CN109735544B - Method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA - Google Patents
Method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA Download PDFInfo
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Abstract
A novel method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA belongs to the technical field of biology. The method takes cellulose synthetase A3 of arabidopsis thaliana biotrophic oomycetes as a target gene to synthesize antisense sRNAs with the length being more than or equal to 25nt, and inhibits the germination of arabidopsis thaliana biotrophic oomycete spores through the antisense sRNAs, so that the arabidopsis thaliana biotrophic oomycete downy mildew infection is inhibited. The invention has reliability and high efficiency, and the simple and effective sRNA method has potential in deciphering gene function in the obligate biological nutrition pathogenic bacteria and controlling plant diseases by R-gene.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inhibiting arabidopsis living body nutritional type oomycete downy mildew infection based on small RNA.
Background
Non-coding small rnas (srnas) of 20 to 30 nucleotides (nt) in length are known to be involved in the regulation of gene expression and defense in eukaryotes. Different types of RNAs, such as double-stranded RNA (dsRNA) and small interfering RNA (siRNA), can trigger homologous RNA degradation or inhibit mRNA translation. This process, known as RNA silencing, plays an important role in a variety of biological processes, including innate immunity and development.
In plant and microorganism interactions, plants and microorganisms can exchange RNA molecules and then integrate into RNA silencing mechanisms in interacting recipient cells. This transboundary RNA transfer was demonstrated for the first time between fungi and plants. Botrytis cinerea, an ascomycete fungus infected with over 200 plants, transports sRNAs by hijacking the plant cell gene silencing machinery, silencing arabidopsis and tomato genes. On the other hand, large quantities of cotton sRNAs were found in verticillium wilt hyphae extracted from infected plant tissues, where some cotton-derived sRNAs may be targeted against essential mycotoxicity genes.
The movement of sRNAs from plants to pathogens was studied using host-induced gene silencing (HIGS) technology. This technique is usually the production of double-stranded rna (dsRNA) in transgenic plants by agrobacterium or by dsRNA replicating viruses. HIGS has been successfully used to inhibit the basic pathogen genes in a variety of plant-pathogen interaction systems, including barley-Fusarium, Arabidopsis and tomato-verticillium, barley and wheat-Blumeria and Bremia. Similarly, HIGS has been used against nematodes in Arabidopsis.
In another variant, Mitter et al use clay nanoflakes to deliver dsRNA to plants to silence homologous viral RNA.
Living body nutritional type oomycete (Hpa) is a highly specific nutritional type oomycete pathogen, and can cause downy mildew of Arabidopsis thaliana. The Hpa-Arabidopsis system has been used as a model to study the effects of pathogenic bacteria and plant immunity. This pathogen is both sexually and asexually propagated. For infestation, asexual conidia germinate on the surface of plant leaves to form adherent cells, and then a penetrating hyphae grow between the walls of adjacent epidermal cells. In susceptible plants, hyphae branch into the intercellular spaces and form haustoria in the epidermis and mesophyll cells. Within one to two weeks, conidia develop through stomata and carry conidia to start a new round of infection. Sexual spores, called oospores, are produced on cotyledons or leaves of infected plants.
To our knowledge, there is no reliable and efficient genetic transformation method for Hpa. Here, we used in vitro sRNAs synthesized against the HPA-CesA3 gene, and reported that antisense SRNAs of 25nt or longer could inhibit spore germination, thereby inhibiting infection.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA.
The method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on the small RNA is characterized in that the cellulose synthetase A3 of the arabidopsis thaliana biotrophic oomycete is used as a target gene to synthesize antisense sRNAs with the length being more than or equal to 25nt, and the antisense sRNAs is used for inhibiting the germination of arabidopsis thaliana biotrophic oomycete spores, so that the arabidopsis thaliana biotrophic oomycete downy mildew infection is inhibited.
The method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on the small RNA is characterized in that the length of the antisense sRNAs is 25nt or 30 nt.
The method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA is characterized in that antisense sRNAs are Hpa _ CesA3_ RNA _ AS _25 and Hpa _ CesA3_ RNA _ AS _30, the nucleotide sequence of Hpa _ CesA3_ RNA _ AS _25 is shown AS SEQ ID NO.2, the nucleotide sequence of Hpa _ CesA3_ RNA _ AS _30 is shown AS SEQ ID NO.4,
the method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on the small RNA is characterized in that the 5' end of the antisense sRNAs has a capping structure.
The small RNA for inhibiting the arabidopsis thaliana biotrophic oomycete downy mildew infection is characterized in that the small RNA takes cellulose synthetase A3 of arabidopsis thaliana biotrophic oomycete as a target gene to synthesize antisense sRNAs with the length of more than or equal to 25 nt.
The small RNA, wherein the antisense sRNAs are 25nt or 30nt in length.
The small RNA is characterized in that the antisense sRNAs are Hpa _ CesA3_ RNA _ AS _25 and Hpa _ CesA3_ RNA _ AS _30, the nucleotide sequence of Hpa _ CesA3_ RNA _ AS _25 is shown AS SEQ ID No.2, and the nucleotide sequence of Hpa _ CesA3_ RNA _ AS _30 is shown AS SEQ ID No. 4.
The small RNA, wherein the 5' end of the antisense sRNAs has a capping structure.
The small RNA is applied to the prevention and treatment of arabidopsis thaliana biotrophic oomycete downy mildew infection.
The exogenous sRNA of the pathogenic bacteria biotrophic oomycete conserved CesA3 of the arabidopsis downy mildew can inhibit the infection of the arabidopsis HPA, and the method has reliability and high efficiency. The simple and effective sRNA method has potential in the aspects of deciphering the gene function in the obligate biological nutrition pathogenic bacteria and controlling plant diseases by the R-gene.
Drawings
FIG. 1 is a drawing of the use of antisense sRNA targeted Hpa-CesA3 to inhibit sporulation, in FIG. 1a) control (no antisense sRNA), b) seedlings inoculated with 5. mu.M antisense sRNA spore suspension, c) seedlings inoculated with 10. mu.M antisense sRNA spore suspension, d) seedlings inoculated with 20. mu.M antisense sRNA spore suspension;
FIG. 2 shows that antisense sRNA, but not sense sRNA, inhibited sporulation, and in FIG. 2 indicates that control inoculation had a significant difference p <0.05 over the corresponding sRNA;
FIG. 3 shows the pathogen inhibition at the infection stage, in FIG. 3 (a) spores were normally infected, oospore development was observed in control, (b, c) seedlings inoculated with 20. mu.M antisense sRNA were not infected or hyphal developed;
FIG. 4 shows that the Hpa-CesA3 gene targeting antisense sRNA inhibits germination, in FIG. 4 the control (a, b) germination tubes are longer, 20. mu.M antisense sRNA (c) incubated spores do not germinate, or germination tubes are inhibited for a certain time (d), with a 10-fold magnification, a and c, and b and d 40-fold;
FIG. 5 shows that capping sRNA has an effect on gene silencing, and in FIG. 5 Arabidopsis seedlings were inoculated with Hpa-Emoy2 spore suspensions without sRNA (a), 20. mu.M uncovered antisense sRNA (b), and 20. mu.M covered antisense sRNA (c);
FIG. 6 shows that sRNA length has an effect on the development of pathogenic bacteria;
FIG. 7 shows the expression pattern of Hpa-CesA3, and Cs: conidia, dpi: days after inoculation;
FIG. 8 shows that sense and antisense DNA strands do not inhibit sporulation.
Detailed Description
The invention is further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1: experimental methods
1. And (3) separating and breeding strains and pathogenic bacteria: emoy2 and Cala2 isolated from Arabidopsis thaliana biotrophic oomycetes were maintained on Ws-eds 1. Sporulation assays were performed 7 days after inoculation, at the end of the Hpa life cycle. To quantify sporulation, 10 infected seedlings were removed from each replicate and placed in a container containing 250. mu. l H20 in Eppendorf tubes. Vortex sampling and counting the number of conidia by using a blood counting plate.
Synthesis of sRNA and DNA oligonucleotides: in the method, Hpa-CesA3 gene (HpaG810051) is used as a target gene, and sense and antisense sRNAs with different lengths are designed as follows:
hpa _ CesA3_ RNA _ AS _ 245 '-GCCGCAUCGCACGUACCUCAGUAC-3' (shown in SEQ ID NO. 1);
hpa _ CesA3_ RNA _ AS _ 255 '-GCCGCAUCGCACGUACCUCAGUACG-3' (shown in SEQ ID NO. 2);
hpa _ CesA3_ RNA _ S _ 255 '-CGUACUGAGGUACGUGCGAUGCGGC-3' (shown in SEQ ID NO. 3);
hpa _ CesA3_ RNA _ AS _ 305 '-AGUGCCGCAUCGCACGUACCUCAGUACGAC-3' (shown in SEQ ID NO. 4);
similarly, sense and antisense DNA oligonucleotides are designed to the same region. Are respectively as
Hpa _ CesA3_ DNA _ S _ 255 '-GCCGCATCGCACGTACCTCAGTACG-3' (as shown in SEQ ID NO. 5) and
hpa _ CesA3_ DNA _ AS _ 255 '-CGTACTGAGGTACGTGCGATGCGGC-3' (shown in SEQ ID NO. 6). These are deoxynucleotides or nucleotides synthesized from Sigma or Eurofin.
Use of sRNAs in pathogen spore and plant inoculation: from infected Arabidopsis thalianaWs-eds1Hpa spores were collected from seedlings, washed twice in sterile distilled water, and the spore concentration was adjusted to 5X10 using a hemocytometer4And/ml. The sRNAs were finally added to the spore suspension to a final concentration of 5, 10, 20. mu.M, and the spore droplets were then inoculated onto 7-day-old seedlings. As a control, seedlings were inoculated with 20. mu.M DNA oligonucleotides or spores which were not inoculated with sRNAs or DNA oligonucleotides in the same manner. The seedlings were spore-cultured 3 days after inoculation. The number of sporulations is as described above.
4. Spore germination test:
1.1MS Medium prepared 13(Sigma, 84100) using 4.3g/l MS basic salt mixture (Sigma, M5524), agar (1.5%) (Sigma, A1296), sucrose (10 g/l) and distilled water. The MS powder was dissolved in sterile distilled water, adjusted to pH 5.7 with 1N NaOH/HCL, and agar and sugar were added. Autoclaved at 15psi and 121 ℃ for 15 min. In the clean bench, about 20 ml of medium was accurately placed in each sterile petri dish.
1.2 cutting off 1.5 cm long glass paper from the common glass paper, and steaming and pressing in distilled water. After autoclaving, the zona pellucida was placed on MS medium in petri dishes on a clean bench and stored in a refrigerator for a long period.
1.3Hpa spore suspension Collection, washed twice in sterile distilled water, adjusted to spore concentration 5X10 with a hemocytometer4Number of spores per ml. Approximately 10. mu.l spore suspension, containing 0 or 20uM antisense sRNA, was dropped perA piece of cellophane. The dishes were incubated at 16 ℃ in 12h light/12 h dim light. After 48h the spores were observed under a light microscope for incubation and the germinated spores were counted.
5. And (3) dyeing plant tissues: infected and uninoculated control seedlings were stained with a solution of phenol, lactic acid, glycerol and water (1:1:1:1) plus 1mg/ml trypan blue, destained in chloral hydrate and observed under a multiplex microscope.
6. Statistical analysis: for statistical analysis, pairs of students performed t-tests on the data obtained from the plant infection analysis.
7. Bioinformatics: IICB Genomics and transactions Resources (http:// emuicrobedb. org) and EnsemblProperty (http:// promoters. ensemblebl. org) databases are used for Hpa information. Hpa-CesA3 was analyzed using Web servers like InterPro (http:// www.ebi.ac.uk/Interpro /) and Pfam (Punta et al, 2012) (http:// Pfam. wustl. edu /). The 14Hpa-CesA3 gene was searched for nucleotide and amino acid similarity to sequences of Arabidopsis thaliana and oomycetes. Primer design used Geneious (v10.0) (Kearse et al, 2012).
Example 2: screening of sRNA-mediated silencing target genes in HPA
The main components of the oomycete cell wall are β -glucan and cellulose, we use cellulose synthase gene as target for sRNA mediated silencing, Pfam is utilized, we identify that M4BU64 in Hpa belongs to cellulose synthase gene family, the gene is found in computer simulation analysis to correspond to HpaG810051 in Emoy2 genome and exist in the form of single copy gene, EnsemblProtists gene annotation indicates that HpaG810051 has no intron, open reading frame encodes 1144 amino acids (molecular mass 127.028 kDa) prediction protein, BLASTX search of database indicates that HpaG810051 has high similarity with CesA 8656 protein of other oomycetes, therefore we name HpaG810051 HpaG-CesA 3. then we obtain albugo, Leucopora keratoderma, Lactuca, Phytophthora disporum, Pediophora, Pepper, potato, Phytophyta, potato, Phytophthora capsicum, and Neurophysalospora sp, the homology of CesA gene sequence is found by comparison of CesA-DNA homology between CesA-DNA sequence and exogenous nucleotide sequence of exogenous gene expression of CesA-DNA sequence of Geobacillus, the gene expression of the gene homology of Hpah 2-Geotrichu 27% HPA 2-P-S2, the gene expression of the gene, the gene expression of the gene, the gene expression of the gene of.
We then used the published transcriptome data to study the expression pattern of Hpa-CesA3 in Arabidopsis thaliana Col-0 inoculated with either avirulent or virulent Hpa isolates, Emoy2 or Waco 9. It is clear that HPA-CesA3 is highly expressed in spores and the expression level is significantly reduced during mycelium development (FIG. 7).
Example 3: Hpa-CesA3 antisense sRNA inhibits Hpa sporulation
Since Hpa is an obligate biotrophic pathogen that grows on arabidopsis, we examined whether Hpa-CesA3 has any homology to the arabidopsis CesA gene. BLASTN searches of the Arabidopsis database did not reveal significant similarities. Subsequently, we designed 25-nt sense and antisense RNA oligonucleotides from the 5' region of the gene without any homology among other genes in the Hpa genome. Positive and antisense sRNAs were added to HPA spores at concentrations of 5, 10 and 20. mu.M, respectively, and mixed to inoculate 7-day-old Arabidopsis seedlings. At day 7 (dpi) after inoculation, Hpa sporulation was detected (FIG. 1), with no significant difference between control plants (FIG. 1a) and plants inoculated with 5, 10, 20. mu.M sRNA spore suspensions. Inoculation of spore suspensions containing 5 and 10M antisense RNA (FIGS. 1b and 1c) significantly reduced sporulation. Interestingly, inoculation of a spore suspension containing 20. mu.M of antisense RNA had no sporulating effect on the inoculated plants (FIG. 1 d). Quantitative data analysis further showed that sporulation of inoculated plants was significantly reduced or not increased after inoculation with Hpa spores and antisense sRNAs mixed (figure 2). Experiments were repeated at least 5 times, each at least 3 times, with similar results and verified. We also designed 25nt sense and antisense DNA oligonucleotides from the same gene region and performed similar vaccination experiments with 20. mu.M DNA oligonucleotides. There was no significant difference in sporulation between control plants inoculated with either sense or antisense DNA oligonucleotides and plants inoculated with antisense DNA oligonucleotides (FIG. 8).
Example 4: Hpa-CesA3 antisense sRNA inhibits spore germination
To investigate the inhibitory effect of Hpa-cesA3 antisense sRNA on sporulation, we inoculated 7-day-old Arabidopsis seedlings with a spore suspension containing 20. mu.M antisense sRNA. The inoculated leaves were stained with trypan blue on day 7, and the degree of development of Hpa in the tissues was observed (fig. 3). Normal pathogen development was observed in control leaf tissue (fig. 3a), but no hyphae or pathogen spores were present on cotyledons inoculated with spore suspensions containing 20 μ MsRNA (fig. 3b, c). Suggesting that antisense sRNA may inhibit spore germination, thereby preventing infestation. In trypan blue staining, spores that did not germinate may be washed away. To further investigate this problem, we set up germination tests with cellophane strips. After 8h, spore germination was observed under a light microscope. As shown in fig. 4, untreated, control spores were bright, producing germ tubes of different lengths within two days (fig. 4a, b). However, the antisense sRNA treated spores turned dark brown, and the germination tubes were either mainly missing or in few cases captured (fig. 4 c, d). We repeated this experiment 5 times and we observed 100% inhibition of germination in all experiments, see table 1.
Table 1: determination of spore germination by antisense sRNA method
Example 5: Hpa-cesA3 antisense sRNA non-ethnicically inhibits Hpa infection
Hpa-CesA3 antisense sRNA-mediated inhibition of Hpa infection was performed using the Hpa-Emoy2 isolate. To determine if our observed inhibition of infection was isolate-specific, we performed a similar study using the Hpa-Cala2 isolate. The homology between Emoy2 Hpa-CesA3 gene and Cala2 gene was 99.971% and the homology in the sRNA target region was 100% as determined by BLASTN. Subsequently, we inoculated Hpa-Cala2 spores with 20. mu.M antisense sRNA in a mixture with 7-day-old Arabidopsis seedlings. As we expect, when inoculated with a spore suspension containing 20. mu.M antisense sRNA, sporulation was normal in control seedlings (data not shown), indicating that Hpa-CesA3 antisense sRNA inhibition of Hpa infection was not race specific.
Example 6: it is crucial that capped antisense sRNA inhibits Hpa infection
The above experiment was performed with 25nt antisense sRNAs. To determine whether capping (N7 methylated guanosine) affected silencing of Hpa-CesA3, we obtained an unlimited version of the same antisense sRNA and performed a similar vaccination study. After 7 days, control Arabidopsis seedlings and Arabidopsis seedlings inoculated with no upper-limiting antisense 25nt sRNA spores developed typical HPA infection (FIG. 5), which was the result of normal sporulation and germination (FIGS. 5a, b). In the same experiment, spores inoculated with capped antisense sRNA neither germinated nor sporulated (fig. 5 c). These results indicate that the sRNA cap has important biological activity for inhibiting plant Hpa infestation.
Example 7: sRNA length Effect Hpa-CesA3 antisense sRNA inhibition of Hpa infection
The length of sRNA may influence gene silencing (Vargason et al, 2003). In addition to 25nt sRNA, we also examined the effect of 24 and 30nt antisense Hpa-CesA3 sRNAs on Hpa infection. After inoculating 20 mu MsRNAs with 24nt SRNAs, the plant generates normal sporulation phenomenon and is infected with Hpa. However, 30nt Arabidopsis seedlings inoculated with 20. mu.M antisense RNA were not sporulated and the inoculated Arabidopsis plants remained healthy (FIG. 6).
Sequence listing
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Claims (5)
1. A method for inhibiting arabidopsis thaliana biotrophic oomycete downy mildew infection based on small RNA is characterized in that cellulose synthetase A3 of arabidopsis thaliana biotrophic oomycetes is used AS a target gene to synthesize antisense sRNAs with the length being more than or equal to 25nt, and germination of arabidopsis thaliana biotrophic oomycete spores is inhibited through the antisense sRNAs, so that arabidopsis thaliana biotrophic oomycete downy mildew infection is inhibited, wherein the antisense sRNAs are Hpa _ CesA3_ RNA _ AS _25 and Hpa _ CesA3_ RNA _ AS _30, the nucleotide sequence of the Hpa _ CesA3_ RNA _ AS _25 is shown in SEQ ID No.2, and the nucleotide sequence of the Hpa _ CesA3_ RNA _ AS _30 is shown in SEQ ID No. 4.
2. The method of claim 1, wherein the 5' end of the antisense sRNAs has a capping structure, and wherein the method comprises inhibiting an Arabidopsis thaliana biotrophic oomycete downy mildew infection using a small RNA.
3. The small RNA is characterized in that the small RNA is antisense sRNAs with the length being more than or equal to 25nt, which are synthesized by taking cellulose synthetase A3 of arabidopsis thaliana biotrophic oomycetes AS a target gene, wherein the antisense sRNAs are Hpa _ CesA3_ RNA _ AS _25 and Hpa _ CesA3_ RNA _ AS _30, the nucleotide sequence of the Hpa _ CesA3_ RNA _ AS _25 is shown AS SEQ ID No.2, and the nucleotide sequence of the Hpa _ CesA3_ RNA _ AS _30 is shown AS SEQ ID No. 4.
4. The small RNA of claim 3, wherein the 5' end of the antisense sRNAs has a capping structure.
5. The use of the small RNA of claim 3 or 4 for the prevention and treatment of an Arabidopsis thaliana biotrophic oomycete downy mildew infection.
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