Application of sulforaphane and derivatives thereof as bacterial effector protein transcription inhibitor
阅读说明：本技术 萝卜硫素及其衍生物在作为细菌效应蛋白转录抑制剂中的应用 (Application of sulforaphane and derivatives thereof as bacterial effector protein transcription inhibitor ) 是由 周俭民 雷晓光 王伟 杨靖 张健 于 2019-09-27 设计创作，主要内容包括：本发明公开了萝卜硫素及其衍生物在作为细菌效应蛋白转录抑制剂中的应用。本发明提供了萝卜硫素及其衍生物在如下1)-7)中任一种中的应用：1)防治病原细菌；2)提高植物对病原细菌的抗性；3)抑制病原细菌的致病力；4)抑制病原细菌III型分泌系统的功能；5)抑制病原细菌III型分泌系统效应蛋白相关基因的表达；6)抑制病原细菌转录因子hrpL的表达；7)作为病原细菌效应蛋白转录抑制剂。通过实验证明：萝卜硫素及其衍生物可通过特异抑制病原细菌III型分泌系统的转录来抑制病原细菌III型分泌系统的功能,既可抵御病原细菌的入侵又不破坏植物与有益微生物的互作,在防治植物病原细菌中具有广泛的应用前景。(The invention discloses application of sulforaphane and a derivative thereof as a bacterial effector protein transcription inhibitor. The invention provides application of sulforaphane and derivatives thereof in any one of the following 1) -7): 1) preventing and treating pathogenic bacteria; 2) increasing the resistance of plants to pathogenic bacteria; 3) inhibiting the pathogenicity of pathogenic bacteria; 4) inhibiting the function of pathogenic bacteria type III secretion system; 5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes; 6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL; 7) as an inhibitor of the transcription of effector proteins by pathogenic bacteria. Experiments prove that: the sulforaphane and the derivatives thereof can inhibit the function of a pathogenic bacterium III type secretion system by specifically inhibiting the transcription of the pathogenic bacterium III type secretion system, can resist the invasion of pathogenic bacteria without destroying the interaction between plants and beneficial microorganisms, and have wide application prospect in preventing and treating plant pathogenic bacteria.)
1. Use of sulforaphane or a sulforaphane derivative in at least one of the following 1) to 7);
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL;
7) as an inhibitor of the transcription of effector proteins by pathogenic bacteria.
2. Use of sulforaphane or a sulforaphane derivative for the preparation of a product having at least one of the following functions 1) to 6);
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL.
3. A product contains sulforaphane or sulforaphane derivatives as active ingredient;
the product has at least one function of 1) to 6) below;
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL.
4. The use according to claim 1 or 2 or the product according to claim 3, characterized in that: the improvement in the resistance of the plant to pathogenic bacteria is a reduction in the number of bacteria in the leaves of the plant.
5. Use or product according to any of claims 1 to 4, characterized in that: the sulforaphane or the sulforaphane derivative inhibits the expression of pathogenic bacteria III type secretion system effector protein related genes by targeting the 209 th cysteine of HrpS.
6. Use or product according to any of claims 1 to 5, characterized in that: the expression of the gene related to the effect protein of the pathogenic bacterium type III secretion system is used for reducing the relative transcription level of the gene related to the effect protein of the pathogenic bacterium type III secretion system;
and/or, the type III secretory system effector protein related gene is at least one of: avrPto, hopAM1, hopH1, hrpW1, hrpT1, hopC 1;
and/or, the inhibition of the expression of the pathogenic bacterial transcription factor hrpL is to reduce the relative transcription level of the pathogenic bacterial transcription factor hrpL.
7. Use or product according to any of claims 1 to 6, characterized in that: the pathogenic bacteria are pseudomonas syringae.
8. A method of increasing the resistance of a plant to pathogenic bacteria comprising the step of treating the plant with sulforaphane or a sulforaphane derivative.
9. The use or product according to any one of claims 1 to 7 or the method according to claim 8, characterized in that: the sulforaphane derivative is any one of the following: sulforaphane derivative AS1, sulforaphane derivative AS2, sulforaphane derivative AS3, sulforaphane derivative AS4, sulforaphane derivative AS5, sulforaphane derivative AS6, sulforaphane derivative AS7, and sulforaphane derivative AS 8.
10. The sulforaphane derivative AS7 or sulforaphane derivative AS8 of claim 9.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of sulforaphane and derivatives thereof as a bacterial effector protein transcription inhibitor.
Background
In the long-term co-evolution process of the plants and the pathogenic microorganisms, a series of complex and efficient protection mechanisms are gradually formed to resist the infection of the pathogenic microorganisms, and secondary metabolites of the plants play an important role. After the immune system of the plant is activated, the synthesis and release of secondary metabolites with antibacterial activity in the body of the plant can be induced, and the plant can be helped to resist the invasion of pathogenic microorganisms. The secondary metabolites involved in natural plant immunity are large in number and different in structure, and are classified into two major classes according to the synthetic mechanism and the mode of action: the antibacterial secondary metabolites constitutively stored in plants are called phytoanthracipins; and the antibiotic secondary metabolites, which are re-synthesized in response to the immune signal, are called phytoalexins.
The antibacterial secondary metabolites of the phytopanicillins class are stored in healthy plants in the form of active or inactive precursors, which are released and act upon the invasion of pathogenic microorganisms. Saponins (saponins), glucosinolates (glucosinolates) and cyanines (cyanogenins) are the major phytoanthracIPins in plants. Saponins are glycosides whose aglycones are triterpenes or spirostanols and are widely present in flowering plants. Avena sativa root saponins (Avenacins) derived from Avena sativa and alpha-lycoside (alpha-tomato) derived from Lycopersicon esculentum have in vitro inhibitory activity against the growth of fungi and oomycetes. Glucosinolates are important secondary metabolites of cruciferous plants, are derived from different amino acids, and are classified into three major classes, aliphatic, aromatic and indole, according to their side chains. Isothionaphthenate (isothiocyanurate) is an aliphatic glucosinolate derived from methionine and has activity in inhibiting the growth of pathogenic microorganisms such as fungi, bacteria and insects in vitro. Meanwhile, the isothiohydrogen ester also plays an important role in resisting the infection of Arabidopsis non-host bacteria. PEN2 and the monooxygenase CYP81F2 are key proteins catalyzing the synthesis of indole glucosinolates, and double mutants thereof show a phenotype of lacking resistance to pathogenic microorganisms such as barley whitefly (Blumeria graminis f.sp.hordei), pea powdery mildew (Erysiphe pisi) and soybean epidemic enzyme (Phytophthora brassicae).
Phytoalexins are a class of secondary metabolites that are resynthesized after stress of a pathogenic microorganism in a plant. At present, about 44 phytoalexins are found in cruciferae plants, including calalexin, spirobransin, rutalexin and brassinin, and the like, wherein the synthesis pathway and action mechanism of calalexin are deeply researched. Camalexin is an important plant protection agent, can be induced by various pathogenic microorganisms (such as bacteria, oomycetes, fungi and viruses), is derived from tryptophan and plays an important role in inhibiting the growth of pathogenic microorganisms such as fungi in plant cells and the expansion of the pathogenic microorganisms to peripheral cells like indole thioglucoside, and the production of the Camalexin depends on monooxygenases CYP71B15 and CYP71A 13.
Although the currently discovered plant protection agents have various varieties and different structures, synthetic routes and action objects, the basic action mechanism is similar, namely pathogenic bacteria are killed by inhibiting the basic life activities of pathogenic microorganisms, and the target is single. Such as: camalexin is a membrane interferon which can destroy the integrity of bacterial and eukaryotic cell membranes, and brassin is a mitochondrial inhibitor and antioxidant which can inhibit mitochondrial function. The traditional plant protection essence has the defects of no selectivity on acting objects, destruction of beneficial microorganisms in the environment while killing pathogenic microorganisms and easy drug resistance.
The type III secretion system (TTSS) of gram-negative bacteria such as salmonella and pseudomonas syringae is the major weapon of causing disease. The type III secretion system of pseudomonas syringae is coded by an allergic response and pathogenicity (hrp) gene cluster, and once bacteria feel exogenous signals, transcription of the hrp gene and assembly of the type III secretion system are started, so that the bacteria obtain pathogenicity. The bacterial type III secretion system is a syringe-like structure consisting of a substrate ring embedded in the inner membrane of bacteria and needle-like filaments protruding from the surface of bacteria, and can transport toxic proteins of bacteria into plant cells. These toxic proteins interfere with the plant's immune receptor recognition and signal transduction systems, disrupt the plant vesicle trafficking system, manipulate phytohormones to alter plant physiological activities, thereby aiding pathogen invasion.
Disclosure of Invention
An object of the present invention is to provide a novel use of sulforaphane or a sulforaphane derivative.
The invention provides the application of sulforaphane or sulforaphane derivatives in at least one of the following 1) to 7);
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL;
7) as an inhibitor of the transcription of effector proteins by pathogenic bacteria.
The invention also provides the application of the sulforaphane or the sulforaphane derivative in preparing a product with at least one function of the following 1) -6);
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL.
It is another object of the present invention to provide a product.
The active ingredient of the product provided by the invention is sulforaphane or a sulforaphane derivative;
the product has at least one function of 1) to 6) below;
1) preventing and treating pathogenic bacteria;
2) increasing the resistance of plants to pathogenic bacteria;
3) inhibiting the pathogenicity of pathogenic bacteria;
4) inhibiting the function of pathogenic bacteria type III secretion system;
5) inhibiting the expression of pathogenic bacteria type III secretory system effector protein related genes;
6) inhibiting the expression of the pathogenic bacteria transcription factor hrpL.
In the above use or product, the improvement in the resistance of the plant to pathogenic bacteria is a reduction in the number of bacteria in the leaves of the plant.
In the above application or product, the sulforaphane or the sulforaphane derivative inhibits expression of a pathogenic bacterium type III secretion system effector protein-related gene by targeting the cysteine at position 209 of the HrpS.
In the above application or product, the inhibiting of the expression of the pathogenic bacterium type III secretory system effector protein-associated gene is reducing the relative transcription level of the pathogenic bacterium type III secretory system effector protein-associated gene.
In the above application or product, the type III secretory system effector protein-related gene is at least one of: avrPto, hopAM1, hosph 1, hrpW1, hrpT1, hopC 1.
The invention finally provides a method for increasing the resistance of a plant to pathogenic bacteria.
The method of the present invention for increasing the resistance of a plant to pathogenic bacteria comprises the step of treating the plant with sulforaphane or a sulforaphane derivative.
In any of the above uses or products or methods, the pathogenic bacteria is a pathogenic bacteria having a type III secretion system, such as pseudomonas syringae. In a specific embodiment of the invention, the pathogenic bacteria is Pseudomonas syringae Δ saxAB/F/D/G.
In any of the above uses or products or methods, the plant may be a monocot or a dicot; the dicotyledonous plant may specifically be arabidopsis thaliana.
In any of the above applications or products or methods, the sulforaphane derivative is any one of: sulforaphane derivative AS1, sulforaphane derivative AS2, sulforaphane derivative AS3, sulforaphane derivative AS4, sulforaphane derivative AS5, sulforaphane derivative AS6, sulforaphane derivative AS7, and sulforaphane derivative AS 8.
The sulforaphane has the following structural formula:
the structural formulas of the sulforaphane derivatives AS1, AS2, AS3, AS4, AS5, AS6, AS7 and AS8 are respectively AS follows:
the sulforaphane or sulforaphane derivative of the present invention may be artificially synthesized or may be obtained by purchase.
The sulforaphane derivative AS7 or the sulforaphane derivative AS8 also belong to the protection scope of the invention. The preparation of the sulforaphane derivative AS7 is specifically described in example 1. The preparation of the sulforaphane derivative AS8 is specifically described in example 2.
The research of the invention finds that the sulforaphane and the derivative thereof can specifically inhibit the transcription of the effector protein related gene of the bacterial III type secretion system; the sulforaphane synthesis deficient mutant myb28/29 was less resistant to bacteria. Further studies found that sulforaphane specifically targets cysteine at position 209 of HrpS to inhibit transcription in the bacterial type III secretion system. Experiments prove that: the sulforaphane and the derivatives thereof can inhibit the function of a pathogenic bacterium III type secretion system by specifically inhibiting the transcription of the pathogenic bacterium III type secretion system, can resist the invasion of pathogenic bacteria without destroying the interaction between plants and beneficial microorganisms, and have wide application prospect in preventing and treating plant pathogenic bacteria.
Drawings
FIG. 1 is a structural formula of sulforaphane and its derivatives. FIG. 1a is the structural formula of sulforaphane. FIG. 1b is a structural formula of sulforaphane derivative.
FIG. 2 shows the synthesis of sulforaphane derivatives AS7 and AS 8.
FIG. 3 shows the expression of a gene related to the inhibition of type III secretory system effector protein by sulforaphane. and a is the expression of the sulforaphane inhibiting Pst DC3000 delta saxAB/F/D/G effector protein related gene in vitro. b is the expression level of the effector protein related gene in Pst DC3000 delta saxAB/F/D/G inoculated with the myb28/29 mutant is increased compared with the wild type, and the phenotype of the Pst DC3000 delta saxAB/F/D/G effector protein related gene expression level in the myb28/29 mutant can be restored by supplementing 20 mu M sulforaphane to the myb28/29 mutant.
FIG. 4 shows the reduced resistance of the myb28/29 mutant to Pst DC3000 Δ saxAB/F/D/G.
FIG. 5 shows that sulforaphane specifically targets cysteine 209 of HrpS. a is the transcription of the hpL specifically inhibited by the sulforaphane. b and c are the covalent conjugation of sulforaphane to cysteine 209 of HrpS. d and e are the inhibition of the expression of pseudomonas syringae effector protein related genes mediated by cysteine at position 209 of HrpS. F is the resistance of the cysteine at the position 209 of HrpS to the pseudomonas syringae mediated by sulforaphane, wherein delta sax represents Pst DC3000 delta saxAB/F/D/G, and delta sax C209A represents Pst DC3000 delta saxAB/F/D/G/HrpSC209AΔ sax C278A represents Pst DC3000 Δ saxAB/F/D/G/HrpSC278A。
FIG. 6 shows the inhibition of Pseudomonas syringae effector protein-related gene expression by sulforaphane and its derivatives. a is the relative transcription level of sulforaphane and sulforaphane derivatives AS1, AS2, AS3, AS4, AS5 and AS6 for inhibiting effector protein related genes avrPto and hrpL; b is the relative transcription level of sulforaphane derivatives AS7 and AS8 for inhibiting effector protein related genes avrPto and hrpL.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The sulforaphane in the following examples is a product of sigma aldrich trade ltd, and the formula is shown in fig. 1 a. Sulforaphane derivatives AS1, AS2, AS3, AS4, AS5, AS6 are described in Khiar, N.et al, environmental research and biology activities, J.Org.Chem.74,6002-6009(2009) "and" Chen, X.et al, New method for the synthesis of sulforesearch and related disorders. Synthesis 2011,3991 and 3996(2011) ". The structural formula of the sulforaphane derivative AS1-AS6 is shown in figure 1b, and the characterization data are AS follows:
AS1：1H NMR(400MHz,CDCl3)δ3.55(t,J＝6.4Hz,2H),2.53(t,J＝6.9Hz,2H),2.10(s,3H),1.86-1.77(m,2H),1.76-1.68(m,2H).13C NMR(101MHz,CDCl3)δ44.7,33.3,28.8,25.8,15.4。
AS2：1H NMR(400MHz,CDCl3)δ3.61(t,J＝6.3Hz,2H),3.06(t,J＝7.5Hz,2H),2.94(s,3H),2.06-1.96(m,2H),1.95-1.85(m,2H).13C NMR(101MHz,CDCl3)δ53.6,44.5,40.8,28.6,19.8.ESI+-MS:[M+H]+calculated value C6H12NO2S2 +194.0304; found 194.0302.
AS3：1H NMR(400MHz,CDCl3)δ7.67-7.58(m,2H),7.58-7.45(m,3H),3.60-3.45(m,2H),2.90-2.73(m,2H),1.93-1.69(m,4H).13C NMR(101MHz,CDCl3)δ157.1,143.4,131.1,129.3,123.9,115.1,55.9,44.6,28.9,19.5.ESI+-MS:[M+H]+Calculated value C11H14NOS2 +192.0511; found 192.0508.
AS4：1H NMR(400MHz,CDCl3)δ3.58(t,J＝6.1Hz,2H),2.77-2.60(m,4H),1.98-1.83(m,4H),1.32(t,J＝7.5Hz,3H).13C NMR(101MHz,CDCl3)δ50.5,45.8,44.6,29.0,20.1,6.7.ESI+-MS:[M+H]+Calculated value C7H14NOS2 +192.0511; found 192.0508.
AS5：1H NMR(400MHz,CDCl3)δ3.82-3.67(m,2H),2.86-2.73(m,2H),2.63(s,3H),2.26-2.17(m,2H).13C NMR(101MHz,CDCl3)δ51.0,44.1,38.9,23.6.ESI+-MS:[M+H]+Calculated value C5H10NOS2 +164.0198; found 164.0194.
AS6：1H NMR(400MHz,CDCl3)δ3.53(t,J＝6.4Hz,2H),2.77-2.60(m,2H),2.56(s,3H),1.86-1.77(m,2H),1.77-1.68(m,2H),1.67-1.51(m,2H).13C NMR(101MHz,CDCl3)δ54.1,44.7,38.6,29.5,25.8,21.9.ESI+-MS:[M+H]+Calculated value C7H14NOS2 +192.0511; found 192.0511.
Pseudomonas syringae Pst DC3000 Δ saxAB/F/D/G in the following examples is described in the literature "Fan, J.et al. Pseudomonas syringae Pst DC3000 delta saxAB/F/D/G is a bacterium obtained by knocking out saxAB/F/D/G gene in Pseudomonas syringae pv tomato DC3000 (Pst DC 3000). The saxAB/F/D/G can degrade the sulforaphane or the derivatives thereof and release the action of the sulforaphane, so that the pseudomonas syringae Pst DC3000 delta saxAB/F/D/G cannot degrade the sulforaphane or the derivatives thereof.
The sulforaphane synthesis-deficient mutant myb28/29 in the following examples is described in the literature "In E.E.et al.A systems biology approach derivatives a R2R3 MYB gene with a differentiation and overlapping functions in the regulation of antigenic glucosinolates PLoS ONE 2, e1322(2007) "and" Beekwilder, J., et al.the experiment of the sensitivity of the antigenic glucosinolates on selected heterologous proteins Arabidopsis thaliana PLoS ONE 3, e2068(2008) ", sulforaph synthesis-deficient mutant MYB28/29 is a mutant with loss of function of the Myb28 and Myb29 genes in the context of wild-type Arabidopsis thaliana (Columbia ecotype ). Sulforaphane is aliphatic glucosideThe Myb28 and Myb29 are key transcription factors for controlling the synthesis enzyme of the aliphatic glucoside pathway, and the content of the sulforaphane in the Myb28/29 mutant is extremely low.
The puc19 vector in the following examples is described in "Li, L.et al, the FLS2-associated kinase BIK1 direct phosphorous amides the NADPH oxidase RbohD to control plant immunity. cell Host Microbe 15, 329-338 (2014)".
pK18mobsacB in the following examples is described in "Feng, F., Yang, F., Rong, W., Wu, X.G., Zhang, J., Chen, S., He, C.Z., and Zhou, J. -M. (2012),. AXANTHOMONAS uridine 5' -monophophate transfer enzyme inhibitors plant immumkinases. Nature 485, 114. snake 118.".
The pet28a vector in the following examples is described in the literature "Li, L.et al, the FLS2-associated kinase BIK1 direct phosphorus amides the NADPH oxidase RbohD to control plant immunity, cell Host Microbe 15, 329-338 (2014)".
The KB liquid medium (1L) formulation in the following examples is as follows: peptone 29g, glycerol 8mL, dipotassium hydrogen phosphate 1.96g, heptahydrate and magnesium sulfate 1.52 g. KB solid medium (1L) was prepared by mixing 15g of agar with KB liquid medium (1L). KB-sucrose solid Medium (1L) KB solid medium (1L) was prepared by mixing 15g of agar and 100g of sucrose with KB liquid medium (1L).
The minimal medium (1L) formulation in the following examples is as follows: 6.8g of monopotassium phosphate, 1g of ammonium sulfate, 3.45mg of magnesium chloride hexahydrate, 100mg of sodium chloride and 1.8g of fructose, and the pH value is 5.7.
The formulation of LB medium (1L) in the following examples is as follows: 10g of tryptone, 5g of yeast extract and 5g of sodium chloride. LB solid medium (1L) was prepared by mixing 15g of agar with LB liquid medium (1L).
The genes and their Genbank numbers referred to in the following examples are shown in Table 1.
TABLE 1 genes and their Genbank numbers
Name of gene
Genbank number
saxA
PSPTO_1858
saxB
PSPTO_1859
saxF
PSPTO_3100
saxD
PSPTO_4304
saxG
PSPTO_2592
avrPto
PSPTO_4001
hopAM1
PSPTO_1022
hopH1
PSPTO_0588
hrpW1
PSPTO_1373
hrpT1
PSPTO_1390
hopC1
PSPTO_0589
hrpL
PSPTO_1404
hrpR
PSPTO_1379
hrpS
PSPTO_1380
trp
PSPTO_0159
Example 1 sulforaphane derivative AS7 and Process for its Synthesis
AS7 was synthesized according to the following steps:
(1) synthesis of compound 8:
mercaptoethylamine (compound 7) (300mg, 3.88mmol, 1eq) (purchased from Togaku, cat. No.: 60-23-1) and sodium hydroxide (233mg, 5.82mmol, 1.5eq) were dissolved in ethanol (7.5mL) and iodomethane (290. mu.L, 4.66mmol, 1.2eq) was added at 0 ℃. The mixture was stirred at room temperature for 3 hours, then spun dry and filtered to redissolve in chloroform to give the crude compound 8 (fig. 2A).
(2) Synthesis of compound 9:
the crude compound 8 was dissolved in ethyl acetate (30mL) and saturated sodium bicarbonate (20mL) and o-nitrobenzenesulfonyl chloride (1.29g, 5.82mmol, 1.5eq) was added at 0 ℃. The mixture was stirred at room temperature for 3 h, the organic phase washed with water and concentrated by rotary evaporation to give the crude product which was purified by column chromatography (petroleum ether/ethyl acetate 6:1) to give compound 9(900mg, 84%) as a colourless oil (fig. 2B).
(3) Synthesis of compound 10:
the crude product of compound 9(900mg, 3.26mmol, 1eq) was dissolved in tetrahydrofuran (10mL) and water (10mL), and sodium periodate (976mg, 4.56mmol, 1.4eq) was added at 0 ℃. The mixture was stirred at room temperature for 8 hours, spin-dried, water and ethyl acetate were added, ethyl acetate was extracted, the organic phase was washed with saturated brine, and dried over anhydrous sodium sulfate. Purification by column chromatography (petroleum ether/ethyl acetate ═ 6:1) gave compound 10(877mg, 92%) as a white solid (fig. 2C).
(4) Synthesis of compound 12:
compound 10(188mg, 0.64mmol, 1eq) was dissolved in 4mL of dimethylformamide, potassium carbonate was added at 0 ℃ and then stirred at room temperature for 15 minutes, cooled to 0 ℃ and compound 11(333.7mg, 1.38mmol, 1.5eq) dissolved in 2mL of dimethylformamide was added and stirred at room temperature for 15 hours. Ethyl acetate (10 mL. times.3) was extracted, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was evaporated under reduced pressure. Purification by column chromatography (dichloromethane/methanol 50:1) gave compound 12(242mg, 92%) as a white solid (fig. 2D).
(5) Synthesis of compound 13:
compound 12(242mg, 0.59mmol) was dissolved in 6mL of acetonitrile, and cesium carbonate and thiophenol were added at 0 ℃ and then stirred at normal temperature for 2 hours. The mixture was filtered through celite, and the solvent was evaporated under reduced pressure. Purification by column chromatography (dichloromethane/methanol ═ 10:1) gave compound 13(89mg, 86%) as a colorless liquid (fig. 2E).
(6) Synthesis of compound 14:
compound 13(40mg, 0.22mmol) was dissolved in 2mL of ethanol, and 4-pentyne formaldehyde, sodium cyanoborohydride, and acetic acid were added at 0 ℃ and then stirred at room temperature for 36 hours. Adding 40% sodium hydroxide to alkalinity, extracting with dichloromethane, drying with anhydrous sodium sulfate, filtering to remove the drying agent, and evaporating the solvent under reduced pressure. Purification by column chromatography (dichloromethane/methanol ═ 50:1) gave compound 14(20mg, 37%) as a colourless liquid (fig. 2F).
(7) Synthesis of compound AS 7:
compound 14(10.2mg, 0.042mmol) was dissolved in 2mL of diethyl ether, triphenylphosphine was added, and the mixture was stirred at 40 ℃ for 4 hours. Cooled to room temperature, 2mL of carbon disulfide was added and refluxed for 5 hours. The solvent was distilled off under reduced pressure. Purification by column chromatography (dichloromethane/methanol ═ 50:1) gave compound AS7(5.5mg, 51%) AS a colourless liquid (fig. 2G).
Compound AS7 characterization data are specifically AS follows:1H NMR(400MHz,CDCl3)δ3.70-3.52(m,2H),3.10-3.00(m,1H),2.93-2.84(m,2H),2.83-2.64(m,5H),2.63(s,3H),2.42-2.24(m,2H),1.95(t,J＝2.5Hz,1H),1.78-1.56(m,2H).13C NMR(101MHz,CDCl3)δ84.1,77.2,69.0,54.3,53.7,52.2,47.4,43.8,39.0,25.8,15.6.ESI+-MS:[M+H]+calculated value C11H19N2OS2 +259.0933; found 259.0932.
Example 2 sulforaphane derivative AS8 and Synthesis thereof
AS8 was synthesized according to the following steps:
(1) synthesis of compound 15:
compound 13(40mg, 0.22mmol) was dissolved in 2mL of diethyl ether, and 3-bromopropyne and cesium carbonate were added at 0 ℃ and then stirred at room temperature for 15 hours. Quenched with 5mL of water, extracted with dichloromethane (10 mL. times.3), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was evaporated under reduced pressure. Purification by column chromatography (dichloromethane/methanol ═ 50:1) gave compound 15(20mg, 43%) as a colourless liquid (fig. 2H).
(2) Synthesis of compound AS 8:
compound 15(10.7mg, 0.05mmol) was dissolved in 2mL of diethyl ether, triphenylphosphine was added, and the mixture was stirred at 40 ℃ for 4 hours. Cooled to room temperature, 2mL of carbon disulfide was added and refluxed for 5 hours. The solvent was distilled off under reduced pressure. Purification by column chromatography (dichloromethane/methanol ═ 50:1) gave compound AS8(6.2mg, 54%) AS a colourless liquid (fig. 2I).
Compound AS8 characterization data are specifically AS follows:1H NMR(400MHz,CDCl3)δ3.68-3.58(m,2H),3.51(t,J＝2.4Hz,2H),3.12-3.05(m,2H),2.94-2.79(m,4H),2.66(s,3H),2.27(t,J＝2.3Hz,1H).13C NMR(126MHz,CDCl3)δ77.4,74.0,53.4,53.1 46.8,43.6,42.3,39.0.ESI+-MS:[M+H]+calculated value C9H15N2OS2 +231.0620; found 231.0619.
Example 3 application of sulforaphane in inhibition of expression of Pseudomonas syringae Effector related genes
Sulforaphane for inhibiting expression of pseudomonas syringae effector protein
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ and 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected.
2. After completion of step 1, the obtained cells were washed twice with water, and then resuspended in a minimal medium until the OD became 0.4, to obtain resuspended cells.
3. After completion of step 2, sulforaphane was added to the resuspended cells to a final concentration of 20. mu.M, and after 6 hours, the expression of avrPto, hopAM1, hoppH 1, hrpW1, hrpT1 and hopC1 genes related to effector proteins (effector proteins secreted by type III secretory system) in Pst DC 3000. delta. saxAB/F/D/G cells were detected by a real-time fluorescent quantitative PCR method. The detection primer sequences are shown in table 2.
TABLE 2 primer sequences
Primer name
Primer sequences
16S-RT-F
CATTGAGACAGGTGCTGCAT
16S-RT-R
CACCGGCAGTCTCCTTAGAG
hopH1-RT-F
GCAGAGGAAGCACTTGACCA
hopH1-RT-R
TGCTTGCTTTGTCTGGTGAC
hopC1-RT-F
ATGGCATCAGTCAGCGTG
hopC1-RT-R
TAAACTTTCTGTGCCAGCCATCG
hopAM1-RT-F
GGCGTAAAGAACATTGTCTC
hopAM1-RT-R
GAAATCAGAACCCAGCCA
hopT1-RT-F
ATGATGATTCGTAGCCTAACG
hopT1-RT-R
CTAATCCTTTAACTGCACGACA
hrpW1-RT-F
CACTGCCGACAAATCTATG
hrpW1-RT-R
AGATTAGTGACCGCTGCG
avrPto-RT-F
GCCTGAGAAATCGCTACAACA
avrPto-RT-R
CGTTCGGGTTCATAGTCGCA
The results show that: 20 μ M sulforaphane reduced the expression level of Pst DC3000 Δ saxAB/F/D/G effector-related genes avrPto, hopAM1, hopH1, hrpW1, hrpT1, and hopC1 to about 30% of the original level (FIG. 3 a).
Secondly, the sulforaphane inhibits the expression of pseudomonas syringae effector protein related genes in arabidopsis thaliana
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ at 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected. The obtained cells were washed twice with water, and then diluted with sterile water until the OD was 0.1.
2. After completion of step 1, Pst DC3000 Δ saxAB/F/D/G bacterial suspension with OD ═ 0.1 was inoculated into mature leaves of four-week-old wild-type arabidopsis thaliana Col-0 and myb28/29 mutants, respectively (bacterial suspension was injected over the entire leaves), and 24 hours after inoculation, expression of Pst DC3000 Δ saxAB/F/D/G effector-related genes avrPto, hom 1, hopH1, hrpW1, hrpT1, and hopC1 in Col-0 and myb28/29 mutants, respectively, was examined.
The results show that: the expression level of the Pst DC3000 Δ saxAB/F/D/G effector-associated gene in the myb28/29 mutant inoculated with Pst DC3000 Δ saxAB/F/D/G was significantly higher than that of the wild type inoculated with Pst DC3000 Δ saxAB/F/D/G (FIG. 3 b).
Thirdly, exogenous application of sulforaphane can inhibit expression of Pst DC3000 delta saxAB/F/D/G effector protein related genes in myb28/29 mutant
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ at 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected. The obtained cells were washed twice with water, and then diluted with sterile water until the OD was 0.1.
2. After step 1 was completed, the following sets of experiments were performed:
col-0: pst DC3000 delta saxAB/F/D/G bacterial liquid with OD being 0.1 is inoculated to wild type arabidopsis thaliana Col-0, and expression of Pst DC3000 delta saxAB/F/D/G effector protein related genes avrPto, hopAM1, hopH1, hrpW1, hrpT1 and hopC1 in Col-0 is detected 24 hours after inoculation.
myb 28/29: pst DC3000 delta saxAB/F/D/G bacterial liquid with OD being 0.1 is inoculated into myb28/29 mutant, and 24 hours after inoculation, expression of Pst DC3000 delta saxAB/F/D/G effector related genes avrPto, hopAM1, hopH1, hrpW1, hrp 1 and hopC1 in the myb28/29 mutant is detected.
myb28/29+20 μ M sulforaphane: sulforaphane was added to Pst DC3000 Δ saxAB/F/D/G bacterial suspension with OD ═ 0.1 to a final concentration of 20 μ M, to obtain a bacterial suspension containing sulforaphane. The bacterial suspension containing sulforaphane was inoculated with myb28/29 mutant, and 24 hours after inoculation, the expression of Pst DC3000 Δ saxAB/F/D/G effector-related genes avrPto, hopAM1, hopH1, hrpW1, hrpT1 and hopC1 in myb28/29 mutant was tested.
The results show that: exogenous application of 20. mu.M sulforaphane to myb28/29 mutant inoculated with Pst DC 3000. delta. saxAB/F/D/G bacterial suspension inhibited the expression of Pst DC 3000. delta. saxAB/F/D/G effector-related genes in the mutant (FIG. 3 c).
Example 4 reduction of resistance of the myb28/29 mutant to Pseudomonas syringae
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ at 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected. The obtained cells were washed twice with water, and then diluted with sterile water until the OD was 0.001.
2. After completion of step 1, Pst DC3000 Δ saxAB/F/D/G strain with OD ═ 0.001 was inoculated with wild type arabidopsis thaliana Col-0 and myb28/29 mutants, respectively. The inoculation method was the same as in example 3.
3. After completion of step 2, the number of bacteria in the leaves of the Col-0 and myb28/29 mutants inoculated on day 0 and day 3, respectively, was determined. The method comprises the following specific steps: the area obtained by the punch was 0.5652cm2The leaf of (4) was placed in a centrifuge tube containing 100. mu.L of sterile water, gently ground into a homogenate, and the number of bacteria was titrated by a dilution method.
The results show that: the average number of bacteria in Col-0 inoculated with Pst DC 3000. delta. saxAB/F/D/G was 14541313/cm2(ii) a The average number of bacteria in the Pst DC3000 Δ saxAB/F/D/G inoculated mutant was 40273355/cm2(ii) a The number of bacteria in the myb28/29 mutant inoculated with Pst DC3000 Δ saxAB/F/D/G was significantly greater than the wild type inoculated with Pst DC3000 Δ saxAB/F/D/G (FIG. 4). Illustrating that the deletion mutant of sulforaphane synthesis (myb28/29 mutant) has reduced resistance to Pst DC3000 delta saxAB/F/D/GSulforaphane can improve the resistance of plants to pathogenic bacteria.
Example 5 specific targeting of sulforaphane to cysteine 209 of HrpS
Expression of sulforaphane inhibition transcription factor hrpL
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ at 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected.
2. After completion of step 1, the obtained cells were washed twice with water, and then resuspended in a minimal medium until the OD became 0.4, to obtain resuspended cells.
3. And (3) after the step 2 is finished, adding sulforaphane into the resuspended thalli to enable the final concentration of the sulforaphane in the resuspended thalli to be 20 mu M, and detecting and controlling the expression conditions of Pst DC3000 delta saxAB/F/D/G effector protein transcription related genes hrpR, hrpS, lon-1, rpoN, RhpR, RhpS and hrpL by using a real-time fluorescent quantitative PCR method after 6 hours. The primer sequences are shown in Table 3.
TABLE 3 primer sequences
Primer name
Primer sequences
16S-RT-F
CATTGAGACAGGTGCTGCAT
16S-RT-R
CACCGGCAGTCTCCTTAGAG
lon1-RT-F
GCAACTGGGCAAGAAAGTC
lon1-RT-R
GCCTTCATCTGCTCGTTCA
rpoN-RT-F
AAACCCTATGCTGGAACGGC
rpoN-RT-R
CCACTCGTCATCATCGTTGCT
hrpR-RT-F
ACTGCTGCGTGTGTTGGAGA
hrpR-RT-R
AAGGCTGGCAAGTGAAGCGT
hrpS-RT-F
ATTGCTGAGGGTGCTGGAAA
hrpS-RT-R
AACATCGGGAACGGGAACA
rphR-RT-F
TCACCGATGAAACCTTCG
rphR-RT-R
AAGACTCTGTGCGGTCGTTC
rphS-RT-F
TGACACAGGACCTCAACGAC
rphS-RT-R
CGCTGTCTTCAATCACGATG
hrpL-RT-F
CTGCGTAACGAGCACAAGTT
hrpL-RT-R
GCGACACTTCCAGCACTTT
The results show that: sulforaphane reduced the expression level of the effector protein transcription-related factor hrpL to about 30% (FIG. 5a), without affecting the expression of other genes. Since hrpR and hrpS form a heterothermic polymer to activate transcription of hrpL, sulforaphane may regulate the functions of hrpR and hrpS.
II, the sulforaphane is covalently bonded with the 209 th cysteine of HrpS
1. Mu.g of his-hrpS protein purified in vitro was reacted with 0.25. mu.M Sulforaphane (SFN) for 2 hours at room temperature. The preparation of the in vitro purified his-hrpS protein was as follows:
1) extracting the genomic DNA of the Pst DC3000 delta saxAB/F/D/G strain, amplifying by using the extracted genomic DNA as a template and adopting hrpS-HIS-BamHI-F and hrpS-HIS-XhoI-R primers (the primer sequences are shown in Table 4) to obtain an hrpS gene fragment, and constructing the hrpS gene fragment between BamHI and XhoI sites in a pet28a vector to obtain an HrpS expression vector pet28 a-hrpS.
TABLE 4 primer sequences
Primer name
Primer sequences
hrpS-HIS-BamHI-F
AGCAAATGGGTCGCGGATCCATGAGTCTTGATGAAAGGTTT
hrpS-HIS-XhoI-R
CAGTGGTGGTGGTGGTGGTGTCAGATCTGCAATTCTTTGATG
2) The HrpS expression vector pet28a-hrpS obtained in 1) was transformed into BL21 expression strain (Beijing holotype gold organism), and a single clone was selected and cultured overnight at 37 ℃ in LB solid medium containing 50. mu.g/mL kanamycin to obtain a transformant.
3) The monoclonal transformant obtained in 2) was picked up and cultured at 37 ℃ in 2mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and when a certain cell concentration (OD ═ 1) was reached, 100. mu.L of the transformant was taken out and plated on an LB plate containing 50. mu.g/mL of kanamycin and cultured overnight at 37 ℃.
4) The bacteria on the plate obtained in 3) were resuspended in 10mL of LB medium, and transferred to 1L of LB liquid medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ under shaking at 200 g.
5) When the concentration reached OD 0.4-0.6, IPTG (4mM) was added. The cells were cultured at 16 ℃ and 200g for 16 hours with shaking, and the cells were collected.
6) The cells collected in 5) were resuspended in 30mL of PBS buffer (containing 1 Xthe protease inhibitor cocktail, Roche).
7) The cells were disrupted by sonication at 35% intensity for 2 minutes, and suspended for 10 seconds every 5 seconds. Sonication was performed on ice.
8) The homogenate of the sonicated cells was centrifuged at 45,000g at 4 ℃ for 30 minutes to obtain a supernatant.
9) Balancing: nickel columns (NI-NTA, GE)1mL affinity columns were equilibrated with at least 5mL (5 column volumes) of PBS.
10) Loading: the supernatant obtained in 8) was filtered through a 0.45 μm filter, and the filtered sample was sufficiently bound to a nickel column using AKATA protein chromatograph (GE).
11) And (3) eluting the hybrid protein: the hetero-proteins adsorbed on the nickel column were eluted with 5mL of a washing solution (1 XPBS, 25mM imidazole).
12) Eluting the target protein: the target protein was eluted with 5mL of an eluent (1 XPBS, 300mM imidazole) and the eluent was collected.
13) Regenerating a column: the column was washed with 5mL of eluent, 5mL of equilibration solution and 5mL of 20% ethanol in sequence and finally stored in 20% ethanol.
14) Pouring the eluent obtained in the step 12) into a concentration tube, centrifuging at 4 ℃ and 4000g until the volume of the protein solution is 1-1.5mL, adding 15mL of PBS buffer (containing 1 Xprotease inhibitor cocktail (Roche) and 10% of glycerol), centrifuging at 4 ℃ and 4000g until the volume of the protein solution is less than 0.5mL, storing the protein solution for subsequent tests at 4 ℃ after concentration measurement, and storing the rest of the protein solution at-80 ℃ after quick freezing in liquid nitrogen.
2. After completion of step 1, 50. mu.M of the thiol-reactive probe IPM (Kerafast) was added to the reaction mixture and incubated at room temperature for one hour.
3. After completion of step 2, the precipitate was digested with pancreatin (enzyme to substrate ratio 1: 50 by volume) at room temperature for 2 hours.
4. After completion of step 3, the digested peptide fragment was desalted using a C18 column and the product was dried by suspension.
5. After completion of step 4, redissolve with pH 6.030% CAN.
6. After completion of step 5, the peptide fragments were biotinylated and 20. mu.L ddH was added to each 1mg peptide sample2O and 10. mu.L of acetonitrile, vortexed sufficiently, pH was measured to be around 6.0, then 1. mu.L of 40mM light/heavy-azide-UV-biotin reagent was added to each of the peptide fragments of the NaHS treatment and blank control, vortexed sufficiently, followed by 4. mu.L of sodium ascorbate at stock concentration of 100mM, 1. mu.L of TBTA ligand at stock concentration of 50mM, and 4. mu.L of copper sulfate at stock concentration of 100mM, vortexed sufficiently, and reacted at room temperature in the dark for 2 hours, during which vortexed once every 20 minutes.
7. After step 6 was completed, the light and heavy isotope-labeled peptide fragments were mixed together and subjected to strong cation exchange using a microspin SCX column to remove residual click chemistry reagents. The method comprises the following specific steps:
1) the SCX column was placed in a 2mL centrifuge tube, 500. mu.L of HPLC grade methanol was added, 100g was centrifuged for 1 min, and the eluate was discarded.
2) Add 500. mu.L of HPLC grade water, centrifuge for 4 min at 100g, discard the eluate and repeat once.
3) Add 500. mu.L of SCX equilibration buffer, centrifuge at 100g for 2 minutes to allow the SCX equilibration to soak through the filler, and allow to stand at room temperature for 1 hour.
4) Centrifuge at 100g for 2 min and discard the eluate.
5) Add 500. mu.L SCX loading buffer, centrifuge for 4 min at 100g, discard the eluate and repeat once.
6) Add 150. mu.L of sample, centrifuge at 100g for 20 seconds, and discard the eluate.
7) Add 150. mu.L SCX loading buffer, centrifuge at 100g for 20s, and discard the eluate.
8) The SCX column was placed in a new 2mL centrifuge tube, 150 μ L of SCX eluent was added, 100g was centrifuged for 90s, the eluent was collected and transferred to a 15mL centrifuge tube containing 10mL of streptavidin binding buffer.
8. The streptomycin avidin agarose gel bead enrichment comprises the following specific steps:
1) 200 μ L of streptavidin magnetic beads were suspended in 10mL of streptavidin binding buffer, centrifuged at 25 ℃ at 1700g for 3 minutes, the supernatant was discarded, and the procedure was repeated once and finally resuspended in 200 μ L of streptavidin binding buffer for further use.
2) Adding the resuspended streptavidin beads to the product obtained in step 7-8), reacting at room temperature in the dark for 2 hours, centrifuging at 1700g for 3 minutes, and discarding the supernatant.
3) Add 10mL streptavidin binding buffer heavy suspension, 1700g centrifugation for 3 minutes, discard the supernatant, repeat three times.
4) Add 10mL HPLC grade water heavy suspension, 1700g centrifugation for 3 minutes, discard the supernatant, repeat.
9. Photolysis, comprising the following steps: with 5 volumes of photolysis buffer (25mM NH)4HCO3) Resuspending the streptavidin beads from 4) of step 8, transferring them to a glass vial, and adding a magnetic stirring rotor, placing on a magnetic stirrer, and photolyzing for 2 hours under 365nm ultraviolet light. After photolysis was complete, all liquid in the vial was transferred to a new EP tube and centrifuged at 1700gAfter 3 minutes, the supernatant was collected. Concentrate to about 200. mu.L with a rotary evaporator.
10. Desalting the peptide segment by using an HLB SPE desalting column, and specifically comprising the following steps:
1) 1mL of acetonitrile was added to activate the desalting column and flow through.
2) The column was washed with 2mL HPLC grade water and run through.
3) And (3) adding the product obtained in the step (9) to a desalting column and flowing through.
4) The column was washed with 1mL HPLC grade water and run through.
5) Adding 1mL of HLB solvent A to elute the peptide segment, and collecting the eluent.
6) The peptide-containing eluate was evaporated to dryness using a rotary evaporator, and the evaporated peptide sample was dissolved in 12. mu.L of 0.1% formic acid, centrifuged at 12,000rpm for 10 minutes, and 5. mu.L of the supernatant was subjected to LC-MS/MS analysis.
The results show that: sulforaphane was covalently conjugated to cysteine 209 of HrpS (fig. 5b and 5 c).
Thirdly, the sulforaphane inhibits the expression of the Pst DC3000 delta saxAB/F/D/G III type secretion system related gene by targeting the HrpS 209 th cysteine
To further verify whether sulforaphane inhibits the expression of the Pst DC3000 Δ saxAB/F/D/G type III secretion system-related genes by targeting cysteine at position 209 of HrpS. Firstly, mutating the 209 th cysteine and the 278 th cysteine of HrpS in Pst DC3000 delta saxAB/F/D/G by a homologous recombination method to obtain Pst DC3000 delta saxAB/F/D/G/HrpSC209AAnd Pst DC3000 delta saxAB/F/D/G/HrpSC278AA mutant; and then the sulforaphane is used for treating the mutant strain and detecting the expression condition of the III type secretion system effector protein related gene in the mutant strain. The method comprises the following specific steps:
1、Pst DC3000ΔsaxAB/F/D/G/HrpSC209Aand Pst DC3000 delta saxAB/F/D/G/HrpSC278AConstruction of mutants
1) Extracting the genomic DNA of the Pst DC3000 delta saxAB/F/D/G strain, amplifying by using the extracted genomic DNA as a template and using primers hrpS-KpnI-puc-F and hrpS-HA-puc-SalI-R (the primer sequences are shown in Table 5) to obtain an HrpS gene fragment (sequence 1), and constructing the hrpS gene fragment between KpnI and SalI sites of an intermediate vector puc19 to obtain a recombinant vector.
2) The recombinant vector was used as a template, and the HrpS-209A-F and HrpS-209A-R primers (the primer sequences are shown in Table 5) were used for amplification to obtain HrpSC209AFragment (HrpS)C209AThe fragment is obtained by mutating TGC to GCT at the 625-627 position of the HrpS gene fragment); the recombinant vector is used as a template, and HrpS-278A-F and HrpS-278A-R primers (the primer sequences are shown in Table 5) are adopted for amplification to obtain HrpSC278AFragment (HrpS)C278AThe fragment is obtained by mutating TGT into GCC at positions 823-825 of the HrpS gene fragment).
3) Mixing HrpSC209AThe fragment is constructed on a suicide plasmid pK18mobsacB to obtain pK18mobsacB-HrpSC209A(ii) a Mixing HrpSC278AThe fragment is constructed on a suicide plasmid pK18mobsacB to obtain pK18mobsacB-HrpSC278A。
4) Respectively converting pK18mobsacB-HrpS by using an electric shock transformation methodC209AAnd pK18mobsacB-HrpSC278ATransferring the recombinant bacteria into Pst DC3000 delta saxAB/F/D/G to respectively obtain recombinant bacteria, then respectively incubating the recombinant bacteria at 28 ℃ for 4 hours, spreading the recombinant bacteria on a KB plate containing 100 mu G/mL rifampicin and 50 mu G/mL kanamycin, culturing at 28 ℃ for 48 hours, and screening positive clones.
5) Positive single colonies were picked and placed in 1mL KB liquid medium, 28 ℃, 220rpm culture overnight.
6) The culture solution in 5) was spread on a KB plate containing 10% sucrose and cultured at 28 ℃ for 48 hours.
7) The clones obtained in 6), streaked on both the KB (containing rifampicin resistance) and KB (containing rifampicin and kanamycin resistance) plates, were candidate positive clones which could grow on KB (containing rifampicin resistance) but could not grow on KB (containing rifampicin and kanamycin resistance).
8) DNA of candidate positive clones was extracted, and the hrpS gene was amplified using HrpS-mosB-F-BamHI and HrpS-mosB-R-HindIII primers (see Table 5 for primer sequences), sequenced, and the final positive clone was determined after sequencing.
The primer sequences are shown in Table 5.
TABLE 5 primer sequences
Primer name
Primer sequences
hrpS-KpnI-puc-F
ACGGGGGACGAGCTCGGTACCATGAGTCTTGATGAAAGGT
hrpS-HA-puc-SalI-R
AACATCGTATGGGTAGTCGACGATCTGCAATTCTTTGATGC
HrpS-209A-F
GTTCCCGTTCCCGATGTTGCTCCACTGCTGCACAAAG
HrpS-209A-R
CTTTGTGCAGCAGTGGAGCAACATCGGGAACGGGAAC
HrpS-278A-F
CTCAAGCGCCACGACAATGCCGTGGATTCGGTAAGCCTGG
HrpS-278A-R
CCAGGCTTACCGAATCCACGGCATTGTCGTGGCGCTTGAG
HrpS-mosB-F-BamHI
AGCTCGGTACCCGGGGATCCATGAGTCTTGATGAAAGGTT
HrpS-mosB-R-HindIII
CGACGGCCAGTGCCAAGCTTTCAGATCTGC AATTCTTTGA
Pst DC3000ΔsaxAB/F/D/G/HrpSC209AThe mutant strain (marked as delta sax C209A in the figure) is a strain obtained by mutating TGC to GCT at the 625-627 position of an HrpS gene fragment in the genome of the Pst DC3000 delta saxAB/F/D/G strain. Pst DC3000 delta saxAB/F/D/G/HrpSC209AThe HrpS protein expressed in the mutant strain is obtained by mutating cysteine C at position 209 of wild type HrpS protein into alanine A.
Pst DC3000ΔsaxAB/F/D/G/HrpSC278AThe mutant strain (marked as delta sax C278A in the figure) is a strain obtained by mutating TGT from 823-825 site of HrpS gene fragment in genome of Pst DC3000 delta saxAB/F/D/G strain to GCC. Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe HrpS protein expressed in the mutant strain is obtained by mutating cysteine C at position 278 of wild type HrpS protein into alanine A.
2. Expression of the response protein in the mutant strains
The expression of effector proteins in each of the mutant strains constructed in step 2 was examined according to the method in example 3.
The results show that: in a minimal medium, Pst DC3000 delta saxAB/F/D/G/HrpSC209AThe transcription levels of the effector proteins hrpT1 and hrpW1 in the mutant strain were substantially identical to those in Pst DC3000 Δ saxAB/F/D/G strain, indicating that the mutation of cysteine at position 209 of the HrpS protein to alanine did not affect the function of the HrpS protein. However, Pst DC3000 delta saxAB/F/D/G/HrpSC209AThe mutant strain is insensitive to sulforaphane, namely 20 mu M sulforaphane can not inhibit Pst DC3000 delta saxAB/F/D/G/HrpSC209AExpression of the effector proteins hrpT1 and hrpW1 in the mutant strain. While Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe transcription levels of the effective proteins hrpT1 and hrpW1 in the mutant strain were weaker than those in the Pst DC3000 Δ saxAB/F/D/G strain, indicating that the mutation of the 278 th cysteine of the HrpS protein to alanine affected the function of the HrpS protein. However, the Pst DC 3000. delta. saxAB/F/D/G wild strain and Pst DC 3000. delta. saxAB/F/D/G/HrpS were treated with 20. mu.M sulforaphaneC278AAfter the mutant strain, the expression levels of effector proteins hrpT1 and hrpW1 were reduced to about 30% (FIG. 5D and FIG. 5e), indicating that Pst DC3000 Δ saxAB/F/D/G wild strain and Pst DC3000 Δ saxAB/F/D/G/HrpSC278AThe mutant strains all responded to sulforaphane treatment.
In conclusion, sulforaphane inhibits the expression of effector protein-related genes of pathogenic bacteria type III secretion systems by targeting the cysteine at position 209 of HrpS.
Fourthly, sulforaphane inhibits the resistance of plants to Pst DC3000 delta saxAB/F/D/G by targeting cysteine at position 209 of HrpS.
1. Respectively picking Pst DC3000 delta saxAB/F/D/G and Pst DC3000 delta saxAB/F/D/G/HrpSC209AAnd Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe single colonies of (4) were cultured overnight at 28 ℃ and 220rpm in KB medium, centrifuged at 4000rpm for 10 minutes, and the cells were collected. The obtained cells were washed twice with water, and then diluted with sterile water until the OD was 0.001.
2. After completion of step 1, Pst DC 3000. delta. saxAB/F/D/G/HrpS with OD ═ 0.001C209AAnd Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe wild type Arabidopsis thaliana Col-0 and myb28/29 mutants were inoculated with the bacterial solutions, respectively. The inoculation method was the same as in example 3.
3. After step 2 is completed, Pst DC3000 delta saxAB/F/D/G and Pst DC3000 delta saxAB/F/D/G/HrpS in Col-0 and myb28/29 mutant leaves inoculated on days 0 and 3 respectively are detectedC209AAnd Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe number of bacteria. The method comprises the following specific steps: the area obtained by the punch was 0.5652cm2The leaf of (4) was placed in a centrifuge tube containing 100. mu.L of sterile water, gently ground into a homogenate, and the number of bacteria was titrated by a dilution method.
The results show that: 1) the average number of bacteria in Col-0 inoculated with Pst DC 3000. delta. saxAB/F/D/G was 7939667/cm2(ii) a The average number of bacteria in myb28/29 mutant inoculated with Pst DC3000 delta saxAB/F/D/G was 19185686/cm2. 2) Inoculation of Pst DC3000 delta saxAB/F/D/G/HrpSC209AThe average value of the number of bacteria in Col-0 (b) was 6767516/cm2(ii) a The average number of bacteria in myb28/29 mutant inoculated with Pst DC3000 delta saxAB/F/D/G was 7088199/cm2. 3) Inoculation of Pst DC3000 delta saxAB/F/D/G/HrpSC278AThe average value of the number of bacteria in Col-0 (b) was 1179523/cm2(ii) a The average number of bacteria in myb28/29 mutant inoculated with Pst DC3000 delta saxAB/F/D/G was 4385300/cm2。
Although mutant strain Pst DC3000 delta saxAB/F/D/G/HrpSC209AAnd Pst DC3000 delta saxAB/F/D/G/HrpSC278AIs reduced, but the two bacteria respond differently to SFN. Inoculation of Pst DC3000 delta saxAB/F/D/G and Pst DC3000 delta saxAB/F/D/G/HrpSC278AAt this time, the number of bacteria in the myb28/29 mutant was significantly greater than that in the wild type, whereas the Pst DC3000 Δ saxAB/F/D/G/HrpS was inoculatedC209AThe number of bacteria in the myb28/29 mutant was comparable to the wild type (FIG. 5 f). It is said that cysteine 209 of HrpS mediates resistance of sulforaphane to Pst DC3000 Δ saxAB/F/D/G.
Example 6 application of sulforaphane derivatives to inhibition of Pseudomonas syringae Effector protein expression
1. Single colonies of Pst DC 3000. delta. saxAB/F/D/G were picked and placed in KB medium, incubated overnight at 28 ℃ at 220rpm, centrifuged at 4000rpm for 10 minutes, and the cells were collected.
2. After completion of step 1, the obtained cells were washed twice with water, and then resuspended in a minimal medium until the OD became 0.4, to obtain resuspended cells.
3. After completion of step 2, Sulforaphane (SFN) and sulforaphane derivatives (AS1, AS2, AS3, AS4, AS5, AS6, AS7 and AS8) were added to the resuspended cells, respectively, so that the final concentration of the sulforaphane derivatives in the resuspended cells was 100. mu.M (AS1, AS2, AS3, AS4, AS5 and AS6) or 400. mu.M (AS7 and AS8), and expression of the corresponding protein-related genes avrPto, pfhrL and trp in Pst DC 3000. delta. saxAB/F/D/G cells was examined after 6 hours. Samples with DMSO added were also used as controls (Mock).
The results show that: sulforaphane (SFN), sulforaphane derivatives (AS1, AS2, AS3, AS4, AS5, AS6, AS7 and AS8) all can inhibit the transcription of Pst DC3000 delta saxAB/F/D/G effector related genes avrPto and hrpL, without affecting the relative transcriptional expression of trp (FIG. 6a and FIG. 6 b).
Sequence listing
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120> application of sulforaphane and derivatives thereof as bacterial effector protein transcription inhibitor
<160>1
<170>PatentIn version 3.5
<210>1
<211>909
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atgagtcttg atgaaaggtt tgaggatgat ctggacgagg agcgggttcc gaatctgggg 60
atagttgccg aaagtatttc gcaactgggt atcgacgtgc tgctatcggg tgagaccggc 120
acgggcaaag acacgattgc ccgacggatt catgagatgt caggccgcaa agggcgcctg 180
gtggcgatga attgcgcggc cattccggag tccctcgccg agagcgagtt attcggcgtg 240
gtcagcggtg cctacaccgg cgctgatcgc tccagagtcg gttatgtcga agcggcgcag 300
ggcggcacgc tgtacctgga tgagatcgat agcatgccgc tgagcctgca agccaaattg 360
ctgagggtgc tggaaacccg agcgcttgaa cggctgggtt cgacgtcgac gatcaagctg 420
gatatctgcg tgatcgcctc cgcccaatgc tcgctggacg acgccgtcga gcgggggcag 480
tttcgtcgcg atctgtattt tcgcctgaac gtcctgacac tcaagcttcc tccgctacgt 540
aaccagtctg atcgcatagt tcccctgttc acacgtttta cggccgccgc cgcgagggag 600
ctcggtgttc ccgttcccga tgtttgccca ctgctgcaca aagtgctgct gggccacgac 660
tggcccggca atatccgtga gctcaaggct gcagccaaac gccatgtgct gggtttcccc 720
ttgctgggcg ccgagccgca gggcgaagag cacttggcct gtgggctcaa atcgcaattg 780
cgagtgatcg aaaaagccct gattcaggag tcgctcaagc gccacgacaa ttgtgtggat 840
tcggtaagcc tggaactgga cgtgccacgc cgtacgctct atcgacgcat caaagaattg 900
cagatctga 909
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