Patent Application
20240099311
This application is based upon and claims priority to Chinese Patent Application No. 202211125577.6, filed on Sep. 16, 2022, the entire contents of which are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBRZBC079_Sequence_Listing.xml, created on Mar. 13, 2023, and is 31,814 bytes in size.
The present invention relates to the technical field of genetic engineering and in particular, to a virulence factor FsPL gene from the sugarcane pokkah boeng disease and use thereof.
Pokkah boeng disease (PBD), a widespread fungal disease of sugarcane caused by Fusarium sacchari and other Fusarium species with a relatively high incidence, mainly attacks the young leaves at the top of sugarcane. It causes, in a mild case, chlorosis and distortion of the leaves at the top of sugarcane and, in a severe case, a deformity of and necrosis in the whole top part, which lead to top rot and even the death of the whole sugarcane plant. In recent years, as large areas of sugarcane cultivars susceptible to the pokkah boeng disease have been planted, the incidence of the pokkah boeng disease in sugarcane areas has been increasing and trending upward, causing heavy yield losses (about 10-40%) in sugarcane production.
The plant cell wall is the initial physical and defensive barrier against a variety of biological stresses. As an important site where a host interacts with pathogens, the plant cell wall is often dynamically remodeled to produce immune responses, preventing pathogen infection. It also participates in sensing external stressors and transmits corresponding signals to stimulate defense responses. The plant cell wall is a complex network of polysaccharides such as cellulose, hemicellulose and pectin. To successfully infect a host, plant pathogens produce a series of cell-wall degrading enzymes (CWDEs), including cellulases, pectinases, xylanases, xyloglucanases, and the like, which physically disintegrate the plant cell wall, helping themselves penetrate host cells and spread throughout the plant's tissues. The oligosaccharides such as cellodextrins, oligogalacturonic acids (OGAs) and xyloglucan oligosaccharides produced from the action of CWDs on the cell walls of the plant can serve as elicitors to trigger the plant's defense responses against the infection with pathogens. Plant's defense responses include PTI and ETI. The immune response triggered on the recognition of PAMPs or DAMPs by the pattern recognition receptors (PRRs) on plant cell surfaces is a basic defense response in a plant termed PTI. Typical PTI includes bursts of reactive oxygen species, callose accumulation, electrolyte leakage, etc., by which microorganisms are typically confined to the site of infection. As typical DAMPs, the oligogalacturonic acids (OGAs) produced by the pectate lyase cleavage of polygalacturonic acids via a β-elimination reaction can be recognized by WAK1, a plasma membrane-associated protein on the plant-cell surface, activating the MAPK cascade; the MAPK cascade increases free cytosolic calcium levels and promotes ROS generation, triggering PTI responses. However, pathogens can interfere with or disrupt PTI by secreting effector proteins that enhance pathogen virulence. In the process of evolution, plants utilize R protein to directly or indirectly recognize these effectors, thereby triggering the second-line defense response—ETI (effector-triggered immunity) response. One of the most notable features of ETI is to trigger the hypersensitive response (HR) in plants—that is, producing defense proteins that induce rapid programmed cell death at the site of pathogen attack to limit pathogen transmission. This division between PAMPs and effectors, or between PTI and ETI, is, however, blurred and has been challenged recently.
Pectate lyase is an important enzyme secreted by plant pathogens and has been reported in a variety of microorganisms. Pectate lyase was found earlier in a variety of bacteria such as Erwinia carotovora, Enwinia chrysanthemi, Erwinia chrysanthemi and Pseudomonas solanacearum. The pectate lyase genes have also been studied in fungi. BcSpl1 contributes to the virulence of Botrytis cinerea and elicits hypersensitive responses in the host. VdPL1-4 is an important virulence factor for Verticillium dahliae; it plays an important role in the pathogenic process of the pathogenic bacterium. Vmpl4 is involved in the pathogenic process of Valsa mali. The deletion of the CcpelA gene affects the infectivity of Colletotrichum coccodes.
Therefore, the research on the role the pectate lyase gene FsPL plays in the pathogenic process of Fusarium sacchari is of great significance for the further understanding of the pathogenic mechanism of the bacterium and provides a theoretical basis for the prevention and treatment of PBD.
The present invention aims to provide a virulence factor FsPL gene from the sugarcane pokkah boeng disease and use thereof. The present invention reveals a gene FsPL that is related to the pathogenicity of F. sacchari. Preliminary analysis shows that the deletion of the gene affects the ability of hyphae to penetrate and further affects the pathogenicity of pathogens. This provides a basis for the molecular mechanism of the pathogen infection and pathogenic process.
To achieve the aim described above, the present invention provides the following technical solutions.
The present invention provides a virulence factor FsPL gene from the sugarcane pokkah boeng disease, which is from the common pathogens of pokkah boeng disease, and the virulence factor FsPL gene from the sugarcane pokkah boeng disease has a sequence set forth in SEQ ID NO: 1.
The present invention also provides a protein encoded by a virulence factor FsPL gene from the sugarcane pokkah boeng disease, and the protein has an amino acid sequence set forth in SEQ ID NO: 2.
The present invention also provides a silencing vector, which comprises an original vector and the FsPL gene.
The present invention also provides a silent recombinant bacterium, which is transformed or transfected with the silencing vector.
The present invention also provides a primer pair for cloning the sequence of the virulence factor FsPL gene from the sugarcane pokkah boeng disease, and the primer pair is one of a PVX-FsPL primer pair, a PVX-FsPLΔSP primer pair, a pCAMBIA2300-FsPL primer pair, and a pCAMBIA2300-FsPLΔSP primer pair;
The present invention further provides use of the FsPL gene, the protein, the silencing vector and the silent recombinant bacterium in manufacturing a peptide medicament against sugarcane pokkah boeng fungi.
Compared to the prior art, the present invention has the following advantages:
In order to more clearly illustrate the technical solutions in the examples of the present invention or in the prior art, the drawings required to be used in the description of the examples or the prior art are briefly introduced below. It is obvious that the drawings in the description below are merely examples of the present invention, and those of ordinary skilled in the art can obtain other drawings according to the drawings provided without creative efforts.
Agrobacterium carrying a recombinant vector is allowed to permeate into tobacco leaves by injection, and the sites of injection are marked; after 7 d, the symptoms are observed and photographed (photographic records are made before and after decoloring) (in
The present invention provides a virulence factor FsPL gene from the sugarcane pokkah boeng disease, which is from the common pathogens of pokkah boeng disease, and the virulence factor FsPL gene from the sugarcane pokkah boeng disease has a sequence set forth in SEQ ID NO: 1.
The present invention also provides a protein encoded by a virulence factor FsPL gene from the sugarcane pokkah boeng disease, and the protein has an amino acid sequence set forth in SEQ ID NO: 2.
The present invention also provides an upstream nucleotide sequence and a downstream nucleotide sequence of FsPL as set forth in SEQ ID NO: 27 and SEQ ID NO: 28.
The present invention also provides an Hph sequence as set forth in SEQ ID NO: 29.
The present invention also provides a silencing vector, which comprises an original vector and the FsPL gene.
The present invention also provides a silent recombinant bacterium, which is transformed or transfected with the silencing vector.
The present invention also provides a primer pair for cloning the sequence of the virulence factor FsPL gene from the sugarcane pokkah boeng disease, and the primer pair is one of a PVX-FsPL primer pair, a PVX-FsPLΔSP primer pair, a pCAMBIA2300-FsPL primer pair, and a pCAMBIA2300-FsPLΔSP primer pair;
The present invention further provides use of the FsPL gene, the protein, the silencing vector and the silent recombinant bacterium in manufacturing a peptide medicament against sugarcane pokkah boeng fungi, in particular to use of the gene sequence, the protein, and the like to develop related anti-disease fungus peptide medicaments.
The technical schemes provided by the present invention will be described in detail below with reference to the examples, which, however, should not be construed as limiting the scope of the present invention.
1 Materials and Methods
1.1 Experimental Materials
Test plants: the test sugarcane cultivar was “Zhongzhe 1” cultivated by Guangxi University; this particular cultivar is susceptible to the pokkah boeng disease, and the photoperiod was 16 h:8 h (light: dark); the test tobacco (Nicotiana benthamiana) was cultivated in a greenhouse with a 16:8 (light:dark) photoperiod. Test strains: E. coli Top10 competence, purchased from Shanghai Weidi Biotech Co., Ltd.; Trans1-T1 competence, purchased from Beijing TransGen Biotech Co., Ltd., cultured in an LB medium at a constant temperature of 37° C.; Agrobacterium competence GV3101, purchased from Shanghai Weidi Biotech Co., Ltd.; the GV3101 strain contains the pJIC SA_Rep concomitant factor so normal replication of PVX plasmid can be ensured in Agrobacterium and the GV3101 strain is cultured in an LB medium at a constant temperature of 28° C.
Test vector: Potato Virus X (PVX), gifted by academician Zhensheng Kang from Northwest A&F University (Chunlei Tang. Identification and Functional Analysis of Effector Proteins and Genes Inducing Host Cell Necrosis in Interactions of Puccinia striiformis with Wheat [D]. Northwest A&F University, 2013;
Likun Wang, Xin Fan, Chunlei Tang, et al. Inhibition of Accumulation of Callose and Reactive Oxygen Species in Plants by Puccinia striiformis Effector Pst30 [J]. Acta Phytopathologica Sinica, 2020(2):9).
1.2 Experimental Methods
1.2.1 The conserved domain of the protein encoded by the gene of interest was predicted and analyzed using an online tool Conserved Domain Search (website: https://www.ncbi.nlm.nih.gov/). The signal peptide region of the protein was predicted and analyzed using online tools SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/) and SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi? NORMAL=1). Primer design was performed using the Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) tool; the qRT-PCR primer was designed using the IDT (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) tool. Homology analysis was performed on the cloned target fragment by using BLAST alignment analysis software, and multi-sequence alignment analysis was performed by using DNAMAN software.
1.2.2 Agrobacterium-Mediated Transient Expression of the Gene in Nicotiana benthamiana
Transient expression of FsPL was performed in tobacco leaves to verify the function of the gene. Different fragments (FsPL-FL, FsPL-Δsp) of FsPL1 were amplified using the cDNA of the F. sacchari wild-type strain as a template and were inserted into the PVX vector at the ClaI and NotI sites, and the vector was transformed into Agrobacterium GV3101. Agrobacterium permeation tests were performed on tobacco leaves. Sites into which PVX empty vector and PVX-GFP were injected were used as negative controls, and a site into which PVX-Bax was inoculated was used as a positive control. After 7 d, the symptoms were observed and photographed, during which attention was paid to symptom changes, and each treatment was repeated at least 3 times. Gene expression was detected by qRT-PCR and Western blot.
1.2.3 Elicitation of PTI response in hosts by FsPL Agrobacterium carrying the gene of interest was injected into 4-6 week old Nicotiana benthamiana leaves. To study ROS accumulation, tobacco leaves that had been infected for 48 h were collected and stained with 1 mg/mL 3′,3-diaminobenzidine (DAB), and chlorophyll in the leaves was removed with absolute ethanol. Reactive oxygen species deposition was observed and photographed. To measure the leakage of electrolytes in the tobacco leaves, 48 h after Agrobacterium infection, the leaves in the infection area are collected using a 1 cm diameter hole punch. The leaf punches were placed in sterile deionized water and kept for 2 h at a rotational speed of 165 rpm at a temperature of 25° C. The ionic conductivity was measured on FiveEasy FP30 (Mettler-Toledo, Shanghai). To observe callose deposition, leaves that had been infected for 48 h were stained with 0.1% aniline blue, and callose deposition was observed under a fluorescence microscope. All the experiments were performed three times in total.
1.2.4 Gene Gun-Mediated Transient Expression of the Gene in Maize Leaves
To further analyze the function of the FsPL gene, overexpression of FsPL was performed by gene gun bombardment in the leaves of maize B73, which is a very close relative to the sugarcane host for F. sacchari.+1 position leaves of the 5-leaf-stage maize plants with the same growth pattern were cut into 6 cm long leaf segments, and the main veins were removed. The leaves were pasted in parallel on Petri dishes. The maize leaves were bombarded with Bio-Rad PDS-1000/He at a distance of 6 cm under a pressure of 1100 psi. The bombarded leaves were cultured in the dark at 28° C. for 2 d, stained with GUS staining solution and decolored in 100% ethanol, and then the blue spots were counted. The experiment was repeated 3 times.
1.2.5 Gene Knock-Out Mutant Construction
To obtain a mutant strain in which the FsPL gene is deleted, a homologous recombinant fragment comprising both flanks of the gene of interest and the hygromycin (Hph) gene sequence was constructed. Both flank sequences upstream and downstream of the FsPL gene were amplified from the genome DNA of the F. sacchari wild-type strain by using primers FsPL-AF/AR and BF/BR, respectively, about 1000 bp each. The two fragments were fused with the hygromycin gene by fusion PCR and the fused fragment (about 4200 bp) was transferred into F. sacchari protoplasts by PEG-mediated fungal genetic transformation. The transformed strains were screened for hygromycin resistance. The transformed strains with resistance were identified by PCR and purified by monospore isolation or protoplast regeneration.
1.2.6 Mutant Biophenotype and Pathogenicity Analysis
(1) Colony morphology and growth rate determination: Bacterial cakes of the wild-type WT and mutant ΔFsPL were collected using a 6 mm diameter hole punch and each was transferred to a plate containing 15 mL of PDA medium. The cakes were cultured inverted at 28° C. in the dark for 7 days. The colony diameter was measured every 24 h, and the colony morphology was observed and recorded. Hypha growth curves were drawn based on the recorded data.
(2) Conidium count: The bacterial cakes (6 mm) of the test strains were each transferred to a CMC medium and cultured at 28° C. with shaking for 3 d, then conidium suspensions were obtained, and conidia were counted using a hemocytometer.
(3) In-vitro leaf inoculation to sugarcane: +1 position leaves of the 5-leaf-stage maize plants with the same growth pattern were selected and cut into 6 cm long leaf segments with disinfected scissors, and 2 cm cuts were made in the same positions on both sides of the leaf vein. The leaves were placed in Petri dishes (with filter paper wet with sterile water in them). Bacterial cakes of the wild-type strain and knock-out mutant strain that had grown in PDA media for 7 d were collected using a 6 mm diameter hole punch and inoculated to the cuts on Zhongzhe 1 leaves. After 3 d of incubation in the dark in a moist environment at 28° C., the formation of lesion spots on the leaves was observed and photographed. The inoculation experiment was repeated 3 times, 10 leaves inoculated each time.
(4) Cellophane penetration tests: Bacterial cakes of the wild-type strain (WT) and mutant strain (ΔFsPL) from the same batch of culture were collected using a 6 mm diameter hold punch and inoculated on PDA media covered in cellophane. Three replicates were set. After 3 d of inverted incubation in an incubator at a constant temperature of 28° C., photographic records of colony growth were made. The cellophane (containing hyphae) was removed from the surface of the media, placed back in the incubator and cultured for another 3 days. Whether colonies were able to grow on PDA media was observed and photographic records were made.
1.2.7 Determination of the Activity of Pectate Lyase Bacterial cakes of the wild type and mutant were collected using a hole punch, placed into 100 mL of culture media for pectate lyase and cultured at 28° C. with shaking at 210 rpm. After 5 d, their crude enzyme solutions after boiling inactivation were used as controls, the absorption of the crude enzyme solutions at the wavelength of 540 nm was measured by DNS colorimetric method, and the D-galacturonic acid content was calculated from the standard curve. At 50° C. at pH 4.8, 1 mL of pectinase decomposes pectin for 1 h to produce 1 mg of D-galacturonic acid as one enzymatic activity unit (U). Three replicates were set for the experiment, and the experiment was repeated 3 times.
2 Results and Analysis
2.1 Results of Sequence Analysis
FsPL ORF is 702 bp in full length, encodes 233 amino acids and has a signal peptide of 19 aa at the N terminus, and aa at positions 23 to 222 form the pectate lyase domain of the gene (
2.2 FsPL can Induce Tobacco Cell Necrosis
The activity of the gene of interest to induce cell necrosis was measured through its transient expression in Nicotiana benthamiana leaves. The results are shown in A of
2.3 FsPL Triggers PTI Response
One of the immune responses plants produce against invasions of pathogens is the production of a large amount of reactive oxygen species (ROS). The present invention used DAB for staining, and the results show that the sites where GFP and PVX were injected could not be stained brown, serving as a negative control; the site where Bax was injected was stained brown, serving as a positive control. At the site infected with FsPL, significant brown deposits can be seen; this is consistent with the positive control, indicating that FsPL caused a burst of reactive oxygen species in Nicotiana benthamiana leaf tissue. After the deletion of the signal peptide, the site was less brown, indicating that the ability of FsPL to cause a burst of reactive oxygen species was reduced (in
After staining with aniline blue, callose deposited on the cell wall can be stained blue and is visible under a fluorescence microscope. In the present invention, callose accumulation caused after tobacco leaves were infected with FsPL was detected using aniline blue. The results show that compared to the negative control GFP, the infiltration areas of the positive control Bax and FsPL presented large areas of blue fluorescence. After the deletion of the signal peptide, callose deposition decreased to some extent (in
2.4 Transient Expression of the FsPL Gene Leads to Induction of Cell Necrosis in Maize Leaves
To further verify the function of the FsPL gene, maize leaves were bombarded with a gene gun, and overexpression of the FsPL gene was performed in the leaves of maize, which is a very close relative to sugarcane (the host for F. sacchari). The results of gene gun bombardment show that many blue spots appeared on the leaves bombarded with GUS+EV, indicating that the gene gun-mediated transient expression in the maize leaves was successful, serving as a negative control. The number of blue spots on the leaves bombarded with GUS+Bax decreased by about 88.36%; Bax can elicit PCD response in plants, serving as a positive control. The number of blue spots on the leaves bombarded with GUS+FsPL decreased by about 76.72%, indicating that FsPL can induce cell necrosis in maize leaves. The number of blue spots on the leaves bombarded with GUS+FsPLΔsp decreased by about 51.64%, indicating that after removal of the signal peptide, the gene could still elicit PCD response, but its ability to induce PCD decreased to some extent (
2.5 FsPL is a Virulence Factor for F. sacchari
To investigate the function of the FsPL gene in F. sacchari, the FsPL gene in the F. sacchari strain was knocked out by homologous recombination, yielding a mutant strain ΔFsPL. The mutant strain was thinner than the wild-type colonies, and produced significantly fewer aerial hyphae (
2.6 Deletion of the FsPL gene affects the extracellular pectinase activity of F. sacchari
A standard curve was drawn for D-galacturonic acid, and the regression equation yielded was y=1.5617x−0.0182, R2=0.997 (in
Pectin is the most complex and dominant component of the plant cell wall. Pathogenic bacteria have to break through this barrier when invading plants. Pectate lyase is an important cell wall-degrading enzyme, which can help pathogens to successfully invade and colonize host plants and expand. Pectate lyase is widely sourced and usually exists in multigene family form. In 1989, a pectate lyase gene was reported to be found in Aspergillus nidulans. After that, pectate lyase genes were also found in pathogens such as Colletotrichum gloeosporioides, Nectria hematococca, Trichoderma viride, Aspergillus niger, Alternaria brassicicola, Phytophthora capsici, Verticillium dahliae. The present invention reports the pectate lyase gene FsPL in F. sacchari. The Agrobacterium-mediated transient expression in tobacco leaves and gene gun-mediated transient expression in maize leaves indicates that FsPL can elicit PCD response in Nicotiana benthamiana and maize leaves. The pectate lyase genes in pathogens can cause damage to the cell walls of their host plants by degrading pectin, the major component in the cell walls, and finally cause cell death. FsPL has a signal peptide at the N terminus. When the signal peptide was deleted from FsPL, the PCD response elicited by the gene in plants was found to be weakened, which indicates that the signal peptide played some role in the functioning of the gene. Since signal peptides are an important component of secretory proteins, the deletion of the signal peptide may affect the recognition of proteins by plants; this requires further experimental investigations.
PTI, as a nonspecific defense response in plants, resists invasions of pathogens through a series of physiological reactions such as bursts of reactive oxygen species, electrolyte leakage and callose accumulation. According to the present invention, the measurement of these physiological indicators indicates that FPL could elicit typical PTI response in plants and the signal peptide played some role in this process. Previous studies show that the pectate lyase family members can elicit immune responses in hosts; for example, the pectate lyase gene MoPL1 of Magnaporthe grisea can serve as a DAMP to elicit PTI response in tobacco leaves, and both the pectate lyase gene VdPEL1 of Verticillium dahliae and the pectate lyase gene BcSpl1 of Botrytis cinerea can elicit immune defense responses in plants. It was speculated from these results that FsPL can not only be used as a pectate lyase to directly participate in degrading plant cell walls but also be used as a DAMP to elicit PTI response in plants.
In the present invention, a knock-out mutant of F. sacchari was constructed by knocking out the FsPL gene, and the results indicate that the knock-out mutant produced significantly fewer aerial hyphae and had a slightly slower hyphal growth rate while the spore morphology and sporulation quantity did not significantly change, which indicates that the gene may play a role in the vegetative growth of pathogens but not in the regulation of sporulation. In-vitro inoculation experiments show that the pathogenicity of the mutant strain significantly decreased. In terms of the relationship between the pectate lyase gene and the pathogenicity of pathogens, the deletion of the pectate lyase gene may cause a decrease in the pathogenicity of the pathogens on plants.
To further analyze the mechanism of the FsPL gene affecting the pathogenicity of pathogens, the present invention tested the ability of mutant hyphae to penetrate through cellophane penetration experiments, and the results show that the mutant ΔFsPL could not penetrate cellophane—the ability to penetrate decreased. Therefore, it was speculated that the deletion of the FsPL gene affected the ability of F. sacchari hyphae to penetrate. The pectinase secreted by pathogens participates in a pathogenic process by degrading the pectin of plant cell walls. The present invention measured the extracellular pectate lyase activity by the DNS method, and the results show that the extracellular enzymatic activity of the mutant significantly decreased. In conclusion, the FsPL gene affects the degradation of plant cell walls by affecting the extracellular enzymatic activity of F. sacchari, further affects bacterial invasion and colonization, and finally affects the pathogenicity of pathogens.
The above descriptions are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall fall within the protection scope of the present invention. See the attached sequence listing for details.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211125577.6 | Sep 2022 | CN | national |
