Key genes for mouthguard development in Southern root-knot nematodes and their applications

Abstract

This invention discloses a key gene for stylet development in the southern root-knot nematode and its application, belonging to the fields of genetic engineering and biological control. This invention is the first to discover that three genes, Mi_16868.1, Mi_38955.1, and Mi_39174.1, are essential for stylet development. Silencing any one of these genes can lead to stylet loss or malformation, reducing egg hatching rate to below 30%; co-silencing of all three genes results in a hatching rate of less than 10% and a stylet abnormality rate of 60.3%. This invention further constructs a dsRNA expression vector targeting the above genes, and transforms tobacco using Agrobacterium-mediated transformation to obtain transgenic plants stably expressing dsRNA. Pot experiments show that the number of root knots in transgenic tobacco roots is significantly reduced, exhibiting clear nematode resistance. This invention provides a novel molecular target and technical solution for the green control of root-knot nematodes, and can be widely applied to the breeding of nematode-resistant varieties in economic crops such as tobacco, tomato, and cucumber.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biological control technology. Specifically, it relates to a key gene for the development of the mouthparts of the southern root-knot nematode and its application. Background Technology

[0002] Root-knot nematodes are among the most widespread and damaging plant parasitic nematodes in the world, infecting thousands of economic crops such as tomatoes, tobacco, cucumbers, and cotton, causing an average annual economic loss of over US$100 billion globally. Existing control methods for root-knot nematodes mainly include: (1) chemical nematicides (such as aldicarb and chlorpyrifos), which are effective but highly toxic and prone to residues, and have been restricted or banned in many places; (2) agricultural measures (crop rotation, soil improvement, and resistant rootstocks), which are slow to take effect and are constrained by climate and farming systems; (3) resistance breeding, as most crops lack natural resistance sources, and commercially available resistance genes (such as Mi-1) face the risk of breakthroughs in nematode toxicity races; (4) biological control (Paecilomyces lilacinus, Bacillus, etc.) - poor field stability and large fluctuations in control efficacy.

[0003] In recent years, RNAi control strategies, represented by host-induced gene silencing (HIGS), have been considered promising green alternatives. Currently reported transgenic nematode-resistant targets mainly focus on effector protein genes secreted by the esophageal glands of nematodes, such as the conserved secretory peptide 16D10 of root-knot nematodes, which can mimic plant transcription factor ligands to promote root growth. RNAi silencing in Arabidopsis, grapes, and cucurbits can achieve broad-spectrum resistance across species. US patent application US20150259700A1 discloses a dsRNA transgenic plant targeting Mc16D10L; Korean patent KR20180045161A also discloses an RNAi rootstock cucurbitaceous plant targeting M. incognita16D10.

[0004] The stylet is a specialized parasitic and feeding organ evolved in plant-parasitic nematodes. Located within the nematode's oral cavity, it is a needle-like structure. Structurally, it is a hollow organ with openings at both ends, consisting of three main parts: a cone, a stalk, and a basal bulb. The basal bulb connects to the stylet and esophageal gland cells. Functionally, the stylet plays a central role in initiating infection; the nematode establishes an infection channel by mechanically piercing the host cell wall. Furthermore, it serves as a pathway for transmitting effector proteins that can regulate host cells and suppress plant immune responses. The stylet is the only way for nematodes to obtain nutrients from host cells, thus supporting their growth, development, and reproduction. Because the formation and function of the stylet directly affect the success of parasitism, it is a core determinant of nematode parasitism. Stylet formation begins in the embryonic development stage and is influenced by various environmental factors, including pheromone signals, nutrient supply, and culture conditions. Due to its unique morphological characteristics, the stylet structure has long been a key feature in nematode classification and identification. However, its tiny size presents significant technical challenges to isolating and observing this structure, hindering in-depth research into its developmental regulation and molecular composition. Therefore, elucidating the molecular mechanisms controlling stylet development is of great importance for identifying new targets for controlling nematode infections and enhancing plant resistance.

[0005] Currently, most research on nematode feeding behavior focuses on esophageal glands and their secreted effector proteins, while studies on the stylet itself are mainly limited to phenotypic observations. Microscopic anatomy and electron microscopy have revealed the basic structure of the stylet and its changes during development. However, some fundamental questions remain unanswered, such as which genes are involved in regulating stylet formation, its precise developmental timing, the core gene regulatory networks involved, and the molecular mechanisms upon which its function depends. These knowledge gaps limit a comprehensive understanding of nematode parasitism. Therefore, a systematic analysis of key genes involved in stylet development is crucial for elucidating the developmental and evolutionary mechanisms of this unique parasitic organ and advancing our understanding of nematode parasitic adaptations and organ specialization. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a key gene for the stylet development of southern root-knot nematodes and its application, so as to solve the problem of root-knot nematode control.

[0007] To achieve the above objectives, the inventors first sought to identify key molecular targets for stylet development in *Symplocos spp.* By observing the phenotype of *Symplocos spp.*, we confirmed that stylet development involves two independent stages: the initial formation during the embryonic period and the secondary reconstruction during the J3 / J4 molting period. Therefore, we performed Smart-seq2 single-needle transcriptome sequencing on six developmental stages: 1.5-fold, 2-fold, moving (embryonic period), and S4, S5, and S6 (reconstruction period), obtaining 31 high-quality transcriptome datasets. Through expression pattern clustering, we identified 1,423 upregulated genes during the embryonic period and 887 upregulated genes during the reconstruction period. Based on the inventors' research experience, we screened 45 candidate genes and performed in vitro RNAi screening. Ultimately, we found that silencing three genes, Mi_16868.1, Mi_38955.1, and Mi_39174.1, led to complete stylet absence or malformation, significantly reducing egg hatching rate (<30%).

[0008] Based on the above research results, the first aspect of the present invention is to provide a key gene for the development of the stylet of the southern root-knot nematode, which is Mi_16868.1, Mi_38955.1 or Mi_39174.1; the coding sequence of the Mi_16868.1 gene is shown in SEQ ID NO:1, the coding sequence of the Mi_38955.1 gene is shown in SEQ ID NO:2, and the coding sequence of the Mi_39174.1 gene is shown in SEQ ID NO:3.

[0009] It should be noted that the Mi_16868.1 gene encodes a protein containing a collagen domain; the Mi_38955.1 gene encodes a protein containing both a collagen domain and a Col_cuticle_N domain; and the Mi_39174.1 gene encodes a protein containing a ground-like domain.

[0010] A second aspect of this invention involves providing the application of the aforementioned key gene as a target in the preparation or screening of agents, RNAi formulations, or transgenic plants for controlling root-knot nematodes. More preferably, the agent or formulation comprises dsRNA, siRNA, or a recombinant expression vector capable of transcribed to produce the dsRNA, which can silence the key gene, thereby disrupting the normal development of the stylet of the root-knot nematode by inhibiting the expression of the key gene. More preferably, the root-knot nematodes include *Syzygium sulphureum*, *Soybean cyst nematode*, and *Syzygium sulphureum*.

[0011] A third aspect of the present invention is to provide a dsRNA selected from any one of (a1)-(a3):

[0012] (a1) A double-stranded RNA consisting of the nucleotides shown in SEQ ID NO:4 and their reverse complementary sequences;

[0013] (a2) A double-stranded RNA consisting of the nucleotides shown in SEQ ID NO:5 and their reverse complementary sequences;

[0014] (a3) A double-stranded RNA consisting of the nucleotide shown in SEQ ID NO:6 and the nucleotide of its reverse complementary sequence.

[0015] A fourth aspect of this invention is to provide the application of the aforementioned dsRNA in the control of root-knot nematodes or in the preparation of products for the control of root-knot nematodes. Specifically, the application involves introducing the aforementioned dsRNA into root-knot nematodes, thereby achieving the control of root-knot nematodes. More preferably, the root-knot nematodes include southern root-knot nematodes, soybean cyst nematodes, and sweet potato rot nematodes.

[0016] A fifth aspect of the present invention provides a method for controlling root-knot nematodes, the method comprising the steps of: contacting a root-knot nematode or its eggs or a host plant with one, two or three of the aforementioned dsRNAs, wherein the contact method is selected from any one or a combination of (b1)-(b3):

[0017] (b1) Apply the solution containing the dsRNA directly to the soil or plant roots;

[0018] (b2) The dsRNA is coated in seed coating or granular formulation and applied to the soil;

[0019] (b3) The inverted repeat sequences of the above key genes are integrated into the plant genome, and the plant cells continuously transcribe the dsRNA to produce a host-induced gene silencing effect on root-knot nematodes.

[0020] More preferably, the root-knot nematode or its eggs or host plant are contacted with two or three of the above-mentioned dsRNAs, because the co-silencing of two and three genes further exacerbates the stylet defect, and the hatching rate is reduced to at least <10%.

[0021] In a preferred embodiment of the present invention, the interference vector (pBWA(V)BS backbone) for the three target genes was transformed into tobacco using Agrobacterium-mediated transformation to obtain positive plants stably expressing dsRNA. Then, in a pot resistance test, after inoculation with J2 nematodes for 35-40 days, the number of root nodules in the transgenic tobacco was significantly reduced compared to the wild type. The principle is that when nematodes feed on plant roots, dsRNA is absorbed into the body through the intestines, triggering systemic RNAi, achieving cross-species gene silencing of "plant producing the drug, nematode suffering damage."

[0022] Compared with existing technologies, the key gene for mouthpart development of *Sinonovacula contortus* and its application provided by this invention have the following advantages and advancements:

[0023] (1) This invention pioneers a new mechanism for nematode resistance by "targeting stylet structure development". Existing RNAi anti-nematode technologies (such as targeting 16D10, Mi-CRT, MiCTL1a, MgRGF, etc.) all focus on effector proteins secreted by the esophageal glands of nematodes. The essence of the function of these targets is to interfere with the interaction signals between nematodes and hosts, such as mimicking plant hormones, suppressing immunity, and regulating ROS. Although nematodes may be temporarily suppressed, their stylets remain intact, and theoretically they still have the ability to infect. However, this invention is the first to shift the target of attack from "the user of the parasitic tool" to "the parasitic tool itself", by directly destroying the structural integrity of the stylet, thus physically depriving nematodes of the prerequisite for infection. This fundamental change in thinking means that even if nematodes may evolve strategies to bypass the interference of a certain effector in the future, they will not be able to bypass the structural barrier of "not being able to feed without a stylet". Therefore, the resistance mechanism of this invention is theoretically more durable and thorough than existing technologies.

[0024] (2) This invention is the first to achieve molecular analysis and target identification of the entire process of stylet development. Although stylets have been used as a taxonomic marker for nematodes for over a century, the molecular basis of their development has always been a black box. This invention is the first to map the genetic blueprint of stylet development at the molecular level and identify three essential genes with confirmed functions. This achievement not only provides a new target for nematode control but also provides an important theoretical basis for understanding the organ specialization and evolutionary origin of plant parasitic nematodes, and has significant academic and applied value.

[0025] (3) The synergistic effect of multiple targets in this invention provides a flexible control scheme. This invention found that single-gene silencing can significantly inhibit nematodes, while co-silencing of two or three genes can increase the stylet abnormality rate to 60.3% and reduce the hatching rate to below 10%. This obvious synergistic effect means that in practical applications, target combinations can be flexibly selected according to resistance requirements, i.e., single targets are used for routine control, and multiple targets are used for enhanced control in high-risk areas. At the same time, since the possibility of nematodes simultaneously mutating three essential genes is extremely low, the combination of multiple targets can also greatly reduce the probability of nematodes developing resistance mutations, giving the technology a longer field lifespan.

[0026] (4) This invention has the potential to be rapidly applied to other economic crops. Unlike many patents that remain at the in vitro RNAi stage, this invention completes a closed loop of "target discovery → in vitro functional verification → transgenic plant construction → pot resistance testing". The transgenic tobacco showed a significant reduction in the number of root knots and exhibited clear resistance, directly proving the feasibility of the HIGS route in this technical solution. Given that tobacco, tomato, cucumber, cotton, and other crops are all susceptible to root-knot nematodes and that their genetic transformation systems are mature, the technical solution of this invention has the potential to be rapidly applied to other economic crops, and its industrialization prospects are broad.

[0027] (5) The safety advantages of this invention are outstanding. The target gene is derived from the southern root-knot nematode itself and has no homology with the plant genome or the mammalian genome, so there is no risk of off-target effects. At the same time, the HIGS strategy enables the continuous production of dsRNA in the plant, which is passively ingested by the nematode when feeding, without the need for artificial application of exogenous dsRNA, thus avoiding the environmental residues and ecological risks of chemical pesticides, which is in line with the development direction of green and sustainable agriculture.

[0028] In summary, this invention surpasses existing technologies in four dimensions: theoretical discovery, technical approach, prevention and control effect, and application prospects, demonstrating significant creativity and industrial applicability. Attached Figure Description

[0029] Figure 1 The process of mouthpart formation in Southern root-knot nematodes.

[0030] Figure 2 PCA analysis of Smart-seq2 data during the first formation of the mouthpiece.

[0031] Figure 3 PCA analysis of Smart-seq2 data during the needle reshaping process.

[0032] Figure 4 Two highly expressed gene sets during mouthpiece formation and remodeling.

[0033] Figure 5 Venn diagram of the high-expression gene set of the oral needle.

[0034] Figure 6 : Amplification of the T7 promoter dsRNA template of the target gene.

[0035] Figure 7 : dsMi_16868.1, dsMi_38955.1 and dsMi_39174.1 in vitro RNAi oral phenotypes.

[0036] Figure 8 Results of in vitro interference egg hatching rate of 40 candidate genes.

[0037] Figure 9 The hatching rate results were verified by mixed interference of dsMi_16868.1, dsMi_38955.1 and dsMi_39174.1.

[0038] Figure 10 Detection of the efficiency of individual and mixed RNAi interference qRT-PCR for dsMi_16868.1, dsMi_38955.1 and dsMi_39174.1.

[0039] Figure 11Information on conserved protein domains of Mi_16868.1, Mi_38955.1, and Mi_39174.1.

[0040] Figure 12 Recombinant plasmid vector digestion.

[0041] Figure 13 The process of constructing genetically modified tobacco.

[0042] Figure 14 Identification of positive strains of transgenic tobacco.

[0043] Figure 15 Detection of target gene expression levels in transgenic tobacco.

[0044] Figure 16 Root morphology of T0 generation transgenic tobacco.

[0045] Figure 17 Statistics on the number of tobacco root knots transfected with different target genes. Detailed Implementation

[0046] To better understand the technical content of this invention, specific embodiments are provided below for further detailed explanation. Unless otherwise specified, the methods involved in the embodiments are conventional methods in the art and can be operated with reference to the general techniques described in the literature. Reagents or instruments whose manufacturers are not specified can all be obtained commercially.

[0047] Example 1:

[0048] method

[0049] 1. Nematode culture and egg collection

[0050] Thirty-five days after infection of tobacco plants, the WHF4-1 strain of *M. incognita* was isolated from tobacco roots. Infected roots were thoroughly rinsed with tap water and cut into small fragments, then incubated in a 10% (v / v) sodium hypochlorite solution for 8 minutes to release nematode eggs. The resulting suspension was filtered through a 500 μm filter and rinsed thoroughly with tap water. The material was then washed with 0.01% Triton X-100 to remove residual impurities. Finally, the purified nematode eggs were collected by centrifugation in a 35% (w / v) sucrose solution.

[0051] 2. Samples were collected during the formation and reconstruction stages of the mouthpiece.

[0052] Two distinct stylet developmental processes in *M. incognita* were investigated: stylet formation during the embryonic period and stylet remodeling after the embryonic period. Embryos at the 1.5-fold, 2-fold, and moving stages were collected individually under a microscope. Sampling criteria for stylet remodeling stages were based on the stylet development process, and sampling was divided into four different morphological stages: (1) Molt: during molting, when the nematode no longer has a stylet; (2) Growing: after molting, the stylet is being re-established, and the stylet can be observed at this time; (3) Regain: after J4, the stylet has been established, and a fully functional stylet is regained. Individual nematodes were immediately transferred to lysis buffer and stored at −80°C before Smart-seq2 library construction. Three to six biological replicates were collected for each developmental stage.

[0053] 3. Discovery of genes related to mouthpiece development

[0054] This study used two datasets related to the two stages of stylet development in *M. incognita*. One part was the stage of the first stylet formation during the embryonic development of *M. incognita*, which is reflected in the typical phenotype of stylet formation from absence to appearance during the 1.5-fold to moving stage. Therefore, we specifically selected 15 samples from the three stages of embryonic development (1.5-fold, 2-fold, and moving) for subsequent analysis. After removing the adapters from the original data, we performed correlation tests, aligned the filtered final dataset to the reference genome, and quantified gene expression. We conducted global co-expression pattern analysis on the two datasets from the embryonic development and J3J4 stages of *M. incognita*, specifically screening for differentially expressed genes with high expression during stylet formation. Finally, we took the intersection of the two datasets of differentially expressed genes as the target genes.

[0055] 4. Collection of early-stage eggs from the female insect.

[0056] After collecting J3J4, the residue remaining on the 60-mesh sieve is the female insect. After collecting the female insects in centrifuge tubes, remove as much plant tissue as possible, then remove most of the water, leaving only about 150 μL of total volume. Then, use pointed tweezers to crush the female insects as much as possible, and resuspend them in 1 mL of water with tritons. Vigorously pipette several times, and let them settle naturally for 5-10 seconds. Gently aspirate 600 μL of the upper white solution into a new centrifuge tube. Repeat the process of crushing and adding water five times with pointed tweezers until only insect fragments remain. Use a grinding rod to rotate and squeeze a few times, add water one last time, suspend, pipette, and collect the supernatant. Combine all the supernatants and centrifuge at 1000 g for 30 seconds. The precipitate is the desired insect eggs.

[0057] 5. dsRNA in vitro interference experiment

[0058] After incubation for 60 h in an interference system (110 μL dsRNA, 55 μL eggs, 2 μL Triton-x 100, 33 μL H2O), samples were added to 96-well plates for observation. Each gene treatment group was identified using 8 wells. In each well, 100 μL sterile H2O and 2 μL of 1% sterile Triton were added. 2-3 μL (50-100 insects) of the interfered eggs were collected and their phenotype and hatching rate were analyzed under a microscope. The remaining interfered eggs were washed 2-3 times with water containing Triton to completely remove the dsRNA. The eggs at the bottom of the centrifuged tubes were used to extract RNA for qRT-PCR to verify the interference efficiency.

[0059] Hatching rate statistics method: The number of insect eggs added to each hole and the number of J2 larvae hatched were counted every two days. After 10 days, the hatching rate of insect eggs after different disturbance times was calculated.

[0060] 6. Construction of RNA sequencing libraries from single embryos and single nematodes

[0061] Traditional RNA-seq protocols require a relatively large amount of input RNA for library amplification and construction, while it is difficult to obtain a synchronized embryo culture system for plant parasitic nematodes. Smart-seq2 is a low-throughput single-cell RNA sequencing method that has been successfully used for transcriptome sequencing of single *C. elegans* nematodes, but has not yet been applied to PPN embryo samples. In this study, we successfully constructed a single embryo RNA sequencing library from early *M. incognita* embryos using the Smart-seq2 protocol.

[0062] 7. Construction of the transgenic system

[0063] (1) Construction of interference vector

[0064] Two restriction enzyme sites were selected on the upper and lower arms of the intron of the BWA(V)BS overexpression vector, and amplification was performed using PFU. Three 50 μL amplification systems were prepared according to Table 1:

[0065] Table 1

[0066]

[0067] Reaction program: 94℃ for 5 min; 94℃ for 30 s, 50℃ for 45 s, 72℃ for 15 s (30 cycles); 72℃ for 10 min.

[0068] Incubate at 16℃ for 30 min. After the reaction, the target fragment is excised and recovered using a NanoDrop2000 to measure the concentration of the recovered fragment. The vector digestion system is shown in Table 2.

[0069] Table 2

[0070]

[0071] The product was digested in a 37°C water bath for 1 h.

[0072] (3) The target fragment is subjected to double digestion according to the vector enzyme digestion system. After the reaction is completed, the vector enzyme digestion product and the target gene product are purified for ligation reaction.

[0073] (4) The enzyme ligation system of the vector and the target fragment is shown in Table 3.

[0074] Table 3

[0075]

[0076] The reaction solution was placed in a 20°C water bath for 1 hour for connection.

[0077] (5) Transform 5-10 μL of the ligation product into competent Escherichia coli cells (see the standard method for transformation of competent Escherichia coli cells), transform into kanamycin-resistant plates, incubate at 37°C for 12 hours, and perform colony PCR identification.

[0078] (6) Extract 10 transformants for colony PCR identification, take positive band samples for sequencing, and inoculate samples with correct sequencing for plasmid extraction and preservation.

[0079] (7) Take 1 μL of the correctly identified plasmid and transform it into Agrobacterium GV3101 competent cells. Mix thoroughly and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 30℃ and 180 rpm for 30 min on a shaker. Inoculate 50 µL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30℃ for 48 h. After a positive PCR test, proceed with the genetic transformation of tobacco.

[0080] (8) Disinfect tobacco seeds with 75% alcohol for 30 seconds, rinse with sterile water for 1 minute, then disinfect with 84 disinfectant for 3-5 minutes, and rinse with sterile water 3 times, 1 minute each time. Sow the disinfected tobacco seeds on germination medium and culture at 23℃ for 16 / 8 h light / dark for 4-5 weeks. Cut sterile tobacco leaves into small pieces with a scalpel and inoculate them on pre-culture medium.

[0081] (9) Pick Agrobacterium and place it in the infection solution to prepare OD. 600 Tobacco leaves that have been pre-cultured for 2-3 days were inoculated into the Agrobacterium suspension at a concentration of 0.2 for 10-15 min. The inoculated tobacco leaves were then inoculated onto filter paper, dried, and then inoculated onto a co-culture medium and incubated in the dark for 48-72 h.

[0082] (10) Transfer the co-cultured explants to an induction medium and culture for 2-3 weeks to grow callus tissue.

[0083] (11) Select callus tissues with good growth and inoculate them into culture machines with corresponding resistance levels, and culture for 15-30 days. Inoculate vigorous positive callus tissues from the second screening onto differentiation culture machines and culture for 15-30 days under 16 / 8 h light / dark conditions. If seedlings form from the callus tissues during differentiation, inoculate them onto seedling growth medium and grow for 7-10 days. Then transplant them into soil for subsequent pot experiments.

[0084] 8. Transgenic Potted Plant Experiment

[0085] (1) Nematode inoculation and culture: Tobacco seedlings were cultured to a height of about 15 cm, and 8000-1000 pre-hatched J2 juvenile southern root-knot nematodes were inoculated into the roots of the tobacco plants; GFP and wild-type control groups were set up for inoculation at the same time. The eggs were collected after about 35 days of culture under greenhouse conditions.

[0086] (2) Tobacco root knot count: After the tobacco is taken back, the roots are washed under running water. The number of root knots of each plant is counted and evaluated according to the size of the root knots. Extra large root knots are counted as 6-8, medium root knots as 3-5, small root knots as 1-2, and single root knots as 1.

[0087] 9. Detection of interference gene transcription levels

[0088] (1) The extraction procedure for RNA from nematodes and transgenic tobacco was performed according to the TransZol™ UP Plus RNA Kit. Tissue samples frozen in liquid nitrogen were removed, and 300 μL of TransZol™ Up from the kit was added. The samples were homogenized using a homogenizer (pre-freezing module; nematode homogenization parameters: 70 Hz; plant leaf homogenization parameters: 65 Hz). 0.2 mL of chloroform was added, and the mixture was vigorously shaken for 30 s and incubated at room temperature for 3 min. The remaining steps were performed according to the kit instructions.

[0089] (2) In vitro reverse transcription experiment:

[0090] The extracted RNA was digested and reverse transcribed to obtain cDNA. This experiment was performed using the ABScript III RT MasterMix for qPCR with gDNA Remover kit. An appropriate amount of RNA starter template was added according to the specific concentration of the extracted RNA. Reverse transcription conditions: 37℃ for 2 min, 55℃ for 15 min, 85℃ for 5 min, 4℃ Hold. The cDNA product can be used for subsequent qRT reactions and should be stored at -20℃ for later use.

[0091] (3) qRT-PCR reaction:

[0092] The experiment was performed using ABClonal's 2×Universal SYBR green Fast qPCR Mix. This experiment required protection from light. Each reaction was performed in triplicate. The reaction mixture was aliquoted into a dedicated qPCR plate according to the label, sealed with an optical sealing film, centrifuged at 2500 rpm, and then placed in a dedicated quantitative PCR instrument to set the reaction program.

[0093] result

[0094] 1. Morphological characteristics during the formation of the mouthpiece

[0095] To determine the developmental stages associated with the formation of the mouthpiece structure, we first conducted a detailed morphological observation of *M. incognita* during embryonic and post-embryonic development. Figure 1 It was observed that no identifiable stylet structures were observed during the 1.5-fold stage of embryonic development. The 2-fold stage indicated the initiation of stylet development. As embryonic development progressed into the movement stage, complete stylet structures were established, suggesting that functional stylet structures were formed before hatching. In addition to embryonic development, a second stylet development event was observed during molting in J3–J4. Morphological observations showed that the primitive stylet structure degenerated in late J3 (S4), leading to the disappearance of the stylet in the infected organ. Subsequently, in the remodeling stage (S5), new stylet structures began to form. By the early J4 stage (S6), a complete stylet structure with typical morphology had been successfully reconstructed. These observations suggest that the development of *Sinus spp.* involves two independent stages: primary formation during embryonic development and secondary remodeling in the post-embryonic stage. Based on these developmental characteristics, the embryonic stages of 1.5-fold, 2-fold, and moving, as well as the remodeling stages of S4, S5, and S6, were selected for subsequent transcriptome analysis. These six stages represent the beginning and completion of two mouthpiece developments, providing experimental evidence for identifying genes involved in mouthpiece formation.

[0096] 2. Acquisition of transcriptome data during mouthpiece formation

[0097] After identifying the key developmental stages associated with stylet formation and remodeling, researchers generated stage-specific transcriptome datasets to characterize gene expression dynamics during these two developmental periods. Based on the morphological observations, the inventors selected embryos at the 1.5-fold, 2-fold, and moving stages, as well as larvae at the S4, S5, and S6 stages, for Smart-seq2 transcriptome sequencing. To ensure comparability between datasets, all samples underwent the same RNA amplification, library preparation, and sequencing methods. A total of 31 single-needle nematode transcriptome datasets were obtained, including 15 samples representing the stylet formation stage and 16 samples representing the stylet remodeling stage. After quality control and expression quantification, principal component analysis (PCA) was performed to assess sample reproducibility and overall transcriptional activity. Figure 2 It can be seen that samples collected during the mouthpiece formation process in the embryonic stage are clearly grouped according to developmental stage, with PC1 and PC2 explaining 47% and 28% of the total variance, respectively. Figure 3 The transcriptomes associated with mouthpin remodeling also exhibited a clear clustering pattern, with PC1 and PC2 explaining 72% and 9% of the variance, respectively. The close clustering of repetitive samples from the same developmental stage indicates high reproducibility and quality of the data. Overall, these datasets provide a comprehensive transcriptional resource covering the primary and secondary remodeling processes of the mouthpin and lay the foundation for subsequent identification of genes involved in spine development.

[0098] 3. Identify candidate genes highly expressed during mouthpiece formation.

[0099] Cluster analysis of expression patterns revealed multiple gene clusters exhibiting high expression in two independent mouthpiece developmental events. Figure 4 It was found that 1,423 genes showed increased expression levels during mouthpiece formation in the embryonic stage, while 887 genes were specifically upregulated during mouthpiece remodeling. To identify the core regulatory genes shared by these two developmental processes, we performed an intersection analysis of the two gene sets. Figure 5 The results showed that 55 overlapping genes were present during both stylet formation and remodeling. Since these genes were activated in both independent stylet developmental events, they are considered to play important roles in stylet biosynthesis.

[0100] To evaluate the function of the identified candidate genes, in vitro RNA interference (RNAi) screening was performed. Homology analysis was conducted on all candidate genes before designing the experiment. For genes with highly similar sequences, only one representative homologous gene was selected for functional validation. Therefore, a total of 45 genes were selected for subsequent analysis. A total of 45 pairs of T7-labeled primers were designed, and double-stranded RNA corresponding to 41 genes was successfully synthesized, including dsGFP as a control. Figure 6 ).

[0101] This study selected early embryos lacking visible stylet structures for RNAi treatment to ensure gene silencing occurred before stylet formation. After treatment with gene-specific dsRNA, newly hatched J1 larvae were examined to observe abnormalities in their feeding organ development. Phenotypic screening showed that most candidate gene silencing mediated by RNAi had little or no detectable effect on stylet morphology. In contrast, knockout of three genes, Mi_16868.1, Mi_38955.1, and Mi_39174.1, resulted in severe stylet development defects. Most larvae from these RNAi-treated larvae exhibited complete stylet absence or structural malformation. Furthermore, related feeding structures, including the pharyngeal glands, esophagus, and basal bulb, were also absent. Similarly, when two target genes (dsMi_16868.1+dsMi_38955.1, dsMi_16868.1+dsMi_39174.1, dsMi_38955.1+dsMi_39174.1) and three target genes (dsMi_16868.1+dsMi_38955.1+dsMi_39174.1) were simultaneously silenced, a large number of early J1 larvae exhibited stylet absence or malformation. These abnormally developed larvae also showed significant defects in the pharyngeal glands, digestive tract, esophagus, and basal bulb. In contrast, nematode embryos treated with dsGFP developed normally, with well-developed stylet, basal bulb, and pharyngeal gland structures. Figure 7 Phenotypic analysis showed that RNAi-mediated target gene silencing resulted in severe defects in stylet formation, including abnormal or complete stylet morphology in unhatched eggs.

[0102] To quantify the frequency of stylet-related developmental abnormalities, 50 to 70 nematodes were randomly selected from each treatment group for phenotypic analysis. Table 4 shows that in the dsGFP-treated control group, most nematodes developed normally, with an extremely low abnormality rate (<1%). In contrast, RNAi treatment of Mi_16868.1, Mi_38955.1, and Mi_39174.1 resulted in developmental defect frequencies ranging from 20% to 60%. The stylet developmental abnormality rates of these three genes were significantly higher than those in the control group. In conclusion, these results identify Mi_16868.1, Mi_38955.1, and Mi_39174.1 as key regulators of nematode stylets and provide a basis for further research on their function.

[0103] Table 4. Statistics on the number of oral sting phenotypes

[0104]

[0105] 4. Silencing three candidate genes can severely affect egg hatching and embryonic development.

[0106] To determine whether the three developmental genes are involved in embryonic development, the hatching rate of eggs treated with RNAi interference was evaluated. Statistical analysis showed that among the 40 candidate genes, the hatching rate of most RNAi-treated eggs was between 40% and 60%, with no significant difference compared to the control group, indicating no significant effect on egg hatching. Figure 8 a–d). In contrast, knocking out Mi_16868.1, Mi_38955.1, and Mi_39174.1 via RNA interference significantly reduced the egg hatching rate to below 30%, a decrease of approximately 50% compared to the control group. Figure 8 (b and d). Of these three genes, Mi_38955.1 produces the most intense phenotype, reducing the hatching rate to below 20%.

[0107] To further investigate the potential functional interactions among these genes, RNA interference was performed on two-gene and three-gene sequences. Figure 9 Simultaneous silencing of two genes further reduced the hatching rate of eggs, with the dsMi_16868.1 + dsMi_38955.1 treatment showing the most significant effect, reducing the hatching rate to below 20%. Notably, triple gene silencing resulted in the most severe developmental defects, with less than 10% of embryos successfully hatching after six days of incubation. These results indicate that Mi_16868.1, Mi_38955.1, and Mi_39174.1 are essential for normal embryonic development and collectively promote successful egg hatching.

[0108] To assess the efficiency of RNAi, the transcriptional level of the target gene was quantitatively analyzed by qRT-PCR. Figure 10 The results showed that single RNAi treatment reduced the transcription level of the target gene by approximately 60% compared to the dsGFP control group, confirming effective gene silencing. Dual-gene RNAi treatments targeting Mi_16868.1 + Mi_38955.1 and Mi_16868.1 + Mi_39174.1 significantly suppressed both target genes. In the Mi_38955.1 + Mi_39174.1 treatment, the transcriptional level of Mi_39174.1 decreased more significantly than that of Mi_38955.1. Similarly, triple-gene RNAi significantly reduced the expression levels of all target genes. Although the silencing efficiency varied among genes, these results indicate that the observed developmental defects are closely related to the effective suppression of target gene expression.

[0109] To further explore the potential functions of these genes, a conserved domain analysis was conducted. Figure 11The prediction results showed that both Mi_16868.1 and Mi_38955.1 contain collagen domains, indicating that they belong to the collagen superfamily. Collagen is an extracellular structural protein that plays a crucial role in the formation and maintenance of connective tissue. Furthermore, Mi_38955.1 also contains a Col_cuticle_N domain, located at the N-terminal region of keratinocytes secreted by subepidermal cells in nematodes. Although the exact function of this domain is unclear, it is associated with the nematode cuticle, a rigid and elastic extracellular matrix secreted by basement membrane cells, primarily composed of collagen. The Mi_39174.1 gene was predicted to contain a "Ground-like" domain, which is found only in *C. elegans* and its closely related species. Studies have suggested that proteins containing such "Ground-like" domains may bind to and regulate the activity of patched molecules on the membrane, and may also be involved in the regulation of neuropeptides. The function of proteins containing "Ground-like" domains in PPNs has not yet been reported. These structural features support the hypothesis that stylet formation requires coordinated regulation between extracellular structural components and developmental signaling factors.

[0110] In summary, these findings indicate that Mi_16868.1, Mi_38955.1, and Mi_39174.1 are crucial for embryonic development and egg hatching, and are key regulators associated with stylet development in Southern root-knot nematodes.

[0111] 5. Successfully constructed Mi_16868.1, Mi_38955.1, and Mi_39174.1 transgenic tobacco varieties.

[0112] The use of genetic engineering techniques to transfer key genes involved in the growth and development of nematodes into target plants is of great significance for nematode research and is also considered a green engineering approach to nematode control. This study aimed to better reveal the effects of target genes within nematodes and explore their influence on nematode development under host conditions. We attempted to heterologously transform three validated target genes into a tobacco system to investigate the feasibility of nematode control. We constructed a pBWA(A)BS vector containing the target genes. Enzyme digestion confirmed the successful construction of recombinant vectors for the three genes Mi_16868.1, Mi_38955.1, and Mi_39174.1. Figure 12 ).

[0113] The next step in this study is to transform the extracted recombinant vector plasmid into Agrobacterium competent cells, and screen for positive strains for genetic transformation experiments in tobacco. The process involves pre-culture, Agrobacterium infection and co-culture, induction, screening, differentiation, and rooting. Figure 13We obtained varying numbers of transgenic tobacco seedlings, and further marked the rooted plants. Positive plants for the target gene were then identified using PCR. Figure 14 Based on the identification results, 10 strongly positive plants for each target gene were selected for subsequent pot experiments.

[0114] 6. Tobacco plants transfected with the target gene exhibit M. incognita resistance.

[0115] The study transplanted positive plants into individual pots for pot experiments. After 15 days of growth, approximately 2000 J2-stage nematodes were introduced into each seedling to test their infectivity. After 35-40 days of culture, the number and size of root knots in the T0 generation were counted to assess whether transgenic tobacco affected the infectivity of nematodes. We first randomly selected 12 transgenic plants and extracted RNA from their leaves. After in vitro reverse transcription, qRT-PCR amplification was performed to identify the expression level of the target gene in the plants. The results showed that Mi_16868 2#, Mi_16868 4#, Mi_38955 1#, Mi_16868 4#, Mi_39174 1#, and Mi_39174 2# exhibited higher target gene expression levels, which were 20-200 times higher than those in wild-type tobacco plants. Figure 15 This indicates that the target gene was successfully transferred into the tobacco plant and can be stably expressed.

[0116] We then observed the root characteristics of tobacco plants after nematode infection. Morphologically, tobacco plants transfected with the target gene had fewer root knots and lower root knot density compared to GFP and wild-type plants. Wild-type and GFP plants had larger root knots and a greater number of root knots, with Mi_38955.1 showing the most significant effect. Figure 16 These results indicate that reduced expression levels of target genes within nematodes can affect their reproduction and infection behavior, thus endowing the plants with a certain degree of nematode resistance.

[0117] We further analyzed the number of root knots infected by nematodes in the T0 generation of transgenic tobacco. Calculations showed no significant difference in the number of root knots between the CK and GFP groups. However, compared to the control group, the number of root knots in all transgenic plants decreased to some extent, with the most significant decrease observed in tobacco plants transgenic with the Mi_38955 gene. Figure 17 The results indicate that inhibiting the expression levels of Mi_16868.1, Mi_38955.1, and Mi_39174.1 in nematodes can reduce the growth, development, and reproductive capacity of nematodes, thus endowing transgenic tobacco with M. incognita resistance characteristics.

Claims

1. The key gene for stylet development in the southern root-knot nematode is Mi_16868.1, Mi_38955.1, or Mi_39174.1; the coding sequence of the Mi_16868.1 gene is shown in SEQ ID NO:1, the coding sequence of the Mi_38955.1 gene is shown in SEQ ID NO:2, and the coding sequence of the Mi_39174.1 gene is shown in SEQ ID NO:

3.

2. The use of the key gene described in claim 1 as a target in the preparation or screening of agents, RNAi preparations or transgenic plants for the control of root-knot nematodes.

3. The application according to claim 2, characterized in that, The agent or formulation contains dsRNA, siRNA, or a recombinant expression vector capable of transcribed to produce the key gene, thereby disrupting the normal development of the root-knot nematode's mouthparts by inhibiting the expression of the key gene.

4. The application according to claim 2, characterized in that, The root-knot nematodes mentioned include Southern root-knot nematode, soybean cyst nematode, and sweet potato rot nematode.

5. A dsRNA, selected from any one of (a1)-(a3): (a1) A double-stranded RNA consisting of the nucleotides shown in SEQ ID NO:4 and their reverse complementary sequences; (a2) A double-stranded RNA consisting of the nucleotides shown in SEQ ID NO:5 and their reverse complementary sequences; (a3) A double-stranded RNA consisting of the nucleotide shown in SEQ ID NO:6 and the nucleotide of its reverse complementary sequence.

6. The use of the dsRNA according to claim 5 in the control of root-knot nematodes or in the preparation of products for the control of root-knot nematodes.

7. The application according to claim 6, characterized in that, The application involves introducing the dsRNA described in claim 5 into root-knot nematodes to achieve control of root-knot nematodes.

8. The application according to claim 6, characterized in that, The root-knot nematodes mentioned include Southern root-knot nematode, soybean cyst nematode, and sweet potato rot nematode.

9. A method for controlling root-knot nematodes, characterized in that, The method includes the following steps: contacting a root-knot nematode or its eggs or a host plant with one, two, or three of the dsRNAs described in claim 5, wherein the contact method is selected from any one or a combination of (b1)-(b3): (b1) Apply the solution containing the dsRNA directly to the soil or plant roots; (b2) The dsRNA is coated in seed coating or granular formulation and applied to the soil; (b3) The inverted repeat sequence of the key gene described in claim 1 is integrated into the plant genome, and the plant cells continuously transcribe the dsRNA to produce a host-induced gene silencing effect on root-knot nematodes.

10. The method according to claim 9, characterized in that, The root-knot nematodes mentioned include Southern root-knot nematode, soybean cyst nematode, and sweet potato rot nematode.

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