Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice

A technology of blast fungus and effector protein, which is applied in vitro to detect the translocation of blast fungus effector protein in rice root tissue, and achieves the effect of simple method

Inactive Publication Date: 2012-10-24
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, there is no direct in vitro detection of the prokaryotic expression product of the rice blast fungus effector gene that can autonomously transport into the rice root tissue without the presence of pathogenic bacteria, so as to quickly identify whether the rice blast fungus effector gene can be transported autonomously like the oomycete effector Report on the method of entry into host cells

Method used

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  • Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice
  • Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice
  • Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice

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Experimental program
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Effect test

Embodiment 1

[0015] 5.0mg / ml of GFP fusion protein was treated at 0.5cm of rice root tip for 12h, rinsed with sterilized water for 4h, and the transport of GFP fusion protein in rice root tissue was directly observed by confocal microscope.

[0016] The specific method is:

[0017] The expression product of the rice blast fungus effector protein gene (the process of obtaining the expression product by using the method of IPTG chemical induction will not be repeated) was centrifuged at 4°C to collect the bacterial cells, and the freshly prepared buffer solution (20mM Tris-HCl, pH7.4 ; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol) suspended cells. The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The prokaryotic expression product was purified using a GST purification column (purchased from Amersia Company), and the purified expression product was treated with the root of the susceptible rice variety Lijiang Xintuan Heigu at a c...

Embodiment 2

[0021] 5.0mg / ml of GFP fusion protein was treated at 0.5cm of rice root tip for 24h, rinsed with sterilized water for 4h, and the transport of GFP fusion protein in rice root tissue was directly observed by confocal microscope.

[0022] Centrifuge the expression product of the effector protein gene of Magnaporthe grisea to collect the cells, and suspend the cells with freshly prepared buffer (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol) . The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The treatment is to treat the root tissue of the susceptible rice variety Lijiang Xintuan Heigu at a concentration of 5.0mg / ml (including 25mM MES) after purification of the prokaryotic expression product of the gene for 24 hours, rinse with sterilized water for 4 hours, and observe the effector protein with a confocal microscope Gene transport in rice root tissue. As a control, after the prokaryotic expres...

Embodiment 3

[0026] The GFP fusion protein of 5.0 mg / ml was treated at the root tip of rice at 0.5 cm for 24 hours, rinsed with sterilized water for 4 hours, and the translocation of GFP fusion protein in rice root tissue was directly observed by confocal microscopy.

[0027] Centrifuge the expression product of the effector protein gene of Magnaporthe grisea to collect the cells, and suspend the cells with freshly prepared buffer (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol) . The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The treatment is to treat the root tissue of the susceptible rice variety Lijiang Xintuan Heigu at a concentration of 5.0mg / ml (including 25mM MES) after purification of the prokaryotic expression product of the gene for 36 hours, rinse with sterilized water for 4 hours, and observe the effector protein with a confocal microscope Gene transport in rice root tissue. As a control, a...

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Abstract

The invention discloses a method for in vitro detection of the transport of Magnaporthe grisea effector proteins in root tissues of rice. The transport of GFP fused proteins in the root tissues of rice is directly observed through a confocal microscope after disease-resistant rice cultivar Lijiangxintuanheigu root tips of 0.5cm are processed with 5.0mg / ml of prokaryotic expression products of Magnaporthe grisea effector proteins fusing GFP for 12-72h. The method is simple, accurate and rapid.

Description

technical field [0001] The present invention relates to a method for detecting the translocation of rice blast fungus effector protein in rice root tissue in vitro, in particular to a method for treating rice root tissue with prokaryotic expression product of blast fungus effect protein gene, and detecting effect protein in vitro without blast fungus oryzae A method for autonomous transport into rice tissue in the presence of the present invention belongs to the fields of plant protection and biotechnology. Background technique [0002] Pathogen effector proteins have dual roles of making plants pathogenic and inducing plant defense responses. Pathogenic bacteria transport effectors through type Ⅲ secretion system, and its transport mechanism has been studied clearly. But for filamentous fungi, how effector proteins are transported into host cells remains unknown, although specialized infection structures such as haustoria are involved in the transport of effector proteins....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/63
Inventor 杨静李成云朱有勇刘林施竹凤
Owner YUNNAN AGRICULTURAL UNIVERSITY
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