CN118027163A - Apple alternaria leaf spot effector gene and application thereof - Google Patents

Abstract

The invention provides an apple alternaria leaf spot pathogenic bacteria effector gene and application thereof, and belongs to the technical field of molecular disease resistance. The effector AaAlt a1 can induce the immune response of tobacco and apples; further, after AaAlt a of the alternaria mali is knocked out, the pathogenicity of the alternaria mali is weakened. The invention provides a theoretical basis for researching pathogenic mechanism and host interaction of apple alternaria leaf spot. The method is favorable for elucidating the infection mechanism of pathogenic fungi to hosts in the occurrence process of apple alternaria leaf spot symptoms from the molecular mechanism, and has positive guiding effect on further realizing the control of apple alternaria leaf spot.

Description

An apple leaf spot disease effector gene and its application

Technical Field

The invention relates to the technical field of molecular disease resistance, and in particular to an apple leaf spot disease effector AaAlt a1 gene and an application thereof.

Background technique

Apple early leaf fall disease seriously threatens the sustainable development of the global apple industry. Among them, Alternaria alternata sp. Mali is a common pathogen causing apple leaf spot disease in apple producing areas, which causes dark brown spots on apple leaves and premature leaf fall.

Apple leaf spot pathogen is a biotrophic parasitic fungus, which is often highly specialized and can change the host's physiological and defense state through specific effector proteins to promote its own survival and reproduction. This type of effector protein is also called effector factor, which is a type of secretory protein secreted by pathogens into host cells during the process of pathogen infection of plants to inhibit the host's defense response.

At present, the most economical and effective crop disease prevention and control measure recognized internationally is to plant disease-resistant varieties, and plant resistance genes that have the function of identifying non-toxic effector factors are important members of this measure. The study of fungal effector factors is expected to reveal the nutritional mode of pathogenic fungi and the molecular mechanism of the co-evolution of plants and pathogenic fungi, which may bring innovation to plant disease control.

Summary of the invention

Aiming at the defects in the existing control methods of apple leaf spot disease, the present invention aims to provide an effector factor of apple leaf spot disease and its application. The effector factor (AaAlt a 1) is the key to the pathogenicity of apple leaf spot disease.

In order to achieve the above purpose, the technical solution adopted by the present invention is:

An effector factor of apple leaf spot disease pathogen (AaAlt a 1), characterized in that the amino acid sequence of the effector factor is as shown in SEQ ID NO: 2, and the effector factor is encoded by the effector factor gene sequence of the leaf spot disease pathogen as shown in SEQ ID NO: 1. The apple leaf spot disease pathogen is Alternaria applea.

The amino acid sequence of the effector factor of the apple leaf spot pathogen with the signal peptide (amino acids 1-18) removed is shown in SEQ ID NO:3.

The application of the above-mentioned effector factor gene of the apple leaf spot pathogen is characterized in that the pathogenicity of the apple leaf spot pathogen is reduced by knocking out the above-mentioned effector factor gene of the apple leaf spot pathogen.

The method for knocking out the effector gene of the apple leaf spot pathogen comprises the following steps:

The front and back sequences of the effector gene of the apple leaf spot pathogen were amplified and then constructed into the initial expression vector to obtain a recombinant vector;

The obtained recombinant vector was transformed into the pathogenic bacteria of apple leaf spot disease to achieve gene knockout.

A recombinant vector for knocking out the effector gene of the above-mentioned leaf spot pathogen, characterized in that the recombinant vector comprises:

Initial expression vector;

The upstream sequence fragment of the effector gene of the leaf spot pathogenic bacteria has a length of 0.5 kbp to 1.5 kbp;

And a downstream sequence fragment of the effector factor gene of the leaf spot pathogen, the length of the downstream sequence fragment is 0.5k bp to 1.5k bp.

The above initial expression vector is the PCX62 vector.

The apple leaf spot disease effector and application thereof of the present invention have the following beneficial effects:

The invention provides an effector factor AaAlt a 1 gene of the pathogenic bacteria of apple leaf spot disease, which is beneficial to clarify the infection mechanism of pathogenic fungi on the host during the occurrence of apple leaf spot disease symptoms from the perspective of molecular mechanism, and has a positive effect on further realizing the prevention and treatment of apple leaf spot disease. The effector factor AaAlt a 1 of the pathogenic bacteria of apple leaf spot disease can induce an immune response in the leaves of the host plant; after the effector factor is knocked out, the pathogenicity of the pathogenic bacteria of apple leaf spot disease can be reduced, which is beneficial to the molecular breeding of disease resistance of apple or tobacco.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention has the following accompanying drawings:

Figure 1 shows the characteristics of the effector AaAlt a 1: the evolutionary relationship between AaAlt a 1 and other fungal homologous genes was determined using the maximum likelihood method;

Figure 2 shows the characteristics of the effector AaAlt a 1: SignalP predicted the signal peptide structure of AaAlt a 1;

Figure 3 shows the characteristics of the effector AaAlt a 1: TMHMM predicted the transmembrane structure of AaAlt a 1;

Figure 4 shows the characteristics of the effector AaAlt a 1: Alt a 1 (GenBank: QOJ44335.1) and the 3D structure of the protein encoded by AaAlt a 1;

FIG5 is a pathogenicity analysis of the effector factor AaAlt a 1 on Nicotiana benthamiana in Example 2 of the present invention;

Figure 6 is the detection of AaAlt a 1 gene expression in plants before and after transient overexpression of AaAlt a 1: A is a schematic diagram of the AaAlt a 1 overexpression vector; B is a gel diagram of gene expression detection; C is the qRT-PCR detection of the AaAlt a 1 gene expression in the susceptible variety Jinguan overexpression; D is the qRT-PCR detection of the AaAlt a 1 gene expression in the susceptible variety Gala 3 overexpression; E is the qRT-PCR detection of the AaAlt a 1 gene expression in the resistant variety Hanfu overexpression; F is the qRT-PCR detection of the AaAlt a 1 gene expression in the resistant variety M26 overexpression; the same letters in C-F indicate that the difference is not significant (P＞0.05), and the different letters indicate that the difference is significant (P＜0.05);

Figure 7 shows the resistance of Gala 3 to ALT7 after overexpression of AaAlt a 1: phenotypic images and lesion statistics of tissue culture seedlings of the susceptible variety Gala 3 mediated by Agrobacterium-mediated transient expression 3 days after inoculation with ALT7 and 2 days after inoculation;

Figure 8 shows the resistance of "Golden Crown" to ALT7 after overexpression of AaAlt a 1: phenotypic images and lesion statistics of the susceptible variety "Golden Crown" tissue culture seedlings 3 days after Agrobacterium-mediated transient expression and two days after inoculation with ALT7;

Figure 9 shows the resistance of "Hanfu" to ALT7 after overexpressing AaAlt a 1: phenotypic images and lesion statistics of the tissue culture seedlings of the disease-resistant variety "Hanfu" mediated by Agrobacterium-mediated transient expression and inoculated with ALT7 two days later;

Figure 10 shows the resistance of "M26" to ALT7 after overexpressing AaAlt a 1: Phenotypic images and lesion statistics of the tissue culture seedlings of the disease-resistant variety "M26" mediated by Agrobacterium-mediated transient expression three days after inoculation with ALT7 two days later

FIG11 is the subcellular localization of AaAlt a 1 described in Example 2 of the present invention;

FIG12 shows the gene overexpression and knockout of AaAlt a 1 of ALT7 in Example 4 of the present invention: A shows the overexpression and knockout strategy of AaAlt a 1; B shows the PCR verification of two AaAlt a 1 overexpression mutants using primer pair hyg-F/hyg-R; C shows the PCR verification of two AaAlt a 1 knockout mutants using primer pairs P1/P2 and A1/A2;

FIG13 is the experimental result of overexpression and knockout mutants infecting disease-resistant apples Hanfu and M26 and susceptible apples Gala 3 and Golden Delicious in Example 4 of the present invention: phenotype diagram of mutant-infected apples;

Figure 14 is the experimental results of overexpression and knockout mutants infecting disease-resistant apples Hanfu and M26 and susceptible apples Gala 3 and Golden Crown in Example 4 of the present invention: A is the lesion area statistics of "Hanfu", B is the lesion area statistics of "M26", C is the lesion area statistics of "Gala 3", and D is the lesion area statistics of "Golden Crown".

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings.

The present invention selects Alternaria alba ALT7 as experimental material, combines existing relevant theoretical basis and experimental conditions, and screens out effect factor AaAlt a 1 for research.

First, the base sequence and the amino acid sequence encoded by the effector AaAlt a 1 were screened out through the ALT7 secreted protein spectrum results, and then AaAlt a 1 was cloned from Alternaria alba ALT7, and then AaAlt a 1 was subjected to bioinformatics analysis. The effector AaAlt a 1 was connected to the Super1300 GFP vector, and the transient expression technology of Agrobacterium was used to transiently express it in apple tissue culture seedlings and inoculate Alternaria alba to explore its function, and the expression of the gene was detected by qRT-PCR technology. The results showed that the effector AaAlt a 1 could be normally expressed in apples and enhance the pathogenicity of Alternaria alba. After transiently expressing the effector AaAlt a 1 in tobacco (Nicotiana benthamiana) and apple (Gala 3) tissue culture seedlings, its subcellular localization was observed under a confocal microscope, and the results showed that the effector AaAlt a 1 was located in the cell membrane and cytoplasm.

To further explore the function of the effector AaAlt a 1, the Agrobacterium tumefaciens-mediated transformation (ATMT) technique was used to overexpress and knockout the effector in Alternaria alba ALT7, and overexpression and knockout mutant strains were obtained, respectively. The biological characteristics and pathogenicity of the overexpression and knockout AaAlt a 1 mutant strains were analyzed, and it was found that the growth rate and hyphae morphology of the overexpression mutant strains were not significantly changed compared with ALT7 and empty vector transformants. ALT7, empty vector transformants, overexpression mutants and knockout mutants were inoculated in susceptible and resistant apple varieties, respectively. The results showed that the lesions of apples infected by the overexpression mutant strains were significantly enlarged, while the morbidity of the plants caused by the knockout AaAlt a 1 mutant strains was greatly reduced, indicating that overexpression of AaAlt a 1 significantly enhanced the pathogenicity of the apple leaf spot pathogen, while knockout of AaAlt a 1 could reduce the pathogenicity of the apple leaf spot pathogen and improve the disease resistance of the plants.

The results showed that the function of the effector AaAlt a 1 can cause cell death in tobacco and play a role in the cell membrane and cytoplasm. By analyzing the pathogenicity of the effector AaAlt a 1, the infectivity of the overexpressed mutant was enhanced, and the pathogenicity of the knockout mutant was greatly weakened. This shows that AaAlt a 1 plays a key role in infecting host plants, providing a theoretical basis for studying the function and pathogenic mechanism of the effector factor of apple leaf spot pathogen.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods and operating procedures involved in the examples are all routine techniques in the art (see the third edition of Molecular Cloning Experiment Guide, the seventh edition of Microbiology, etc.); or according to the conditions recommended by the manufacturer's instructions. For parts not specifically described in the present invention, ordinary technicians in the field can refer to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of the present invention for implementation.

The competent E. coli Trans 5α and the competent Agrobacterium strain GV3101 used in the following examples were purchased from Shanghai Weidi Biotechnology Co., Ltd. The vector Super1300 GFP used for transient expression and the vector pCX62 used for gene overexpression were both commercially available. Primers involved in the following examples: According to the principle of primer design, the software DNAMAN and the primer design website (https://sg.idtdna.com/primerquest/home/index) were used for primer design, and the full-length primers for cloning the AaAlt a 1 gene of the effector factor of Alternaria apple were designed, and the primers with restriction sites required for constructing the Super1300 GFP vector and the pCX62 vector and the primers for verifying the overexpression transformant PCR were designed. The designed primer sequences are shown in Table 1.

Table 1 Primer sequences required for each experiment

Example 1 Discovery and Cloning of AaAlt a 1

1. Preparation of fungal secretory protein samples and mass spectrometry analysis

ALT7 culture medium was prepared in a clean bench, and the new mycelium and spores on the bacterial plate were picked into 100 ml of liquid PDB medium, and cultured at 28°C and 180 r/min for 20 days. After 20 days, the mycelium in the culture medium was filtered out with a magic filter cloth, and the conidia and other impurities were removed with a 0.22 μm filter membrane, and the culture medium was concentrated. The small peptides below 10 kDa were filtered out with an Amicon Ultra-15 centrifugal filter, and the secretion fluid was concentrated again, and the obtained secretion fluid was analyzed by liquid chromatography-mass spectrometry (LC-MS). From the results, 13,466 amino acid sequences were identified to match the fungal database in SwissProt, and 132 protein sequences were obtained that exceeded the identification threshold.

Then, SignalP (https://services.healthtech.dtu.dk/services/SignalP-6.0/) was used to screen 132 protein sequences, and 75 protein sequences contained signal peptide structures. In addition, TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) was used to screen 75 protein sequences, and 7 proteins (as shown in Table 2) were found to contain not only signal peptides but also transmembrane structures, and were confirmed to be secreted proteins. The top-ranked secreted protein (Accession: ALTA1_ALTAL) was selected and named AaAlt a 1 as the effector described in the present invention.

Table 2 ALT7 secretory protein profile screening results

2. Fungal Total RNA Extraction

(1) ALT7 mycelial spore samples were quickly ground in liquid nitrogen;

(2) Add 990 μL of preheated CTAB solution and 10 μL of β-mercaptoethanol to the sample, vortex for 30 seconds, and then place in a 65°C water bath for 10 minutes;

(3) Add 1000 μL CI (chloroform/isoamyl alcohol volume ratio = 24:1) and mix by inverting;

(4) 4°C, 12,000 rpm, 10 min;

(5) Pipette 800 μL of supernatant, add an equal volume of CI, and mix by inverting;

(6) 4°C, 12,000 rpm, 10 min;

(7) Aspirate about 650 μL of supernatant and add 1000 μL of isopropanol;

(8) Precipitation at -20°C for 30 min;

(9) 4°C, 12,000 rpm, 10 min;

(10) Pour off the supernatant and add 1 mL of 75% ethanol to wash the precipitate;

(11) 4°C, 12000 rpm, 10 min;

(12) Pour off the supernatant and incubate at 4°C, 12,000 rpm for 2 min;

(13) Aspirate the supernatant and add 40 μL RNase-free H ₂ O to dissolve the precipitate;

(14) 1% agarose gel electrophoresis was used to detect the integrity, and the absorbance at 260 nm was measured by UV spectrophotometer to calculate the RNA concentration;

(15) RNA samples were stored at -80°C.

3. Reverse transcription reaction system and steps ( 1st Strand cDNA Synthesis Kit, gDNA Purge, E042)

(1) Add the following reactants in order

Mix gently and centrifuge.

(2) Incubate at 42°C for 15-30 minutes (if the RNA template does not contain poly A structure, incubate at 25°C for 5 minutes and then incubate at 42°C for 15-30 minutes).

(3) Place on ice.

Primers were designed based on the Alt a 1 sequence provided by NCBI (https://www.ncbi.nlm.nih.gov/).

The primer sequences used for AaAlt a 1 are as follows:

Upstream primer: 5’-ATGCAGTTCACCACCATCGCCTCT-3’

Downstream primer: 5’-TTAAGAGCTCTTGGGGAGAGTGAC-3’

PCR reaction system (CW0682L)

The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; 34 cycles at 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min; and finally extension at 72°C for 2 min.

PCR product detection: Prepare 1% agarose gel according to the size of the target fragment, use 0.1% TAE electrophoresis buffer, perform electrophoresis at 70-110V for about 15 minutes, stain with ethidium bromide, and detect the size of the PCR product fragment under ultraviolet light.

The band size is consistent with expectations. The target band was cut off with a blade under ultraviolet light and recovered by gel. The gene recovery product was constructed into the pEASY-T3 vector and transformed into Escherichia coli. The single clone colony was picked and shaken for culture and then colony PCR amplification was performed. After the sequencing results were aligned correctly, the plasmid was shaken and stored at -20°C for standby use. The gene sequence of the effector factor AaAlt a 1 is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO:2. The basic biological information of the effector was analyzed using DNAMAN software. Through sequence analysis, it can be seen that the sequence of the effector factor AaAlt a 1 of the apple leaf spot pathogen is 534bp in length, the open reading frame ORF is 474bp, encoding 157 amino acids, and the molecular weight of the encoded protein is predicted to be 17.01kD, without transmembrane structure, and the 1st to 18th amino acids are signal peptides. The protein structure is similar to the NCBI sequence protein structure, and the basic properties are shown in Figure 1. Example 2 Study on the function of effector factor AaAlt a 1 of apple leaf spot pathogen 1. Construction of Agrobacterium-mediated transient expression system

AaAlt a 1 with signal peptide and AaAlta1-ΔSP without signal peptide were constructed into Super1300-GFP (between XbaⅠ and SwaⅠ restriction sites) respectively. According to the restriction sites in Super1300-GFP expression vector, primers with restriction sites were designed. After cloning into the gene fragment, gel recovery was performed to obtain the recovered product of the gene. The effector, AaAlt a 1, was constructed into the Super1300-GFP expression vector by restriction digestion, ligation and transformation. After the transformation, a single clone Agrobacterium plaque was picked for colony PCR verification. The primers used were Super1300-GFP vector primers. The results showed that the effector Super1300-GFP had been successfully transformed into Agrobacterium, and the bacterial solution was stored at low temperature for subsequent activation or inoculation experiments.

The specific operations are as follows:

(1) Take a tube of competent cells (Shanghai Weidi Biotechnology, CAT#: AC1001), completely dissolve on ice and gently suspend the cells;

(2) Add 5 μL of plant expression vector plasmid, mix gently, and place on ice for 5 min;

(3) Cold shock in liquid nitrogen for 5 min;

(4) Heat shock at 37°C for 5 min;

(5) Place on ice for 5 min.

(6) Add 500 μL YEP medium and culture at 28°C with shaking at 180 rpm for 2-3 h;

(7) Centrifuge at 10,000 rpm for 1 min at room temperature, remove approximately 400 μL of supernatant, and use the remaining culture medium to suspend the cells.

(8) The bacteria were spread on solid YEP medium supplemented with antibiotics (50 mg/L Kana, 20 mg/L Rif).

(9) Incubate the plate upside down at 28°C for 24-48 hours.

2. Analysis of the autopathogenicity of the effector AaAlt a 1

The inoculation scheme for exploring the function of the effector itself is as follows: In the same tobacco (Nicotiana benthamiana) leaves, inoculate the induction solution containing AaAlt a 1-GFP and free-GFP (the composition of the induction solution: 100mL of 10mM MgCl ₂ , 2mL of 0.5M MES, 200μL of 0.1M AS), observe the symptoms for about a week and take photos. The Agrobacterium inoculation method is as follows:

(1) Add the Agrobacterium transformed with the recombinant plasmid to 50 mL of liquid LB containing Rif and Kana resistance, and culture at 28°C with shaking at 260 rpm for 6-8 h until the OD600 is approximately 1.0.

(2) Centrifuge at 3500 rpm for 5 min at room temperature, discard the supernatant (keep a small amount for resuspension), collect the cells, and wash them 2-3 times with 10 mM _MgCl2 by shaking.

(3) Prepare Agrobacterium induction solution and suspend the bacteria in an appropriate amount of the induction solution so that the OD600 of the bacterial solution reaches about 0.5, and then culture in a 28°C incubator in the dark for 3-5 hours.

(4) Use Nicotiana benthamiana with good growth, and select leaves with good growth and basically the same size to inoculate Agrobacterium. Use a syringe to suck up the bacterial solution and inject the bacterial solution into the leaf from the back of the leaf.

(5) Observe the symptoms of the inoculated leaves for about a week and take photos of the leaves to record them.

Agrobacterium-mediated transient expression technology was used to verify the pathogenicity of the effector AaAlt a 1. The empty GFP was used as a negative control and inoculated into Nicotiana benthamiana leaves of the same growth in the same period. 15 to 20 leaves were inoculated each time and repeated 3 to 4 times to ensure the reliability of the experimental results and avoid accidental occurrence. The results were observed 7 days after inoculation, and photographed and recorded. The inoculation results are shown in Figure 5. The results showed that compared with the empty GFP, the effector AaAlt a 1 could cause necrosis of Nicotiana benthamiana leaves 7 days after inoculation.

3. Pathogenicity analysis of effector factor AaAlt a 1 to different apple varieties

Apple susceptible varieties Jinguan, Gala 3 and resistant varieties Hanfu, M26 tissue culture seedlings were cultured in a light incubator at 25±1℃ and 60% humidity, with a day and night length of 16-8h and a light intensity of 200μmol·m ^-2 ·s ^-1 . Agrobacterium injection was performed when 5-6 true leaves of the plant were unfolded. Leaves with good growth and basically the same size were selected for Agrobacterium injection. The gene expression was detected 3 days after injection and ALT7 was inoculated.

(1) Pre-culture: Add 2 mL of YEP (50 mg/L Km, 20 mg/L Rif) to Agrobacterium plaques or glycerol culture and culture overnight at 28°C and 180 rpm;

(2) Main culture: 4 mL of YEP medium (with corresponding antibiotics and 10 μM acetosyringone), add 1/50 volume of bacterial solution (80 μL), and culture at 28°C and 180 rpm for 12-16 h;

(3) Centrifuge at room temperature at 10,000 rpm for 1 min, remove the culture medium, and suspend the bacteria in 1-2 mL of suspension solution (you can vortex). Take 10 μL of bacterial solution and add it to 990 μL of suspension solution. Measure OD600 with a spectrophotometer and adjust the bacterial suspension to OD600 = 1.0. Let stand at room temperature for 2-5 hours;

Suspension: (10 mM MES-KOH (pH 5.2), 10 mM MgCl ₂ , 100 μM acetosyringone).

(4) Vortex the bacterial solution or use a pipette to suspend the bacteria before use, and use a 1 mL syringe without a needle to draw up the bacterial solution;

(5) Avoid the leaf veins and use the syringe needle to make a small hole on the leaf. Press the small hole with the syringe filled with bacterial solution. Use the fingers of the other hand to press the small hole in the opposite direction of the leaf. Slowly apply force to inject the bacterial solution into the leaf. Inject 2 holes on each leaf.

The expression level was detected by fluorescence quantitative PCR 3 days after injection. The fluorescence quantitative PCR reaction system and steps are as follows:

Design of AaAlt a 1 specific primers:

F5’-ACTACAACAGCCTCGGCTTC-3’

R5’-GTCGTCGCTAACCTTCTGCT-3’

Fluorescence quantification was performed on Applied Biosystems 7500 using SYBR Green fluorescence quantitative premix (TIANGEN, FP121221). The PCR reaction program was as follows: 95°C, 15mins; 95°C 10s, 60°C 30s, for a total of 40 cycles. The results were statistically analyzed using the 2-ΔΔCt method (Livak and Schmittgen, 2001). The test results are shown in Figure 6 (Agrobacterium-mediated transient expression of susceptible varieties ‘Golden Crown’ and ‘Gala 3’ tissue culture seedlings after 3 days of AaAlt a 1 expression, WT represents ‘Golden Crown’ and ‘Gala 3’ tissue culture seedlings without transient expression; EV represents ‘Golden Crown’ and ‘Gala 3’ tissue culture seedlings with no vector for transient expression; OE-AaAlta 1 represents ‘Golden Crown’ and ‘Gala 3’ tissue culture seedlings with overexpression of AaAlt a 1 vector for transient expression).

The apple leaves were inoculated with leaf spot pathogen ALT7 3 days after injection. The operation was as follows:

(1) ALT7 was cultured in PDA medium in the dark for 7 days.

(2) Add 2 mL of sterile water, gently scrape off the spore suspension with a spreading stick, and place it into a 2 mL centrifuge tube.

(3) Use a 1 mL syringe without a needle to draw up the spore suspension.

(4) Avoid the leaf veins and use the syringe needle to make a small hole on the leaf. Press the hole with the syringe filled with bacterial solution. Use the fingers of your other hand to press the hole in the opposite direction of the leaf. Slowly apply force to inject the bacterial solution into the leaf. The color of the injected part will become lighter. Inject 1-2 holes on each leaf.

(5) After two days of dark culture, observe the phenotype.

(6) ImageJ software was used to count the lesion area. The results are shown in Figures 7-10 (Three days after the AaAlt a1-transiently expressed AaAlt a 1-mediated tissue culture seedlings of the susceptible varieties ‘Jinguan’ and ‘Gala 3’ and the resistant varieties ‘Hanfu’ and ‘M26’ were inoculated with ALT7, and 48 hours later, the lesion area statistical graph and phenotype graph were shown; in each figure, WT represents the tissue culture seedlings of ‘Jinguan’ without transient expression; EV represents the tissue culture seedlings of ‘Jinguan’ with no vector transient expression; OE-AaAlt a 1 represents the tissue culture seedlings of the susceptible varieties ‘Jinguan’ and ‘Gala 3’ and the resistant varieties ‘Hanfu’ and ‘M26’ with the vector overexpressing AaAlt a 1 for transient expression).

Agrobacterium-mediated transient expression of the effector factor AaAlt a 1 in the leaves of apple tissue culture seedlings of susceptible varieties ‘Golden Crown’ and ‘Gala 3’ and resistant varieties ‘Hanfu’ and ‘M26’ showed that the expression level of AaAlt a 1 in apples inoculated with OE-AaAlt a 1 overexpression transformants was significantly increased compared with that inoculated with empty vectors, and the incidence of leaves increased significantly and the symptoms were severe 2 days after inoculation with ALT7. This indicates that overexpression of AaAlt a 1 in susceptible varieties ‘Golden Crown’ and ‘Gala 3’ and resistant varieties ‘Hanfu’ and ‘M26’ will reduce disease resistance and enhance the pathogenicity of apple leaf spot pathogen.

4. Subcellular localization of effector factor AaAlt a 1

The plant expression vector super1300-GFP used in the present invention shows green fluorescence under a confocal microscope and can be used to detect the location of protein expression in cells. The induction solution containing the effector factors AaAlt a 1GFP, AaAlta1-ΔSP-GFP and empty GFP was inoculated on the leaves of Nicotiana benthamiana and apple variety Gala 3 respectively, and a preliminary observation was made with a fluorescence microscope after 48 hours to determine whether the gene was normally expressed, and then leaf slices were made and subcellular localization was observed using a confocal microscope. The observation results are shown in Figure 11. The results show that the effector factor AaAlt a 1 is normally expressed in plant (Nicotiana benthamiana and Gala 3 apple) cells, and its subcellular localization is located in the cell membrane and cytoplasm, while AaAlta1-ΔSP without the signal peptide is located on the cell membrane.

Example 3 Overexpression and knockout of the AaAlt a 1 gene of the apple leaf spot pathogen

1. Construction of overexpression vector of effector factor AaAlt a 1 of apple leaf spot pathogen

The AaAlt a 1 gene was constructed into the filamentous fungus gene editing vector pCX62 (between the XhoI and HindⅢ restriction sites), and primers with restriction sites were designed according to the restriction sites in the pCX62 expression vector. After cloning into the gene fragment, the gel was recovered to obtain the recovered product of the gene. The effector, AaAlt a 1, was constructed into the pCX62 expression vector by restriction, connection and transformation, and transformed into the Agrobacterium competent cell EHA105. After completion, the monoclonal Agrobacterium plaque was picked for colony PCR verification, and the primers used were pCX62 vector primers. The results show that the effector pCX62 has been successfully transformed into Agrobacterium, and the bacterial solution is stored at low temperature for subsequent activation or inoculation experiments. The specific operation steps are consistent with the specific steps for constructing the Agrobacterium-mediated transient expression system in Example 2.

2. Construction of knockout vector for the effector factor AaAlt a 1 of apple leaf spot pathogen

(1) Amplify the upstream and downstream 500 bp sequences of the AaAlt a 1 gene on the ALT7 genome, construct the upstream 500 bp into the 5' end of the Hyg fragment of the filamentous fungus gene editing vector pCX62 (between the XhoI and HindⅢ restriction sites), and construct the downstream 500 bp into the 3' end of the Hyg fragment of the filamentous fungus gene editing vector pCX62 (between the BamHI and XbaI restriction sites). The construction strategy is shown in Figure 12A. According to the restriction sites in the pCX62 expression vector, primers with restriction sites are designed, and after cloning into the gene fragment, gel recovery is performed to obtain the recovered product of the gene. The upstream and downstream 500 bp fragments of the AaAlt a 1 gene on the ALT7 genome are constructed into the pCX62 expression vector by restriction digestion, ligation and transformation, and transformed into the Agrobacterium competent cell EHA105. After completion, single clone Agrobacterium plaques are picked for colony PCR verification. The primers used are the pCX62 vector primers. The results showed that the effector AaAlta 1 knockout vector had been successfully transformed into Agrobacterium, and the bacterial solution was stored at low temperature for subsequent activation or inoculation experiments. The specific operation steps were consistent with the specific steps for constructing the Agrobacterium-mediated transient expression system in Example 2.

(2) Referring to the above steps, 1500 bp sequences upstream and downstream of the AaAlt a 1 gene on the ALT7 genome were amplified to construct the AaAlt a 1 knockout vector.

(3) Referring to the above steps, the upstream 820 bp and downstream 790 bp sequences of the AaAlt a 1 gene on the ALT7 genome were amplified to construct the AaAlt a 1 knockout vector.

(4) Referring to the above steps, the upstream 1300 bp and downstream 1280 bp sequences of the AaAlt a 1 gene on the ALT7 genome were amplified to construct the AaAlt a 1 knockout vector.

3. Agrobacterium-mediated genetic transformation, screening and verification

For fungal transformation methods, see Gao F et al. (Gao F, Zhou B-J, Li G-Y, Jia P-S, Li H, et al. (2010) A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity. PLoS ONE 5(12):e15319.doi:10.1371/journal.pone.0015319). Agrobacterium containing the target vector and ALT7 spores were mixed at a ratio of 1:1, and the mixture (200 μL per plate) was co-cultured on IM solid medium containing 200 mM acetosyringone and 40 mM 2-morpholineethanesulfonic acid for 24-48 h, and induced on a composite definition plate containing 200 μg/mL cefotaxime sodium and 75 μg/mL hygromycin. Transformants were selected and screened on PDA medium containing 200 μg/ml hygromycin. Transformants with colonies were selected for subculture and single spore separation on PDA plates, and then DNA was extracted from transformants with stable traits for PCR verification. Primers hyg-F and hyg-R were designed with the hygromycin phosphotransferase gene to verify the overexpression strain. ALT7 was used as a control, and the PCR band size was in line with expectations, indicating successful transformation. The test results are shown in Figure 12. Finally, two overexpression transformants S1 and S2 and one empty vector transformant were obtained through screening.

4. Pathogenicity analysis of overexpression and knockout transformants of the effector factor AaAlt a 1 of apple leaf spot pathogen

Select consistent spore suspensions of ALT7, empty vector transformants, overexpression mutants and knockout mutants for inoculation. For the inoculation method, refer to the inoculation method recorded in the analysis of the pathogenicity of effector AaAlt a 1 on different apple varieties in Section 2 of Example 2. The disease-resistant varieties Hanfu and M26 and the susceptible varieties Gala 3 and Golden Crown were selected for inoculation, and leaves with the same growth were selected and pierced with a syringe needle, and the spore suspension was injected for infection. ALT7 was used as a control and the experiment was repeated 3 times. After 2 days, the symptoms were observed and photographed for record. The results are shown in Figures 13 and 14. The results show that compared with ALT7 and empty vector transformants, the size of the lesions of the disease-resistant varieties of apples infected by the overexpression mutants was significantly increased, indicating that the overexpression of the effector AaAlt a 1 increased the ability of the apple leaf spot pathogen to infect apples and enhanced the pathogenicity of the apple leaf spot pathogen. At the same time, the size of lesions in susceptible apple varieties infected by knockout mutants (knockout mutants after amplification of 500bp sequences upstream and downstream of the effector gene) was also greatly reduced (Gala 3 was reduced by about 80%, and Golden Crown was reduced by about 90%). Knocking out AaAlt a 1 in ALT7 can significantly reduce its pathogenicity, protecting apples from leaf spot pathogens.

For the knockout mutants obtained after amplification of 1500 bp sequences upstream and downstream of the effector gene described in Section 2, subsection (2) above, the reduction rates of lesion size in infected apple varieties were close to the above experimental results, namely: Gala 3 was reduced by about 79% and Golden Delicious was reduced by about 88%.

For the knockout mutants obtained after amplification of the 820 bp upstream and 790 bp downstream sequences of the effector gene described in Section 2, subsection (3) above, the reduction rates of lesion size in infected apple varieties were close to the above experimental results, namely: Gala 3 was reduced by about 80% and Golden Delicious was reduced by about 87%.

For the knockout mutants obtained after amplification of the 1300bp upstream and 1280bp downstream sequences of the effector gene described in Section 2, subsection (4) above, the reduction rates of lesion size in infected apple varieties were close to the above experimental results, namely: Gala 3 was reduced by about 78% and Golden Crown was reduced by about 90%.

The present invention verifies the function of the effector factor AaAlt a 1 of apple leaf spot pathogen, and determines the position of the signal peptide by predicting its bioinformatics. On this basis, the gene sequence without the signal peptide is used to construct the gene onto the super1300-GFP expression vector, and the subcellular localization is preliminarily explored using the Agrobacterium-mediated transient expression system. At the same time, AaAlt a 1 is transiently overexpressed in apple tissue culture seedlings, and AaAlt a 1 is overexpressed and knocked out in apple leaf spot pathogen, respectively, and it is found that it cannot affect the normal growth of apple alternaria, but it is a key factor in the pathogenicity of apple leaf spot pathogen, overexpression can enhance the pathogenicity of apple leaf spot pathogen, and the pathogenicity of apple leaf spot pathogen is greatly weakened after knocking out, thereby determining that the effector factor AaAlt a 1 plays a role in the process of infecting the host plant, and provides a theoretical basis for the exploration of the pathogenic mechanism of apple alternaria and the host interaction.

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims (5)

1. The apple alternaria leaf spot pathogenic bacteria effector is characterized in that the amino acid sequence of the effector is shown as SEQ ID NO. 2, and the effector is encoded by the alternaria leaf spot pathogenic bacteria effector gene sequence shown as SEQ ID NO. 1.

2. Use of an apple alternaria leaf spot pathogenic effector gene according to claim 1, wherein apple alternaria leaf spot pathogenic pathogenicity is reduced by knocking out the apple alternaria leaf spot pathogenic effector gene according to claim 1.

3. Use of an apple alternaria leaf spot pathogenic effector gene as claimed in claim 2 wherein the method of knocking out an apple alternaria leaf spot pathogenic effector gene comprises the steps of:

amplifying sequences before and after the apple alternaria leaf spot pathogenic bacteria effector genes, and constructing the amplified sequences into an initial expression vector to obtain a recombinant vector;

And transforming the recombinant vector into pathogenic bacteria of apple alternaria leaf spot to realize gene knockout.

4. A recombinant vector for knocking out the spot-leaf pathogen effector gene of claim 1, the recombinant vector comprising:

An initial expression vector;

the length of the upstream sequence fragment of the spot defoliation pathogenic bacteria effector gene is 0.5k bp to 1.5k bp;

and a downstream sequence fragment of the Spot defoliation pathogenic effector gene, the downstream sequence fragment having a length of 0.5 kbp to 1.5 kbp.

5. The recombinant vector for knocking out a pathogenic bacterial effector gene of apple alternaria leaf spot of claim 4, wherein the expression vector is a PCX62 vector.