CN113278722A

CN113278722A - Method for screening solo-LTR virulence candidate strains harboring Inago2 in AvrPiz-t

Info

Publication number: CN113278722A
Application number: CN202110509167.0A
Authority: CN
Inventors: 李进斌; 王群; 陆琳; 何成兴
Original assignee: Institute of Agricultural Environment and Resources of Yunnan Academy of Agricultural Sciences
Current assignee: Institute of Agricultural Environment and Resources of Yunnan Academy of Agricultural Sciences
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2021-08-20

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a primer and a method for quickly identifying a toxic strain of a solo-LTR fragment inserted into an Inago2 retrotransposon in a rice blast fungus avirulence gene AvrPiz-t by using a specific primer. The invention develops a pair of specific primers for the first time, and the primers can quickly detect whether the promoter region of the AvrPiz-t gene of the rice blast fungus is inserted into the solo-LTR fragment of the Inago2 of the retrotransposon, thereby providing guarantee for quickly screening toxic candidate strains, providing technical support for dynamic change detection of field strains, providing guidance information for rice blast resistance breeding and rice blast comprehensive prevention and control, and providing materials for developing rice blast pathogenesis research. The primer has strong specificity and high sensitivity, can quickly screen out toxic candidate strains of rice blast germs to IRBLzt-T, and has important scientific research and application values.

Description

Method for screening solo-LTR virulence candidate strains harboring Inago2 in AvrPiz-t

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a method for quickly identifying a toxic strain by using a specific primer to determine whether a rice blast bacterium avirulence gene AvrPiz-t is inserted into a solo-LTR fragment of an Inago2 retrotransposon.

Background

The rice blast is one of the most devastating fungal diseases in rice production. The use of Resistance genes (R) is one of the most economical and ecologically friendly measures to control rice blast. However, resistant varieties of rice "lose" resistance after 3-5 years of planting due to the high, rapid mutation of the rice blast fungus (Magnaporthe oryzae). The interaction of the Resistance gene (R) of rice and the avirulence gene (Avirulance gene, Avr) of rice blast bacteria conforms to the gene-to-gene theory of Flor, namely, the Resistance reaction of rice can be triggered only by the interaction of the two genes when the two genes exist at the same time, otherwise, the immune reaction of rice can not be triggered by the lack of any one of the two genes, and the rice is infected by diseases.

The rice blast germ Piz-t gene is a broad-spectrum resistance gene of rice blast, is a resistance gene with higher utilization value in production and breeding, and has been successfully cloned in 2006. The corresponding avirulence gene AvrPiz-t was also cloned successfully in 2009. It is possible to detect whether the rice blast fungus strain has AvrPiz-t gene and whether mutation occurs by using PCR amplification reaction and sequencing technology. The avirulence gene of rice blast fungus determines the effectiveness of the corresponding resistance gene in rice. The presence or absence of avirulence genes and variations can affect the "loss" of resistance genes. Insertion of bases in the coding region, upstream and downstream of the avirulence gene is one way of variation of the avirulence gene. The inserted base may be a transposon, an inverted transposon, a repetitive DNA element, or may be a single base insertion. Transposons are moveable DNA sequences in a genome that can "jump" from one location to another in the genome through a series of processes, such as cutting, reintegration, and the like. It has great plasticity to the structure and function of gene. At present, related reports indicate that the transposon has an effect on the function transformation of the avirulence gene of rice blast fungi. Such as: the insertion of the sequences of the AVR-Pia flanking transposons Pot3 and of the AVR-Pii flanking Maggy confers on the pathogenic bacteria a great plasticity during evolution, resulting in the ready availability or loss of avirulence genes (Yoshida et al, 2009). Variation of high or low copy repetitive DNA sequences such as MGR583, MGR586 and MGR608(Hamer et al, 1989) in the sequences flanking the PWL2 gene resulted in the loss of non-toxic function of PWL2 (Sweigard et al, 1995). Insertion of the Pot3 transposon 462bp upstream of the AVRPiz-t start codon changed strain Guy11 to a virulent strain (Li et al, 2009). The insertion of the coding sequence of AVR-Pita (Zhou et al, 2007) and the 5' non-coding sequence into transposon Pot3(Dai et al, 2010; Kang et al, 2001) resulted in a non-toxic loss of function. These findings indicate that under certain specific conditions, reverse elements can be inserted into the genome, which may be targets for overcoming pathogen mutations in phytopathology studies. Identifying new reverse transcription factors and researching the activity of the reverse transcription factors are helpful for clarifying the pathogenic essence of the rice blast germs and provide a thought for the comprehensive prevention and control of the rice blast.

Inago2 is a retrotransposon newly found in Japanese strain 9439009 in 2011 interspersed in the Magnaporthe genome, belonging to the retrotransposable Ty3/gypsy family, and the activity of the retrotransposon is still unknown (Sanchez, Asano, & Sone, 2011). At present, the inventors found that 198bp of the solo-LTR highly homologous fragment of Inago2 was inserted into the AvrPiz-t promoter region of the avirulent gene of 1 strain among 100 rice blast strains in Yunnan. Pathogenicity analysis shows that the mutation of the strain AvrPiz-T can cause the disease of the isogenic line IRBLzt-T with the Piz-T gene. This indicates that the insertion of the solo-LTR fragment of Inago2 evolved the toxicity of the avirulence gene AvrPiz-t of Pyricularia oryzae from avirulence to virulence. This is the first report of the variation of the toxic function of the rice blast fungi by the insertion of the transposon element. The discovery of pathogenic sites can promote the development of other fields and lead the gene detection to become mature. Conventional methods for determining virulent candidate strains require PCR amplification and sequencing of genes, followed by comparison with the sequence of avirulence genes, and subsequent selection of variant strains as virulent candidate strains. Finally, the toxicity candidate strain needs pathogenicity determination to judge whether toxicity is mutated or not, and if the toxicity is not mutated, the toxicity candidate strain is a nontoxic strain; otherwise, the strain is a virulent strain. The solo-LTR fragment strain with the retrotransposon Inago2 inserted in the Magnaporthe grisea AvrPiz-t gene can be used as a virulence candidate strain. The invention designs a pair of primers by utilizing the characteristic that the size of a strain gene fragment is increased after the retrotransposon is inserted, and the primers can amplify both wild type genes and mutant genes inserted into the retrotransposon. The invention can judge whether the strain is a toxic candidate strain according to the electrophoresis result of the PCR product after PCR amplification, thereby reducing the time and the fund of sequencing reaction and leading the gene detection technology of the toxic strain to be improved day by day. The invention not only provides materials for the interaction of avirulence genes AvrPiz-t and Piz-t genes and the research of pathogenesis thereof, but also can quickly master the dynamic change of field strains and provide guidance information for rice blast resistance breeding and comprehensive prevention and control thereof.

At present, no literature, primers or methods have been searched for screening and identifying primers and methods for virulent strains harboring the Inago2 transposon solo-LTR fragment in AvrPiz-t.

Reference to the literature

Dai,Y.,Jia,Y.,Correll,J.,Wang,X.,&Wang,Y.(2010).Diversification and evolution of the avirulence gene AVR-Pita1 in field isolates of Magnaporthe oryzae.Fungal Genetics&Biology,47(12),973-980.

Hamer,J.E.,Farrall,L.,Orbach,M.J.,Valent,B.,&Chumley,F.G.(1989).Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.Proceedings of the National Academy of Sciences of the United States of America,86(24),9981-9985.

Kang,S.,Lebrun,M.H.,Farrall,L.,&Valent,B.(2001).Gain of virulence caused by insertion of a Pot3transposon in a Magnaporthe grisea avirulence gene.Molecular Plant Microbe Interact,14(5),671-674.

Li,W.,Wang,B.,Wu,J.,Lu,G.,Hu,Y.,Zhang,X.,...Wang,Z.(2009).The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t.Molecular Plant Microbe Interact,22(4),411-420.

Sanchez,E.,Asano,K.,&Sone,T.(2011).Characterization of Inago1 and Inago2 retrotransposons in Magnaporthe oryzae.Journal of General Plant Pathology,77(4),239-242.

Sweigard,J.A.,Carroll,A.M.,Kang,S.,Farrall,L.,Chumley,F.G.,&Valent,B.(1995).Identification,cloning,and characterization of PWL2,a gene for host species specificity in the rice blast fungus.Plant Cell,7(8),1221-1233.

Yoshida,K.,Saitoh,H.,Fujisawa,S.,Kanzaki,H.,Matsumura,H.,Yoshida,K.,...Win,J.(2009).Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae.Plant Cell,21(5),1573-1591.

Zhou,E.,Jia,Y.,Singh,P.,Correll,J.C.,&Lee,F.N.(2007).Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence.Fungal Genetics&Biology,44(10),1024-1034.

Disclosure of Invention

Aiming at the technical problems, the invention provides a pair of primers Inago2-F1/Inago2-R1 for PCR amplification so as to detect whether the rice blast fungus strain is inserted into the solo-LTR fragment of the Inago2 retrotransposon, and provide guarantee for quick screening of toxic candidate strains.

Therefore, aiming at the characteristic that the resistance of the Piz-t gene can be lost due to the fact that the mutant of the solo-LTR fragment of the transposon Inago2 is inserted into the promoter region of the Magnaporthe grisea AvrPiz-t gene, a pair of primers Inago2-F1/Inago2-R1 is developed for PCR amplification, so that whether the Magnaporthe grisea strain is inserted into the solo-LTR fragment of the transposon Inago2 can be detected, and the rapid screening of toxic candidate strains is guaranteed.

Specific primers identified by screening of virulent strains harboring Inago2 retrotransposon solo-LTR fragment in AvrPiz-t, the primer sequences:

Inago2-F1：5‘-TGGAGTTATCCTCGACAC-3’

Inago2-R1：5‘-CCGAATTCCAGCCGAAGATAC-3’。

the invention also provides a method for screening and identifying toxic strains with Inago2 retrotransposon solo-LTR fragments in the rice blast germ avirulence gene AvrPiz-t by using the specific primers, which comprises the following steps:

s1: extracting total DNA of a strain to be detected;

s2, constructing a special PCR reaction system by taking the sample DNA obtained in the step (1) as a template, and carrying out PCR amplification by using the specific primer in claim 1;

s3, detecting the PCR amplification product by agarose gel electrophoresis, wherein when the PCR product is 800bp, the rice blast fungus strain is mutated, namely the rice blast fungus strain is inserted into the solo-LTR fragment of the retrotransposon; when the PCR product was 600bp, the rice blast strain was not mutated, i.e., the rice blast strain did not insert the solo-LTR fragment of Inago2 transposon.

Further, the PCR reaction system was 25. mu.l of 2 Xpfu PCR MasterMix (Tiangen Biotech Co. LTD, Beijing, China), 1. mu.l of each of 10. mu.M primers, 2. mu.l of total DNA of Pyricularia oryzae, and 21. mu.l of sterilized double distilled water.

Further, the PCR amplification reaction conditions are as follows: the PCR reaction products were detected by pre-denaturation at 95 ℃ for 3min, followed by 35 cycles comprising denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and final extension at 72 ℃ for 7 min.1% agarose gel electrophoresis at 100V for 50 min.

The invention develops a pair of specific primers for the first time, and the primers can quickly detect whether the promoter region of the AvrPiz-t gene of the rice blast fungus is inserted into the solo-LTR fragment of the Inago2 of the retrotransposon, thereby providing guarantee for quickly screening toxic candidate strains, providing technical support for dynamic change detection of field strains, providing guidance information for rice blast resistance breeding and rice blast comprehensive prevention and control, and providing materials for developing rice blast pathogenesis research. The primer has strong specificity and high sensitivity, and can quickly screen out toxic candidate strains causing IRBLzt-T.

Drawings and description of the drawings

FIG. 1 is an agarose gel electrophoresis image of AvrPiz-t gene amplified by Inago2-F1/Inago2-R1 primer, wherein M is DL2000 DNA marker, and the sizes of the fragments are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; 1-13 are PCR amplification products of rice blast fungus strains in each rice area of Yunnan province, and CK is negative control.

FIG. 2 is a diagram showing the pathogenic reaction of rice blast strains to resistant variety IRBLzT-T (holding Piz-T gene) and susceptible variety Lijiang New York Black grain (LTH), and 1-4 correspond to strains No. 1-4 in FIG. 1.

Detailed Description

1. Procedure and validation of specific primer design

Designing a primer: comparing sequences of upstream regions of insertion sites of AvrPiz-t gene promoters between the rice blast strains with and without the insertion, and selecting a small segment of oligonucleotide sequence (about 50 percent of GC content) as a front primer in a conserved region; then, a conserved oligonucleotide sequence (about 50% GC content) is selected downstream of the gene coding region (CDS), and the reverse complementary sequence is used as a post-primer. Finally, comparing whether primer sequences before and after are easy to form primer dimers, and redesigning the primer sequences easy to form primer dimers. The designed positions of the front and rear primers include the upstream promoter region and CDS region of the insertion site. And (3) verification: the designed primer sequences are synthesized by a sequencing company, and then the synthesized primers are used for carrying out PCR amplification on the rice blast fungus strains. And carrying out agarose gel electrophoresis on the PCR product to detect the size of the amplified fragment. The sizes of the amplification products of the inserted strain and the uninserted strain are consistent with the size of the predicted product, so that the pair of primers are specific primers, and the sequence of the amplification product can be further verified to be consistent with the sequence of the predicted product through sequencing.

In total, 3 primers were designed, as shown in Table 1 below.

Table 1 primer sequences designed for the solo-LTR insertion of Inago2 in AvrPizt

2. AvrPiz-t gene detection and pathogenicity verification by using primers in the table 1

2.1 amplification of AvrPiz-t Gene detection Using Inago2-F1/Inago2-R1 primer

13 total DNA of strains in a Yunnan province rice planting area and sterilized deionized water (negative control) are used as PCR amplification reaction templates, Inago2-F1 (5'-TGGAGTTATCCTCGACAC-3') and Inago2-R1 (5'-CCGAATTCCAGCCGAAGATAC-3') are specially designed amplification primers, and PCR amplification products of a solo-LTR fragment inserted into an inversion seat are about 800bp, and the PCR amplification products are not inserted into the inversion seat and are about 600 bp. The 50. mu.l PCR amplification reaction components were as follows: 2 Xpfu PCR MasterMix (Tiangen Biotech Co. LTD, Beijing, China) 25. mu.l, 10. mu.M primers 1. mu.l each, rice blast fungus Total DNA 2. mu.l, sterilized double distilled water 21. mu.l. PCR amplification reaction procedure: the PCR reaction products were detected by pre-denaturation at 95 ℃ for 3min, followed by 35 cycles comprising denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and final extension at 72 ℃ for 7 min.1% agarose gel electrophoresis at 100V for 50 min, and DL2000(Tiangen Biotech Co. LTD, Beijing, China) was used as a molecular Marker for DNA to estimate the size of the PCR products.

As a result: the electrophoresis results of the PCR amplification products of 13 strains are shown in FIG. 1. In FIG. 1, the fragment of strain No. 1 is about 800bp, the fragment of strain No. 2-13 is about 600bp, and CK is a negative control and is not amplified to obtain a product. This result indicated that the strain No. 1 inserted a fragment of about 200bp, which was sequenced to indicate that the solo-LTR fragment of Inago2 was inserted.

Similarly, the primers Inago2+ F2/Inago2+ R2, Inago2+ F3/Inago2-R1 and Inago2+ F3/Inago2+ R2 are used for amplifying the AvrPiz-t gene for detection, and no effective product is amplified.

The results show that:

1) the F1& R1 (expected fragment > fragment containing Inago2+ CDS) primers were amplified in most of the DNA templates selected, and the amplification product was about 200bp larger for strains with an Inago2 insertion than for strains without an insertion. Consistent with the expected results.

2) The amplification results of F2& R2, F3& R1, F3& R2 (the fragment was expected to be amplified only by the strain containing the Inago2 insertion) differed from the expected results that not only the strain with the Inago2 insertion was amplified, but also some strains without the Inago2 insertion had amplification products and the background was relatively noisy. Indicating that the primer specificity is not strong.

2.2, pathogenicity verification: the pathogenicity of the rice blast strain to IRBLzt-T is determined by adopting a conventional spray inoculation method. The method comprises the following steps: culturing with oat agar culture medium; after the culture dish is full of mycelia, washing off the aerial mycelia with tap water, and placing under a fluorescent lamp for 48 hours to promote spore production; washing spore with distilled water to obtain 5 × 10 powder⁵Adding 0.02% Tween-20 (increasing the adhesive force of the spore suspension to leaves) into the spore suspension per ml before inoculation, and uniformly spraying and inoculating the spore suspension to the rice seedlings in the 3-4 leaf stage after shaking; after inoculation, the seedlings are placed in an incubator at 28 ℃ and the humidity is 90 percent for dark culture for 24 hours, the seedlings are taken out of the incubator and are cultured for 5 to 6 days at high temperature and high humidity in a greenhouse, and then the disease condition is investigated. The survey criteria and disease grade were as follows: and (4) taking LTH as a control variety, investigating the disease level of the penultimate leaf according to the size and the shape of the lesion, and grading the disease level into 6. Wherein, the

grades

0, 1 and 3 are anti, and the

grades

5, 7 and 9 are feeling. The classification criteria are as follows: grade 0, no scab; stage 1, brown spot with needle point size; grade 3, the circular lesion is less than 2mm, and no obvious center exists; 5-grade gray scab with round to oval shape, brown edge and water immersion shape, wherein the diameter of the scab is more than 2 mm; 7, more spindle-shaped water-immersed lesions, larger diameter of the lesions, connected with some lesions, and more than 50 percent of the area of the lesions; 9-grade, spindle-shaped water-soaked scabs are connected into pieces, and the leaves are withered.

Pathogenicity validation in FIG. 2 also indicates that strain No. 1 is pathogenic to IRBLzt-T, and that the remaining 12 strains are not pathogenic to IRBLzt-T. The pathogenicity results for some strains are shown in FIG. 2. It is also shown that whether the solo-LTR fragment of the transposon Inago2 is inserted into the promoter region of the Magnaporthe grisea AvrPiz-t gene can be rapidly detected by using the method and the specific primer of the invention.

SEQUENCE LISTING

<110> research institute of agricultural environmental resources of agricultural academy of sciences of Yunnan province

<120> method for screening solo-LTR virulence candidate Strain harboring Inago2 in AvrPiz-t

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<170> PatentIn version 3.5

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ccgaattcca gccgaagata c 21

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Claims

Primers for the screening identification of strains virulent with the Inago2 retrotransposon solo-LTR fragment held in AvrPiz-t, characterized in that: the primer sequence is as follows:

Inago2-F1：5‘-TGGAGTTATCCTCGACAC-3’

Inago2-R1：5‘-CCGAATTCCAGCCGAAGATAC-3’。
2. method for the screening identification of virulent strains of AvrPiz-t harboring the Inago2 retrotransposon solo-LTR fragment according to the primers of claim 1, comprising the following steps:

s1: extracting total DNA of a strain to be detected;

s2, constructing a special PCR reaction system by taking the sample DNA obtained in the step (1) as a template, and carrying out PCR amplification by using the specific primer in claim 1;

s3, detecting the PCR amplification product by agarose gel electrophoresis, wherein when the PCR product is 800bp, the rice blast fungus strain is mutated, namely the rice blast fungus strain is inserted into the solo-LTR fragment of the retrotransposon; when the PCR product was 600bp, the rice blast strain was not mutated, i.e., the rice blast strain did not insert the solo-LTR fragment of Inago2 transposon.
3. Method for the screening and identification of virulent strains of AvrPiz-t harboring the Inago2 retrotransposon solo-LTR fragment according to the primers of claim 2, characterized in that: the PCR reaction system comprises 25 mu L of 2 Xpfu PCR MasterMix (Tiangen Biotech Co. LTD, Beijing, China), 1 mu L of each 10 mu M primer, 2 mu L of rice blast germ total DNA and 21 mu L of sterilized double distilled water.
4. Method for the screening and identification of virulent strains of AvrPiz-t harboring the Inago2 retrotransposon solo-LTR fragment according to the primers of claim 2, characterized in that: the PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, followed by 35 cycles comprising denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 7min, and detection of PCR reaction products by 1% agarose gel electrophoresis at 100V for 50 min.