Effectors protein xopaa mutants and their use in rice xanthomonas oryzae resistance

Abstract

The application discloses an effector protein XopAA mutant and application thereof in Xanthomonas oryzae resistance, and belongs to the technical field of molecular biology. The application finds that the effector protein XopAA mutant can significantly affect the pathogenicity of Xanthomonas oryzae for the first time; spraying the effector protein XopAA mutant can significantly induce early immune response of rice and reduce the generation of bacterial leaf blight; the rice plants heterogeneously expressing the effector protein XopAA are more resistant to diseases than wild type plants. Therefore, the effector protein XopAA mutant has a good prospect in green prevention and treatment of bacterial diseases.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the XopAA mutant of the effector protein and its application in resistance to bacterial leaf streak in rice. Background Technology

[0002] Rice (Oryza sativa) is one of the most important food crops. Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola (Xoc), can cause up to 30% yield loss in rice every year, seriously restricting the development of the rice industry.

[0003] In current rice production, the control of rice diseases still relies mainly on the spraying of chemical agents. However, the overuse of pesticides not only damages the ecological environment, causing air, water, and soil pollution, but also leads to the residue and accumulation of toxic substances on the surface of crops. These harmful substances pose a potential threat to chronic diseases in humans. Therefore, developing green immune inducers or cultivating and applying broad-spectrum resistant varieties are effective alternatives to chemical control.

[0004] In an environment where rice and pathogens have long coexisted, plants have evolved pattern recognition receptors on their cell membrane surfaces. These receptors trigger primary immune responses by recognizing pathogen-related molecular patterns, including bursts of reactive oxygen species, calcium ion influx, callose deposition, and expression of defense genes. Therefore, seeking effective plant-derived formulations that can enhance rice resistance to bacterial leaf streak by triggering innate immune responses offers a green, environmentally friendly, precise, and efficient new approach to the control of this disease. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an effector protein XopAA mutant and its application in resistance to bacterial leaf streak of rice. The effector protein XopAA mutant of this invention is a phosphorylated and inactivated form of the effector protein XopAA (Xoc_2511) of Xanthomonas oryzae leaf streak pathogen, XopAA(S / A), which can significantly induce an early immune response in rice, reduce the occurrence of bacterial leaf streak, and has broad application prospects in the green control of bacterial diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an effector protein XopAA mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The XopAA mutant of the present invention exists in the phosphorylated inactivated form of the XopAA (Xoc_2511) effector protein of Xanthomonas rice leaf streak pathogen. Specifically, some phosphorylation sites (serine and threonine) in the effector protein XopAA are mutated to alanine through site-directed mutagenesis.

[0009] In a second aspect, the present invention provides the use of the above-described effector protein XopAA mutant in any one of the following (1)-(3):

[0010] (1) Activate rice immune signals;

[0011] (2) Improve rice's resistance to bacterial leaf streak;

[0012] (3) Prepare agents for the prevention and control of bacterial leaf streak disease in rice.

[0013] In the above applications, the XopAA mutant of the effector protein enhances the resistance of rice to bacterial leaf streak fungus through at least one of the following pathways (a)-(b):

[0014] (a) Inducing the expression of defense genes in rice;

[0015] (b) Triggers an outbreak of reactive oxygen species in rice.

[0016] A third aspect of the invention provides the use of a gene encoding a mutant of the effector protein XopAA in either (1) or (2) below:

[0017] (1) Improve the resistance of rice to bacterial leaf streak;

[0018] (2) Develop rice varieties resistant to bacterial leaf streak disease.

[0019] In the above applications, the gene encoding the XopAA mutant effector protein is a nucleic acid molecule as shown in i) or ii) below:

[0020] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.3;

[0021] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.4.

[0022] In a fourth aspect, the present invention provides the use of a recombinant expression vector or engineered bacteria containing a gene encoding a mutant of the effector protein XopAA in the following (1) or (2):

[0023] (1) Improve the resistance of rice to bacterial leaf streak;

[0024] (2) Develop rice varieties resistant to bacterial leaf streak disease.

[0025] In the above applications, the recombinant expression vector can be constructed using existing plant expression vectors or prokaryotic expression vectors. The vectors include binary Agrobacterium vectors and vectors for prokaryotic expression, such as pCXUN, pCAMBIA1300, pTA7001, pTA7002, pBin, PET-30a, PMAL-C2X, pGEX-4T, or other derived vectors. When constructing a recombinant expression vector using the effector protein XopAA or its non-phosphorylated form XopAA (S / A), any one or more enhancers and / or promoters can be combined, including translational enhancers or transcriptional enhancers, and enhancing, constitutive, tissue-specific, or inducible promoters, such as the 35S promoter, Ubiquitin promoter, and dexamethasone inducible promoter.

[0026] A fifth aspect of the present invention provides a method for improving the resistance of rice to bacterial leaf streak, comprising the step of heterologously expressing an effector protein XopAA mutant in rice.

[0027] In the above method, the XopAA mutant effector protein can be heterologously expressed in rice by exogenously transferring the gene encoding the XopAA mutant effector protein.

[0028] In a sixth aspect, the present invention provides an immune inducer for the prevention and control of bacterial leaf streak in rice, wherein the immune inducer uses the above-mentioned effector protein XopAA mutant as its active ingredient.

[0029] Preferably, the concentration of the XopAA mutant effector protein in the immune inducer is 0.01-0.5 mg / ml.

[0030] A seventh aspect of the present invention provides a method for controlling bacterial leaf streak of rice, comprising the following steps:

[0031] Before the onset of bacterial leaf streak in rice, the XopAA mutant of the effector protein or the aforementioned immune inducer is sprayed onto the rice leaves.

[0032] The beneficial effects of this invention are:

[0033] This invention is the first to discover that the XopAA mutant effector protein can significantly affect the pathogenicity of rice leaf streak causal agent; exogenous spraying of the XopAA mutant effector protein can significantly induce an early immune response in rice, reducing the occurrence of rice leaf streak disease; rice plants heterologously expressing the XopAA mutant effector protein show greater resistance to the disease than wild-type plants. Therefore, the XopAA mutant effector protein shows promising potential for the green control of bacterial diseases. Attached Figure Description

[0034] Figure 1The effector protein XopAA and its phosphorylated inactivated form XopAA (S / A) can alter the pathogenicity of XopAA.

[0035] Figure 2 Exogenous application of the effector protein XopAA or its phosphorylated inactivated form XopAA (S / A) can stimulate reactive oxygen species in rice.

[0036] Figure 3 Exogenous application of the effector protein XopAA or its phosphorylated inactivated form XopAA (S / A) can enhance rice resistance to RS105.

[0037] Figure 4 Rice expressing the effector protein XopAA is more resistant to bacterial leaf streak than wild-type rice. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] As mentioned earlier, rice is one of the most important food crops, and the occurrence of bacterial leaf streak disease severely restricts the improvement of rice yield and quality. Spraying chemical pesticides is currently one of the most commonly used control methods on the market, but the continuous accumulation of pesticides can cause irreversible damage to the ecological environment and human health. Developing green immune inducers or cultivating and applying broad-spectrum resistant varieties are effective alternatives to chemical control.

[0040] Therefore, the purpose of this invention is to provide the application of the effector protein XopAA in rice disease resistance. XopAA is a key component influencing the pathogenicity of Xanthomonas oryzae leaf streak pathogens and can also serve as an active ingredient in immune inducers. Exogenous application of XopAA can not only be recognized by rice receptors, triggering a burst of reactive oxygen species, but also enhance rice resistance. Therefore, utilizing molecular biology techniques for disease control and crop improvement is an effective measure consistent with the concept of green development.

[0041] The nucleotide sequence of the effector protein XopAA gene is shown in SEQ ID NO.1, as follows:

[0042]

[0043] The amino acid sequence of the effector protein XopAA is shown in SEQ ID NO.2, as follows:

[0044] MQIKTASHSPNAHPHAPVAGDGSLPADHSIEIEAGHGSGTVRPADGTQARACPSPASETKVHLTDLLSISSQRPSRPEVIPAIVQAGGDEGVPDVLPHPARSGASQPRLANPGEARDLESRLLMRDCSSRIAAFLGNLSVVQELRQFDDSAFLGKLRNELVTEPGCAEPKTLL AAMGEKVAELWSHLDKKASTPEEAEFLAEARRYLEPVMISLETDIHTLQHDASAAKRIYQGALTLLLYPLPLATLFTQKTGTYAAFNIASYTYTAIQLVSLMRRPTTDAKLFMKHAINRHSLVFFISLIYAVPTFYAKASPLQRNAGFTAGAAVAQGAMMFGLRLGQDLMDSMR LRFNGAFNRKRDLPDGFRDAIEGVVGDLRTGLSNVNRSVGEFQQDRRITPHMDRQLTFFKQDLSRVVTGLERLLATGTRNETPVAARDDAPGSLEAVRRTLEASFANNPDLKGKLALATVAFAVLGSNIALMRNNGLALPDFIADAVVSSTFLLSEALSPHVTHAGMNDSVSDT VGGMTIGLPFSVAAVMSSYMDDPRANPSGFIAGTVGYTAAYLLFGRVAGDVLSKGLMATSVALGWGQQQAIRLGRSAMALGFELVAGHSSPAAADVHDVAGVEMAGVPHEPLFFSPSQRASAERTLVGTLEQIGDEWHDARDEWEGESEATAGRDDPWVDAPAELPMHPAHQSE

[0045] To further enhance the efficacy of the effector protein XopAA, this invention unexpectedly discovered that by mutating some phosphorylation sites of the effector protein XopAA, a non-phosphorylated form XopAA (S / A) is obtained. Compared with the effector protein XopAA, the non-phosphorylated form XopAA (S / A) can further reduce the pathogenicity of bacterial leaf streak and improve the crop's resistance to bacterial leaf streak.

[0046] The nucleotide sequence of the effector protein XopAA(S / A) gene is shown in SEQ ID NO.3, as follows:

[0047]

[0048] The amino acid sequence of the effector protein XopAA(S / A) is shown in SEQ ID NO.4, as follows:

[0049] MQIKTASHAPNAHPHAPVAGDGSLPADHSIEIEAGHGSGTVRPADGTQARACPSPASETKVHLTDLLSISSQRPARPEVIPAIVQAGGDEGVPDVLPHPARSGASQPRLANPGEARDLESRLLMRDCSSRIAAFLGNLSVVQELRQFDDSAFLGKLRNELVTEPGCAEPKTLL AAMGEKVAELWSHLDKKASTPEEAEFLAEARRYLEPVMISLETDIHTLQHDASAAKRIYQGALTLLLYPLPLATLFTQKTGAYAAFNIASYTYTAIQLVALMRRPAADAKLFMKHAINRHSLVFFISLIYAVPTFYAKASPLQRNAGFTAGAAVAQGAMMFGLRLGQDLMDSMR LRFNGAFNRKRDLPDGFRDAIEGVVGDLRTGLSNVNRSVGEFQQDRRIAPHMDRQLTFFKQDLSRVVTGLERLLAAGARNETPVAARDDAPGSLEAVRRTLEASFANNPDLKGKLALATVAFAVLGSNIALMRNNGLALPDFIADAVVSSTFLLSEALAPHVTHAGMNDAVADT VGGMTIGLPFSVAAVMSAYMDDPRANPSGFIAGTVGYTAAYLLFGRVAGDVLSKGLMATSVALGWGQQQAIRLGRSAMALGFELVAGHSSPAAADVHDVAGVEMAGVPHEPLFFSPSQRAAAERTLVGTLEQIGDEWHDARDEWEGESEATAGRDDPWVDAPAELPMHPAHQSE

[0050] This invention obtained the effector protein XopAA and its non-phosphorylated form XopAA (S / A) through prokaryotic expression and purification. Exogenous spraying of the effector protein XopAA or its non-phosphorylated form XopAA (S / A) revealed that both the effector protein XopAA and its non-phosphorylated form XopAA (S / A) could significantly stimulate the burst of reactive oxygen species in rice.

[0051] The present invention describes the application of exogenous spray of effector protein XopAA or its non-phosphorylated form XopAA (S / A) followed by inoculation of RS105. It was found that both effector protein XopAA and its non-phosphorylated form XopAA (S / A) can significantly improve the resistance of rice to RS105.

[0052] By constructing transgenic plants that overexpress the effector protein XopAA, it was found that they exhibited a significant phenotype resistant to bacterial leaf streak.

[0053] Based on the above experimental results, it can be determined that the effector protein XopAA and its non-phosphorylated form XopAA (S / A) and their encoding genes have the effect of preventing and controlling bacterial leaf streak in rice, thus proposing this invention.

[0054] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels. The pathogen RS105 used in this invention is described in Tal2btargets and activates the expression of OsF3H. 03g to hijack OsUGT74H4 and synergistically interfere with rice immunity. New Phytol, 233:1864-1880 (Wu, T., Zhang, H., Yuan, B., Liu, H., Kong, L., Chu, Z. and Ding, X. (2022)).

[0055] Example 1:

[0056] 1. Experimental crop: Rice (Zhonghua 11)

[0057] 2. Test methods:

[0058] The upstream and downstream 300bp fragments of XopAA were fused into a single fragment and ligated into the pk18 mob sacB vector. Positive clones with correct sequencing were electroporated into RS105 cells and screened on NAN medium (containing Kana antibiotic, 5 g / L polypeptone, 1 g / L yeast extract, 3 g / L beef extract, 15 g / L agar) and NAS medium (containing no antibiotic, 5 g / L polypeptone, 1 g / L yeast extract, 3 g / L beef extract, 100 g / L sucrose, 15 g / L agar) at 28°C. After single-cell growth, PCR was used to detect the absence of the XopAA fragment. Those that were verified to be XopAA-deficient RS105 mutants (RS105_ΔXopAA) were identified.

[0059] XopAA and non-phosphorylated XopAA were ligated into the pVSP61 vector, respectively. Positive clones with correct sequencing were electroporated into RS105_ΔXopAA and cultured at 28°C for 3 days on PSA medium containing kana antibiotic (10 g / L peptone, 10 g / L sucrose, 1 g / L glutamate, 15 g / L agar). After single colonies grew, PCR detection was performed. Those that were verified were the complement strains carrying XopAA (RS105_ΔXopAA(XopAA)) and the complement strains carrying non-phosphorylated XopAA (RS105_ΔXopAA(S / A)).

[0060] RS105, the RS105 mutant lacking XopAA (RS105_ΔXopAA), the complement strain carrying XopAA (RS105_ΔXopAA(XopAA)), and the complement strain carrying non-phosphorylated XopAA (RS105_ΔXopAA(S / A)) were inoculated on potato sucrose agar (PSA) medium and cultured at 28°C for 3 days. The inoculated strains were then applied to leaves of 4-week-old Zhonghua 11 rice plants, and the length of the lesions was counted after 10 days.

[0061] Experimental results: Both the effector protein XopAA and the non-phosphorylated form XopAA (S / A) negatively regulate the pathogenicity of the physiological race RS105 of *Pseudomonas stylosa*; and compared with the effector protein XopAA, the non-phosphorylated form XopAA (S / A) has a more significant negative regulatory effect on the pathogenicity of the physiological race RS105 of *Pseudomonas stylosa*. Figure 1 ).

[0062] Example 2:

[0063] The full-length cDNA sequences of the effector protein XopAA and its non-phosphorylated form XopAA (S / A) (see SEQ ID NO. 1 and SEQ ID NO. 3) were amplified and ligated into the prokaryotic expression vector pMAL-C2X. The constructed vector was transformed into competent BL21 cells, and positive clones with correct sequencing results were selected for prokaryotic expression. BL21 cells transformed with the empty vector, XopAA, and non-phosphorylated XopAA (S / A) were induced to express at 37°C for 3 h in LB liquid medium containing 1 mM IPTG. After expression, the cells were resuspended in buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 1% NP-40), sonicated for 10 min, centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was collected. The obtained protein was purified using a kit (Beijing Polymer M5HiPer MBP protein purification chromatography medium) to obtain purified empty vector, XopAA, and unphosphorylated XopAA (S / A).

[0064] 5 mg of purified empty vector (CK), XopAA, and unphosphorylated XopAA (S / A) were evenly sprayed onto the leaves of Zhonghua 11 rice. Two hours after spraying, the leaves were cut off and soaked in a 0.5 mg / mL DAB solution. After culturing under light for 8 hours, the DAB solution was replaced with 95% ethanol and decolorized at 95°C.

[0065] Experimental results: Both the effector protein XopAA and its unphosphorylated form XopAA (S / A) can significantly stimulate the burst of reactive oxygen species in rice. Figure 2 ).

[0066] Example 3:

[0067] 5 mg of purified empty vector, XopAA, and non-phosphorylated XopAA (S / A) were evenly sprayed onto the leaves of Zhonghua 11 rice. RS105 was inoculated 2 hours after spraying, and the length of lesions was counted 14 days later.

[0068] Experimental results: Both the effector protein XopAA and its non-phosphorylated form XopAA (S / A) significantly improved the resistance of rice to RS105; and compared with the effector protein XopAA, the non-phosphorylated form XopAA (S / A) had a more significant effect on improving the resistance of rice to RS105. Figure 3 ).

[0069] Example 4

[0070] Genetic transformation of XopAA transgenic rice was carried out using the dexamethasone inducible promoter. The specific procedures are as follows:

[0071] XopAA was ligated into the pTA7001 vector, and the correctly sequenced clone was transformed into Agrobacterium EHA105. Mature Zhonghua 11 seeds were dehulled, sterilized with 75% alcohol, and placed on callus induction medium under 30°C light for one week. EHA105 cells carrying XopAA and callus tissue were co-cultured for 3 days. After co-culture, the callus was washed with sterile distilled water and placed on selection medium, incubated at 30°C light for 20 days, and then transferred to differentiation and rooting medium until complete plantlets formed. Seeds from positive PCR-detected plants were harvested, and the T1 generation was used for subsequent experiments.

[0072] XopAA expression was induced for 12 hours before being inoculated onto RS105 cells, and lesion length was measured 14 days later. The experiment was repeated 3 times.

[0073] Experimental results: The XopAA transgenic line exhibited significant resistance to RS105. Figure 4 ).

[0074] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mutant of the effector protein XopAA, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

4.

2. The use of the XopAA mutant of the effector protein according to claim 1 in any one of the following (1)-(3): (1) Activate rice immune signals; (2) Improve the resistance of rice to bacterial leaf streak; (3) Prepare agents for the prevention and control of bacterial leaf streak disease in rice.

3. The application according to claim 2, characterized in that, The XopAA mutant of the effector protein enhances rice resistance to bacterial leaf streak fungus through at least one of the following pathways (a)-(b): (a) Inducing the expression of defense genes in rice; (b) Triggers an outbreak of reactive oxygen species in rice.

4. Use of the gene encoding the XopAA mutant of the effector protein of claim 1 in either (1) or (2): (1) Improve the resistance of rice to bacterial leaf streak; (2) Cultivate rice varieties resistant to bacterial leaf streak.

5. The application according to claim 4, characterized in that, The gene encoding the XopAA mutant effector protein is a nucleic acid molecule as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.3; ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.

4.

6. The use of a recombinant expression vector or engineered bacteria containing a gene encoding the XopAA mutant effector protein of claim 1 in either (1) or (2) below: (1) Improve the resistance of rice to bacterial leaf streak; (2) Cultivate rice varieties resistant to bacterial leaf streak.

7. A method for improving the resistance of rice to bacterial leaf streak, characterized in that, include: The steps of heterologously expressing the XopAA mutant of the effector protein of claim 1 in rice.

8. An immune inducer for the control of bacterial leaf streak in rice, characterized in that, The immune inducer uses the XopAA mutant of the effector protein as described in claim 1 as its active ingredient.

9. The immune inducer according to claim 8, characterized in that, The concentration of the XopAA mutant effector protein in the immune inducer is 0.01-0.5 mg / ml.

10. A method for controlling bacterial leaf streak of rice, characterized in that, Includes the following steps: Before the occurrence of bacterial leaf streak disease in rice, the XopAA mutant of the effector protein as described in claim 1 or the immune inducer as described in claim 8 or 9 is sprayed onto the rice leaves.

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