CN115029373A

CN115029373A - Application of metarhizium CFEM85 protein and method for improving resistance of plants to botrytis cinerea and aphids

Info

Publication number: CN115029373A
Application number: CN202210693952.0A
Authority: CN
Inventors: 农向群; 蔡霓; 刘蓉; 涂雄兵; 王广君; 张泽华
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2022-06-19
Filing date: 2022-06-19
Publication date: 2022-09-09
Anticipated expiration: 2042-06-19
Also published as: CN115029373B

Abstract

The invention relates to the field of prevention and control of agricultural and forestry pests, in particular to application of metarhizium anisopliae CFEM85 protein and a method for improving resistance of plants to botrytis cinerea and aphids. The application comprises the steps of performing permeation injection on the back of tobacco leaves by using active metarhizium CFEM85 protein, and staining trypan blue to observe allergic necrosis; meanwhile, a transient expression vector is constructed, the agrobacterium tumefaciens is used for transforming the tobacco transient expression to verify the activation reaction of the tobacco to plant resistance, aphid and botrytis cinerea are inoculated, and the induction effect of the tobacco to the plant resistance is evaluated. The results show that protein injection and transient expression show that the metarhizium CFEM85 protein can activate the anaphylactic reaction of tobacco and improve the resistance of the tobacco to botrytis cinerea and aphids. The application verifies that the metarhizium CFEM85 protein has the function of activating plant resistance, and provides reference for subsequent functional research and utilization of the protein.

Description

Application of metarhizium CFEM85 protein and method for improving resistance of plants to botrytis cinerea and aphids

Technical Field

The invention relates to the field of prevention and control of agricultural and forestry pests, in particular to application of metarhizium anisopliae CFEM85 protein and a method for improving resistance of plants to botrytis cinerea and aphids.

Background

Metarhizium anisopliae is an important entomopathogenic fungus and is used as a biological pesticide for preventing and controlling various agricultural and forestry pests. The life style is various, and the insect parasitism, soil saprophytosis and plant endophytic life can be realized. A great deal of research has been focused on the pathogenicity to hosts, lethal mechanism and biological control, and in recent years, researchers have focused on the interaction with plants. A plurality of scientific reports prove that the metarhizium anisopliae can be planted in various host plant tissues, the nutrient absorption and resistance level of plants are improved, and how to activate the resistance level of the plants under the condition of being friendly and beneficial to the plants is explored, so that the plant diseases and insect pests are prevented and controlled, and the method has important significance for realizing continuous prevention and control of the metarhizium anisopliae in application.

The protein domain of CFEM (common in partial fungal extracellular membrane protein) has about 60 amino acids, usually 8 cysteines. Unlike other cysteine-rich proteins, this protein domain is only present in fungi and is common to and hence the name of the extracellular membrane proteins of fungi. Different CFEM sources are different, functions are different, some CFEM sources are involved in immunity inhibition, some CFEM sources are beneficial to maintaining the structure of fungi, and the diversity of functions of the CFEM sources is related to the sources and the protein structure. In Verticillium dahliae of Verticillium dahliae, VDPSCP 76,77 can inhibit the cell necrosis of tobacco triggered by effector and inhibit the immunity of tobacco. In phytopathogens, the CFEM protein was studied as a elicitor, and the wheat leaf rust fungus Puccinia triticina CFEM effector PTTG 08198 enhances cell death of wheat, promoting Reactive Oxygen Species (ROS) accumulation. In Magnaporthe oryzae, Pth11 is a G protein-coupled receptor-like protein with 7 transmembrane domains. After the gene Pth11 is knocked out, the attachment cells on the hydrophobic surface are weakened and cannot penetrate through the infected rice leaves. This indicates that Pth11 is involved in cell wall integrity and hydrophobicity. Botrytis cinerea (BcCFEM 1) is significantly upregulated early in bean leaf infestation, and Agrobacterium-mediated transient expression of tobacco shows leaf chlorosis in Nicotiana benthamiana. However, the function of the protein has not been reported in the interaction research of entomopathogenic fungi and plants.

Disclosure of Invention

The invention aims to provide application of metarhizium CFEM85 protein.

It is a further object of the present invention to provide a method for increasing the resistance of plants to botrytis cinerea and aphids.

The invention provides the following applications of metarhizium CFEM85 protein:

activating the allergic reaction of the plant;

enhancing the resistance of plants to botrytis cinerea; or

The resistance of the plant to the aphids is enhanced,

wherein the amino acid sequence of the metarhizium CFEM85 protein is shown as SEQ ID NO. 1 or 2.

SEQ ID NO:1：

SEQ ID NO:2 (sequence of Metarrhizium anisopliae CFEM85 protein after removal of Signal peptide)

According to the use of the invention, the plant is tobacco.

The method for improving the resistance of the plant to botrytis cinerea and aphids comprises the step of expressing a coding gene of the metarhizium anisopliae CFEM85 protein in the plant, wherein the amino acid sequence of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1.

The method for improving the resistance of plants to botrytis cinerea and aphids, according to the present invention, wherein the plants are tobacco.

The method for improving the resistance of plants to botrytis cinerea and aphids, disclosed by the invention, wherein the nucleotide sequence of the coding gene of the metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 3 or 4.

SEQ ID NO:3：

SEQ ID NO. 4 (coding sequence of Metarrhizium anisopliae CFEM85 protein without signal peptide)

The method takes Metarhizium anisopliae (Metarhizium anisopliae) cDNA as a sample, clones to obtain a CFEM85 gene, constructs a recombinant expression vector after sequencing verification and comparison analysis are correct, performs in-vitro expression by using an escherichia coli prokaryotic expression system, and obtains active protein after protein renaturation. Metarhizium anisopliae CFEM domain-containing secretory protein (CFEM 85) has a typical CFEM structure, and 8 conserved cysteines are contained in the CFEM domain. Injecting active metarhizium CFEM85 protein into the back of tobacco leaves, and staining trypan blue to observe allergic necrosis; meanwhile, a transient expression vector is constructed, agrobacterium is used for transforming tobacco transient expression to verify that the agrobacterium converts the tobacco transient expression vector to the plant resistance activation reaction, and RT-qPCR is used for detecting the tobacco resistance related gene. The results show that protein injection and transient expression show that the metarhizium CFEM85 protein can activate the anaphylactic reaction of tobacco and improve the resistance of the tobacco to botrytis cinerea and aphids. The application verifies that the metarhizium CFEM85 protein has the function of activating plant resistance, and provides reference for subsequent functional research and utilization of the protein.

Drawings

FIG. 1 is the full-length amplification of Metarrhizium anisopliae CFEM85 gene and the electrophoresis chart of the vector construction, wherein, the lane: m: DL2000 plus DNA marker, left 1: CFEM85 full length, medium 1: PET-21b-CFEM85, right 1: pYBA-CFEM 85;

FIG. 2-1 is an SDS-PAGE pattern of CFEM85 protein, wherein M: marker; 1: cell disruption solution supernatant, 2: precipitating cell disruption solution;

fig. 2-2 is a denatured SDS-PAGE map of CFEM85 protein, in which M: marker, 1: supernatant of the crushing liquid, 2: buffer I wash, 3: buffer a wash, 4: first buffer C wash, 5: second buffer C wash, 6: 8M Urea Wash 1, 7: 8M Urea Wash 2, 8: 8M Urea Wash 3;

FIGS. 2-3 are SDS-PAGE patterns of CFEM85 protein purification, wherein M: marker, 1: protein dialysate, 2: protein flow-through, 3: 20Mm imidazole, 4: 30Mm imidazole, 5: 50Mm imidazole, 6: 150Mm imidazole elute 1, 7: 150Mm imidazole elute 2;

fig. 3-1 shows the results of CFEM85 recombinant protein treatment of tobacco, wherein CK: 20mM Tris-HCl; and (3) treatment: treatment with 200ug/mL CFEM85 protein;

FIGS. 3-2 show the results of the treatment of tobacco with pYBA1132 CFEM85, wherein note: CK: pYBA1132, GFP and treatment: pYBA1132: CFEM 85;

FIG. 4 shows the DAB staining results after treatment of tobacco with CFEM85 recombinant protein;

FIG. 5 shows the trypan blue staining results after CFEM85 recombinant protein treatment of tobacco;

FIG. 6 shows the results of tobacco cell callose detection, wherein CK: 20mM Tris-HCl, Treatment: CFEM85 protein treatment;

fig. 7 shows that transient expression of CFEM85 induced plant resistance, wherein, after a.12h infiltration of maccfem 85 in tobacco, the leaf botrytis leaf spot size on day 2, b. aphid feeding of tobacco transiently expressing maccfem 85 for 24h, 48h, 72h mean post passage numbers, values of mean ± standard deviation, n 20.f, different letters on the bar represent significant differences at the 0.05 level between different treatments, P < 0.05.

Detailed Description

Test plants and fungi:

the test strain is Metarhizium anisopliae (Metarhizium anisopliae) Ma9 strain, and the test plant Nicotiana benthamiana (Nicotiana benthamiana). Inoculating metarhizium anisopliae on a PDAY culture medium plate, inverting the culture medium plate in a constant-temperature incubator at 26 ℃ for two weeks, and collecting spores to store at 4 ℃ for later use.

Example 1 cloning, expression and purification of Metarrhizium anisopliae CFEM85 Gene

1. Cloning of genes

Dissolving the newly collected spore powder in 0.1% Tween-80, inoculating to hypha-producing liquid culture medium, and culturing at 28 deg.C for 3 d. The mycelia were vacuum-filtered through a vacuum pump, then the dried mycelia were immediately put into liquid nitrogen for quick freezing, and then total RNA was extracted according to the following method. The sample RNA was reverse transcribed into cDNA using the reverse transcription kit 5 × All-In-One RT MasterMix (abm Co.).

By using the full-length specific primers CFEM85-F and CFEM85-R (shown in the following table 1) designed and amplified, an Open Reading Frame (ORF) of the CFEM85 gene is cloned, the full length is 513bp, and the stop codon is TAA, as shown in FIG. 1. The gene codes 170 amino acids, and the theoretical molecular weight of the protein is 16.3 KD.

And (3) carrying out agarose gel electrophoresis detection on the PCR product, recovering a target strip, connecting the recovered and purified PCR product with a PMD19-T vector, placing the PCR product at 16 ℃ for 10 hours, then transforming escherichia coli competent Tans1-T1, and culturing. And (3) selecting a positive transformant, extracting plasmids from the bacterial liquid of the positive transformant, sending the plasmids to a company for sequencing, checking whether the sequence is correct, and determining that the CFEM85 has a signal peptide cutting site at the 18AA position. Removing a signal peptide sequence, adding a front joint sequence at the 5 'end of the gene to form a primer 21b-CFEM85-F, adding a joint sequence at the 3' end to form a primer 21b-CFEM85-R, carrying out double enzyme digestion on a pET-21b vector by EcoRI and SalI, and then utilizing a seamless cloning kit to construct a prokaryotic expression vector. Similarly, a linker sequence is added to the 5 'front end of the complete open reading frame of the CFEM85 gene to form a primer pYBA-CFEM85-F, a linker sequence is added to the 3' rear end to form a primer pYBA-CFEM85-R, and the Pyba-1132 vector is subjected to EcoRI and SalI double enzymesAfter cutting, a prokaryotic expression vector is constructed by using a seamless cloning kit. Then transferred into BL21(DE3) and Trelif respectively ^TM 5a (Beijing Optimus department biology, Ltd.) competence, after overnight growth of the resistant plate, picking single clone, and obtaining the positive clone successfully recombined through bacteria liquid PCR and sequencing. And extracting pYBA-CFEM85 recombinant plasmid to prepare agrobacterium transformation.

TABLE 1 specific primers

Expression and purification of CFEM85 protein

The recombinant positive clone is induced at 37 ℃ by IPTG with the induction concentration of 0.5mM, the rotation speed of 200rpm and the induction time of 4 h. CFEM85 protein was successfully induced to express. The recombinant protein from which the signal peptide was removed was approximately 17kDa (FIG. 2-1), and through protein denaturation, it was found that CFEM85 protein was largely solubilized in the supernatant under 8M urea wash (FIG. 2-2, line6,7, 8). After dialysis and Ni column purification, a large amount of CFEM85 protein was eluted at 150mM imidazole, and after desalting the protein, the protein solution was dissolved in 20mM Tris-HCl (pH 8.0) and left at-20 ℃ for further use, and the SDS-PAGE pattern of CFEM85 protein purification is shown in FIGS. 2-3.

The recombinant protein comprising the signal peptide was also purified.

Example 2 functional study

The purified recombinant protein of example 1 or was used for the following study.

Induction of tobacco cell necrosis reaction by CFEM85 protein

Diluting CFEM85 protein to 200 μ g/mL, slightly scratching the back of the plant leaf with a 1mL disposable syringe needle, pressing the front of the injured leaf with a finger, injecting diluted CFEM85 protein with the volume of about 50 μ l from the wound on the back of the plant leaf by the syringe with the needle removed, and repeating the treatment three times for five times. The injected plants were returned to the greenhouse for further cultivation, and after 24 hours, the cell necrosis reaction of the plant leaves was observed and photographed. After the observation and recording of the plant leaves are finished, dyeing is carried out on one part of the leaves, liquid nitrogen is rapidly frozen after the other part of the leaves is cut off, and the sample is stored at the temperature of 80 ℃ below zero.

As shown in FIG. 3-1, after the CFEM85 protein after expression and purification is injected into tobacco leaves, obvious tobacco cell chlorosis can be observed after 24 h. After 5 days, significant allergic necrosis was observed at the site of protein injection.

As shown in FIG. 3-2, CFEM85 was used to construct transient expression vector pYBA-CFEM85 and transform Agrobacterium, and the empty vector transformed Agrobacterium pYBA1132 was used as control, GFP was used as control, and the treated group leaves were wilted and necrosed 48h after the tobacco was injected, while the control group leaves were not necrosed significantly.

CFEM85 protein induces tobacco live ROS bursts

Plant Reactive Oxygen Species (ROS) mainly include: h ₂ O ₂ ，O ^2- And a hydroxyl radical. Wherein, peroxide H ₂ O ₂ Can be stained brown by DAB and can be used for detecting the accumulation of ROS in plants induced by CFEM85 protein.

Detecting ROS outbreak conditions by using a Diaminobenzidine (DAB) tissue staining method: and respectively picking up the recombinant protein and tobacco leaves treated by the buffer solution for 24 hours, washing the tobacco leaves by distilled water, putting the tobacco leaves into DAB-HCL staining solution (1mg/mL, pH 3.8), vacuumizing to enable the staining agent to immerse the tobacco cells, and incubating for 1-2 hours in the dark at room temperature. Pouring off the staining solution, soaking the leaves in 95% ethanol, boiling in water bath for 10min, repeating for 1-2 times to remove chlorophyll from the leaves, and observing.

The control buffer treated tobacco lamina appeared white by DAB staining, whereas the CFEM85 protein treated tobacco lamina was able to observe a significant brick-red-like accumulation of material (fig. 4), indicating that the recombinant protein CFEM85 treated tobacco was able to produce reactive oxygen species (primarily hydrogen peroxide) in the tobacco which bind DAB to produce a reddish-brown material.

Induction of tobacco allergic necrosis by CFEM85 protein

Trypan blue dye is a cell reactive dye and is commonly used to detect the integrity of cell membranes and to detect whether cells are viable. Live cells will not be stained blue, while dead cells will be stained pale blue. As shown in FIG. 5, tobacco leaves in the CFEM85 protein treated area were stained with blue spots compared with the control group by trypan blue staining, which indicates that the tobacco cells are dead after being treated with CFEM85 protein for 24h, and indicates that the CFEM85 protein can stimulate the allergic necrosis of tobacco.

CFEM85 protein-induced accumulation of tobacco callose

The accumulation of the callose in the tobacco leaves is detected by an aniline blue staining method: the tobacco leaves which are respectively treated with the recombinant protein CFEM85 for 24 hours are taken and placed in a solution of acetic acid/ethanol (1/3 (V/V)) for decolorization, after the leaves are completely decolorized, the leaves are placed in a 0.07M dipotassium hydrogen phosphate-aniline blue (0.1%) solution for dyeing overnight, and finally the leaves are cut into small pieces and placed under a fluorescence microscope for observation.

Callose is glucan bound by beta-1, 3-bonds and plays an important role in regulation of life activities such as sieve tube metabolism and gametophyte development of plants. When plants are stressed by external adverse environment or invaded by pathogens, callose can be rapidly synthesized in sieve tube molecules and deposited on the surface of a sieve plate or blocked in the sieve tube; improving physical defense to hinder pathogen infection or disease spread. The tobacco leaves treated by the CFEM85 protein are bleached and dyed by aniline blue after being subjected to chlorophyll removal, and the punctate fluorescence can be observed under a fluorescence microscope (figure 6), namely callose. The result shows that tobacco callose is accumulated and defense is activated after the CFEM85 protein treats the tobacco.

5. Transient expression of Metarrhizium anisopliae CFEM85(MaCFEM85) improves tobacco resistance to tobacco and Botrytis cinerea

To explore the possible involvement of MaCFEM85 in defense responses to pathogens, it was examined whether overexpression of MaCFEM85 in tobacco would increase resistance to Botrytis.

One week prior to the start of the bioassay, 150 adult aphids were inoculated onto 3 native tobacco plants, 50 per plant. After 72h, all adults were removed with a soft brush and the nymphs continued to feed for 4 days before transferring to Agrobacterium-infiltrated B.benthamiana.

To assess resistance to myzus persicae, gray mold, Agrobacterium carrying PYBA-GFP, PYBA-MaCFEM85 was grown in LB at 28 ℃ for 36 hours. Cells were washed three times in infiltration buffer (10mM MgCl) ₂ 10mM MES, 100. mu.M acetosyringone, pH 5.6) toOD ₆₀₀ 0.6. Bacteria were infiltrated into fully spread leaves with a 1ml needleless syringe, three leaves per treatment and three plants per treatment. There were 12 plants in each experiment.

In the aspect of aphid performance measurement, 20 adult aphids are applied to one infected leaf in a clamping cage 12 hours after the agrobacterium is infected, each treatment is repeated for 5 times, and the death rate of the aphids and the number of newly produced nymphs are recorded every day for 3 days continuously.

In the determination of disease resistance to Botrytis cinerea, newly cultured Botrytis cinerea is infected by Agrobacterium for 12h, and then inoculated into a fungus cake with a diameter of 5mm, and the growth surface of hypha is close to the infiltration area on the surface of the leaf. Placing the inoculated leaves and plants in a humidity condition of more than 70%, and covering a plastic film in a tray at 22 ℃ to promote disease occurrence. After 48h, disease progression for the leaf vaccination trial was estimated by measuring lesion size.

After transient expression of the constructed vectors in naive tobacco, the lesions on the accEM 85-infiltrated leaves were significantly smaller than those of the control plants infiltrated with eGFP vector (panel a in FIG. 7), with a reduction of lesion size of about 30% at 2 dpi. These data indicate that transient expression of MaCFEM85 in nicotiana benthamiana enhances resistance to botrytis cinerea.

6, influence of MaCFEM85 on myzus persicae population after transient expression

To study the effect of MaCFEM85 on the Myzus persicae population after transient expression. In transient expression of MaCFEM85 into tobacco leaf. The Green Fluorescent Protein (GFP) expression was used as a control to monitor aphid fertility. 12h after infiltration, 20 peach adults were confined to each infiltrated leaf, and the infiltrated area was exposed to aphids. After 3d, the death rate of adult aphids and juvenile aphids is recorded, and nymphs born each day are removed. The results show that after MaCFEM85 infiltrated the nicotiana benthamiana plants, the average number of adult aphid offspring per aphid was significantly lower at 24h, 48h, and 72h than in eGFP-infiltrated nicotiana benthamiana (panel b in fig. 7). The transient expression of MaCFEM85 in Nicotiana benthamiana is shown to enhance resistance to aphids.

The above embodiments are only used for understanding the technical solutions of the present application, and do not limit the scope of the present application.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

Application of <120> metarhizium anisopliae CFEM85 protein and method for improving resistance of plants to botrytis cinerea and aphids

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Claims

1. The following applications of Metarrhizium anisopliae CFEM85 protein:

activating the allergic reaction of the plants;

enhancing the resistance of plants to botrytis cinerea; or

Enhancing the resistance of the plants to aphids,

2. Use according to claim 1, wherein the plant is tobacco.

3. A method for improving the resistance of plants to botrytis cinerea and aphids, which is characterized by comprising the step of expressing a coding gene of Metarhizium anisopliae CFEM85 protein in the plants, wherein the amino acid sequence of the Metarhizium anisopliae CFEM85 protein is shown as SEQ ID NO. 1.

4. A method of increasing plant resistance to botrytis cinerea and aphids according to claim 3, wherein said plant is tobacco.

5. The method for increasing plant resistance to Botrytis cinerea and aphids according to claim 3, wherein the nucleotide sequence of the gene encoding the Metarhizium anisopliae CFEM85 protein is shown in SEQ ID NO. 3 or 4.