Peanut disease-resistant gene AhCN34, method for identifying same and use thereof

Abstract

The application discloses a peanut disease-resistant gene, named AhCN34 gene, wherein the DNA sequence of the AhCN34 gene is shown as SEQ ID NO:1, and the amino acid sequence encoded by the AhDef2.2 gene is shown as SEQ ID NO:2. By overexpressing the AhCN34 gene in peanut leaves, the resistance of the peanut to bacterial wilt can be enhanced, and the peanut has important application value for cultivating a new peanut variety with resistance to bacterial wilt.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a peanut AhCN34 gene and an identification method and application thereof. BACKGROUND

[0002] Peanut is an important oil crop and economic crop, and is widely planted in more than 100 countries and regions in the world. The global peanut planting area is about 33 million hectares; the average yield of peanut is 1648 kg / ha, and the total annual yield is 53.93 million tons (FAO.2021). Compared with other domestic oilseed crops, peanut has good characteristics, so the peanut yield in many areas is increasing. These characteristics include high total yield, high unit area yield, high planting efficiency per unit yield, high total value, and high export volume. However, peanut is susceptible to fungal, bacterial, and viral pathogens. The biggest threat to production comes from Ralstonia solanacearum (RS), which is the pathogen of bacterial wilt (BW) (Jiang et al.2017; Chen et al.2020). Once introduced into the soil, this pathogen is difficult to eliminate from the soil and has earned the nickname of "plant cancer" due to its devastating effects on growth and development (Zhang et al.2017a; Luo et al.2019).

[0003] The increase in peanut planting in China has led to the gradual spread of BW in major peanut-producing areas, which is particularly common in high-temperature and high-humidity areas (Elsayed et al.2020). The incidence rate is usually between 10-30%, but in severe cases, the infection rate exceeds 80%, leading to complete crop failure (Zhang et al.2017b). The use of BW-resistant peanut varieties (such as 'Zhonghua No.2', 'Yuanza 9102', and 'Xieheqing No.19') has successfully reduced the incidence rate in major affected areas to below 8%; therefore, this strategy has played a crucial role in stabilizing and developing peanut production in these areas (Jiang et al.2017). In order to ensure adequate supply of oilseeds and proteins, increase peanut-based income, and strengthen competitiveness in international trade, further progress must be made in the genetic improvement of peanut BW resistance.

[0004] To enter the plant, RS exhibits positive chemotaxis towards peanut root metabolites in the soil, enabling it to move towards the root surface (Alvarez et al., 2010). It then invades the root through wounds or cracks and enters the vascular bundle through the cortex and parenchyma (Lowe Power et al., 2018). Once in the vascular bundle, RS causes physical blockage by secreting extracellular polysaccharides, cell wall-degrading enzymes, adhesion proteins, and surface appendages, which prevent xylem and phloem transport and lead to water deficiency within the plant (Yu et al., 2020). In the early stages of infection, the uppermost leaves of the peanut plant droop due to water loss, but remain green. However, later stages bring more extreme damage, with the roots rotting and the entire plant dying.

[0005] In species with RS resistance traits, it behaves as a typical quantitative trait; it is controlled by multiple genes that inhibit pathogen proliferation and slow down the host’s wilting and death. Previous studies have identified quantitative trait loci (QTLs) associated with RS resistance. For example, Ren et al. (2010) located two markers associated with RS resistance at regions of 8.12 cM and 11.46 cM. Peng et al. (2010) generated a linkage map consisting of 98 amplified fragment length polymorphism (AFLP) markers, revealing three QTLs for BW resistance (qBWr1, qBWr2, and qBWr3). Chen et al. (2014) used RNA sequencing (RNA-seq) methods and found that most of the differentially expressed genes in peanut in response to RS infection were nucleotide-binding leucine-rich repeat (NBS-LRR) proteins, mitogen-activated protein kinases (MAPKKs), and WRKY factors. Zhao et al. (2016) utilized QTL mapping techniques and identified two micro-QTLs (qBW-1 and qBW-2) associated with BW resistance in the resistant variety “Yueyou 92,” which together explained 12% of the phenotypic variation. In the same study, the function of qBW-1 was verified using a recombinant inbred line (RIL) population, which revealed four single nucleotide polymorphisms (SNPs) associated with qBW-1. Other studies have cloned peanut homologs of the Arabidopsis disease resistance genes RRS1-R (Deslandes et al. 2003) and ERECTA (Godiard et al. 2003) (AhRLK1 (Zhuang et al. 2019) and AhRRS5 (Zhang et al. 2017a), respectively); ectopic overexpression of these genes in tobacco indicated that they enhanced resistance to BW. Analysis of QTL allelic variation for peanut BW resistance revealed that a primary QTL (qBWB02.1) detected in three environments was located in the B02 chromosome region, which is rich in NBS-LRR-type disease resistance genes (Wang et al., 2018).

[0006] The role of NBS-LRR genes (also known as NLRs) in plant immune responses has been well characterized; they encode plant-specific immune receptors that directly participate in pathogen recognition (Macho & Zipfel, 2015; Maruta et al. 2022). Most plant genomes contain hundreds of NBS-LRR genes with extensive sequence diversity (Griebel et al. 2014; Kourelis et al. 2021). Based on differences in N-terminal domains, NBS-LRRs can be divided into two subfamilies: CC-NB-LRRs (CNLs) and TIR-NB-LRRs (TNLs). Previous studies have provided detailed mechanisms of NBS-LRR function in model plant species. For example, in Arabidopsis thaliana, the NLR gene RPW8 encodes only a coiled-coil (CC) domain, yet still provides resistance to the powdery mildew pathogen (Xiao et al. 2001). In rice (Oryza sativa L.), only when both genes are present, the CNL genes PI5-1 and PI5-2 confer resistance to blast disease due to the requirement for interaction between the CC domains of the encoded proteins (Lee et al. 2009). The potato protein Rx contains NBS and LRR domains that mediate disease resistance (Rairdan et al. 2008); the CC and LRR domains co-regulate the signaling activity of the NBS domain in a recognition-dependent manner (Tameling & Baulcombe, 2007; Cai et al. 2021).

[0007] Bacterial wilt (BW) of peanut (Arachis hypogaea L.) is a devastating soil-borne disease caused by Ralstonia solanacearum (RS) that poses a significant threat to yield and quality of peanut. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins are a class of plant-specific immune receptors that recognize pathogen-secreted effectors and activate immune responses to resist pathogen infection. However, the exact function of AhCN genes (CNs are a class of NLR genes that lack LRR domains) in peanut plants is not fully understood. Breeding disease-resistant varieties is the most economical, environmentally friendly, and effective method to control BW in peanut. However, despite previous studies on RS resistance in peanut, there has been no systematic study of different BW resistance genes in existing peanut germplasm; characterization of differences in resistance mechanisms; and assessment of the potential of germplasm complementary crosses for different resistance methods (Sheng et al. 2022). Furthermore, although many NBS-LRRs have been functionally studied in model plant species, they have not been comprehensively identified and characterized in peanut. SUMMARY

[0008] The technical problem to be solved by the present application is that there is no peanut AhCNs resistance gene in the prior art that can resist bacterial wilt.

[0009] To solve the technical problem, the application provides a peanut AhCN34 gene and an identification method and application thereof.

[0010] The object of the application is achieved by a peanut disease-resistant gene AhCN34, wherein a DNA sequence of the AhCN34 gene is shown as SEQ ID NO: 1.

[0011] An amino acid sequence encoded by the AhCN34 gene is shown as SEQ ID NO: 2.

[0012] The identification method of the peanut disease-resistant gene AhCN34 comprises the following steps:

[0013] (1) A peanut genome is downloaded from PEANUTBASE, and 150 putative peanut AhCNs are identified in the peanut genome by BLAST homology matching and SMART analysis based on sequence homology of known CNs in Arabidopsis thaliana, rice, tomato and wheat;

[0014] (2) Expression levels of the AhCNs are detected at several time points after inoculation of RS in healthy susceptible peanut variety H107 and healthy resistant peanut variety H108, and 15 AhCNs that are significantly differentially expressed in the susceptible peanut variety H107 and the resistant peanut variety H108 are selected;

[0015] (3) The healthy susceptible peanut variety H107 and the healthy resistant peanut variety H108 are each divided into five groups, a control group is sprayed with sterile water, and one group in an experimental group is inoculated with RS, and the other three groups are sprayed with plant hormones SA, MeJA and ABA, respectively;

[0016] (4) The gene that is significantly up-regulated after inoculation of RS and spraying of the three plant hormones SA, MeJA and ABA is the AhCN34 gene.

[0017] The verification method of the peanut disease-resistant gene AhCN34 having the function of resisting bacterial wilt is as follows:

[0018] (1) An overexpression vector of the AhCN34 is constructed: the pCambia1300 YFP vector is linearized by incubation at 37°C for 2 hours and incubation at 65°C for 20 minutes, and then a fragment encoding the AhCN34 is inserted;

[0019] (2) A plasmid encoding an AhCN34-YFP fusion protein is injected into healthy H107 leaves, a plasmid encoding a YFP protein is injected into healthy H107 leaves as a control, and then bacterial wilt is inoculated;

[0020] (3) observing the leaf disease symptoms of step (2) and performing trypan blue (Solarbio) staining, measuring superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activity and malondialdehyde (MDA) content;

[0021] (4) compared with only overexpressing YFP, the leaves overexpressing AhCN34 YFP remained healthy, reduced cell death, and the POD and CAT activities in the leaves were significantly increased, indicating that AhCN34 improved the resistance of peanuts to RS.

[0022] Each AhCNs includes two highly conserved CC and NB-ARC domains.

[0023] The expression method in step (2) is:

[0024] Gene-specific primers are designed using Primer Premier 5.0;

[0025] RNA is extracted using TRIzol reagent;

[0026] The concentration and purity of RNA are determined using NanoDrop ND1000 instrument;

[0027] and TransScript II multiplex probe one-step qRT-PCR is used;

[0028] RNA is used for first-strand cDNA synthesis using SuperMix UDG kit;

[0029] Gene expression analysis is performed using FastKing RT kit with gDNase;

[0030] The specificity of primers is verified by the presence of a single peak melting curve and the check of PCR product size;

[0031] qRT-PCR is performed using TB Green Premix Ex-Taq kit on CFX96 real-time fluorescent PCR instrument;

[0032] The expression level of target genes is normalized to the internal reference gene ACTIN7 using the 2–ΔΔCt method;

[0033] The expression values of each sample and three technical replicates of target genes are averaged;

[0034] Statistically significant differences between samples are determined using Student's t-test in Graphpad Prism 8.0;

[0035] The method for identifying the putative peanut AhCNs is:

[0036] Key domains in each putative AhCN were verified using the NCBI-CDD database;

[0037] The physicochemical properties of the putative AhCNs were predicted using ExPASy;

[0038] Subcellular localization was predicted using Cell PLoc 2.0;

[0039] The amino acid sequences of CN proteins in Arabidopsis thaliana, Oryza sativa, Glycine max, Triticum aestivum L. and Lycopersicon esculentum L. were downloaded from the NCBI database and aligned with ClustalX;

[0040] Phylogenetic trees were constructed in MEGA 7.0;

[0041] The analysis of protein alignment and prediction of segmental duplication events were performed using the Python version of MCScan.

[0042] Application of peanut disease-resistant gene AhCN34 in peanut engineering against Ralstonia solanacearum.

[0043] Compared with the prior art, the technical effect of the present application is that the present application provides a peanut AhCN34 gene, and the resistance to Ralstonia solanacearum can be enhanced by overexpressing AhCN34 in peanut leaves, which has important application value for cultivating new peanut varieties with resistance to Ralstonia solanacearum. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 are the phenotypes and physiological characteristics of peanut plants after infection with Ralstonia solanacearum (RS): A, representative cross-sections and longitudinal sections showing the microstructure of the main stem of peanut varieties H108 and H107, and red arrows indicate the areas where the vascular bundles are invaded by RS; 5 to 25 days after inoculation (dpi), the quantification of chlorophyll content (B), plant height (C), main stem length (D), stem diameter (E), plant fresh weight (F) and plant dry weight (G) in H107 and H108, and the data are represented as mean ± standard value (n=9) *p<0.05, **p<0.01 (Student's t-test).

[0045] Figure 2A, Systematic tree showing the relationship between peanut and other selected plant species CN proteins. This tree revealed nine CN subfamilies; B, Linear relationship between the number of CN genes reported in Arabidopsis, maize, potato, and other 21 plants and genome size; C, Subcellular localization prediction of 150 AhCNs; D, Comprehensive analysis showing the evolutionary relationship between 150 AhCN genes in wild Arachia species (A. duranensis, A. ipansis, and A. monticola) and cultivated peanut (Arachis hypogaea).

[0046] Figure 3 A, Composition of coiled coil (CC) and NB-ARC domains in AhCN proteins; Frequency of amino acid residues in five common conserved sequences among 150 AhCNs; B, Amino acid sequence alignment of selected AhCNs and AtCNs, with conserved regions of amino acids in blue; C, Three-dimensional structure prediction of AtCN1 (top panel) and AhCN34 (bottom panel), with the spatial location of five conserved sequences indicated, 1-5 representing EDVID, P-loop, kinase-2, RNBS-B, and GLPL, respectively.

[0047] Figure 4 A, Expression levels of AhCNs in several peanut tissues (e.g., root, stem, leaf, flower, and pod), with gene expression values calculated as fragments per kilobase of transcript per million mapped reads (FPKM), then transformed and normalized, with values shown as log10(FPKM+0.01); B, Expression levels of defense-related genes in peanut varieties H108 and H107 after inoculation with Ralstonia solanacearum (RS); C, Three major gene expression trends in H108 and H107 at 7 days post inoculation (dpi) with RS, with gene expression data normalized, with red lines representing trends; Each blue line represents the expression of one gene with the corresponding expression profile; D, Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathway enrichment analysis of differentially expressed AhCN genes; E, Correlation between 15 AhCNs that were significantly differentially expressed in plants infected with RS, with red indicating positive correlation coefficients and blue indicating negative correlation coefficients.

[0048] Figure 5Expression of selected AhCNs in response to bacterial infection and hormone treatment: A, expression of AhCNs at 0, 0.5, 1.0, and 7.0 days post inoculation (dpi) with Ralstonia solanacearum (RS); B, expression of AhCNs after treatment of RS-inoculated plants with the indicated concentrations of salicylic acid (SA), methyl jasmonate (MeJA), or abscisic acid (ABA), target gene expression levels were analyzed using quantitative reverse transcription polymerase chain reaction and normalized to the internal control gene ACTIN7, data are presented as mean ± standard error (n = 3) *p < 0.05, **p < 0.01 (Mann-Whitney U test).

[0049] Figure 6 Transient overexpression and functional validation of AhCN34 in peanut defense against Ralstonia solanacearum (RS): A, schematic diagram of the strategy for vector construction of transient expression; B, subcellular localization of yellow fluorescent protein (YFP) control and AhCN34-YFP fusion protein transiently overexpressed in tobacco leaves, the fluorescence of the fusion protein overlapped with the plasma membrane (PM) marker, indicating that AhCN34 was localized to the PM; C, disease symptoms of peanut plants with transient overexpression of YFP (left) or AhCN34-YFP (right) at 3 days post inoculation (dpi) with RS.d, trypan blue staining of peanut leaves transiently overexpressing YFP control or AhCN34-YFP at 3 dpi with RS.E-J, quantification of superoxide dismutase activity (E), peroxidase (POD) activity (F), catalase (CAT) activity (G), and malondialdehyde (MDA) content (H) of peanut leaves transiently overexpressing YFP control or AhCN34-YFP at 3 dpi with RS. Statistical significance was assessed using the Mann-Whitney U test and indicated with asterisks (**, P < 0.01).

[0050] Figure 7 Schematic diagram of gene ontology (GO) term enrichment analysis for DEGs.

[0051] Figure 8 Schematic diagram of uneven distribution of AhCNs on all peanut chromosomes except 7 and 17.

[0052] Figure 9 Schematic diagram of members of each subfamily of AhCNs showing similarity in the presence of motifs.

[0053] Figure 10 Schematic diagram of various plant hormones and resistance-related elements involved in CNs. DETAILED DESCRIPTION

[0054] The application will be further described below in connection with specific embodiments.

[0055] I. Biological materials and reagents

[0056] The high-resistant peanut variety 'H108' and the high-susceptible peanut variety 'H107' bred by the peanut research group of Henan Agricultural University are near-isogenic lines. The RS strain 180731-1 is derived from the Plant Protection Institute of Henan Academy of Agricultural Sciences. Salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA) are obtained from Solarbio.

[0057] The DNA sequence of the AhCN gene provided by the application is SEQ ID NO: 1, and the amino acid sequence encoded by the AhCN gene is SEQ ID NO: 2.

[0058] II. RS inoculation

[0059] For the infection assay, whole peanut seeds were soaked in 1% sodium hypochlorite solution for 30 seconds, rinsed three times in water, and placed in 2L hydroponic containers until the root length was 4 cm. Then each container was placed in an incubator at 30°C with a 16 / 8 hour light / dark cycle. RS was grown in 2,3,5-trihenyte-triazole hexamine (TTC) medium at 28°C for 2 days, then diluted to 108colony forming units (CFU) mL-1 in water for plant inoculation. At the third trifoliate stage, 0.5 cm was cut from the main root tip of each plant and the roots were immersed in the RS suspension. The control plants were soaked in distilled water. There were three independent biological replicates for each treatment group.

[0060] III. Identification and bioinformatics analysis of the putative AhCN gene

[0061] The peanut genome (Tifrunner.gnm1 ann1.CCJH) was downloaded from PEANUTBASE (https: / / phytozome-next.jgi.doe.gov / ). The presence of key domains (CC domain, PF18052; NB-ARC domain, PF00931) in each putative AhCN was verified using the NCBI-CDD database (https: / / www.ncbi.nlm.nih.gov / ) with an E-value threshold of 0.01. The physicochemical properties of putative AhCNs were predicted with ExPASy (https: / / web.expasy.org / protparam / ) and subcellular localization was predicted with Cell PLoc 2.0 (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc / ). Amino acid sequences of CN proteins in Arabidopsis thaliana, Oryza sativa, Glycine max, Triticum aestivum L., and Lycopersicon esculentum L. were downloaded from the NCBI database and aligned with ClustalX. Phylogenetic trees were then constructed in MEGA 7.0 using the neighbor-joining method with 1000 bootstrap replicates. Using the Python version of MCScan (JCVI toolkit), we completed the analysis of protein alignment and prediction of segmental duplication events.

[0062] AhCNs were subjected to conserved motif analysis using MEME (https: / / meme-suite.org / meme / doc / meme.html) with a fixed number of motifs of 20. Peanut chromosome AhCN distribution maps were drawn using MG2C (http: / / mg2c.iask.in / mg2c_v2.1 / ). Promoter regions were extracted from each putative AhCN for cis-acting element prediction with PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare). The tertiary structure of each AhCN was predicted using SWISS-MODEL (https: / / swissmodel.expasy.org / ). The expression pattern of AhCNs in various tissues was analyzed in publicly available transcriptomic data downloaded from PeanutBase (https: / / www.peanutbase.org / ) and visualized using TBtools. In the process of plotting the curves, we standardized the gene expression values using the standardization function in the Mfuzz package (Mfuzz_2.54.0).

[0063] IV. Cloning of AhCN and construction of overexpression vectors

[0064] Total RNA was extracted from peanut using the FastPure® Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). cDNA was synthesized using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (FullStyle Gold, Beijing, China) following the manufacturer’s instructions. PCR products were visualized by agarose gel electrophoresis and recovered using the E.Z.N.A.® Gel Extraction Kit (Vazyme, Nanjing, China). Purified target fragments were ligated into the pMD18-T cloning vector (Thermo Fisher, Massachusetts, USA) during a 5 min incubation at 25°C. E. coli DH5a chemically competent cells were transformed with the resulting plasmids following the manufacturer’s instructions (Vazyme, Shanghai, China). Colony PCR was performed using gene-specific primers on a Bio-Rad instrument (Hercules, CA, USA; Table S1). PCR products were visualized by 1% agarose gel electrophoresis to identify E. coli colonies harboring inserts; these were selected for sequencing by Shanghai Biotechnology Co. (Shanghai, China). Gene cloning was performed using recombinant TA / Brent-Zero plasmids containing the target fragment as a template; gene-specific primers with homology arms at both ends were used to purify and recover PCR products. To generate plasmids encoding AhCN34-YFP fusion proteins, the Seamless Assembly Cloning Kit (Clone Smarter Technologies, Inc., Houston, TEXAS, USA) was used. The pCambia1300 YFP vector was linearized by incubation with Sac I and Kpn I for 2 hours at 37°C and 20 minutes at 65°C, then the fragment encoding AhCN34 was inserted. Agrobacterium tumefaciens EHA105 chemically competent cells were transformed with the plasmids, then used to transform peanut plants following the manufacturer’s instructions (Vazyme, Shanghai, China).

[0065] V. RS stress assay and hormone treatment

[0066] H108 and H107 seedlings were grown to the three-leaf stage, then 60 plants of uniform size were selected. RS was prepared at a concentration of 108 CFU mL1; working solutions of 3 mmol L1SA, 100 mmol L1MeJA and 10 mg L1ABA were generated in 10 mM 2-morpholinoethanesulfonic acid buffer solution. H108 and H107 were each divided into five groups, the control group was sprayed with sterile water, one group was inoculated with RS, and the other three groups were sprayed with exogenous plant hormones SA, MeJA, ABA respectively, 15 seedlings in each group. The sprayed plants were placed in an incubator and incubated at 30 °C under a 16 / 8 h light / dark cycle for 0.5, 1 and 7 days (Zhang et al., 2017a). Roots, stems and leaves were collected at 0, 0.5, 1 and 7 days after treatment, rapidly frozen in liquid nitrogen and stored at -80 °C before further analysis.

[0067] Six, AhCN Quantitative reverse transcription polymerase chain reaction (qRT-PCR) verification of expression

[0068] To verify AhCNsAt the expression level in RS and hormone treatment, we selected four genes (AhCN5 gene sequence as SEQ ID NO: 3, AhCN28 gene sequence as SEQ ID NO: 4, AhCN34 gene sequence as SEQ ID NO: 1 and AhCN73 gene sequence as SEQ ID NO: 5). The gene-specific primers were designed using Primer Premier 5.0 (Table 1). RNA was extracted using TRIzol reagent (Biotech Bio, Shanghai, China) according to the manufacturer’s instructions. The RNA concentration and purity were determined using a NanoDrop ND1000 instrument (Wilmington, DE, USA), and 1.0 ug of RNA per sample was used for first-strand cDNA synthesis using TransScript® II Multiplex One-Step qRT-PCR SuperMix UDG Kit (Full Style Gold, Beijing, China). Gene expression analysis was then performed using a FastKing RT Kit with gDNase (Tiangen, Beijing, China). The specificity of primers was verified by the presence of a single peak melting curve and the check of PCR product size. qRT-PCR was performed on a CFX96 Real-Time Fluorescent PCR instrument (Bio-Rad, Hercules, CA, USA) using a TB Green Premix Ex-Taq Kit (TaKaRa, Dalian, China). The thermal cycling conditions were as follows: 95°C for 30 s; 40 cycles of 5 s at 95°C and 30 s at 60°C. The melting curve was generated at 94°C for 15 s, 60°C for 60 s and 94°C for 15 s. The expression level of target genes was normalized to the internal reference gene AhCN73 using the 2 – The target gene expression level was normalized to the internal reference gene AhCN73 using the 2 ACTIN7 (Livak & Schmittgen 2001). The expression values for each sample and target gene were averaged over three technical replicates. Statistical significant differences between samples were determined using Student’s t-test in Graphpad Prism 8.0 (San Diego, CA, USA). t (Livak & Schmittgen 2001). The expression values for each sample and target gene were averaged over three technical replicates. Statistical significant differences between samples were determined using Student’s t-test in Graphpad Prism 8.0 (San Diego, CA, USA).

[0069] Table 1 Primer sequences

[0070]

[0071] Seven, Peanut AhCN34 Subcellular localization in tobacco.

[0072] Agrobacterium cells carrying pCambia 1300 YFP or AhCN34 YFP plasmids were grown on solid yeast extract peptone (YEP) medium with 50 ug mL"1kanamycin (kan) and 25 ug mL"1rifampicin (rif) at 28 °C in the dark for 48 h. Then single colonies were picked and added to 2 mL liquid YEP medium with the same concentration of antibiotics and incubated at 28 °C with 200 rpm shaking for 12 h. Bacteria were collected and transferred to fresh YEP medium (1 :500 medium:YEP) with the same antibiotics and incubated with shaking until OD600 reached 0.8. Cells were collected by centrifugation at 4000 g for 15 min. Bacteria were injected to the back of leaves of four-week-old tobacco plants using a sterile syringe. The leaves were blotted dry with sterile paper to remove residual culture. 36-48 h after injection, the subcellular localization of YFP and AhCN34 YFP was observed under a laser confocal microscope. OD600 Bacterial cells were collected by centrifugation at 4000 g for 15 min. Bacteria were injected to the back of leaves of four-week-old tobacco plants using a sterile syringe. The leaves were blotted dry with sterile paper to remove residual culture. 36-48 h after injection, the subcellular localization of YFP and AhCN34 YFP was observed under a laser confocal microscope.

[0073] Eight, overexpression of AhCN34 in peanut

[0074] To investigate the effects of AhCN34 overexpression, plasmids encoding YFP or AhCN34-YFP fusion proteins were injected into healthy H107 leaves. Peanut plants were then incubated in the dark at 28 °C for 12 h before being observed for fluorescence under a LSM710 confocal laser scanning microscope (Carl Zeiss, GmbH, Jena, Germany). Two days post-inoculation (dpi), leaf disease symptoms were observed and stained with trypan blue (Solarbio). Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities and malondialdehyde (MDA) content were measured as described by Dazy et al. (2009).

[0075] Nine, pathological analysis of peanut RS resistance

[0076] To visually analyze the physiological differences between resistant and susceptible peanut varieties (H108 and H107, respectively), we first examined the cross-sections and longitudinal sections of the main stem at several time points (5, 10, 15, 20 and 25 dpi) using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). The main stem was cut into 1 mm thick sections using a microtome and the sections were stained with trypan blue (Solarbio) and observed under a laser scanning confocal microscope. Figure 1 ​A). At 10 dpi, the main stem of H108 showed no obvious disease symptoms, while the vascular tissue of H107 showed slight browning. At 15 dpi, the main stem of H108 began to show slight browning symptoms, and the vascular tissue of H107 showed severe browning. At 25 dpi, the vascular bundle of the main stem of H107 was severely damaged, rotten and deformed, while the tissue structure of the main stem of H108 remained intact. Compared with the mock-inoculated plants, the leaf chlorophyll content of the inoculated plants of both genotypes decreased (Figure 1) Figure 1 B). In addition, these plants showed obvious growth retardation; the growth of H107 reached a plateau at 5 dpi, although H108 continued to grow very slowly (Figure 2) Figure 1 C). There was no significant difference in the length of the first main stem node between H108 and H107 (Figure 3) Figure 1 D). However, at 20 dpi, H108 plants showed significantly larger first main stem node diameter and higher fresh weight than H107 plants (Figure 4) Figure 1 E, Figure 1 F). There were also significant differences in dry weight between the two genotypes at several time points, although the differences between different time points were not consistent (Figure 5) Figure 1 G). These preliminary results suggest that the difference in BW resistance between H108 and H107 may be due to the blockage of the main stem vascular system induced by RS in the susceptible variety.

[0077] Ten、 AhCN Characteristics of the gene family

[0078] To determine the transcriptomic differences between H108 and H107 in different RS resistance, we analyzed the transcriptomic data of both varieties at three time points after RS inoculation (Zhao et al., 2022). We identified differentially expressed genes (DEGs) in each variety of inoculated plants compared to non-inoculated plants. This resulted in a total of 1744 disease-related DEGs in both varieties with a threshold of ≥2-fold change in expression p <0.05). Gene Ontology (GO) term enrichment analysis of DEGs showed that the following terms were significantly enriched: stress response, fungal defense, oxidative stress response, SA response, and jasmonic acid response pathways Figure 7 ).

[0079] Using the peanut genome database, a total of 150 peanut AhCN genes were obtained through BLAST homology matching and SMART analysis, named AhCN1-150. Notably, some were significantly up-regulated in response to RS infection in H108 and H107 (p<0.05). To understand the evolutionary history of peanut AhCNs, a phylogenetic tree was constructed to demonstrate their relationship with CNs of species such as Arabidopsis thaliana, Oryza sativa, Glycine max, and Triticum aestivum (Figure 6).Figure 2 A). Cluster analysis identified 9 clusters, with the vast majority of peanut AhCN and soybean GmCN clustering together. Among these, soybean GmCN4 showed some resistance to Phytophthora infestation (Gao...). et al., 2005). Gene annotation data from the peanut genome were used to physically map the location of AhCNs, which are unevenly distributed across all peanut chromosomes except 7 and 17. Figure 8 The highest concentration of AhCNs was found on chromosome 12 (32), with AhCNs clustered at its ends. This may be related to the expansion and functional diversification of gene families. To assess relative family size, we analyzed the number of CN genes in 21 plant species and found a linear relationship with genome size. p <0.05, Figure 2 B). Compared to other plant species, the peanut genome contains a relatively large amount of... AhCN With wild peanut species Durar Peanut (43) Yi Pahut Peanut (65) and moat peanuts ( A. mon The kinship analysis in (64) revealed possible causes AhCN Segmental repetition events in family expansion ( Figure 2 D).

[0080] Next, we characterized the predicted physicochemical properties of the newly identified AhCNs. Significant differences in protein size were observed; the longest AhCN was 1510 amino acids (aa) long (AhCN145), while the shortest was only 304 amino acids (AhCN97). Molecular weights ranged from 3.46 to 17.11 kDa, and theoretical isoelectric points ranged from 4.93 (AhCN146) to 9.41 (AhCN108). Conserved domain analysis revealed that all AhCN proteins contained two conserved domains (CC and NB-ARC), which are believed to have similar biological functions. AhCNs were predicted to be distributed in several organelles, including the nucleus, mitochondria, cytoplasm, extracellular space, plasma membrane, and chloroplasts. The highest percentage (26%) of AhCNs were expected to be located in the cytoplasm, with slightly fewer AhCNs located in the nucleus (24%). Figure 2 C).

[0081] To investigate conserved domains in the CN family, we performed amino acid sequence comparisons based on known CN domains in Arabidopsis thaliana. The AhCN family has at least five conserved domains, including EDVID, P-ring (FIVGMGGIGKTTLAKA), kinase 2 (KRYLVLDDVLKG), RNBS-B (GSRILITRY), and GLPL (IV+YCGGLPLALTVLGP). Figure 3 A, Figure 3B). We selected AhCN34, which is evolutionarily close to GmCN, and compared its three-dimensional structure with that of the putative AhCNs Arabidopsis thaliana The three-dimensional structures of AtCN1 were compared and found to be very similar ( Figure 3 C). Next, we identified 20 unique motifs in the putative AhCNs; members of each subfamily showed similarity in the motifs present, suggesting potential similar biological functions ( Figure 9 ). In contrast, differences in subfamily-specific motifs could have led to functional diversity.

[0082] To determine the functions that the 150 AhCNs might be involved in, we performed a predictive analysis of cis-acting elements in each AhCN gene. This analysis revealed that various plant hormone- and resistance-related elements were involved in the expression of CNs ( Figure 10 ). Specifically, 64% of the AhCN contained ABA response elements (ABREs), 41% contained TCA response elements, and 54% contained TGACG response elements. In addition, 44% contained defense response-related elements (rich in TC). Overall, AhCN most of the identified Cis-acting Regulatory element were associated with the plant's response to biotic stress, suggesting AhCN that the gene family might be involved in hormone pathways.

[0083] Eleven、 AhCN Expression analysis

[0084] To further understand the potential functions of AhCNs, we analyzed the expression patterns of the 150 genes in multiple tissues using peanut RNA-seq data. AhCNs were most highly expressed in flower tissues and least highly expressed in root tissues ( Figure 4 A). We then analyzed the expression levels of AhCNs at several time points (0 dpi, 1 dpi, and 7 dpi) after RS inoculation and performed trend analysis to study the dynamic changes in AhCN expression over time under RS stress conditions ( Figure 4 B). The expression patterns of AhCNs could be divided into three main trends ( Figure 4 C). Genes with expression profile 1 and expression profile 2 were strongly upregulated at 7 dpi and 1 dpi, respectively, while genes with expression profile 3 were downregulated at 1 and 7 dpi. Therefore, we hypothesized that genes associated with disease resistance exist in the genome with expression profile 1 and expression profile 2. To better understand the functions of all differentially expressed AhCN genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathway enrichment analysis. The most enriched annotations were spliceosome-related, plant-pathogen interactions, and ribosome synthesis pathways ( Figure 4D). Fifteen AhCNs were significantly differentially expressed in responses to RS treatment. Correlation analysis revealed strong correlations among the expression levels of several AhCNs, including AhCN5, AhCN28, AhCN34, and AhCN73. Figure 4 E), indicating the potential role of these AhCNs in the peanut BW reaction.

[0085] twelve, AhCN Response to stress treatment

[0086] To validate the transcriptomics data, we used qRT-PCR to measure at several time points after RS ​​treatment. AhCN5 , AhCN28 , AhCN34 and AhCN73 Relative expression in roots, stems, and leaves of H108 and H107 seedlings. At one or more time points following RS inoculation, AhCN5 , AhCN28 and AhCN34 AhCN73 was significantly upregulated in all four tissues of H108 and H107. AhCN73 was upregulated in the roots of H108 and H107, but showed no significant change in the stems or leaves. Figure 5 A). Therefore, although AhCN The family as a whole showed a strong response to RS stress, but the response varied across different tissues. Furthermore, qRT-PCR results were consistent with transcriptomic data, validating the sequencing findings.

[0087] Plant stress responses are mediated by key endogenous plant hormones such as SA, MeJA, and ABA (Robert Seilaniantz et al., 2011; Pieterse et al., 2012). Therefore, we analyzed the effects of SA, MeJA, or ABA on RS-inoculated plants. AhCN of Expression patterns. After treatment with any of the three hormones, AhCN28 and AhCN34 were upregulated, while... AhCN5 and AhCN73 Lower ( Figure 5 B). In H108, only AhCN34 It was significantly upregulated (by a factor of 2) by SA, MeJA, and ABA. (Inference) AhCN34 It may play a role in peanut resistance to BW.

[0088] Thirteen, AhCN34 Effects of subcellular localization and overexpression in peanuts

[0089] In order to determine AhCN34 Subcellular localization was used to construct the fusion vector AhCN34-YFP ( Figure 6A) and expressed in tobacco epidermal cells. YFP control expressed in both plasma membrane and nucleus, while AhCN34 YFP mainly expressed on plasma membrane, which is consistent with its putative function as NBS-LRR ( Figure 6 B). To confirm the hypothesized inhibitory effect of AhCN34 on RS infection, we transiently overexpressed the fusion protein AhCN34 YFP in peanut leaves, and then inoculated the plants with RS. At 3 dpi, only the leaves expressing pCambia1300 YFP control were curled and dried, but the leaves overexpressing AhCN34 YFP remained healthy ( Figure 6 C). This was confirmed by trypan blue staining, which showed that AhCN34 YFP overexpression reduced cell death compared to YFP-only overexpressing leaves ( Figure 6 D). We also analyzed SOD, POD, and CAT activities and MDA levels in leaves transiently overexpressing YFP or AhCN34 YFP under 1-4 dpi and RS ( Figure 6 E、 Figure 6 F、 Figure 6 G、 Figure 6 H). At 1 dpi, CAT activity and MDA levels were significantly different between the two treatment groups; at 2 dpi, POD and CAT activities in leaves overexpressing AhCN34 YFP were significantly higher than the control. Overall, SOD activity and MDA levels of both groups first increased and then decreased, while POD levels showed the opposite trend. These results suggest AhCN34 improved resistance of peanuts to RS.

[0090] XIV. DISCUSSION

[0091] Over millions of years of co-evolution with pathogens under different environmental conditions, plants have developed a wide range of disease resistance genes. These enable plants to adapt to the surrounding conditions and counteract various external hazards. During plant breeding, resistance®genes are often used to promote plant disease resistance. Most of the R genes cloned to date belong to the NBS-LRR family (Hammond-Kosack & Parker 2003; Parry et al. 2008; Gururani et al. 2012). Members of this family can be roughly divided into CNL and TNL according to their sequences in the N-terminal domain. Both types exist in dicotyledonous and monocotyledonous plants (Tameling & Baulcombe 2007; Takken & Goverse 2012). Although R genes exhibit extensive diversity in nucleotide sequences between species, the encoded proteins exhibit highly conserved domains similar to those of NBS LRRs (Takken et al. 2006). Studies of CNL transcription factors have mainly focused on major model plants such as Arabidopsis thaliana (Meyers et al. 2003), rice (Zhou et al. 2004) and wheat (Dong et al. 2020), and little exploration has been made of their role in peanuts.

[0092] Some gene recombination events can result in the loss or truncation of NLR domains. Importantly, this truncated NLR is common in a variety of plants and plays a crucial role in plant immunity; despite the incomplete domain, they have similar functions to complete NLRs: direct or indirect effector recognition and induction of plant immunity (Baggs et al. 2017). In this study, we sought to gain a deeper understanding of the expression patterns and functions of CN genes in peanuts. To do this, we comprehensively analyzed the peanut genome to identify CNs, then analyzed the tissue-specific expression patterns and responses to RS infection. This enabled us to identify CNs that specifically respond to BW, thereby improving our understanding of peanut responses to this economically important disease.

[0093] Using sequence homology to known CNs in Arabidopsis, rice, tomato and wheat, we identified 150 putative AhCNs in peanut genome, each of which contains two highly conserved domains: CC and NB-ARC domains. Phylogenetic analysis of CNs from other plants showed that 150 AhCN genes were clearly distributed in 9 subfamilies. The distribution pattern of AhCNs was similar to that of NBS genes in dicotyledonous plants such as Arabidopsis, soybean and alfalfa, but different from that of rice and maize. Although there were wide variations in the predicted gene length, molecular weight and isoelectric point of AhCNs, the gene structure and protein motifs were still relatively conserved, which indicated functional diversity and conservation. The distribution of conserved motifs was consistent with the phylogenetic classification of 9 CN subfamilies.

[0094] For some NLRs, expression of only the N-terminal domain is sufficient to trigger hypersensitive response. Therefore, it is hypothesized that the N-terminal domain of NLR stimulates and transmits downstream immune signals (Collier et al., 2011). The CC domain structure is a frequently observed protein folding pattern, which plays a key role in protein interactions in addition to promoting cell death and facilitating interactions with co-factors (Robert Seilaniantz et al., 2011). For example, effectors identify oligomerization of the entire NLR protein. This has been observed in Arabidopsis protein RPP1 (Schreiber et al., 2016) and tobacco N protein (Mestre & Baulcombe, 2006). RBA1 is an atypical Arabidopsis NLR protein that only has a TIR domain; despite the lack of NBS and LRR, it specifically recognizes and binds to bacterial type III effector HopBA1, triggering hypersensitive response (Nishimura et al., 2017). Therefore, the N-terminal TIR and CC domains play a key role in NLR oligomerization and activation. Analysis of expression profiles helps to determine gene function. We analyzed here the expression of AhCNs in susceptible peanut variety H107 and resistant peanut variety H108 at several time points after RS inoculation. A total of 15 significantly differentially expressed AhCNs, with relative expression levels and expression trends varying over time. Analysis of AhCN expression in various hormone treatments showed that only AhCN34 responded to RS infection and three plant hormone treatments, suggesting that this gene plays a key role in peanut stress response. This was confirmed by overexpression of AhCN34 YFP in peanut leaves, which showed significantly enhanced resistance to peanut BW.

[0095] Overall, the results of this study provide valuable new insights into the role of AhCNs in peanut BW resistance. We identified several AhCNs that were differentially expressed in response to RS treatment; moreover, we found that AhCNs were upregulated in the resistant peanut variety compared to the susceptible variety, suggesting that these genes enhanced resistance. The transcriptional behavior of CNs under RS stress also provides candidate genes for further study of the mechanisms by which this gene family promotes plant disease resistance. Finally, the validation of AhCN34 as a positive regulator of peanut RS resistance can facilitate genetic breeding efforts to produce new BW-tolerant peanut varieties, which will help ensure continued peanut production in the event of BW spread.

[0096] Fifteen. Conclusion

[0097] In this study, we identified 150 AhCN genes from the peanut genome, which were classified into 9 subfamilies. These genes exhibited highly conserved structural features. After inoculation with RS, the highly resistant peanut variety "H108" was significantly superior to the susceptible variety "H107" in terms of plant height, main stem diameter, and fresh weight. Promoter cis-elements indicated that they were involved in plant hormone signaling and defense responses. Through q-PCR experiments, we found that AhCN34 was significantly upregulated in H108 compared to H107. Importantly, overexpression of AhCN34 in peanut leaves enhanced its resistance to BW. Therefore, these findings provide a theoretical basis and help for future molecular breeding of peanuts.

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Claims

1. The use of peanut disease-resistant gene AhCN34 in the genetic engineering of peanut resistance to bacterial wilt, characterized in that, The DNA sequence of the AhCN34 gene is shown as SEQ ID NO: 1; the application in the peanut anti-rhizome bacterial disease genetic engineering is overexpression of the AhCN34 gene to enhance the ability of peanut anti-rhizome bacterial disease.

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