transfer factor inserted into a genom and the other specimen with the 2nd pathogenic microorganism free from transfer factor inserted into a genom wherein the specimen positioning means 80 after the inoculation with the inoculation means 50 are positioned close to the inoculation means, a light detection means 10 to measure the intensities of faint luminescent rays emitted from the 1st and the 2nd specimens and a judging means 40 to judge the presence of pathogenicity of the pathogenic microorganism from the value measured by the light detection means 10.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for screening the pathogenicity of pathogenic microorganisms or screening for pathogenic resistance of plants and an apparatus therefor.

[0002]

2. Description of the Related Art In recent years, useful genes of plants have been used as useful clues for increasing productivity in areas with salt damage and in cold regions and for breeding high value-added crops containing high vitamins and minerals. The search for is widely done.
Among them, genetic information on disease caused by microorganisms and resistance of plants to it is extremely important as it directly affects productivity. Traditionally, pesticides have been used to control plant diseases caused by microorganisms, but the pesticides not only affect the pathogenic microorganisms but also the plants themselves, and cause problems such as residual pesticides on humans and the environment. The adverse effect is also a problem. In order to eliminate such problems, a method for controlling pathogenic microorganisms without adversely affecting plants and the environment is required. In order to meet such demands, studies on peptides that inhibit the metabolism of diseased microorganisms and studies on imparting resistance to diseased microorganisms to plants and enhancing resistance have been conducted.

Inhibition of metabolism of diseased microorganisms by such peptides and properties such as resistance to diseased microorganisms are one of physiological actions, and it is clear that gene products are involved in this. Therefore, it is extremely effective means from the standpoint of disease control to search for and utilize the genes involved in these physiological actions.

Therefore, from such a point of view, various ideas,
Although being carried out, a method of inactivating a gene using a transposon or a retrotransposon is drawing attention as one of the most effective methods. Here, the transposon is a gene unit that moves from one chromosome to another chromosome, and has a structural gene required to show resistance to antibiotics and a repeating sequence called an arm at both ends, It is randomly inserted into the host genome by the action of the repetitive sequence and a transposer (enzyme that acts on the recombination reaction when the transposon transfers).

Transposons such as transposons and retrotransposons have been found in plant pathogenic fungi, fungi and higher plants. Genes involved in pathogenicity to plants by inactivating specific genes using these transposons, There is a technology to search for genes that induce hypersensitive reactions in plants, genes involved in physiological activities of plants, etc. (Creation and utilization of mutant strains by transposon insertion)
Mamoru Sato Plant Protection Vol. 46, No. 11, p26-p29
1992: Recent research trends on pathogenic genes of plant pathogens
Shinji Rohno Plant Protection Vol.44 No.8 p372-p3
76 1990: Development and characterizain of generalized ge
ne tagging system for higherplant using an engineer
ed maize transposon Ac, WPFitzmaurice, LJLehma
n, LVNguyen, WFThomposon, EAWernsman, MAConkl
ing, Plant Mol Biol vol.20, p177 ~ p198,1991: The use of transgenic plants to understand trans
position mechanismsand to develop transposon taggi
ng strategies, MAHaring, CMRommens, HJNijkamp,
J. Hille, Plant Mol Biol vol.20, p177 to p198, 1991).
Some examples of such techniques are given below.

1) hrp of P. solanacearum, P. syringae, etc.
Technology related to genes (hypersensitivity reaction, virulence gene) (Ge
ne Cluster of Pseudomonas syrigae pv. “phaseolico
la ”Controls Patho-genicity of Bean Plants and Hyer
sensitivity on Nonhostplants, PBLindgren, RCPee
t, NJPanopoulos, J. Bacteriology p512 ~ p5221986: Development and characterizain of generalized ge
ne tagging system for higherplant using an enginee
red maize transposon Ac, WPFitzmaurice, LJLehma
n, LVNguyen, WFThomposon, EAWernsman, MAConkli
ng, Plant Mol Biol vol.20, p177-p198,1991) 2) Technology related to the avr gene of P. syringae pv. glycinea (Gene Cluster of Pseudomonas syrigae pv.
icola ”Controls Patho-genicity of BeanPlants and Hy
ersensitivity on Nonhost plants, PBLindgren, RCP
eet, NJ Nanopoulos, J. Bacteriology p512 ~ p522 1986
) 3) Cloning of thebronze locus in maize by a sim
ple and generalizable procedure using the transposa
ble controlling element Activator (Ac), NVFedorof
f, DBFurtek, OENelson Jr., Proc.Natl.Acad.Sci.USA
vol.81, pp3825 ~ 3929 1984: Molecular Analysis of viviparous-1; An Abscisic A
cid-Insensitive Mutantof Maize, DRMcCarty, CBCar
son, PSStinard, DSRobertson, The PlantCell vol 1,
pp523 ~ 532 1989: Cloned avirulence gene of Psudomonas syringae p
v.glycinea determines race-specific incompatibility
on Glycine max (L.) Merr.BJStaskawicz, D.Dahlbec
k, NTKeen, Proc.Natl.Acad.Sci.USA vol 81, pp6024-6
028 1984) In order to screen for the presence of a pathogen-related gene in the genome in which the transposable element has been inserted, it is necessary to confirm whether or not the pathogenicity of the pathogenic microorganism into which the transposable element has been introduced has disappeared. Also, for resistant plants, it is necessary to confirm whether or not the plants have resistance to pathogens. Conventionally, as a means for confirming this, a method of inoculating a plant with a pathogenic microorganism into which a transposable factor has been introduced, and using the presence or absence of a symptom as a morphological change as an index has been used. Similar methods have been used to screen for changes. That is, in the screening method according to the prior art, it was necessary to actually grow the plant to be inspected, inoculate the plant with the pathogenic microorganism to be screened, and perform a screening test to screen the disease symptoms.

[0007]

However, the conventional screening methods as described above have the following problems.

1) First, the first problem is that it takes a long time to obtain a result. With conventional screening methods, depending on the type of plant, the process from inoculation with pathogenic microorganisms to screening for the presence or absence of symptoms may take as long as one week, and may take several months if delayed. Needed a period.

2) The second problem is that in the screening of pathogenicity, statistical processing must be carried out in order to eliminate the effect of individual differences in plants, and for this reason, hundreds to thousands of samples are tested. Since it is necessary to conduct a screening test with
The point is that a vast site is needed to grow these specimens.

Therefore, an object of the present invention is to provide a method for screening the pathogenicity of pathogenic microorganisms or a method for screening disease resistance of plants and an apparatus therefor which solve the above problems.

[0011]

In order to solve the above-mentioned problems, the method according to the present invention is a method for screening the pathogenicity of a pathogenic microorganism in which a transposable element has been inserted in the genome. The sample is divided into at least two, and the first segment is inoculated with the first pathogenic microorganism in which the transposable element is inserted in the genome, and the amount of faint luminescence from the sample of the test plant is measured. The step and the other section of the divided test plant sample are inoculated with the second pathogenic microorganism in which the transposable element is not inserted in the genome, and the amount of weak luminescence from the test plant sample is measured. The second step and the first
And a third step of screening the pathogenicity of the pathogenic microorganism by comparing the amounts of weak luminescence measured in the second step and the second step, respectively.

In order to solve the above problems, the method according to the present invention is a method for screening the pathogenicity of a pathogenic microorganism in which a transposable element is inserted in the genome. The first pathogenic microorganism having a transposable element inserted into the genome is inoculated into one of the two sections, and the amount of faint luminescence from the sample of the test plant is measured in time series. The step and the other section of the divided sample of the test plant are inoculated with the second pathogenic microorganism in which the transposable element is not inserted in the genome, and the amount of weak luminescence from the sample of the test plant is calculated in time series. The second step of quantitatively measuring, and the third step of screening the pathogenicity of pathogenic microorganisms by comparing the amounts of weak luminescence measured in the first and second steps for each time. Special to prepare To.

In order to solve the above-mentioned problems, the apparatus according to the present invention is a screening apparatus for pathogenicity of pathogenic microorganisms in which a transposable element is inserted in the genome. Sample placement means for placing each of the sample and the other sample, and inoculating one sample with the first pathogenic microorganism having the transposable element inserted in the genome, and inserting the transposable element into the other sample The inoculation means for inoculating the second pathogenic microorganism that has not been inoculated and the sample arranging means after inoculation by the inoculation means are provided so as to face each, and each of the amounts of weak luminescence from one and the other sample is measured. It is characterized by comprising a light detecting means and a judging means for judging the presence or absence of pathogenicity of a pathogenic microorganism from a measurement value of the light detecting means.

Further, it is desirable that the screening apparatus for pathogenicity of pathogenic microorganisms record the measurement values of the light detecting means in time series and give the determination means the measurement values recorded in time series. .

In order to solve the above-mentioned problems, the method according to the present invention is a method for screening a disease resistance of a plant having a transposable element inserted in its genome, which is a test plant having a transposable element inserted in its genome. Inoculation with a pathogenic microorganism to measure the amount of faint luminescence from the test plant, and inoculation of the pathogenic microorganism into the test plant in which the transposable element is not inserted in the genome, The second step of measuring the amount of faint luminescence from the test plant, and the amount of faint luminescence measured in the first and second steps are compared to screen the disease resistance of the test plant. And 3 steps.

In order to solve the above-mentioned problems, the method according to the present invention is a method for screening a disease resistance of a plant in which a transposable element is inserted into the genome, wherein the transposable element is inserted into the genome. The first step of inoculating a test plant with a pathogenic microorganism and measuring the amount of faint luminescence from the test plant in time series, and the pathogenic microorganism to the test plant in which the transposable element is not inserted in the genome The second step of inoculating and measuring the amount of weak light emission from the test plant in time series is compared with the amount of weak light emission measured in each of the first and second steps in time series. Accordingly, a third step of screening disease resistance of the test plant is provided.

In order to solve the above-mentioned problems, the device according to the present invention is a plant disease resistance screening device in which a transposable element is inserted in the genome, wherein Test plant arranging means for arranging each of the test plant and a second test plant in which a transposable element is not inserted in the genome, and an inoculating means for inoculating the first and second test plants with a pathogenic microorganism Provided so as to face the sample placement means after being inoculated by the inoculation means,
A light detecting means for measuring each of the amounts of weak luminescence from one and the other sample, and a determining means for determining the presence or absence of pathogenic resistance of the test plant from the measurement value of the light detecting means, To do.

Further, it is desirable that the above-mentioned plant disease resistance screening apparatus record the measured values of the light detecting means in time series and provide the judging means with the measured values recorded in time series. .

[0019]

According to the method described in claim 1, the prepared sample of the test plant is divided into at least two, and the first pathogenic microorganism having a transposable element inserted into the genome is divided into one division. Inoculate and inoculate the other section with a second pathogenic microorganism that has no transposable element inserted in the genome. Therefore, depending on the degree of interaction between the plant and the pathogenic microorganism, the amount of extremely weak luminescence differs between the 1st section of the sample and the other section, and the amount of weak luminescence from these test plants is measured. Then, since the amounts of weak luminescence measured respectively are compared with each other, it is possible to easily screen for pathogenic microorganisms in a short time and objectively.

According to the method described in claim 2, in addition to the method described in claim 1, the amount of faint luminescence from the test plant is further measured in a time series, and each is measured. Since the amounts of weak luminescence are compared for each time period, it becomes possible to easily and objectively screen for pathogenic microorganisms based on temporal changes that cannot be obtained from the amount of weak luminescence alone.

According to the above-mentioned apparatus, the sample arrangement means for arranging one sample and the other sample obtained by dividing the sample of the test plant into two parts, and one sample in the genome. Inoculation means for inoculating the first pathogenic microorganism in which the transposable element is inserted and inoculating the second sample in which the second pathogenic microorganism in which the transposable element is not inserted in the genome is inoculated, and weakness from one and the other sample Since it is equipped with a light detecting means for measuring each of the amounts of luminescence and a judging means for judging the presence or absence of pathogenicity of a pathogenic microorganism from the measured value of the light detecting means, it is said that the transposable element is inserted in the genome. It is possible to easily measure the weak light emission of both the non-inserted object in a short time, and it is possible to perform objective and accurate screening.

According to the method of the above-mentioned claim 5, the pathogenic microorganism is inoculated to both the test plant in which the transposable element is inserted in the genome and the test plant in which the transposable element is not inserted in the genome. . Therefore, depending on the degree of interaction between the plant and the pathogenic microorganism, the amount of extremely weak light emission is different between the test plant in which the transposable element is inserted in the genome and the test plant in which the transposable element is not inserted in the genome. It will be different, since the amount of weak luminescence from these test plants is measured and the amounts of weak luminescence measured respectively are compared, it is possible to easily screen for pathogenic microorganisms in a short time. It can be done objectively.

According to the method described in claim 6, in addition to the method described in claim 5, the amount of faint luminescence from the test plant is further measured in a time series, and each is measured. Since the amount of faint luminescence is compared for each time, it is possible to easily and objectively screen disease resistance of plants based on temporal changes that cannot be obtained from the amount of faint luminescence alone. Become.

According to the apparatus of claim 7, the first test plant having a transposable element inserted into the genome and the second test plant having no transposable element inserted into the genome are respectively used. An arrangement means for arranging a test plant, an inoculation means for inoculating a first and a second test plant with a pathogenic microorganism, a light detecting means for measuring the amount of weak luminescence from one and the other sample, respectively, Since it is provided with a determination means for determining the presence or absence of pathogenic resistance of the test plant from the measurement value of the detection means, the weak luminescence of both the transposable element inserted and the one not inserted in the genome. The measurement can be easily performed in a short time, and the objective and accurate screening can be performed.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Prior to the description of specific embodiments, the principle of the present invention will be briefly described. The present inventors have found that when a pathogenic microorganism invades a plant, luminescence is observed as a result of the plant actively performing a protective reaction. In particular,
When the interaction between the plant and the pathogen is large, it is recognized that there is a point (hereinafter, referred to as “peak”) where the amount of luminescence specifically changes. On the other hand, no peak is observed when the interaction between the plant and the pathogen is small. Further, as described above, a technique is known in which a transposable element is inserted into the genome to inactivate a part of the gene of the pathogenic microorganism to eliminate the pathogenicity of the pathogenic microorganism.

Therefore, the plant solid is physically divided into two,
Alternatively, the same plant is divided into two groups, one is inoculated with a pathogenic microorganism having a transposable element inserted therein, and the other is inoculated with a pathogenic microorganism not having a transposable element inserted. There may or may not be a peak at. According to the research and examination by the present inventor,
It was found that the case where the peak of the above-mentioned luminescence amount did not appear was the case where the pathogenicity of the pathogenic microorganism disappeared, and the case where the peak appeared appeared when the pathogenicity of the pathogenic microorganism did not disappear.

Therefore, according to this principle, the pathogenicity of pathogenic microorganisms can be screened.

Embodiments of the present invention will be described below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

A screening apparatus for pathogenicity of pathogenic microorganisms according to the first embodiment of the present invention will be described with reference to FIG.

This screening apparatus includes a weak photodetector 20 including a two-dimensional detector 10 (for example, a VIM camera manufactured by Hamamatsu Photonics KK), and the two-dimensional detector 1.
Image processor 30 connected to 0, a computer 40 having a function as a controller for controlling the image processor 30 and the two-dimensional detector, and determining the presence or absence of pathogenicity of pathogenic microorganisms, and via the image processor 30 And a monitor 90 for visually displaying the signal sent from the two-dimensional detector 10.

The system is not limited to this, and various systems can be used as long as the device can perform image analysis from the information detected by the two-dimensional detector. An example of such a system is ARGUS-100 manufactured by Hamamatsu Photonics KK
/ VIM etc.

The faint light detector 20 is, as shown in FIG.
The case 21, the two-dimensional detector 10 fixed in a state of penetrating the upper surface of the case 21, the inoculation device transfer mechanism provided at a predetermined position on the upper left side of the case 21, and the inside of the case 21. Two fixed stages 100a, 100b provided on the bottom surface of the
And a sample transport mechanism provided on the bottom surface inside the housing 21. The two-dimensional detector 10 is provided with the detecting portion facing downward. Also, two fixed stages 10
A microtiter plate 110 for culturing microorganisms is placed on one of 0a and 100b, and a heat block 120 for sterilization is provided on the other. These two fixed stages 100a and 100b are arranged along the moving direction of the inoculation device 50. When the inoculation device 50 moves to the rightmost side of the drawing, the slide body 61
These are arranged so that the inoculation device 50 and the two-dimensional detector 10 are lined up in the moving direction of. The two-dimensional detector 10 can detect even a small amount of light, and a photon-counting imaging device is used.
The photon counting imaging means capturing light as a minimum unit, a photon, and imaging.

The casing 21 is made of a material that blocks any light from the outside. On the left side surface of the housing 21, there is provided a door (not shown) that can be opened and closed for loading and unloading the sample. A seal is applied to the gap between the door and the housing 21,
In the closed state, no external light is incident on the structure.

The inoculation device 50 has a plurality of liquid inoculation pipes (not shown) and an inoculation amount adjusting device (not shown).
This inoculation amount adjusting device is controlled by the electronic computer 40. A plurality of liquid inoculation tubes provided in the inoculation device 50 are provided corresponding to the respective accommodating portions of the microtiter plate, and a sterilizing tip is attached to the tip thereof. The inoculation device transfer mechanism has a support member 51 that holds the inoculation device 50 and can be moved in the vertical direction, a motor (not shown) that moves the inoculation device 50 in the horizontal direction, and a gear mechanism (not shown). . The motor (not shown) and the gear mechanism (not shown) are provided in the housing 52.
It is housed inside. The motor is controlled by the electronic computer 40.

The sample transport mechanism comprises a slide body 61.
A feed screw 62 for horizontally moving the slide body 61, a support rod 63 penetrating both ends of the slide body 61 to support the slide body 61, and a feed screw 6
It has a motor (not shown) for rotating 2 and a gear mechanism (not shown). A motor (not shown) and a gear mechanism (not shown) are housed in the housing 22, and the housing 22 also has a role of supporting the feed screw 62 and the support rod 63. The motor is controlled by the electronic computer 40. A stage 70 is fixed on the upper surface of the slide body 61.
The upper part of the is a sample placement part, and a microtiter plate 80 for accommodating plant pieces is placed on the sample placement part.

The image processor 30 performs a process of imaging the photons detected by the two-dimensional detector 10. The image processor 30 can also record the information from the two-dimensional detector 10 in time series.

For the electronic computer 40, for example, a desktop personal computer is used. Here, for example, NEC's PC-9800 series is used.

In this embodiment, the two-dimensional detector 10 is used.
As the device, other means may be used as long as it is a device capable of highly sensitive light detection in the visible region or the near infrared region. Other means include, for example, a high-sensitivity detection element such as an image intensifier. Furthermore, the two-dimensional detector 10
It is also possible to provide an optical fiber directly under the photocathode and intervene this between the sample to be measured and transmit the light emitted from the sample to the two-dimensional detector 10 through the optical fiber.

Next, the screening method for pathogenicity of pathogenic microorganisms according to the first embodiment of the present invention will be described with reference to the flowchart of FIG.

In carrying out this screening, it is necessary to inactivate pathogenic microorganisms in advance and adjust the test plant to a sterile state. Therefore, before describing such a method, these points will be described.

1) Inactivation of Pathogenic Microorganisms In this example, as a pathogenic microorganism, Peseudomaonas mucu
licola was used, and Tn-5 was used as a transposon for inactivating it. Pese
udomaonas muculicola is a gram-negative bacterium that parasitizes Japanese radish and causes the disease of causing brown spots on the leaves of Japanese radish due to the infestation of this bacterium.
Tn-5 is a transposon having a gene encoding an enzyme that detoxifies the antibiotic kanamycin, and having repeat sequences at both ends thereof. Peseudomao
Regarding the method of introducing Tn-5 into nas muculicola,
Method by Sato et al. (Creation and use of mutant strain by transposon insertion Mamoru Sato Plant Protection Vol. 46, No. 11)
p26 to p29 1992), and a brief description of this outline will be given below.

First, Peseudomaonas muculicola was subjected to liquid shaking culture in a peptone medium at 26 ° C. for 24 hours. Escherichia coli having Tn-5 was subjected to liquid shaking culture in LB medium at 37 ° C. for 24 hours.

Next, the concentration of the medium of Peseudomaonas muculicola and the medium of Escherichia coli having Tn-5 were both 10 ⁵
The concentration was adjusted to -10 ⁹ / ml.

Next, a sterilized filter was placed on the plate medium, both culture solutions were placed on the filter in appropriate amounts, and stirred with a Pipetman.

Next, the plate medium prepared above was transferred to an incubator at 28 ° C. and cultured for about one week.

In this way, zygote colonies appear on the plate medium. At this time, culture is carried out in a minimal medium in order to remove the kanamycin-resistant E. coli remaining unconjugated.

As described above, Peseudomaonas muculico
A pathogenic microorganism in which Tn-5 is introduced into la can be obtained. In the present embodiment, the adjustment is made as
Peseudomaonas muculicola introduced with n-5 is referred to as "mutant strain", and Peseudomaonas muculicola which is not viable and maintains pathogenicity is referred to as "wild strain".

2) Preparation of Pathogenic Microorganisms When the wild strain and the mutant strain obtained above were set on the microtiter plate 110 for microbial culture of the weak light detector 20, the following adjustments were made. First, the mutant strain is shake-cultured overnight at 26 ° C. in a peptone medium containing 50 μg / ml of kanamycin to prepare a mutant strain culture solution.

Next, the mutant culture fluids were collected by centrifugation to obtain 10 mM potassium phosphate buffer (pH
Wash twice with 7.0).

Next, the washed mutant strain is adjusted to a concentration of about OD = 3 to prepare a mutant strain solution.

The wild strain is also prepared by the same procedure as the mutant strain to obtain a wild strain solution.

3) Preparation of test plant In this example, radish was used as the test plant. Since it was decided to use a commercially available radish, the following treatments were performed to adjust this to a sterile state.

First, 70% ethanol solution was sprayed on the surface of the radish to disinfect it.

Next, the radish was sliced into appropriate sizes and immersed in a 1% sodium hypochlorite solution for 15 minutes to sterilize the surface of the radish.

Next, the surface of the radish was washed with sterilized water to wash away sodium hypochlorite.

Next, this radish was cut out vertically to a thickness of about 1 cm, and the center portion was cut out on a disc having a diameter of 1 cm with a sterilized cork borer.

Next, absorbent cotton impregnated with sterilized water was placed on a petri dish or a microtiter plate, and the adjusted radish was placed thereon to prepare a sample.

Next, a method for screening the pathogenicity of pathogenic microorganisms according to the first embodiment of the present invention will be described.

First, the radish pieces to be screened are housed in each of a plurality of housing parts provided in the microtiter plate 80 prepared in advance (step 101).

Next, the microtiter plate 80 accommodating these radish pieces is placed in the sample placement portion above the stage 70 provided in the weak light detector 20 (step 102).

Next, in each of the plurality of accommodating portions provided in the microtiter plate 110 for culturing microorganisms,
The mutant strain liquid and the wild strain liquid adjusted as described above are injected (step 103).

Next, the liquids (mutant strain liquid and wild strain liquid) contained in the respective containing parts of the microtiter plate 110 for culturing microorganisms are converted into the radish pieces contained in the respective containing parts of the microtiter plate 80. About 100 for each
Inoculate each μl (step 104). The inoculation device 50 inoculates each piece of radish. In addition to the method of separating the one containing the mutant strain liquid and the one containing the wild strain liquid in each housing unit of one microtiter plate as in this example, the mutant strain is housed in each microtiter plate. It may be divided into those that contain wild strains and those that contain wild strains.

Next, the feed screw 62 of the sample transport mechanism is rotated to move the slide body 61 located at the upper left of FIG. 1 to the lower right, and the microtiter plate 80 arranged on the stage 70 is moved to the second position. It is set so as to be directly below the dimension detector 10 (step 105). This movement is performed by a command from the electronic computer 40.

Next, a shutter (not shown) of the housing 22 is opened in response to a command from the electronic computer 40, and the two-dimensional detector 10 detects weak light from each sample 80 (step 106).

Next, the output signal from the two-dimensional detector 10 is sent to the electronic computer 40, and the electronic computer 40 compares the recorded data to screen the pathogenicity of the disease microorganism (step 107). . This screening is performed as follows. First, when measured as described above, the amount of luminescence changes with time, but if the peak of luminescence does not appear, it means that the pathogenicity of the pathogenic microorganism has disappeared, and if the peak appears, the pathogenic microorganism The program is entered in the electronic computer 40 so that it is judged that the case has not disappeared, and the result is displayed on the monitor. The captured image on the monitor at this time is shown in FIG.

Specifically, in this example, the results shown in FIGS. 3 and 4 were obtained. As shown in these figures, it was confirmed that the difference in the amount of luminescence was obviously different between the wild strain and the mutant strain. That is, FIG.
As shown in (a), it can be seen that the wild strain 2 has a remarkably large amount of luminescence as compared with the mutant strain 3 and the reference control 1. 4 (a) and 4 (b) are a photon-counting image of the same object taken from the same position and a normal image by white light illumination. Of these, FIG. 4 (a) is a photon-counting imaged image. 4B is a normal image captured by white light illumination. In particular, as shown in FIG. 3, two peaks can be read in the wild strain, whereas no peaks are present in the mutant strain, and the first 5
It can be seen that although the amount of light emission sharply decreases with time, it shows a smooth transition thereafter. This result is the result of studies and studies by the present inventors, that is, when the peak of luminescence does not appear, the pathogenicity of the pathogenic microorganism disappears, and when the peak appears, the pathogenicity of the pathogenic microorganism disappears. This is consistent with the conclusion that it is not. Therefore, using the apparatus and method according to the present embodiment,
It can be said that screening for pathogenic microorganisms can be performed in a relatively short time and with a small number of samples.

For reference, the integrated value at the peak of the wild strain read from the graph shown in FIG. 3, that is, the integrated luminescence amount of each of the wild strain and the mutant strain in 1.5 hours to 2.5 hours. 5 and the integrated value of the luminescence amount of each of the wild strain and the mutant strain in 7 to 8 hours.
Shown in. From these results, it is also possible to examine the degree to which a gene is involved in disease.

Furthermore, the following contents can be read from these results. That is, of the peaks observed in the wild strain, the first peak that appears about 1 hour after inoculation corresponds to the hypersensitivity reaction, which is the first-step defense reaction of radish against Peseudomaonas muculicola, and then It can be read that the second peak that appears after about 7 hours corresponds to the hypersensitivity reaction, which is the second-step defense reaction of radish against the full-scale invasion of Peseudomaonas muculicola. That is, from these results, information on strengthening or weakening of pathogenicity can be obtained.

However, in a plant defense reaction such as a hypersensitivity reaction, there are cases in which the amount of change is large depending on the type of plant and the site of the plant body, and cases where it is not. This is because there are cells that are hypersensitive to pathogenic bacteria and cells that are not very reactive to pathogenic bacteria. The Japanese radish used as a sample this time also has such properties, and in some of the experiments conducted several times, the results shown in Fig. 6 were also shown. For reference, the integrated value at the peak of the wild strain read from the graph shown in FIG. 6, that is, the integrated value of the luminescence amount of each of the wild strain and the mutant strain in 1.5 hours to 2.5 hours, FIG. 7 shows the integrated value of the luminescence amount of each of the wild strain and the mutant strain during 7 hours to 8 hours.

As can be seen from FIG. 6, the second peak of the luminescence amount derived from the defense reaction can be read in the same manner as the result shown in FIG. 3 in the sample which does not react much to the pathogenic bacteria. However, the first peak is not clear as compared with the case of FIG. However, even in such a case, it can be confirmed that the difference in the amount of luminescence is obviously different between the wild strain and the mutant strain, and therefore it is still possible to screen for the pathogenic microorganism.

In the plant defense reaction, when the amount of change is large depending on the site of the plant body, it can be solved by the following means.

1) The first is a method of selecting cells sensitive to pathogenic bacteria, and 2) the second is a method of homogenizing plant tissues. Among them, the first method is technically difficult, while the second method is relatively easy. The second method will be explained in detail. First, the plant tissue used as a sample is cut to an appropriate size and mixed, or the plant tissue is treated with an appropriate enzyme to produce individual cells (protoplastization).
The sample can be homogenized by homogenizing the cells and homogenizing the cells.

Next, the plant disease resistance screening method according to the second embodiment of the present invention will be described. Here, the maize transposable element has long been known,
It is known that this factor works not only in corn but also in plants such as tobacco and rice. Therefore, in the present embodiment, description will be given by taking a cigarette as an example (reference: Phenot
ypic assay for excision of the maize controlling el
ement Ac in tabacco B.Baker, G.Coupland, N.Fedorof
f, P.Starlinger, J.Schell, EMBO J. vol 6, pp1547-1554
(1987)).

The apparatus used for screening at this time may be the same as the above-mentioned apparatus for screening pathogenicity of pathogenic microorganisms, that is, the apparatus shown in FIG. 1, and in this embodiment, the apparatus shown in FIG. It was decided to perform screening using.

First, Agrobacterium having a plasmid into which NPTII (antibiotic kanamycin resistance gene) and a maize self-sustaining transposable Ac are incorporated so that it can be expressed in the T-DNA region is prepared.

Then, this Agrobacterium is liquid-cultured in a YBE medium, and the culture solution and tobacco protoplasts or tissue pieces (hereinafter referred to as "tobacco protoplasts") are co-cultured. This culture is continued for 3 to 4 days, and T-D
28 ° C until the NA region is integrated into the tobacco genome,
It is carried out under the condition of 16 hours.

Next, the cultivated tobacco protoplasts and the like are transferred to a medium containing clafon to eradicate Agrobacterium.

Next, the tobacco protoplasts and the like are transferred to a medium containing kanamycin, and tobacco protoplasts and the like in which the kanamycin resistance gene is integrated in the tobacco genome are selected. Then, if necessary, the cells are proliferated by a commonly used method such as a callus forming method.

Next, the tobacco protoplasts or the like to be screened are stored in each of a plurality of storage portions provided in the microtiter plate 80 prepared in advance. A microtiter plate 80 accommodating these tobacco protoplasts and the like is placed in the sample placement portion above the stage 70 provided in the weak light detector 20. Also, a microtiter plate 11 for culturing microorganisms
An aqueous solution of a pathogenic bacterium containing a filamentous fungus or a bacterium to be assayed is injected into each of the plurality of accommodating portions provided in 0.

Next, the pathogen-containing aqueous solution contained in each housing of the microtiter plate 110 for culturing microorganisms is
Approximately 100 μl of each of the tobacco protoplasts and the like stored in the respective storage portions of the microtiter plate 80
Inoculate each one. The inoculation device 50 inoculates tobacco protoplasts and the like.

Next, the feed screw 62 of the sample transport mechanism is rotated to move the slide body 61 located at the upper left of FIG. 1 to the lower right, and the microtiter plate 80 arranged on the stage 70 is moved to the second position. It is set so as to be directly below the dimension detector 10. This movement is performed by a command from the electronic computer 40.

Next, a shutter (not shown) of the housing 22 is opened by a command from the electronic computer 40, and the two-dimensional detector 10 detects weak light from each accommodating portion of the microtiter plate 80.

Next, the same operations as in steps 205 to 208 are performed on tobacco protoplasts and the like in which the transposable element is not incorporated.

Next, the output signal from the two-dimensional detector 10 is sent to the electronic computer 40, and the electronic computer 40 analyzes the recorded data to screen disease resistance.

After determining the presence or absence of disease resistance in this way, it is also possible to clone only the genes involved in disease resistance using the DNA of the test plant that has lost the disease resistance. Also, in the present embodiment, the first
From the results obtained as in the examples, information on the resistance of the plant body to pathogenic microorganisms can be obtained.

As the maize transposable factor, not only the Ac shown in this Example but several other ones have been discovered. For example, a combination of Ds (non-autonomous transposable factor) and Ac is used. You may use. Furthermore, for monocotyledonous plants, the screening method shown in this example can be carried out by using an appropriate vector and a gene transfer method such as particle gun.

With regard to filamentous fungi, a retrotransposon-like factor of blast fungus has been found, and similar effects can be obtained by combining this factor with the transformation method by particle gun and the screening method shown in this Example. be able to.

[0088]

As described in detail above, according to the screening method of the present invention, the amount of extremely weak luminescence is different between 1 and other sections of the sample depending on the degree of interaction between the plant and the pathogenic microorganism. Since the amount of faint luminescence from these test plants is measured and the measured faint luminescence amounts are compared with each other, the screening of pathogenicity of pathogenic microorganisms or screening of disease resistance of plants is performed. Can be performed easily in a short time and can be performed objectively. In addition, by measuring the amount of faint luminescence from the test plant in time series and comparing the measured amounts of faint luminescence for each time, it is possible to obtain a temporal value that cannot be obtained only from the amount of faint luminescence. It becomes possible to easily and objectively screen for pathogenic microorganisms based on changes or to screen disease resistance of plants. Therefore, a bacterial clone in which a disease-related gene has been inactivated can be selected very rapidly using a transposon or the like.

Further, the amount of weak luminescence from the test plant is measured and the measured amounts of weak luminescence are compared with each other, or the amount of weak luminescence from the test plant is measured in time series. , Since the amounts of weak luminescence measured respectively are compared at each time, not only the screening of pathogenicity of pathogenic microorganisms or the screening of disease resistance of plants, but also information on the enhancement or weakening of pathogenicity of pathogenic microorganisms,
It is possible to obtain information on the resistance of plants to pathogenic microorganisms.

Furthermore, since the amount of faint luminescence from the test plant is measured, the degree of disease can be quantitatively evaluated, and the degree of involvement of a gene in the disease or the resistance of the disease to the plant can be evaluated. The strength of can also be tested together.

[Brief description of drawings]

FIG. 1 is a perspective view showing a screening apparatus according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram showing a flowchart of the first embodiment of the present invention.

FIG. 3 is a graph showing the change over time in the light emission amount of the Japanese radish in the first example.

FIG. 4 is an image captured when a Japanese radish is imaged in the first embodiment.

FIG. 5 is an explanatory diagram showing an integrated value of light emission amounts of Japanese radish in a predetermined time in the first embodiment.

FIG. 6 is a graph showing a change over time in the light emission amount of another Japanese radish in the first example.

FIG. 7 is an explanatory diagram showing an integrated value of light emission amounts of other radish in a predetermined time in the first embodiment.

[Explanation of symbols]

10 ... Two-dimensional detector, 20 ... Weak light detector, 30 ... Image processor, 40 ... Computer, 50 ... Inoculation device,
80 ... Microtiter plate, 90 ... Monitor, 11
0 ... Microtiter plate for microbial culture

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12N 5/04

Claims (8)

[Claims] 1. A method for screening the pathogenicity of a pathogenic microorganism in which a transposable element has been inserted into the genome, wherein the prepared sample of a test plant is divided into at least two samples, and 1
In the first step, inoculating the first pathogenic microorganism having a transposable element inserted in the genome, and measuring the amount of faint luminescence from the sample of the test plant; The second step of inoculating the other section of the sample of No. 2 with the second pathogenic microorganism in which the transposable element is not inserted in the genome, and measuring the amount of faint luminescence from the sample of the test plant, And a third step of screening the pathogenicity of the pathogenic microorganism by comparing the amounts of the weak luminescence measured in the first step and the second step, respectively. Method. 2. A method for screening pathogenicity of a pathogenic microorganism having a transposable element inserted in its genome, wherein the prepared sample of the test plant is divided into at least two samples, and
The first step of inoculating the first pathogenic microorganism having a transposable element inserted in the genome into the section of No. 1, and measuring the amount of faint luminescence from the sample of the test plant in time series; A second pathogenic microorganism in which a transposable element is not inserted in the genome is inoculated into another section of the sample of the test plant, and the amount of weak luminescence from the sample of the test plant is measured in time series. A second step; and a third step of screening the pathogenicity of the pathogenic microorganism by comparing the amounts of the weak luminescence measured in the first and second steps for each time period. A method for screening the pathogenicity of a pathogenic microorganism, which comprises: 3. A device for screening pathogenicity of a pathogenic microorganism in which a transposable element is inserted in the genome, wherein a sample arranging means for arranging one sample and another sample obtained by dividing a sample of a test plant into two parts. And a first transposable element inserted into the genome of the one sample
Inoculating the second sample with a second pathogenic microorganism in which the transposable element is not inserted in the genome, and the sample arranging means after being inoculated by the inoculating means. Light detecting means provided so as to face each one of the one and the other sample measuring the amount of weak luminescence, and a determining means for determining the presence or absence of pathogenicity of the pathogenic microorganism from the measurement value of the light detecting means. A screening device for pathogenicity of pathogenic microorganisms, comprising: 4. The recording means for recording the measurement values of the light detecting means in time series and giving the measurement values recorded in time series to the judging means. A screening device for pathogenicity of the described pathogenic microorganism. 5. A method for screening a disease resistance of a plant having a transposable element inserted in its genome, comprising inoculating a test plant having a transposable element inserted in its genome with a pathogenic microorganism, The first step of measuring the amount of faint luminescence, and the second step of measuring the amount of faint luminescence from the plant to be inoculated with a pathogenic microorganism into a test plant in which the transposable element is not inserted in the genome And a third step of screening the disease resistance of the test plant by comparing the amounts of the faint luminescence measured in the first and second steps, respectively. For screening disease resistance of plants. 6. A method for screening a disease resistance of a plant having a transposable element inserted into its genome, comprising inoculating a test plant having a transposable element inserted into its genome with a pathogenic microorganism, The first step is to measure the amount of faint luminescence in time series, and the pathogenic microorganism is inoculated into the test plant in which the transposable element is not inserted in the genome, and the amount of faint luminescence from the test plant is measured. Screening the disease resistance of the test plant by comparing the amount of the weak luminescence measured in each of the first step and the second step in time series with the second step of measuring in series A method for screening disease resistance of a plant, comprising a third step. 7. A screening device for disease resistance of a plant having a transposable element inserted into its genome, wherein the first test plant having the transposable element inserted into the genome and the transposable element not inserted into the genome Test plant arranging means for arranging each of the second test plants, inoculating means for inoculating the first and second test plants with a pathogenic microorganism, and the sample arranging after being inoculated by the inoculating means Means provided respectively facing each other, the light detection means for measuring each of the amount of weak luminescence from the one and the other sample, and the presence or absence of pathogenic resistance of the test plant from the measurement value of the light detection means. A screening device for disease resistance of a plant, comprising: a judging means for judging. 8. The recording means for recording the measurement values of the light detecting means in time series, and for giving the measurement values recorded in time series to the judging means. The plant disease resistance screening apparatus described.