CaHT1 Regulates Pepper Immune Response to Ralstonia solanacearum and Interacts with CaPYR1

CaHT1 Regulates Pepper Immune Response to Ralstonia solanacearum and Interacts with CaPYR1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CaHT1 Regulates Pepper Immune Response to Ralstonia solanacearum and Interacts with CaPYR1 Xiang Zheng, Jinggang Lv, Huifang Shi, Lingxian Yi, Daojin Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9298395/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Aims Pepper ( Capsicum annuum ) is susceptible to bacterial wilt caused by Ralstonia solanacearum under high temperature and high humidity (HTHH) conditions, leading to severe yield losses. Our previous studies revealed that the abscisic acid (ABA) receptor PYR1 negatively regulates bacterial wilt resistance under normal temperature but exhibits positive regulation under HTHH conditions. We discovered that PYR1 interacts with HT1, a member of the AGC protein kinase C family. Methods To investigate the function of CaHT1 and its interaction with CaPYR1, we performed subcellular localization, bimolecular fluorescence complementation (BiFC), pull-down assays, virus-induced gene silencing (VIGS), transient and stable overexpression, qRT-PCR, and disease index evaluation. Results We found that CaHT1 localizes to the plasma membrane and nucleus and physically interacts with CaPYR1. Silencing CaHT1 increased pepper susceptibility to R. solanacearum, whereas stable overexpression of CaHT1 in both pepper and tobacco enhanced disease resistance. Conclusions This study demonstrates that CaHT1 positively regulates pepper resistance to bacterial wilt under both normal and HTHH conditions, indicating that HT1 achieves its positive regulatory role through protein interaction with PYR1. These findings hold significant implications for elucidating pepper disease resistance mechanisms and genetic improvement. Capsicum annuum High temperature and high humidity Protein kinase Bacterial wilt CaHT1 CaPYR1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights CaHT1 positively regulates pepper resistance to bacterial wilt under normal and HTHH conditions CaHT1 physically interacts with ABA receptor CaPYR1 at plasma membrane CaHT1 silencing enhances pepper susceptibility to Ralstonia solanacearum infection CaHT1 overexpression strengthens disease resistance in tobacco and pepper plants CaHT1-CaPYR1 module may mediate immunity-thermotolerance trade-off mechanisms Introduction Pepper ( Capsicum annuum ) is an economic crop originating from tropical and subtropical regions of South America and is one of the most widely cultivated vegetables globally, particularly prevalent in China. However, its production is severely threatened by bacterial wilt, a disease caused by the soil-borne pathogen Ralstonia solanacearum [ 1 , 2 ]. This pathogen invades pepper plants through root wounds, rapidly proliferates and spreads within the host, leading to vascular bundle obstruction. The resulting impairment of water and nutrient transport in stems and leaves ultimately causes rapid wilting and plant death [ 3 ]. Plants have evolved sophisticated immune systems to combat various pathogens [ 4 ]. These systems involve immune receptors (such as pattern recognition receptors PRRs and resistance proteins R) that recognize pathogen-associated molecular patterns (PAMPs) and effector proteins, thereby activating complex plant immune signaling pathways [ 5 ]. These signals are transmitted and amplified through intricate networks, ultimately reprogramming the transcription of numerous disease resistance-related genes in the nucleus, triggering robust defense responses [ 6 ]. This signaling network plays a critical regulatory role in plant defense, typically involving calcium ion messengers, plant hormones (such as salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA)), reactive oxygen species (ROS), and various kinases (such as mitogen-activated protein kinases (MAPKs)) and transcription factors. Specifically, SA primarily participates in combating biotrophic pathogens, while JA mainly mediates responses to necrotrophic pathogens [ 7 – 9 ]. To counteract pathogen invasion, plants have established immune systems at the molecular level, primarily comprising PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). To detect and respond to various biochemical attacks, plants have evolved two intrinsic immune mechanisms [ 10 , 11 ]. Plant defense mechanisms against pathogen invasion rely on both inherent physical and chemical barriers and inducible immune systems [ 12 ]. The latter, namely the inducible immune system, is activated when cell surface and intracellular receptors recognize pathogen-derived or plant-derived molecules. PTI, as an innate immune response mediated by cell surface pattern recognition receptors (PRRs), recognizes pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [ 13 , 14 ]. One of the key roles of protein kinase C (PKC) is precisely to participate in negative feedback regulation within PTI. PRRs on the cell membrane recognize PAMPs and DAMPs, initiating primary immune responses. PTI is a critical factor in resisting pathogen infection and effectively maintains plant physiological and ecological balance. Pathogens can deliver effector proteins into host cell cytoplasm through the type III secretion system (T3SS) to exert their functions. During plant defense against pathogen invasion, nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly interact with effector proteins, thereby inducing stronger effector-triggered responses (ETI) [ 15 ]. Bidirectional regulatory relationships exist between plant immunity and effector-triggered responses. PTI and ETI share common mechanisms, such as reactive oxygen species (ROS) production, mitogen-activated protein kinase (MAPKs) activation, defense hormone pathway initiation, and accompanying transcriptional reprogramming [ 16 ]. Both participate in immediate defense at pathogen invasion sites and can activate defense responses in plant tissue cells. Recent studies have revealed that enhanced PTI is a major characteristic of ETI. ROS produced in immune responses overlapping between PTI and ETI, as key defense and signaling molecules, are induced in both responses [ 17 ]. The initial event in plant response to high temperature stress is high temperature perception. Changes in intracellular calcium ion concentration are perceived by intracellular protein sensors and histone sensors. Meanwhile, many signaling pathways participate in responding to high temperature stress, such as the aforementioned calcium ion signaling pathway, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [ 18 ]. After entering the plant cell nucleus, these signals cooperate with various transcription factors to initiate the expression of defense-related genes, thereby conferring heat tolerance to plants [ 19 ]. On the other hand, plants secrete heat shock proteins (HSPs) under high temperature stress [ 20 ]. Under high temperature stress conditions, HSPs expression levels increase rapidly. Abscisic acid (ABA), also known as natural abscisic acid, is a plant hormone that inhibits plant growth. ABA functions through protein phosphorylation and dephosphorylation. Protein phosphorylation is completed by SnRK2, while dephosphorylation is executed by PP2C. PP2C is currently the largest known protein phosphatase family, with 76 identified members [ 21 ]. This family belongs to the serine/threonine protein kinase family, exists as monomers in cells, and its enzymatic activity depends on Mn²⁺ and Mg²⁺ ions. As a key non-coding RNA with important biological functions, PP2C also participates in cell signal transduction, cell cycle regulation, protein translation, and post-translational modification [ 22 ]. Among these, the ABA signaling pathway is associated with group A PP2C phosphatases, which play negative regulatory roles in the ABA signaling pathway [ 23 ]. The mechanism of ABA action in plant biotic stress responses is complex. As a key plant hormone, its core role in regulating plant responses to abiotic stresses such as drought, low temperature, and high temperature has been widely recognized. Interestingly, recent studies increasingly reveal ABA's involvement in plant immune responses, although its precise mechanism of action remains complex and sometimes contradictory [ 24 , 25 ]. Studies have shown that ABA can exert negative regulatory effects on plant resistance, as demonstrated in tobacco resistance to bacterial wilt and barley resistance to rice blast. Depending on pathogen species and infection routes, ABA can either promote plant tolerance to pathogens or lead to susceptibility. For example, ABA can enhance the protective function of plant guard cells, blocking various pathogens from entering cells [ 26 ]. However, ABA can also regulate SA, JA, and ethylene-dependent responses, thereby inhibiting later defense mechanisms [ 27 ]. Studies have found positive synergistic effects between ABA and JA during Arabidopsis resistance to Rhizoctonia solani . ABA and ethylene (ET) exhibit antagonistic relationships—ET serves as a characteristic signaling molecule promoting viral infection, while ABA plays a positive role in regulating disease resistance [ 28 ]. The interaction mechanisms between ABA and other defense hormones require further in-depth investigation. Materials and Methods 2.1. Plant Materials and Growth Conditions Plant materials included tobacco ( Nicotiana benthamiana ), sweet pepper variety Zunla-1, etc. Strains used included Escherichia coli DH5α, BL21, Rosetta, Agrobacterium tumefaciens GV3101, Ralstonia solanacearum strain FJAT91, etc. Vectors used included Gateway entry vector pDONR207, plant expression vectors pEarleyGate101, pEarleyGate102, pEarleyGate103, virus-induced gene silencing (VIGS) vectors pTRV1 (pYL192) and pTRV2 (pYL279), prokaryotic expression vectors pEZY-Hb and PDEST-15, etc. Pepper cultivation method Seeds stored at 4°C were placed on moist filter paper in petri dishes, covered with perforated plastic film, and germinated in darkness for 3–5 days. Germinated seeds were transplanted into soil, covered again with perforated plastic film, and cultivated in a growth chamber (25°C, 70% humidity, 16 h light/8 h dark) for approximately 10 days. After cotyledon expansion and emergence of the first true leaf, seedlings were transplanted into pots and regularly watered, fertilized, and loosened under the same conditions. When necessary, plants were moved outdoors for acclimatization 30 days after transplantation. Tobacco cultivation method Seeds stored at room temperature were directly sown into soil, covered with perforated plastic film, and cultivated under the same growth chamber conditions for approximately 10 days. After cotyledon expansion, seedlings were transplanted into pots for continued cultivation and underwent outdoor acclimatization after 30 days. 2.2. Bioinformatics Analysis CaHT1 CDS sequences and homologous amino acid sequences were retrieved from the NCBI database. Conserved domains were analyzed using CD-Search and SMART tools, promoter elements were predicted using the PlantCARE database, and homologous protein families in pepper were identified via the Sol Genomics Network. Specific primers were designed using Primer Premier 5.0 software. Homologous sequences of CaHT1 with solanaceous plants (tomato, tobacco, potato, etc.) were obtained through BLAST searches. Multiple sequence alignment and phylogenetic tree construction were performed using DNAMAN 7 software. Transmembrane domain prediction for CaHT1 was completed using TMHMM software. 2.3. Expression Vector Construction CaHT1 gene was amplified from Zunla-1 cDNA using high-fidelity polymerase. PCR products were separated by agarose gel electrophoresis, target bands were excised and purified. BP reaction : Purified fragments and pDONR207 (0.5 µL each) were recombined overnight at 25°C using BP Clonase II enzyme (0.25 µL). Products were transformed into E. coli DH5α strain and plated on LB agar plates containing gentamicin. Single colonies were screened by PCR, and positive clones were sequenced. Verified plasmids were extracted and stored at − 20°C. LR recombination reaction : Target clones were recombined with host vectors (pEarleyGate series) using LR Clonase II recombinase. Reaction products were transformed into DH5α E. coli , and positive clones were obtained through antibiotic resistance screening and PCR detection. After plasmid extraction, they were introduced into Agrobacterium tumefaciens GV3101 strain via freeze-thaw transformation for subsequent experiments. 2.4. E. coli Transformation and Culture First, DH5α competent cells were thawed on ice, mixed with 2 µL plasmid DNA, and incubated on ice for 30 minutes. Cells were heat-shocked at 42°C for 90 seconds, then cooled on ice for 5 minutes. Second, 400 µL LB medium was added, and cells were recovered at 37°C, 180 rpm for 1 hour. Third, cultures were spread on LB agar plates containing corresponding antibiotics and incubated at 37°C for 20 hours. Finally, single colonies were inoculated into LB liquid medium containing antibiotics, cultured for 3–5 hours, and verified by PCR. Positive clones were amplified for subsequent use. 2.5. Agrobacterium tumefaciens Transformation and Culture First, GV3101 competent cells were thawed on ice, mixed with 2 µL plasmid DNA, and incubated on ice for 30 minutes. Cells were frozen in liquid nitrogen for 5 minutes, then thawed at 37°C for 5 minutes. Second, 400 µL LB broth was added, and cells were cultured at 28°C, 180 rpm for 4–5 hours. Third, cultures were spread on LB plates containing antibiotics and incubated at 28°C for 2 days. Finally, single colonies were verified by PCR followed by overnight culture and amplification. 2.6. Ralstonia solanacearum Culture and Inoculation Culture method : FJAT91 strain was inoculated from − 80°C frozen stock onto TTC agar plates and cultured at 28°C for 48 hours. Single white colonies were cultured overnight, then subcultured at 1:1000 ratio for 48 hours. Bacterial cells were collected by centrifugation (4500 rpm, 10 minutes), washed, and resuspended in deionized water to target OD₆₀₀ values. Root inoculation method Wounds were created on lateral roots using scissors, and 15 mL bacterial suspension (OD₆₀₀ = 1.0) was inoculated. Plants were placed in environments at 28°C or 37°C with 80% humidity to monitor disease progression. Leaf infiltration method Bacterial suspension (OD₆₀₀ = 0.4) was infiltrated into the abaxial leaf surface using a 1 mL needleless syringe, ensuring coverage of the midrib region. 2.7. Protein-Protein Interaction Assays Prokaryotic expression optimization GST or His-tagged constructs were transformed into BL21 strain. Expression was induced by IPTG at different temperatures (16°C overnight culture, 28°C for 6–8 hours, 37°C for 3–5 hours). Expression levels were detected by SDS-PAGE analysis. Pull-down assay : Purified CaHT1-GST and CaPYR1-His proteins were incubated with magnetic beads in four combinations: CaHT1-GST + CaPYR1-His, CaHT1-GST + negative control, GST + CaPYR1-His, and GST + negative control. After washing, bound proteins were eluted and detected by Western blot using anti-GST and anti-His antibodies. Bimolecular fluorescence complementation (BiFC) Constructs expressing CaHT1 fused to YFP⁺ and CaPYR1 fused to YFP⁻ were co-infiltrated into tobacco leaves. Fluorescence signals were observed using confocal microscopy 48 hours after infiltration. 2.8. Subcellular Localization CaHT1 was fused with GFP through Gateway cloning technology and introduced into GV3101 strain. Bacterial suspension (OD₆₀₀ = 0.8) was infiltrated into tobacco leaves. GFP fluorescence was observed using confocal microscopy after 48 hours. 2.9. Transient Overexpression CaHT1 overexpression vectors were introduced into pepper leaves via Agrobacterium infiltration. 2.10. Protein Extraction and Western Blot Analysis Leaf tissues were ground in liquid nitrogen and extracted with protein extraction buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, followed by primary and secondary antibody incubation. Signals were detected by chemiluminescence. 2.11. Histochemical Staining Trypan blue staining : Leaves with hypersensitive response lesions were boiled in trypan blue solution (lactic acid:glycerol:ethanol:water = 1:1:1:1) for 15 minutes, incubated for 24 hours, decolorized with boiling ethanol, and photographed. DAB (3,3'-diaminobenzidine) staining Leaves were immersed in 1 mg/mL DAB solution for 24 hours, decolorized with 95% ethanol, and imaged. 2.12. RNA Extraction, Reverse Transcription, and qPCR Detection Total RNA was extracted from frozen tissues using TRIpure reagent. RNA quality was assessed by A₂₆₀/A₂₈₀ ratio (1.9–2.1). cDNA was synthesized using reverse transcription kits. qPCR detection was performed using SYBR Green master mix on a real-time PCR system. CaACTIN (pepper) and NbEF-1α (tobacco) served as internal reference genes. Relative expression levels were calculated using the 2⁻ΔΔCt method. 2.13. Virus-Induced Gene Silencing (VIGS) 300 bp fragments from the 3'UTR or coding region (CDS) of CaHT1 gene were cloned into pTRV2 vector. This construct was co-transformed with pTRV1 into GV3101 strain. When bacterial mixture absorbance (OD₆₀₀) reached 0.8, it was infiltrated into cotyledons and first true leaves of sweet pepper seedlings. Plants were dark-cultured at 16°C for 56 hours before transferring to normal growth conditions. Silencing efficiency was verified by qPCR using gene-specific primers. TRV::PDS served as positive visual control [ 29 ]. 2.14. Disease Index Evaluation Plants were root-inoculated with R. solanacearum (15 mL per plant, OD₆₀₀ = 1.0). Wilting symptoms were recorded daily and scored on a 0–4 scale: 0 = healthy; 1 = ≤ 25% leaf wilting; 2 = 25–50%; 3 = 50–75%; 4 = 75–100%. Disease index (DI) was calculated as: $ $ DI (%) = \frac{\sum(s \times n)}{N \times S} \times 100 $ $ where s = disease score, n = number of plants with that score, N = total number of plants, S = maximum score (4). 2.15. Bacterial Colony Forming Unit (CFU) Assay Leaves were treated with R. solanacearum infiltration (OD₆₀₀ = 0.4) and sampled at 24 and 48 hours post-inoculation. Leaf discs were surface-sterilized, homogenized, serially diluted, and plated on TTC agar plates. Colonies were counted after 48 hours of culture at 28°C [ 30 ]. 2.16. Transgenic Plant Construction Pepper (flamingo beak method) : GV3101 carrying 35S::CaHT1-GFP plasmid infected Zunla-1 seedlings. After co-cultivation, stem segments regenerated from wound sites were selected on medium containing Basta, and verified by PCR and Western blot after rooting [ 31 ]. Tobacco (leaf disc method) Agrobacterium carrying target constructs infected sterile leaf discs, regenerated into whole plants after antibiotic medium selection. Transgenic lines were confirmed by PCR and seed propagation. 2.17. CTAB Method DNA Extraction Leaf tissues were ground in liquid nitrogen, lysed with CTAB buffer, and extracted with chloroform. DNA was precipitated with isopropanol, washed with ethanol, dissolved in deionized water for PCR analysis [ 32 ]. Results 3.1. Subcellular Localization of CaHT1 Protein function is closely related to its subcellular localization. To investigate CaHT1 localization, we transiently expressed 35S::CaHT1-YFP in tobacco leaves, with 35S::YFP as negative control and P2300 as nuclear localization marker. Confocal microscopy observation showed YFP fluorescence present in both cytoplasm and nucleus, indicating CaHT1 localizes to these two compartments (Fig. 1 A). 3.2. Interaction Analysis between CaHT1 and CaPYR1 In BiFC experiments, YFP fluorescence signals were observed at the plasma membrane, indicating CaHT1 interacts with CaPYR1 at the plasma membrane in planta (Fig. 1 B). To verify direct physical interaction in vitro, we expressed CaHT1-GST and CaPYR1-His proteins in E. coli and conducted pull-down assays. After BeaverBeads™ purification, CaHT1-GST was detected in immunoprecipitates from CaHT1-GST + CaPYR1-His samples, confirming direct interaction between the two in vitro as well (Fig. 1 C). Protein-protein interactions often occur when intracellular physical distances are extremely close. To investigate subcellular localization proximity between CaHT1 and CaPYR1, we constructed recombinant plasmids expressing CaHT1-YFP and CaPYR1-CFP. After Agrobacterium tumefaciens -mediated transient expression in tobacco leaves, confocal microscopy observation showed both CaHT1 and CaPYR1 localized to the plasma membrane with overlapping fluorescence signals, indicating spatial proximity between them, supporting their potential interaction in vivo (Fig. 1 D). 3.3. CaHT1 Sequence and Transcriptional Level Analysis CaHT1 belongs to the protein kinase C (PKC) family and contains a MAPKKK-like serine/threonine kinase domain. Transmembrane domain prediction using TMHMM server ( http://services.healthtech.dtu.dk/services ) revealed that CaHT1 lacks transmembrane domains and is primarily located outside the membrane (Fig. 2 A, B). To investigate CaHT1 functional conservation, we performed homology alignment with related genes from other solanaceous species. CaHT1 showed high sequence similarity with potato StHT1 (XP_006347031.1), tobacco NtHT1 (XP_016469683.1), tomato SlHT1 (XP_025886140.1), and potato SpHT1 (XP_015066476.1). SlHT1 (tomato, XP_025886140.1) and SpHT1 (wild tomato, XP_015066476.1) exhibited particularly high sequence similarity (Fig. 2 C, D). 3.4. CaHT1 Promoter Cis-Element Analysis We predicted cis-acting elements in the CaHT1 promoter using the PlantCARE database ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Multiple stress-responsive elements were identified, including abscisic acid-responsive elements (ABRE), wound-responsive elements (WUN-motif), light-responsive modules (Box 4), CCAAT boxes (MYBHv1 binding sites), and cell cycle regulatory elements (MSA-like). The presence of these elements suggests CaHT1 may participate in ABA-mediated defense signaling (Fig. 2 A). 3.5. CaHT1 Protein Structure Prediction CaHT1 primary structure was predicted using ExPASy ProtParam tool ( https://web.expasy.org/protparam/ ). The protein encodes 505 amino acids, with molecular weight of 125,326.60 Da, isoelectric point (pI) of 5.01, and total atom count of 16,113. Molecular formula is C₄₆₂₃H₇₇₃₃N₁₅₁₅O₁₉₃₆S₃₀₆. Grand average of hydropathy (GRAVY) is 0.752, and instability index (II) is 39.61 (Table 1 ). Table 1 Predicted primary structure of CaHT1​​ Primary Structure Feature Predicted Result Number of Amino Acids 505 Molecular Weight (MW)/Da Isoelectric Point (PI) Total Atoms Molecular Formula Average Hydrophilicity Coefficient (GRAVY) Instability Index (II) 125326.60 5.01 16113 C 4623 H 7733 N 1515 O 1936 S 306 0.752 39.61 3.6. CaHT1 Expression Analysis under Normal and HTHH Conditions Our laboratory transcriptome database analysis showed: compared with room temperature control (RTHH), CaHT1 transcription levels increased under room temperature inoculation treatment (RSRT), but decreased under high temperature inoculation (RSHT) and high temperature high humidity (HTHH) treatment conditions (Fig. 3 A). To further elucidate CaHT1 expression patterns at the transcriptional level and its disease resistance mechanism, we used CaHT1-specific primers to perform qRT-PCR detection at 1, 12, 24, and 48 hours post-treatment under four conditions: room temperature (RTHH), room temperature inoculation (RSRT), high temperature (HTHH), and high temperature inoculation (RSHT). Results showed: CaHT1 responded to RSRT treatment at 1 hour, to RTHH treatment at 12 hours, to RSHT treatment at 24 hours, and to both RSRT and RSHT treatments at 48 hours. Notably, CaHT1 transcription levels significantly increased at 24 and 48 hours (Fig. 3 B). 3.7. Effects of CaHT1 Silencing on Pepper Resistance to R. solanacearum Infection We employed virus-induced gene silencing (VIGS) technology to knock down CaHT1 expression in pepper. TRV::CaHT1 (experimental group), TRV::00 (negative control), and TRV::CaPDS (positive control) were inoculated into five-leaf stage pepper seedlings. After 16 hours of dark culture at 16°C, plants were transferred to 25°C growth chamber with 16 h light/8 h dark cycle. Leaf whitening phenomenon in TRV::CaPDS plants confirmed VIGS success (Fig. 4 A). When plants developed 8–10 leaves, total RNA was extracted and reverse transcribed to cDNA. qRT-PCR verification showed CaHT1 transcript levels were significantly reduced in TRV::CaHT1 plants, indicating effective silencing (Fig. 4 B). Subsequently, TRV::CaHT1 (experimental group) and TRV::00 (control group) plants were treated under four conditions (HTHH, RSHH, RSRT, RTHH), and disease progression was monitored. Under RSRT conditions, no significant differences in wilting symptoms were observed between TRV::CaHT1 and TRV::00 plants. However, under RSHH (37°C) conditions, TRV::CaHT1 plants exhibited earlier wilting compared to TRV::00 controls (Fig. 4 C, E, F). Bacterial population detection (CFU) showed that under both RSRT and RSHT conditions, R. solanacearum quantities in TRV::CaHT1 plants were significantly higher than in TRV::00 controls (Fig. 4 D). These results indicate that silencing CaHT1 gene enhances pepper susceptibility to R. solanacearum . Transcriptional levels of defense-related genes reflect plant immune status. We used qRT-PCR technology to detect expression of marker genes ( CabZIP11 , CabHLH35 , CaMgst3 , CaPR1 , CaWRKY22 ) in TRV::CaHT1 and TRV::00 plants under RTHH, RSRT, HTHH, and RSHT treatment conditions. Compared with controls, under RSRT treatment, transcript levels of CaBIZP11 , CaMgst3 , CaPR1 , and CaWRKY22 were significantly reduced in CaHT1-silenced plants; under HTHH treatment, CaWRKY22 expression decreased; while under RSHT treatment, all five marker genes showed downregulated expression (Fig. 5 A, B, C, D, E). These results further indicate CaHT1 functions as a positive regulator of pepper resistance to bacterial wilt. Given that ABA signaling exerts negative regulatory effects on pepper resistance through its receptors, we hypothesize that HT1 participates in the assembly of ABA-CaPYR1-mediated CaPHDy/CaGTEz reading complex for H3K4me3/H3K9ac, thereby activating CaWRKY22 , CabZIP11 , and CabHLH35 expression, conferring specific resistance under high temperature and high humidity conditions. 3.8. Effects of CaHT1 Stable Overexpression on Tobacco Resistance to Bacterial Wilt To further verify the positive regulatory role of CaHT1 in disease resistance, we constructed stable CaHT1-overexpressing tobacco transgenic plants through Agrobacterium -mediated transformation. Western blot analysis of T₂ generation lines (CaHT1-1 and CaHT1-2) using anti-GFP antibody detected bands at 55–70 kDa, confirming successful CaHT1-GFP overexpression (Fig. S2A). T₂ generation transgenic lines (CaHT1-1, CaHT1-2) and wild-type (WT) plants were treated with RTHH, RSRT, HTHH, and RSHT. CFU assays at 24 h and 48 h post-RSRT and RSHT treatment showed colony numbers in CaHT1-#1 and CaHT1-2 were lower than wild-type (Fig. S2B). Under RSRT and RSHT conditions, CaHT1 overexpression lines exhibited lower mortality rates and stronger resistance to R. solanacearum compared to WT plants (Fig. S2C, D). Colony counting at 24 and 48 hours post-inoculation showed bacterial numbers in CaHT1-#1 and CaHT1-2 were lower than WT under both RSRT and RSHT treatment conditions. These data indicate CaHT1 overexpression significantly enhanced tobacco disease resistance. 3.9. Effects of CaHT1 Stable Overexpression on Sweet Pepper Resistance to Bacterial Wilt To verify the positive role of CaHT1 in pepper disease resistance, we constructed CaHT1-overexpressing transgenic pepper plants using Agrobacterium -mediated transformation combined with the flamingo beak method. Western blot analysis of T₂ generation sweet pepper lines (CaHT1-1 and CaHT1-2) using anti-GFP antibody showed bands at 55–70 kDa, confirming CaHT1-GFP overexpression (Fig. 6 A). Colony counting at 24 and 48 hours post-inoculation showed bacterial numbers in CaHT1-1 and CaHT1-2 were significantly lower than wild-type under RSRT and RSHT conditions (Fig. 6 B). T₂ generation transgenic plants and wild-type sweet pepper were treated under RTHH, RSRT, HTHH, and RSHT conditions. Compared with wild-type, CaHT1 overexpression lines exhibited delayed wilting and stronger resistance under RSRT and RSHT treatments (Fig. 6 C). Collectively, these results indicate CaHT1 overexpression enhances pepper resistance to R. solanacearum . 3.10. CaHT1 Transient Overexpression Effect Analysis To further investigate CaHT1's role in immune responses, transient overexpression was performed in pepper leaves. First, qRT-PCR showed: CaHT1 transcript levels in 35S::CaHT1-GFP infiltrated leaves were significantly higher than 35S::GFP controls, showing this trend at 48 hours under both RTHH and HTHH treatment conditions (Fig. 6 D). Similarly, we transiently overexpressed 35S::CaHT1-GFP or 35S::GFP (empty vector) in tobacco leaves. Fluorescence signals were detected by confocal microscopy, and 55–70 kDa bands were detected by Western blot using anti-GFP antibody (Fig. 6 E). The above evidence confirms successful transient overexpression. Subsequently, we detected expression of defense marker gene CaWRKY22 in sweet pepper leaves under RTHH and HTHH conditions. qRT-PCR results showed that under HTHH treatment conditions, CaWRKY22 transcript levels in CaHT1-overexpressing leaves were higher than controls (Fig. 6 F). Plant immune activation often involves salicylic acid accumulation, programmed cell death (PCD), reactive oxygen species (ROS) production, and hypersensitive response (HR)-related gene expression. We observed that 3-day local transient overexpression of CaHT1 in pepper leaves under HTHH conditions induced visible HR lesions, while lesions were less obvious under room temperature conditions. Further detection through white light and UV imaging, as well as DAB and trypan blue staining, indicated that under HTHH conditions, cell death and H₂O₂ accumulation were enhanced in CaHT1 overexpression regions (Fig. 6 G). To investigate functional interaction between CaHT1 and CaPYR1, we silenced CaHT1 gene through VIGS technology (TRV::CaHT1), then transiently overexpressed 35S::CaHT1-GFP or 35S::GFP in TRV::CaHT1 and TRV::00 plants. After treatment at 28°C or 37°C, necrotic response lesions appeared only in TRV::CaHT1 plants overexpressing CaPYR1 under high temperature conditions, while TRV::00 plants did not exhibit such responses. This suggests CaHT1 may not be the sole critical factor in CaPYR1-mediated immune activation in pepper (Fig. 6 H). Discussion This study confirms through multiple experiments that the CaHT1 gene positively regulates pepper immune responses against bacterial wilt. Previous laboratory studies indicated that solanaceous crops activate SA and JA-mediated defense responses to counteract R. solanacearum infection under normal temperature conditions. However, under high temperature and high humidity (HTHH) conditions, these SA and JA-mediated immune responses are suppressed to varying degrees. Instead, cytokinin-mediated immune responses are initiated, characterized by specific upregulation of GST-encoding genes (such as Mgst3 and PRP1 ) [ 33 ]. These findings suggest that immune response mechanisms employed by solanaceous plants under HTHH conditions differ significantly from those under normal temperature conditions. Previous laboratory studies revealed that multiple WRKY transcription factors, including CaWRKY40, CaWRKY6, CaWRKY28, and CaWRKY58, play important regulatory roles in pepper disease resistance. Particularly, CaWRKY40 occupies a central hub position in responses to bacterial wilt under HTHH conditions [ 34 ]. For example, CaCDPK15 positively regulates pepper responses to R. solanacearum through interaction with CaWRKY40 [ 35 ]. CaCDPK29 phosphorylation of Ser137 site in CaWRKY27b nuclear localization signal (NLS) upregulates its expression and promotes its translocation from cytoplasm to nucleus. Although the conserved WRKYGQK motif in CaWRKY27b undergoes mutation (Q→M), resulting in loss of DNA binding ability to W-box in promoters of immune and heat tolerance-related marker genes, CaWRKY27b can still interact with CaWRKY40 within the nucleus. This interaction forms complexes with molecules such as CaNPR1, CaDEF1, and CaHSP24, thereby exerting indirect positive regulatory effects, enhancing pepper immunity to RSI and tolerance to HTHH. CaCDPK29 phosphorylation of CaWRKY27b strengthens CaWRKY40 function in pepper resistance to bacterial wilt [ 36 ]. In summary, early laboratory research primarily focused on protein kinase CaCDPK responses to stress, with less exploration of the protein kinase C (PKC) family. Previous studies revealed CaPYR1 negatively regulates immune responses to R. solanacearum under normal temperature, but positively regulates such responses under high temperature conditions. Subsequent pull-down experiments combined with LC-MS analysis suggested potential interaction between CaHT1 and CaPYR1. Therefore, this study selected protein kinase CaHT1 as research subject to further investigate protein kinase C family functions in pepper bacterial wilt resistance. This study, combining pull-down experiments with liquid chromatography-mass spectrometry analysis, strongly confirmed interaction between CaHT1 and CaPYR1. This finding prompted us to further investigate the mechanism of protein kinase CaHT1 (a protein kinase C family member) in pepper bacterial wilt resistance. Previous studies indicate that abscisic acid binding to its receptors participates in regulating plant stress physiological responses. Two possible abscisic acid receptors include Mg²⁺ chelatase H subunit and protein-coupled receptors. Abscisic acid plays dual roles in plant disease resistance. ABA exerts positive regulatory effects on responses to pathogen-associated molecular patterns (PAMPs) and saprophytic pathogens. However, more research focuses on ABA's negative regulatory role in plant resistance, such as during tobacco resistance to bacterial wilt and barley resistance to rice blast, where ABA weakens plant resistance. Depending on pathogen species and infection modes, ABA can cause plants to exhibit disease tolerance or susceptibility. For example, ABA can promote guard cell function, blocking various pathogens from entering cells. Previous laboratory studies showed that silencing CaPYR1 gene in sweet pepper plants under HTHH or RSHT conditions leads to downregulation of marker genes CaMgst3 and CaPRP1 [ 37 ]. Conversely, CaPYR1 overexpression in tobacco plants results in CaMgst3 and CaPRP1 upregulation. This indicates CaPYR1 or ABA can inhibit SA or JA-mediated immune regulatory pathways, thereby negatively regulating pepper bacterial wilt resistance under normal temperature. CaPYR1 or ABA enhances pepper bacterial wilt resistance under high temperature and high humidity conditions through upregulation of CaMgst3 and CaPRP1. Studies show that CaMgst3 and CaPRP1 transcription upregulation following bacterial wilt inoculation in pepper under HTHH conditions correlates with cytokinin secretion. ABA exhibits antagonistic effects similar to SA and JA. CaPYR1 negatively regulates pepper bacterial wilt resistance under normal temperature, but positively regulates this process under HTHH conditions, and can enter the nucleus to bind and regulate CaPHD4 and CaGTE4 activity. Based on this, we speculate CaHT1 may further mediate disease resistance responses through interaction with CaPYR1. However, whether HT1 participates in assembling ABA-CaPYR1-mediated CaPHDy/CaGTEz open reading frame H3K4me3/H3K9ac modification complex, thereby activating CaWRKY22, CabZIP11, and CabHLH35 expression, and subsequently initiating pepper-specific immune responses to Ralstonia infection under HTHH conditions, requires further in-depth investigation. Protein phosphorylation and dephosphorylation are crucial in disease resistance processes. During early and late stages of plant immune responses, calcium ion signaling molecules can activate protein kinases, thereby regulating transcription processes. Protein phosphorylation is catalyzed by protein kinases. Kinases, as important phosphorylating enzymes, are responsible for transferring high-energy phosphate groups from high-energy small molecules (such as ATP) to specific target substances (such as proteins, lipids, carbohydrates, amino acids, nucleic acids). Previous studies have found protein kinases participate in plant stress responses. For example, mitogen-activated protein kinases (MAPK) participate in plant drought, salt, and cold stress responses [ 38 ]. Specifically, MAPK hierarchical phosphorylation systems can amplify external stimulus signals, activate downstream target genes, and initiate related stress responses [ 39 ]. Among these, MAP-KKK located upstream of MAPK cascade phosphorylation systems undergoes phosphorylation following environmental stress [ 40 ]. Activated MAP-KKK can promote biological functions of downstream protein kinases, such as activating transcription factors related to expression of specific disease resistance genes like PBF1 and G/HBF1. For example, previous studies found AhMPK6 and AhMPK3 expression can enhance tobacco disease resistance, while OsBWMK1 similarly improves tobacco disease resistance through phosphorylation of transcription factor OsEREBP1 [ 41 ]. Taking FLS2 as an example, this protein kinase can recognize flagellin. In rice, protein kinases OsBRR1 and OsWAK1 respond to rice blast, with transcription expression levels significantly elevated especially under ABA induction [ 42 ]. HT1 depends on second messengers: when cell surface receptor kinases receive external stimuli, second messengers including cAMP, cGMP, and various phospholipids are produced, subsequently activating AGC protein kinases, transmitting signals to downstream molecules through phosphorylation. Studies show AGC protein kinases can regulate polar transport of growth factors, respond to biotic and abiotic stresses, and play key roles in polar growth of root hairs and pollen tubes. Protein phosphorylation, as critical post-translational modification, is essential for signal transduction networks composed of interconnected signaling pathways—cells make decisions responding to internal and external stimuli through this network [ 43 ]. ABA receptor CaPYR1 negatively regulates pepper bacterial wilt resistance under normal temperature, but positively regulates this resistance under HTHH conditions [ 44 , 45 ]. Based on the above analysis, we believe: functional differences under different conditions originate from CaPYR1 phosphorylation modifications at different sites, indicating this receptor may be phosphorylated by different protein kinases under different conditions, and these kinases play important roles in regulating pepper disease resistance, with CaHT1 possibly being one such protein. In this study, we first reveal the positive regulatory role of protein kinase CaHT1 in pepper bacterial wilt immune responses, and further confirm its physical interaction with ABA receptor CaPYR1. We have reason to believe CaPYR1's negative regulation of bacterial wilt resistance under normal temperature, transitioning to positive regulation under HTHH conditions, may occur through phosphorylation modifications at different sites responding to different upstream signals, thereby achieving flexible regulation of downstream immune pathways. This echoes our laboratory's previous research on CaCDPK29-CaWRKY27b module: CaCDPK29 promotes CaWRKY27b entry into nucleus through phosphorylation and interaction with CaWRKY40, thereby coordinately activating expression of immune and heat tolerance-related genes [ 46 ]. Therefore, as a protein kinase, interaction between CaHT1 and CaPYR1 likely mediates CaPYR1 functional transition under different environmental conditions through similar phosphorylation events. Notably, under high temperature and high humidity conditions, plants often need to balance immune responses with heat tolerance. Our laboratory's latest research found transcription factor CaKAN3 cooperates with CaHSF8 to activate NLR genes under HTHH conditions, thereby enhancing immunity to bacterial wilt. However, under sustained high temperature or extreme high temperature conditions, CaHSF8 dissociates from CaKAN3,转而 upregulating heat shock protein (HSP) genes to activate heat tolerance. This indicates pepper possesses precise molecular "switches" to trade off between immunity and heat tolerance when responding to complex stresses. The functional transition mechanism of CaHT1-CaPYR1 module may be a key link in this trade-off process. We speculate CaHT1 phosphorylation modification of CaPYR1 may alter CaPYR1 interaction patterns with other regulatory factors (such as CaKAN3, CaHSF8, or CaNAC2c), thereby determining whether to prioritize initiating immune responses or heat tolerance mechanisms. Future research should thoroughly investigate CaHT1 phosphorylation site specificity on CaPYR1, and how these phosphorylation events affect CaPYR1 binding affinity and activity with downstream signaling components, thereby elucidating its specific action mechanisms in immunity-heat tolerance trade-offs. We know that when plant immune systems are activated, they typically exhibit defense gene expression associated with salicylic acid (SA) accumulation, programmed cell death (PCD), reactive oxygen species (ROS) generation, and hypersensitive response (HR). In this experiment, we inoculated TRV::00 and TRV::CaHT1 sweet pepper seedlings with 35S::CaHT1-GFP and 35S::GFP bacterial solutions via Agrobacterium . We observed hypersensitive response spots appeared on TRV::CaHT1 plants overexpressing CaPYR1, while TRV::00 pepper leaves failed to trigger hypersensitive responses or hydrogen peroxide accumulation. Under HTHH treatment conditions, local CaPYR1 overexpression in pepper leaves resulted in hypersensitive response spot formation, accompanied by more severe cell death and hydrogen peroxide accumulation. This indicates CaHT1 can activate plant PTI and ETI immune responses. Initiation of these immune responses is often closely related to rapid and precise gene expression regulation, with chromatin remodeling playing key roles in this process. Our laboratory's previous research revealed CaSWC4 precisely regulates trade-offs between immunity and heat tolerance by recruiting CabZIP63/CaWRKY40 and promoting deposition of H2A.Z, H3K9ac, H4K5ac and other histone modifications, thereby activating chromatin of immune or heat tolerance-related genes [ 47 ]. Given CaHT1 as a protein kinase, its phosphorylation of CaPYR1 may affect downstream gene transcriptional regulation, we have reason to speculate CaHT1-CaPYR1 module may have synergistic effects with CaSWC4-mediated chromatin remodeling mechanisms. Specifically, CaHT1 phosphorylation modification of CaPYR1 may affect CaPYR1 interaction with chromatin remodeling complex components (such as CaSWC4, CaTAF14b, or CaRUVBL2), thereby indirectly regulating chromatin accessibility at target gene promoter regions. For example, phosphorylated CaPYR1 may enhance its binding with CaSWC4, thereby promoting deposition of active histone modifications such as H3K4me3/H3K9ac, subsequently activating expression of immune-related genes CaWRKY22 , CabZIP11 , and CabHLH35 [ 48 ]. Additionally, cross-regulation between CaHT1-CaPYR1 module and WRKY transcription factor family should be considered. This study found CaHT1 silencing significantly downregulates CaWRKY22 expression, and our laboratory has identified multiple WRKY transcription factors playing critical roles in pepper disease resistance (such as CaWRKY40, CaWRKY27b). CaHT1 phosphorylation modification of CaPYR1 may, through regulating CaPYR1 interaction with these WRKY transcription factors, or directly affecting WRKY transcription factor activity and stability itself, collectively shape pepper immune response mechanisms. Future research should employ ChIP-seq and other technologies to investigate whether CaHT1-CaPYR1 module directly or indirectly affects chromatin remodeling factors and WRKY transcription factor binding to target gene promoters, thereby comprehensively elucidating its molecular mechanisms in gene expression regulation. Plant hormones such as abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) play complex cross-regulatory roles in plant immunity and stress responses. This study found CaPYR1, as an ABA receptor, exhibits significantly different functions under different temperature conditions, emphasizing the condition-dependency of ABA signaling in pepper bacterial wilt resistance. Our laboratory's previous research found ABA signaling pathway positively regulates heat tolerance but negatively regulates bacterial wilt resistance capacity, while JA signaling pathway primarily participates in immune responses. Antagonistic and synergistic interactions between hormones constitute refined regulatory networks for plants responding to complex stresses. The discovery of CaHT1-CaPYR1 module provides new entry points for analyzing this network's operating mechanisms. Accordingly, we speculate CaHT1 phosphorylation modification of CaPYR1 may be a key node in "crosstalk" between ABA signaling and JA/SA signaling. Specifically, under high temperature conditions, CaHT1 phosphorylation modification of CaPYR1 may enhance ABA signaling pathway, thereby promoting heat tolerance gene expression; simultaneously, it may finely regulate immune response intensity through antagonistic effects with JA/SA signaling pathways. This regulation may be achieved through affecting activity or stability of downstream key transcription factors (such as CaNAC2c, CaPti1-CaERF3) [ 49 ]. For example, CaNAC2c interacts with CaHSP70 under HTS conditions to activate heat tolerance, while interacts with CaNAC029 under RSI conditions to activate JA-mediated immunity. CaHT1-CaPYR1 module may determine CaNAC2c's tendency toward heat tolerance or immunity by affecting its interaction partner selection. Additionally, CaPti1-CaERF3 module positively regulates bacterial wilt resistance by activating SA-dependent CaPR1 and dehydration tolerance-related genes, and inducing stomatal closure in ABA signaling-dependent manner [ 50 ]. Whether direct or indirect regulatory relationships exist between CaHT1-CaPYR1 module and CaPti1-CaERF3 module (for example, whether CaHT1 affects CaPti1 or CaERF3 activity or coupling with ABA signaling through phosphorylation) will become important directions for future research [ 50 ]. In-depth analysis of how CaHT1-CaPYR1 module integrates ABA, JA, and SA signaling will help construct more comprehensive pepper complex stress response regulatory network models, providing theoretical basis for breeding broad-spectrum resistant pepper varieties. Declarations Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 校对报告 当前使用的样式是 [Plant and Soil] 当前文档包含的题录共0条 有0条题录存在必填字段内容缺失的问题 所有题录的数据正常 Funding This work was supported by the Fujian Provincial Natural Science Foundation [Grant Number: 2024J01311873]. Author Contributions Xiang Zheng : Conceptualization, Methodology, Investigation, Data curation, Writing – original draft. Huifang Shi : Investigation, Validation, Writing – review & editing. Lingxian Yi : Supervision, Writing – review & editing. Daojin Yu : Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Acknowledgements This research was supported by the Fujian Provincial Natural Science Foundation (Project No.: 2024J01311873). We thank Shi Huifang for assistance in completing partial experimental work. We also thank Lecturer Yi Lingxian and Professor Yu Daojin for guidance in research design and valuable suggestions during manuscript preparation. We appreciate all laboratory members for helpful discussions and technical support. 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Int J Mol Sci 24:4849 Cai W, Yang S, Wu R, Cao J, Shen L, Guan D, Shui L (2021) Pepper NAC-type transcription factor NAC2c balances trade-off between growth and defense responses. Plant Physiol 186:2169–2189 Shi L, Li X, Weng Y, Cai H, Liu K, Xie B, Hassan A, Guan D, He S, Liu Z (2022) CaPti1-CaERF3 module positively regulates sweet pepper resistance to bacterial wilt by enhancing immunity and dehydration tolerance. J Plant Biol Cell Mol Biol 111:250–268 Supplementary Files Graphicalabstract.docx SupportingInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 15 Apr, 2026 Editor invited by journal 11 Apr, 2026 Editor assigned by journal 11 Apr, 2026 First submitted to journal 09 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9298395","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623823383,"identity":"f271afe1-cab2-4115-b446-189cfa3b2e1b","order_by":0,"name":"Xiang Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Zheng","suffix":""},{"id":623823384,"identity":"705dc293-60f6-4c10-bb90-4f52db02c3fb","order_by":1,"name":"Jinggang Lv","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinggang","middleName":"","lastName":"Lv","suffix":""},{"id":623823385,"identity":"34dfdd4f-4f39-481e-b987-bf6d9651cc6e","order_by":2,"name":"Huifang Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huifang","middleName":"","lastName":"Shi","suffix":""},{"id":623823386,"identity":"23d31467-0c6d-461d-b51d-a56263334d26","order_by":3,"name":"Lingxian Yi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lingxian","middleName":"","lastName":"Yi","suffix":""},{"id":623823387,"identity":"2cea5027-b2a7-423c-8487-6e556ff9f41c","order_by":4,"name":"Daojin Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDCCAwwMBglAmh/EeQDkEK9FsoGBsSGBWC1gYHCAWC18xw8fKHi443Di5uM95g8SKmyMGdgPH92AT4vkmbQEg8QzhxO3nTlj2JBwJs2MgSct7QY+LQYHcgwMEtuAWm7kGDYAGTYMEjxm+LWcfwPRsnkG0VpuQG3ZIAHRYkZQi+SNZ0C/tKUbzzhzrHAG0C/GbIT8wnc++ZjhzzZr2f725g0fPlTYGPazHz6GVwsQsAGjohmJS0A5CDA/YGCoI0LdKBgFo2AUjFgAAO+HU/Xj42nsAAAAAElFTkSuQmCC","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Daojin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-04-02 05:30:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9298395/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9298395/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107647529,"identity":"c3a240f6-7bc6-4e41-9ce9-24509155283d","added_by":"auto","created_at":"2026-04-23 14:28:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3429891,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of the interaction between CaHT1 and CaPYR1 protein;\u003c/p\u003e\n\u003cp\u003eA) Subcellular localization of CaHT1 in epidermal cells of tobacco Ben Merged: YFP represents yellow fluorescence, RFP represents red fluorescence, Visible represents Visible light, Merged: YFP, RFP and visible light superposition, scale 25 µm.\u003c/p\u003e\n\u003cp\u003eB) BIFC experiment, where YFP represents yellow fluorescence, Visible represents Visible light, Merged represents the superposition of YFP and visible light, the scale is 25 µm;\u003c/p\u003e\n\u003cp\u003eC) The Pull-down experiment verified the protein interaction between CaHT1 and CaPYR1 in vitro.\u003c/p\u003e\n\u003cp\u003eD) In the co-localization experiment, a confocal microscope was used to observe the fluorescence signals of cells overexpressed with corresponding genes, where CFP represents cyan fluorescence, YFP represents yellow fluorescence, Visible represents Visible light, and Merged represents the superposition of CFP, YFP and visible light with a scale of 25 µm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/789b800f5d585584bfc3bcf0.png"},{"id":107868911,"identity":"30a24d67-77e8-4f36-b781-2661102619d4","added_by":"auto","created_at":"2026-04-27 07:34:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3740439,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequence analysis of CaHT1 and CaHT1 sequence alignment with other species genes; A) the domain of CaHT1; B) transmembrane domain of CaHT1; C) Evolutionary tree of CaHT1; D) Sequence alignment of CaHT1 with homologous gene sequences of other species. Multiple sequence comparisons were made between CaHT1 and homologous genes of other species using DNAman software, in which the color of the shaded part represented homology of different species, among which the black shaded part represented homology of 100%, the red shaded part represented homology of 75 %-100 %, and the blue shaded part represented homology of 50 %-75 %.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/9d3efb0973eb992329e44be5.png"},{"id":107707628,"identity":"8e1f81a2-9c45-4139-94d1-193321dbe1f5","added_by":"auto","created_at":"2026-04-24 09:20:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":934986,"visible":true,"origin":"","legend":"\u003cp\u003eA) Transcriptional expression pattern analysis of CaHT1 in pepper,Under the treatment of RTHH, RSRT, HTHH and RSHT, the transcriptional expression level of CaHT1 gene was detected by fluorescence quantitative RT-PCR at 1, 12, 24 and 48 h.\u003c/p\u003e\n\u003cp\u003eB ) Analysis of the FPKM value of CaHT1 and the transcriptional expression pattern of CaHT1 The transcription level of CaHT1 was analyzed under four different treatments: normal temperature (RTHH), normal temperature (RSRT), high temperature (RSHT) and high temperature (HTHH). Different capital letters represent significant differences, and their significance was analyzed using LSD method.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/32b65e6ad81c945b037972c8.png"},{"id":107707233,"identity":"dd9b757f-3c06-4425-9ba9-94ca7a0d3e1a","added_by":"auto","created_at":"2026-04-24 09:19:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1728530,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of silenced CaHT1 on the immune response of Solanacearum in response to pepper ; A) The positive control plants with albinism B) The expression of CaHT1 gene was regulated by real-time fluorescence quantitative PCR under four treatment conditions including RTHH, RSRT, HTHH and RSHT. On this basis, the gene expression level of the control group plants was set as 1, and the mean ± standard deviation of 4 biological replicates was used as the control. LSD test was used for statistical processing in different capital letters (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). C) The phenotypic changes of CaHT1 gene after 10 days of silencing were recorded by RTHH, RTRS, HTHH and RSHT under the same conditions.D) After RSRT and RSHT treatments for 24 hours and 48 hours respectively, the colonies (CFU) of Bacillus thuringiensis in the TRV:00 and TRV:CaHT1 strains of peppers were determined；E F) Under two conditions of RSRT and RSHT, statistical analysis of plant disease indicators was conducted in CaHT1 group and control group, and the disease index was divided into 0 level (no wilting symptoms, All were healthy), primary (0-25% leaf wilt), secondary (25-50% leaf wilt), tertiary (50%-75% leaf wilt), and grade IV (75-100% leaf wilt). d) Pepper plants with TRV::CaHT1 and TRV::00 were inoculated with bacterial bacterial bacillus and placed under RTHH and HTHH conditions, respectively, and the number of each strain was counted.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/0f4a5dec8daee9d11d935dd0.png"},{"id":107647535,"identity":"d94462e4-bf4e-465d-b626-3a20f809f6cd","added_by":"auto","created_at":"2026-04-23 14:28:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":390032,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different treatments of CaHT1 gene silencing on transcription levels of related marker genes in pepper plants; Silent CaHT1 and non-silent pepper plants were treated with RTHH, RSRT, HTHH and RSHT. After 48 h, The transcription levels of disease-resistant marker genes CabZIP11, CabHLH35, CaMgst3, CaPR1 and CaWRKY22 in cDNA samples were detected by qRT-PCR.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/69ebfa2c2339a6ea207fc259.png"},{"id":107647536,"identity":"8e0a11bb-a086-4aaf-955a-26edb109dfbb","added_by":"auto","created_at":"2026-04-23 14:28:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2187135,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of CaHT1 transgenic pepper plants and the effect of different treatments on the resistance of Bacillus anemicus, verification of transient overexpression of CaHT1 and the effect of transient overexpression of CaHT1 on triggering cell death and hydrogen peroxide (H2O2) accumulation in peppers, and the induction of cellular immune response after transient overexpression of CaPYR1 in CAHT1-silenced plants; A）western blot was used to verify the stable overexpression of \u003cem\u003eCaHT1-#1\u003c/em\u003e and \u003cem\u003eCaHT1-#2 \u003c/em\u003etransgenic pepper strains. B) After treatment with RSRT and RSHT for 24 h and 48 h, bacterial colonies (CFU) of WT, \u003cem\u003eCaHT1-#1 \u003c/em\u003eand \u003cem\u003eCaHT1-#2 \u003c/em\u003etransgenic capsicum were determined; C) Phenotypes of WT and \u003cem\u003eCaHT1-#1\u003c/em\u003e and \u003cem\u003eCaHT1-#2 \u003c/em\u003etransgenic peppers under four different treatment conditions of RTHH, RSRT, HTHH and RSHT; D) qRT-PCR was used to analyze the transcription levels of RTHH and HTHH in pepper plants with instantaneous overexpression of \u003cem\u003eCaHT1 \u003c/em\u003eand control plants under two conditions; E) The instantaneous overexpression of CaHT1-GFP and GFP in pepper was detected by western blot using GPF antibody;F) The transcription level of related resistance marker gene\u003cem\u003e CaWRKY22 \u003c/em\u003ein transgenic tobacco plants was detected by qRT-PCR; G) The necrosis of allergic cells and the accumulation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were detected with the four experimental techniques of UV, Trypan blue and DAB respectively. H）Transient overexpression of \u003cem\u003eCaPYR1\u003c/em\u003e in \u003cem\u003eCaHT1\u003c/em\u003e-silenced plants elicits cellular immune responses\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/43764e2e1fcfff9c87b0385e.png"},{"id":107871763,"identity":"340771f8-f1b0-46c5-a503-a86a7eebd914","added_by":"auto","created_at":"2026-04-27 07:54:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13715210,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/428e16ff-606c-4b11-90e8-274bcfbcba24.pdf"},{"id":107647530,"identity":"2e221d80-9f07-4bcf-8699-f91adff997f6","added_by":"auto","created_at":"2026-04-23 14:28:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":792479,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/3a60eb517d91614911035194.docx"},{"id":107647532,"identity":"a28ca908-f385-4082-b0ea-6504366f749b","added_by":"auto","created_at":"2026-04-23 14:28:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":777548,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9298395/v1/dcf13b0b710a49c624c0467f.docx"}],"financialInterests":"","formattedTitle":"CaHT1 Regulates Pepper Immune Response to Ralstonia solanacearum and Interacts with CaPYR1","fulltext":[{"header":"Highlights","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eCaHT1 positively regulates pepper resistance to bacterial wilt under normal and HTHH conditions\u003c/li\u003e\n \u003cli\u003eCaHT1 physically interacts with ABA receptor CaPYR1 at plasma membrane\u003c/li\u003e\n \u003cli\u003eCaHT1 silencing enhances pepper susceptibility to \u003cem\u003eRalstonia solanacearum\u003c/em\u003e infection\u003c/li\u003e\n \u003cli\u003eCaHT1 overexpression strengthens disease resistance in tobacco and pepper plants\u003c/li\u003e\n \u003cli\u003eCaHT1-CaPYR1 module may mediate immunity-thermotolerance trade-off mechanisms\u003cbr clear=\"all\"\u003e\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003ePepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e) is an economic crop originating from tropical and subtropical regions of South America and is one of the most widely cultivated vegetables globally, particularly prevalent in China. However, its production is severely threatened by bacterial wilt, a disease caused by the soil-borne pathogen \u003cem\u003eRalstonia solanacearum\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This pathogen invades pepper plants through root wounds, rapidly proliferates and spreads within the host, leading to vascular bundle obstruction. The resulting impairment of water and nutrient transport in stems and leaves ultimately causes rapid wilting and plant death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlants have evolved sophisticated immune systems to combat various pathogens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These systems involve immune receptors (such as pattern recognition receptors PRRs and resistance proteins R) that recognize pathogen-associated molecular patterns (PAMPs) and effector proteins, thereby activating complex plant immune signaling pathways [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These signals are transmitted and amplified through intricate networks, ultimately reprogramming the transcription of numerous disease resistance-related genes in the nucleus, triggering robust defense responses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This signaling network plays a critical regulatory role in plant defense, typically involving calcium ion messengers, plant hormones (such as salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA)), reactive oxygen species (ROS), and various kinases (such as mitogen-activated protein kinases (MAPKs)) and transcription factors. Specifically, SA primarily participates in combating biotrophic pathogens, while JA mainly mediates responses to necrotrophic pathogens [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo counteract pathogen invasion, plants have established immune systems at the molecular level, primarily comprising PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). To detect and respond to various biochemical attacks, plants have evolved two intrinsic immune mechanisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Plant defense mechanisms against pathogen invasion rely on both inherent physical and chemical barriers and inducible immune systems [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The latter, namely the inducible immune system, is activated when cell surface and intracellular receptors recognize pathogen-derived or plant-derived molecules. PTI, as an innate immune response mediated by cell surface pattern recognition receptors (PRRs), recognizes pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. One of the key roles of protein kinase C (PKC) is precisely to participate in negative feedback regulation within PTI. PRRs on the cell membrane recognize PAMPs and DAMPs, initiating primary immune responses. PTI is a critical factor in resisting pathogen infection and effectively maintains plant physiological and ecological balance. Pathogens can deliver effector proteins into host cell cytoplasm through the type III secretion system (T3SS) to exert their functions. During plant defense against pathogen invasion, nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly interact with effector proteins, thereby inducing stronger effector-triggered responses (ETI) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Bidirectional regulatory relationships exist between plant immunity and effector-triggered responses. PTI and ETI share common mechanisms, such as reactive oxygen species (ROS) production, mitogen-activated protein kinase (MAPKs) activation, defense hormone pathway initiation, and accompanying transcriptional reprogramming [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Both participate in immediate defense at pathogen invasion sites and can activate defense responses in plant tissue cells. Recent studies have revealed that enhanced PTI is a major characteristic of ETI. ROS produced in immune responses overlapping between PTI and ETI, as key defense and signaling molecules, are induced in both responses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe initial event in plant response to high temperature stress is high temperature perception. Changes in intracellular calcium ion concentration are perceived by intracellular protein sensors and histone sensors. Meanwhile, many signaling pathways participate in responding to high temperature stress, such as the aforementioned calcium ion signaling pathway, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After entering the plant cell nucleus, these signals cooperate with various transcription factors to initiate the expression of defense-related genes, thereby conferring heat tolerance to plants [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. On the other hand, plants secrete heat shock proteins (HSPs) under high temperature stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Under high temperature stress conditions, HSPs expression levels increase rapidly.\u003c/p\u003e \u003cp\u003eAbscisic acid (ABA), also known as natural abscisic acid, is a plant hormone that inhibits plant growth. ABA functions through protein phosphorylation and dephosphorylation. Protein phosphorylation is completed by SnRK2, while dephosphorylation is executed by PP2C. PP2C is currently the largest known protein phosphatase family, with 76 identified members [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This family belongs to the serine/threonine protein kinase family, exists as monomers in cells, and its enzymatic activity depends on Mn\u0026sup2;⁺ and Mg\u0026sup2;⁺ ions. As a key non-coding RNA with important biological functions, PP2C also participates in cell signal transduction, cell cycle regulation, protein translation, and post-translational modification [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Among these, the ABA signaling pathway is associated with group A PP2C phosphatases, which play negative regulatory roles in the ABA signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanism of ABA action in plant biotic stress responses is complex. As a key plant hormone, its core role in regulating plant responses to abiotic stresses such as drought, low temperature, and high temperature has been widely recognized. Interestingly, recent studies increasingly reveal ABA's involvement in plant immune responses, although its precise mechanism of action remains complex and sometimes contradictory [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Studies have shown that ABA can exert negative regulatory effects on plant resistance, as demonstrated in tobacco resistance to bacterial wilt and barley resistance to rice blast. Depending on pathogen species and infection routes, ABA can either promote plant tolerance to pathogens or lead to susceptibility. For example, ABA can enhance the protective function of plant guard cells, blocking various pathogens from entering cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, ABA can also regulate SA, JA, and ethylene-dependent responses, thereby inhibiting later defense mechanisms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies have found positive synergistic effects between ABA and JA during \u003cem\u003eArabidopsis\u003c/em\u003e resistance to \u003cem\u003eRhizoctonia solani\u003c/em\u003e. ABA and ethylene (ET) exhibit antagonistic relationships\u0026mdash;ET serves as a characteristic signaling molecule promoting viral infection, while ABA plays a positive role in regulating disease resistance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The interaction mechanisms between ABA and other defense hormones require further in-depth investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant Materials and Growth Conditions\u003c/h2\u003e \u003cp\u003ePlant materials included tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e), sweet pepper variety Zunla-1, etc. Strains used included \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α, BL21, Rosetta, \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, \u003cem\u003eRalstonia solanacearum\u003c/em\u003e strain FJAT91, etc. Vectors used included Gateway entry vector pDONR207, plant expression vectors pEarleyGate101, pEarleyGate102, pEarleyGate103, virus-induced gene silencing (VIGS) vectors pTRV1 (pYL192) and pTRV2 (pYL279), prokaryotic expression vectors pEZY-Hb and PDEST-15, etc.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePepper cultivation method\u003c/strong\u003e \u003cp\u003eSeeds stored at 4\u0026deg;C were placed on moist filter paper in petri dishes, covered with perforated plastic film, and germinated in darkness for 3\u0026ndash;5 days. Germinated seeds were transplanted into soil, covered again with perforated plastic film, and cultivated in a growth chamber (25\u0026deg;C, 70% humidity, 16 h light/8 h dark) for approximately 10 days. After cotyledon expansion and emergence of the first true leaf, seedlings were transplanted into pots and regularly watered, fertilized, and loosened under the same conditions. When necessary, plants were moved outdoors for acclimatization 30 days after transplantation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTobacco cultivation method\u003c/strong\u003e \u003cp\u003eSeeds stored at room temperature were directly sown into soil, covered with perforated plastic film, and cultivated under the same growth chamber conditions for approximately 10 days. After cotyledon expansion, seedlings were transplanted into pots for continued cultivation and underwent outdoor acclimatization after 30 days.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eCaHT1 CDS sequences and homologous amino acid sequences were retrieved from the NCBI database. Conserved domains were analyzed using CD-Search and SMART tools, promoter elements were predicted using the PlantCARE database, and homologous protein families in pepper were identified via the Sol Genomics Network. Specific primers were designed using Primer Premier 5.0 software. Homologous sequences of CaHT1 with solanaceous plants (tomato, tobacco, potato, etc.) were obtained through BLAST searches. Multiple sequence alignment and phylogenetic tree construction were performed using DNAMAN 7 software. Transmembrane domain prediction for CaHT1 was completed using TMHMM software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Expression Vector Construction\u003c/h2\u003e \u003cp\u003eCaHT1 gene was amplified from Zunla-1 cDNA using high-fidelity polymerase. PCR products were separated by agarose gel electrophoresis, target bands were excised and purified. \u003cb\u003eBP reaction\u003c/b\u003e: Purified fragments and pDONR207 (0.5 \u0026micro;L each) were recombined overnight at 25\u0026deg;C using BP Clonase II enzyme (0.25 \u0026micro;L). Products were transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α strain and plated on LB agar plates containing gentamicin. Single colonies were screened by PCR, and positive clones were sequenced. Verified plasmids were extracted and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. \u003cb\u003eLR recombination reaction\u003c/b\u003e: Target clones were recombined with host vectors (pEarleyGate series) using LR Clonase II recombinase. Reaction products were transformed into DH5α \u003cem\u003eE. coli\u003c/em\u003e, and positive clones were obtained through antibiotic resistance screening and PCR detection. After plasmid extraction, they were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 strain via freeze-thaw transformation for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eE. coli\u003c/em\u003e Transformation and Culture\u003c/h2\u003e \u003cp\u003eFirst, DH5α competent cells were thawed on ice, mixed with 2 \u0026micro;L plasmid DNA, and incubated on ice for 30 minutes. Cells were heat-shocked at 42\u0026deg;C for 90 seconds, then cooled on ice for 5 minutes. Second, 400 \u0026micro;L LB medium was added, and cells were recovered at 37\u0026deg;C, 180 rpm for 1 hour. Third, cultures were spread on LB agar plates containing corresponding antibiotics and incubated at 37\u0026deg;C for 20 hours. Finally, single colonies were inoculated into LB liquid medium containing antibiotics, cultured for 3\u0026ndash;5 hours, and verified by PCR. Positive clones were amplified for subsequent use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e Transformation and Culture\u003c/h2\u003e \u003cp\u003eFirst, GV3101 competent cells were thawed on ice, mixed with 2 \u0026micro;L plasmid DNA, and incubated on ice for 30 minutes. Cells were frozen in liquid nitrogen for 5 minutes, then thawed at 37\u0026deg;C for 5 minutes. Second, 400 \u0026micro;L LB broth was added, and cells were cultured at 28\u0026deg;C, 180 rpm for 4\u0026ndash;5 hours. Third, cultures were spread on LB plates containing antibiotics and incubated at 28\u0026deg;C for 2 days. Finally, single colonies were verified by PCR followed by overnight culture and amplification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eRalstonia solanacearum\u003c/em\u003e Culture and Inoculation\u003c/h2\u003e \u003cp\u003e \u003cb\u003eCulture method\u003c/b\u003e: FJAT91 strain was inoculated from \u0026minus;\u0026thinsp;80\u0026deg;C frozen stock onto TTC agar plates and cultured at 28\u0026deg;C for 48 hours. Single white colonies were cultured overnight, then subcultured at 1:1000 ratio for 48 hours. Bacterial cells were collected by centrifugation (4500 rpm, 10 minutes), washed, and resuspended in deionized water to target OD₆₀₀ values.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRoot inoculation method\u003c/strong\u003e \u003cp\u003eWounds were created on lateral roots using scissors, and 15 mL bacterial suspension (OD₆₀₀ = 1.0) was inoculated. Plants were placed in environments at 28\u0026deg;C or 37\u0026deg;C with 80% humidity to monitor disease progression.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLeaf infiltration method\u003c/strong\u003e \u003cp\u003eBacterial suspension (OD₆₀₀ = 0.4) was infiltrated into the abaxial leaf surface using a 1 mL needleless syringe, ensuring coverage of the midrib region.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Protein-Protein Interaction Assays\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eProkaryotic expression optimization\u003c/strong\u003e \u003cp\u003eGST or His-tagged constructs were transformed into BL21 strain. Expression was induced by IPTG at different temperatures (16\u0026deg;C overnight culture, 28\u0026deg;C for 6\u0026ndash;8 hours, 37\u0026deg;C for 3\u0026ndash;5 hours). Expression levels were detected by SDS-PAGE analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePull-down assay\u003c/b\u003e: Purified CaHT1-GST and CaPYR1-His proteins were incubated with magnetic beads in four combinations: CaHT1-GST\u0026thinsp;+\u0026thinsp;CaPYR1-His, CaHT1-GST\u0026thinsp;+\u0026thinsp;negative control, GST\u0026thinsp;+\u0026thinsp;CaPYR1-His, and GST\u0026thinsp;+\u0026thinsp;negative control. After washing, bound proteins were eluted and detected by Western blot using anti-GST and anti-His antibodies.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBimolecular fluorescence complementation (BiFC)\u003c/strong\u003e \u003cp\u003eConstructs expressing CaHT1 fused to YFP⁺ and CaPYR1 fused to YFP⁻ were co-infiltrated into tobacco leaves. Fluorescence signals were observed using confocal microscopy 48 hours after infiltration.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Subcellular Localization\u003c/h2\u003e \u003cp\u003eCaHT1 was fused with GFP through Gateway cloning technology and introduced into GV3101 strain. Bacterial suspension (OD₆₀₀ = 0.8) was infiltrated into tobacco leaves. GFP fluorescence was observed using confocal microscopy after 48 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Transient Overexpression\u003c/h2\u003e \u003cp\u003eCaHT1 overexpression vectors were introduced into pepper leaves via \u003cem\u003eAgrobacterium\u003c/em\u003e infiltration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Protein Extraction and Western Blot Analysis\u003c/h2\u003e \u003cp\u003eLeaf tissues were ground in liquid nitrogen and extracted with protein extraction buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, followed by primary and secondary antibody incubation. Signals were detected by chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Histochemical Staining\u003c/h2\u003e \u003cp\u003e \u003cb\u003eTrypan blue staining\u003c/b\u003e: Leaves with hypersensitive response lesions were boiled in trypan blue solution (lactic acid:glycerol:ethanol:water\u0026thinsp;=\u0026thinsp;1:1:1:1) for 15 minutes, incubated for 24 hours, decolorized with boiling ethanol, and photographed.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDAB (3,3'-diaminobenzidine) staining\u003c/strong\u003e \u003cp\u003eLeaves were immersed in 1 mg/mL DAB solution for 24 hours, decolorized with 95% ethanol, and imaged.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. RNA Extraction, Reverse Transcription, and qPCR Detection\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from frozen tissues using TRIpure reagent. RNA quality was assessed by A₂₆₀/A₂₈₀ ratio (1.9\u0026ndash;2.1). cDNA was synthesized using reverse transcription kits. qPCR detection was performed using SYBR Green master mix on a real-time PCR system. \u003cem\u003eCaACTIN\u003c/em\u003e (pepper) and \u003cem\u003eNbEF-1α\u003c/em\u003e (tobacco) served as internal reference genes. Relative expression levels were calculated using the 2⁻ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Virus-Induced Gene Silencing (VIGS)\u003c/h2\u003e \u003cp\u003e300 bp fragments from the 3'UTR or coding region (CDS) of CaHT1 gene were cloned into pTRV2 vector. This construct was co-transformed with pTRV1 into GV3101 strain. When bacterial mixture absorbance (OD₆₀₀) reached 0.8, it was infiltrated into cotyledons and first true leaves of sweet pepper seedlings. Plants were dark-cultured at 16\u0026deg;C for 56 hours before transferring to normal growth conditions. Silencing efficiency was verified by qPCR using gene-specific primers. TRV::PDS served as positive visual control [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Disease Index Evaluation\u003c/h2\u003e \u003cp\u003ePlants were root-inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e (15 mL per plant, OD₆₀₀ = 1.0). Wilting symptoms were recorded daily and scored on a 0\u0026ndash;4 scale: 0\u0026thinsp;=\u0026thinsp;healthy; 1\u0026thinsp;=\u0026thinsp;\u0026le;\u0026thinsp;25% leaf wilting; 2\u0026thinsp;=\u0026thinsp;25\u0026ndash;50%; 3\u0026thinsp;=\u0026thinsp;50\u0026ndash;75%; 4\u0026thinsp;=\u0026thinsp;75\u0026ndash;100%. Disease index (DI) was calculated as:\u003c/p\u003e \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e\u003cspan\u003e$\u003c/span\u003eDI (%) = \\frac{\\sum(s \\times n)}{N \\times S} \\times 100\u003cspan\u003e$\u003c/span\u003e\u003cspan\u003e$\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere s\u0026thinsp;=\u0026thinsp;disease score, n\u0026thinsp;=\u0026thinsp;number of plants with that score, N\u0026thinsp;=\u0026thinsp;total number of plants, S\u0026thinsp;=\u0026thinsp;maximum score (4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Bacterial Colony Forming Unit (CFU) Assay\u003c/h2\u003e \u003cp\u003eLeaves were treated with \u003cem\u003eR. solanacearum\u003c/em\u003e infiltration (OD₆₀₀ = 0.4) and sampled at 24 and 48 hours post-inoculation. Leaf discs were surface-sterilized, homogenized, serially diluted, and plated on TTC agar plates. Colonies were counted after 48 hours of culture at 28\u0026deg;C [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Transgenic Plant Construction\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePepper (flamingo beak method)\u003c/b\u003e: GV3101 carrying 35S::CaHT1-GFP plasmid infected Zunla-1 seedlings. After co-cultivation, stem segments regenerated from wound sites were selected on medium containing Basta, and verified by PCR and Western blot after rooting [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTobacco (leaf disc method)\u003c/strong\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e carrying target constructs infected sterile leaf discs, regenerated into whole plants after antibiotic medium selection. Transgenic lines were confirmed by PCR and seed propagation.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. CTAB Method DNA Extraction\u003c/h2\u003e \u003cp\u003eLeaf tissues were ground in liquid nitrogen, lysed with CTAB buffer, and extracted with chloroform. DNA was precipitated with isopropanol, washed with ethanol, dissolved in deionized water for PCR analysis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Subcellular Localization of CaHT1\u003c/h2\u003e \u003cp\u003eProtein function is closely related to its subcellular localization. To investigate CaHT1 localization, we transiently expressed 35S::CaHT1-YFP in tobacco leaves, with 35S::YFP as negative control and P2300 as nuclear localization marker. Confocal microscopy observation showed YFP fluorescence present in both cytoplasm and nucleus, indicating CaHT1 localizes to these two compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Interaction Analysis between CaHT1 and CaPYR1\u003c/h2\u003e \u003cp\u003eIn BiFC experiments, YFP fluorescence signals were observed at the plasma membrane, indicating CaHT1 interacts with CaPYR1 at the plasma membrane in planta (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To verify direct physical interaction in vitro, we expressed CaHT1-GST and CaPYR1-His proteins in \u003cem\u003eE. coli\u003c/em\u003e and conducted pull-down assays. After BeaverBeads\u0026trade; purification, CaHT1-GST was detected in immunoprecipitates from CaHT1-GST\u0026thinsp;+\u0026thinsp;CaPYR1-His samples, confirming direct interaction between the two in vitro as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eProtein-protein interactions often occur when intracellular physical distances are extremely close. To investigate subcellular localization proximity between CaHT1 and CaPYR1, we constructed recombinant plasmids expressing CaHT1-YFP and CaPYR1-CFP. After \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transient expression in tobacco leaves, confocal microscopy observation showed both CaHT1 and CaPYR1 localized to the plasma membrane with overlapping fluorescence signals, indicating spatial proximity between them, supporting their potential interaction in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. CaHT1 Sequence and Transcriptional Level Analysis\u003c/h2\u003e \u003cp\u003eCaHT1 belongs to the protein kinase C (PKC) family and contains a MAPKKK-like serine/threonine kinase domain. Transmembrane domain prediction using TMHMM server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://services.healthtech.dtu.dk/services\u003c/span\u003e\u003cspan address=\"http://services.healthtech.dtu.dk/services\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed that CaHT1 lacks transmembrane domains and is primarily located outside the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). To investigate CaHT1 functional conservation, we performed homology alignment with related genes from other solanaceous species. CaHT1 showed high sequence similarity with potato StHT1 (XP_006347031.1), tobacco NtHT1 (XP_016469683.1), tomato SlHT1 (XP_025886140.1), and potato SpHT1 (XP_015066476.1). SlHT1 (tomato, XP_025886140.1) and SpHT1 (wild tomato, XP_015066476.1) exhibited particularly high sequence similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. CaHT1 Promoter Cis-Element Analysis\u003c/h2\u003e \u003cp\u003eWe predicted cis-acting elements in the CaHT1 promoter using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple stress-responsive elements were identified, including abscisic acid-responsive elements (ABRE), wound-responsive elements (WUN-motif), light-responsive modules (Box 4), CCAAT boxes (MYBHv1 binding sites), and cell cycle regulatory elements (MSA-like). The presence of these elements suggests CaHT1 may participate in ABA-mediated defense signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. CaHT1 Protein Structure Prediction\u003c/h2\u003e \u003cp\u003eCaHT1 primary structure was predicted using ExPASy ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The protein encodes 505 amino acids, with molecular weight of 125,326.60 Da, isoelectric point (pI) of 5.01, and total atom count of 16,113. Molecular formula is C₄₆₂₃H₇₇₃₃N₁₅₁₅O₁₉₃₆S₃₀₆. Grand average of hydropathy (GRAVY) is 0.752, and instability index (II) is 39.61 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePredicted primary structure of CaHT1​​\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary Structure Feature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePredicted Result\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of Amino Acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e505\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolecular Weight (MW)/Da\u003c/p\u003e \u003cp\u003eIsoelectric Point (PI)\u003c/p\u003e \u003cp\u003eTotal Atoms\u003c/p\u003e \u003cp\u003eMolecular Formula\u003c/p\u003e \u003cp\u003eAverage Hydrophilicity Coefficient (GRAVY)\u003c/p\u003e \u003cp\u003eInstability Index (II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125326.60\u003c/p\u003e \u003cp\u003e5.01\u003c/p\u003e \u003cp\u003e16113\u003c/p\u003e \u003cp\u003eC\u003csub\u003e4623\u003c/sub\u003eH\u003csub\u003e7733\u003c/sub\u003eN\u003csub\u003e1515\u003c/sub\u003eO\u003csub\u003e1936\u003c/sub\u003eS\u003csub\u003e306\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e0.752\u003c/p\u003e \u003cp\u003e39.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. CaHT1 Expression Analysis under Normal and HTHH Conditions\u003c/h2\u003e \u003cp\u003eOur laboratory transcriptome database analysis showed: compared with room temperature control (RTHH), CaHT1 transcription levels increased under room temperature inoculation treatment (RSRT), but decreased under high temperature inoculation (RSHT) and high temperature high humidity (HTHH) treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo further elucidate CaHT1 expression patterns at the transcriptional level and its disease resistance mechanism, we used CaHT1-specific primers to perform qRT-PCR detection at 1, 12, 24, and 48 hours post-treatment under four conditions: room temperature (RTHH), room temperature inoculation (RSRT), high temperature (HTHH), and high temperature inoculation (RSHT). Results showed: CaHT1 responded to RSRT treatment at 1 hour, to RTHH treatment at 12 hours, to RSHT treatment at 24 hours, and to both RSRT and RSHT treatments at 48 hours. Notably, CaHT1 transcription levels significantly increased at 24 and 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Effects of CaHT1 Silencing on Pepper Resistance to \u003cem\u003eR. solanacearum\u003c/em\u003e Infection\u003c/h2\u003e \u003cp\u003eWe employed virus-induced gene silencing (VIGS) technology to knock down CaHT1 expression in pepper. TRV::CaHT1 (experimental group), TRV::00 (negative control), and TRV::CaPDS (positive control) were inoculated into five-leaf stage pepper seedlings. After 16 hours of dark culture at 16\u0026deg;C, plants were transferred to 25\u0026deg;C growth chamber with 16 h light/8 h dark cycle. Leaf whitening phenomenon in TRV::CaPDS plants confirmed VIGS success (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When plants developed 8\u0026ndash;10 leaves, total RNA was extracted and reverse transcribed to cDNA. qRT-PCR verification showed CaHT1 transcript levels were significantly reduced in TRV::CaHT1 plants, indicating effective silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSubsequently, TRV::CaHT1 (experimental group) and TRV::00 (control group) plants were treated under four conditions (HTHH, RSHH, RSRT, RTHH), and disease progression was monitored. Under RSRT conditions, no significant differences in wilting symptoms were observed between TRV::CaHT1 and TRV::00 plants. However, under RSHH (37\u0026deg;C) conditions, TRV::CaHT1 plants exhibited earlier wilting compared to TRV::00 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, E, F). Bacterial population detection (CFU) showed that under both RSRT and RSHT conditions, \u003cem\u003eR. solanacearum\u003c/em\u003e quantities in TRV::CaHT1 plants were significantly higher than in TRV::00 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicate that silencing CaHT1 gene enhances pepper susceptibility to \u003cem\u003eR. solanacearum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTranscriptional levels of defense-related genes reflect plant immune status. We used qRT-PCR technology to detect expression of marker genes (\u003cem\u003eCabZIP11\u003c/em\u003e, \u003cem\u003eCabHLH35\u003c/em\u003e, \u003cem\u003eCaMgst3\u003c/em\u003e, \u003cem\u003eCaPR1\u003c/em\u003e, \u003cem\u003eCaWRKY22\u003c/em\u003e) in TRV::CaHT1 and TRV::00 plants under RTHH, RSRT, HTHH, and RSHT treatment conditions. Compared with controls, under RSRT treatment, transcript levels of \u003cem\u003eCaBIZP11\u003c/em\u003e, \u003cem\u003eCaMgst3\u003c/em\u003e, \u003cem\u003eCaPR1\u003c/em\u003e, and \u003cem\u003eCaWRKY22\u003c/em\u003e were significantly reduced in CaHT1-silenced plants; under HTHH treatment, \u003cem\u003eCaWRKY22\u003c/em\u003e expression decreased; while under RSHT treatment, all five marker genes showed downregulated expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, C, D, E). These results further indicate CaHT1 functions as a positive regulator of pepper resistance to bacterial wilt. Given that ABA signaling exerts negative regulatory effects on pepper resistance through its receptors, we hypothesize that HT1 participates in the assembly of ABA-CaPYR1-mediated CaPHDy/CaGTEz reading complex for H3K4me3/H3K9ac, thereby activating \u003cem\u003eCaWRKY22\u003c/em\u003e, \u003cem\u003eCabZIP11\u003c/em\u003e, and \u003cem\u003eCabHLH35\u003c/em\u003e expression, conferring specific resistance under high temperature and high humidity conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Effects of CaHT1 Stable Overexpression on Tobacco Resistance to Bacterial Wilt\u003c/h2\u003e \u003cp\u003eTo further verify the positive regulatory role of CaHT1 in disease resistance, we constructed stable CaHT1-overexpressing tobacco transgenic plants through \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Western blot analysis of T₂ generation lines (CaHT1-1 and CaHT1-2) using anti-GFP antibody detected bands at 55\u0026ndash;70 kDa, confirming successful CaHT1-GFP overexpression (Fig. S2A).\u003c/p\u003e \u003cp\u003eT₂ generation transgenic lines (CaHT1-1, CaHT1-2) and wild-type (WT) plants were treated with RTHH, RSRT, HTHH, and RSHT. CFU assays at 24 h and 48 h post-RSRT and RSHT treatment showed colony numbers in CaHT1-#1 and CaHT1-2 were lower than wild-type (Fig. S2B). Under RSRT and RSHT conditions, CaHT1 overexpression lines exhibited lower mortality rates and stronger resistance to \u003cem\u003eR. solanacearum\u003c/em\u003e compared to WT plants (Fig. S2C, D). Colony counting at 24 and 48 hours post-inoculation showed bacterial numbers in CaHT1-#1 and CaHT1-2 were lower than WT under both RSRT and RSHT treatment conditions. These data indicate CaHT1 overexpression significantly enhanced tobacco disease resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Effects of CaHT1 Stable Overexpression on Sweet Pepper Resistance to Bacterial Wilt\u003c/h2\u003e \u003cp\u003eTo verify the positive role of CaHT1 in pepper disease resistance, we constructed CaHT1-overexpressing transgenic pepper plants using \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation combined with the flamingo beak method. Western blot analysis of T₂ generation sweet pepper lines (CaHT1-1 and CaHT1-2) using anti-GFP antibody showed bands at 55\u0026ndash;70 kDa, confirming CaHT1-GFP overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eColony counting at 24 and 48 hours post-inoculation showed bacterial numbers in CaHT1-1 and CaHT1-2 were significantly lower than wild-type under RSRT and RSHT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). T₂ generation transgenic plants and wild-type sweet pepper were treated under RTHH, RSRT, HTHH, and RSHT conditions. Compared with wild-type, CaHT1 overexpression lines exhibited delayed wilting and stronger resistance under RSRT and RSHT treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Collectively, these results indicate CaHT1 overexpression enhances pepper resistance to \u003cem\u003eR. solanacearum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.10. CaHT1 Transient Overexpression Effect Analysis\u003c/h2\u003e \u003cp\u003eTo further investigate CaHT1's role in immune responses, transient overexpression was performed in pepper leaves. First, qRT-PCR showed: CaHT1 transcript levels in 35S::CaHT1-GFP infiltrated leaves were significantly higher than 35S::GFP controls, showing this trend at 48 hours under both RTHH and HTHH treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similarly, we transiently overexpressed 35S::CaHT1-GFP or 35S::GFP (empty vector) in tobacco leaves. Fluorescence signals were detected by confocal microscopy, and 55\u0026ndash;70 kDa bands were detected by Western blot using anti-GFP antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The above evidence confirms successful transient overexpression.\u003c/p\u003e \u003cp\u003eSubsequently, we detected expression of defense marker gene \u003cem\u003eCaWRKY22\u003c/em\u003e in sweet pepper leaves under RTHH and HTHH conditions. qRT-PCR results showed that under HTHH treatment conditions, \u003cem\u003eCaWRKY22\u003c/em\u003e transcript levels in CaHT1-overexpressing leaves were higher than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003ePlant immune activation often involves salicylic acid accumulation, programmed cell death (PCD), reactive oxygen species (ROS) production, and hypersensitive response (HR)-related gene expression. We observed that 3-day local transient overexpression of CaHT1 in pepper leaves under HTHH conditions induced visible HR lesions, while lesions were less obvious under room temperature conditions. Further detection through white light and UV imaging, as well as DAB and trypan blue staining, indicated that under HTHH conditions, cell death and H₂O₂ accumulation were enhanced in CaHT1 overexpression regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo investigate functional interaction between CaHT1 and CaPYR1, we silenced CaHT1 gene through VIGS technology (TRV::CaHT1), then transiently overexpressed 35S::CaHT1-GFP or 35S::GFP in TRV::CaHT1 and TRV::00 plants. After treatment at 28\u0026deg;C or 37\u0026deg;C, necrotic response lesions appeared only in TRV::CaHT1 plants overexpressing CaPYR1 under high temperature conditions, while TRV::00 plants did not exhibit such responses. This suggests CaHT1 may not be the sole critical factor in CaPYR1-mediated immune activation in pepper (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study confirms through multiple experiments that the CaHT1 gene positively regulates pepper immune responses against bacterial wilt. Previous laboratory studies indicated that solanaceous crops activate SA and JA-mediated defense responses to counteract \u003cem\u003eR. solanacearum\u003c/em\u003e infection under normal temperature conditions. However, under high temperature and high humidity (HTHH) conditions, these SA and JA-mediated immune responses are suppressed to varying degrees. Instead, cytokinin-mediated immune responses are initiated, characterized by specific upregulation of GST-encoding genes (such as \u003cem\u003eMgst3\u003c/em\u003e and \u003cem\u003ePRP1\u003c/em\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These findings suggest that immune response mechanisms employed by solanaceous plants under HTHH conditions differ significantly from those under normal temperature conditions.\u003c/p\u003e \u003cp\u003ePrevious laboratory studies revealed that multiple WRKY transcription factors, including CaWRKY40, CaWRKY6, CaWRKY28, and CaWRKY58, play important regulatory roles in pepper disease resistance. Particularly, CaWRKY40 occupies a central hub position in responses to bacterial wilt under HTHH conditions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For example, CaCDPK15 positively regulates pepper responses to \u003cem\u003eR. solanacearum\u003c/em\u003e through interaction with CaWRKY40 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. CaCDPK29 phosphorylation of Ser137 site in CaWRKY27b nuclear localization signal (NLS) upregulates its expression and promotes its translocation from cytoplasm to nucleus. Although the conserved WRKYGQK motif in CaWRKY27b undergoes mutation (Q\u0026rarr;M), resulting in loss of DNA binding ability to W-box in promoters of immune and heat tolerance-related marker genes, CaWRKY27b can still interact with CaWRKY40 within the nucleus. This interaction forms complexes with molecules such as CaNPR1, CaDEF1, and CaHSP24, thereby exerting indirect positive regulatory effects, enhancing pepper immunity to RSI and tolerance to HTHH. CaCDPK29 phosphorylation of CaWRKY27b strengthens CaWRKY40 function in pepper resistance to bacterial wilt [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, early laboratory research primarily focused on protein kinase CaCDPK responses to stress, with less exploration of the protein kinase C (PKC) family. Previous studies revealed CaPYR1 negatively regulates immune responses to \u003cem\u003eR. solanacearum\u003c/em\u003e under normal temperature, but positively regulates such responses under high temperature conditions. Subsequent pull-down experiments combined with LC-MS analysis suggested potential interaction between CaHT1 and CaPYR1. Therefore, this study selected protein kinase CaHT1 as research subject to further investigate protein kinase C family functions in pepper bacterial wilt resistance. This study, combining pull-down experiments with liquid chromatography-mass spectrometry analysis, strongly confirmed interaction between CaHT1 and CaPYR1. This finding prompted us to further investigate the mechanism of protein kinase CaHT1 (a protein kinase C family member) in pepper bacterial wilt resistance.\u003c/p\u003e \u003cp\u003ePrevious studies indicate that abscisic acid binding to its receptors participates in regulating plant stress physiological responses. Two possible abscisic acid receptors include Mg\u0026sup2;⁺ chelatase H subunit and protein-coupled receptors. Abscisic acid plays dual roles in plant disease resistance. ABA exerts positive regulatory effects on responses to pathogen-associated molecular patterns (PAMPs) and saprophytic pathogens. However, more research focuses on ABA's negative regulatory role in plant resistance, such as during tobacco resistance to bacterial wilt and barley resistance to rice blast, where ABA weakens plant resistance. Depending on pathogen species and infection modes, ABA can cause plants to exhibit disease tolerance or susceptibility. For example, ABA can promote guard cell function, blocking various pathogens from entering cells. Previous laboratory studies showed that silencing CaPYR1 gene in sweet pepper plants under HTHH or RSHT conditions leads to downregulation of marker genes CaMgst3 and CaPRP1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Conversely, CaPYR1 overexpression in tobacco plants results in CaMgst3 and CaPRP1 upregulation. This indicates CaPYR1 or ABA can inhibit SA or JA-mediated immune regulatory pathways, thereby negatively regulating pepper bacterial wilt resistance under normal temperature. CaPYR1 or ABA enhances pepper bacterial wilt resistance under high temperature and high humidity conditions through upregulation of CaMgst3 and CaPRP1. Studies show that CaMgst3 and CaPRP1 transcription upregulation following bacterial wilt inoculation in pepper under HTHH conditions correlates with cytokinin secretion. ABA exhibits antagonistic effects similar to SA and JA. CaPYR1 negatively regulates pepper bacterial wilt resistance under normal temperature, but positively regulates this process under HTHH conditions, and can enter the nucleus to bind and regulate CaPHD4 and CaGTE4 activity. Based on this, we speculate CaHT1 may further mediate disease resistance responses through interaction with CaPYR1. However, whether HT1 participates in assembling ABA-CaPYR1-mediated CaPHDy/CaGTEz open reading frame H3K4me3/H3K9ac modification complex, thereby activating CaWRKY22, CabZIP11, and CabHLH35 expression, and subsequently initiating pepper-specific immune responses to \u003cem\u003eRalstonia\u003c/em\u003e infection under HTHH conditions, requires further in-depth investigation.\u003c/p\u003e \u003cp\u003eProtein phosphorylation and dephosphorylation are crucial in disease resistance processes. During early and late stages of plant immune responses, calcium ion signaling molecules can activate protein kinases, thereby regulating transcription processes. Protein phosphorylation is catalyzed by protein kinases. Kinases, as important phosphorylating enzymes, are responsible for transferring high-energy phosphate groups from high-energy small molecules (such as ATP) to specific target substances (such as proteins, lipids, carbohydrates, amino acids, nucleic acids). Previous studies have found protein kinases participate in plant stress responses. For example, mitogen-activated protein kinases (MAPK) participate in plant drought, salt, and cold stress responses [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Specifically, MAPK hierarchical phosphorylation systems can amplify external stimulus signals, activate downstream target genes, and initiate related stress responses [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Among these, MAP-KKK located upstream of MAPK cascade phosphorylation systems undergoes phosphorylation following environmental stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Activated MAP-KKK can promote biological functions of downstream protein kinases, such as activating transcription factors related to expression of specific disease resistance genes like PBF1 and G/HBF1. For example, previous studies found AhMPK6 and AhMPK3 expression can enhance tobacco disease resistance, while OsBWMK1 similarly improves tobacco disease resistance through phosphorylation of transcription factor OsEREBP1 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Taking FLS2 as an example, this protein kinase can recognize flagellin. In rice, protein kinases OsBRR1 and OsWAK1 respond to rice blast, with transcription expression levels significantly elevated especially under ABA induction [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. HT1 depends on second messengers: when cell surface receptor kinases receive external stimuli, second messengers including cAMP, cGMP, and various phospholipids are produced, subsequently activating AGC protein kinases, transmitting signals to downstream molecules through phosphorylation. Studies show AGC protein kinases can regulate polar transport of growth factors, respond to biotic and abiotic stresses, and play key roles in polar growth of root hairs and pollen tubes. Protein phosphorylation, as critical post-translational modification, is essential for signal transduction networks composed of interconnected signaling pathways\u0026mdash;cells make decisions responding to internal and external stimuli through this network [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. ABA receptor CaPYR1 negatively regulates pepper bacterial wilt resistance under normal temperature, but positively regulates this resistance under HTHH conditions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Based on the above analysis, we believe: functional differences under different conditions originate from CaPYR1 phosphorylation modifications at different sites, indicating this receptor may be phosphorylated by different protein kinases under different conditions, and these kinases play important roles in regulating pepper disease resistance, with CaHT1 possibly being one such protein. In this study, we first reveal the positive regulatory role of protein kinase CaHT1 in pepper bacterial wilt immune responses, and further confirm its physical interaction with ABA receptor CaPYR1. We have reason to believe CaPYR1's negative regulation of bacterial wilt resistance under normal temperature, transitioning to positive regulation under HTHH conditions, may occur through phosphorylation modifications at different sites responding to different upstream signals, thereby achieving flexible regulation of downstream immune pathways. This echoes our laboratory's previous research on CaCDPK29-CaWRKY27b module: CaCDPK29 promotes CaWRKY27b entry into nucleus through phosphorylation and interaction with CaWRKY40, thereby coordinately activating expression of immune and heat tolerance-related genes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, as a protein kinase, interaction between CaHT1 and CaPYR1 likely mediates CaPYR1 functional transition under different environmental conditions through similar phosphorylation events.\u003c/p\u003e \u003cp\u003eNotably, under high temperature and high humidity conditions, plants often need to balance immune responses with heat tolerance. Our laboratory's latest research found transcription factor CaKAN3 cooperates with CaHSF8 to activate NLR genes under HTHH conditions, thereby enhancing immunity to bacterial wilt. However, under sustained high temperature or extreme high temperature conditions, CaHSF8 dissociates from CaKAN3,转而 upregulating heat shock protein (HSP) genes to activate heat tolerance. This indicates pepper possesses precise molecular \"switches\" to trade off between immunity and heat tolerance when responding to complex stresses. The functional transition mechanism of CaHT1-CaPYR1 module may be a key link in this trade-off process. We speculate CaHT1 phosphorylation modification of CaPYR1 may alter CaPYR1 interaction patterns with other regulatory factors (such as CaKAN3, CaHSF8, or CaNAC2c), thereby determining whether to prioritize initiating immune responses or heat tolerance mechanisms. Future research should thoroughly investigate CaHT1 phosphorylation site specificity on CaPYR1, and how these phosphorylation events affect CaPYR1 binding affinity and activity with downstream signaling components, thereby elucidating its specific action mechanisms in immunity-heat tolerance trade-offs.\u003c/p\u003e \u003cp\u003eWe know that when plant immune systems are activated, they typically exhibit defense gene expression associated with salicylic acid (SA) accumulation, programmed cell death (PCD), reactive oxygen species (ROS) generation, and hypersensitive response (HR). In this experiment, we inoculated TRV::00 and TRV::CaHT1 sweet pepper seedlings with 35S::CaHT1-GFP and 35S::GFP bacterial solutions via \u003cem\u003eAgrobacterium\u003c/em\u003e. We observed hypersensitive response spots appeared on TRV::CaHT1 plants overexpressing CaPYR1, while TRV::00 pepper leaves failed to trigger hypersensitive responses or hydrogen peroxide accumulation. Under HTHH treatment conditions, local CaPYR1 overexpression in pepper leaves resulted in hypersensitive response spot formation, accompanied by more severe cell death and hydrogen peroxide accumulation. This indicates CaHT1 can activate plant PTI and ETI immune responses. Initiation of these immune responses is often closely related to rapid and precise gene expression regulation, with chromatin remodeling playing key roles in this process. Our laboratory's previous research revealed CaSWC4 precisely regulates trade-offs between immunity and heat tolerance by recruiting CabZIP63/CaWRKY40 and promoting deposition of H2A.Z, H3K9ac, H4K5ac and other histone modifications, thereby activating chromatin of immune or heat tolerance-related genes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven CaHT1 as a protein kinase, its phosphorylation of CaPYR1 may affect downstream gene transcriptional regulation, we have reason to speculate CaHT1-CaPYR1 module may have synergistic effects with CaSWC4-mediated chromatin remodeling mechanisms. Specifically, CaHT1 phosphorylation modification of CaPYR1 may affect CaPYR1 interaction with chromatin remodeling complex components (such as CaSWC4, CaTAF14b, or CaRUVBL2), thereby indirectly regulating chromatin accessibility at target gene promoter regions. For example, phosphorylated CaPYR1 may enhance its binding with CaSWC4, thereby promoting deposition of active histone modifications such as H3K4me3/H3K9ac, subsequently activating expression of immune-related genes \u003cem\u003eCaWRKY22\u003c/em\u003e, \u003cem\u003eCabZIP11\u003c/em\u003e, and \u003cem\u003eCabHLH35\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additionally, cross-regulation between CaHT1-CaPYR1 module and WRKY transcription factor family should be considered. This study found CaHT1 silencing significantly downregulates CaWRKY22 expression, and our laboratory has identified multiple WRKY transcription factors playing critical roles in pepper disease resistance (such as CaWRKY40, CaWRKY27b). CaHT1 phosphorylation modification of CaPYR1 may, through regulating CaPYR1 interaction with these WRKY transcription factors, or directly affecting WRKY transcription factor activity and stability itself, collectively shape pepper immune response mechanisms. Future research should employ ChIP-seq and other technologies to investigate whether CaHT1-CaPYR1 module directly or indirectly affects chromatin remodeling factors and WRKY transcription factor binding to target gene promoters, thereby comprehensively elucidating its molecular mechanisms in gene expression regulation.\u003c/p\u003e \u003cp\u003ePlant hormones such as abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) play complex cross-regulatory roles in plant immunity and stress responses. This study found CaPYR1, as an ABA receptor, exhibits significantly different functions under different temperature conditions, emphasizing the condition-dependency of ABA signaling in pepper bacterial wilt resistance. Our laboratory's previous research found ABA signaling pathway positively regulates heat tolerance but negatively regulates bacterial wilt resistance capacity, while JA signaling pathway primarily participates in immune responses. Antagonistic and synergistic interactions between hormones constitute refined regulatory networks for plants responding to complex stresses. The discovery of CaHT1-CaPYR1 module provides new entry points for analyzing this network's operating mechanisms.\u003c/p\u003e \u003cp\u003eAccordingly, we speculate CaHT1 phosphorylation modification of CaPYR1 may be a key node in \"crosstalk\" between ABA signaling and JA/SA signaling. Specifically, under high temperature conditions, CaHT1 phosphorylation modification of CaPYR1 may enhance ABA signaling pathway, thereby promoting heat tolerance gene expression; simultaneously, it may finely regulate immune response intensity through antagonistic effects with JA/SA signaling pathways. This regulation may be achieved through affecting activity or stability of downstream key transcription factors (such as CaNAC2c, CaPti1-CaERF3) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For example, CaNAC2c interacts with CaHSP70 under HTS conditions to activate heat tolerance, while interacts with CaNAC029 under RSI conditions to activate JA-mediated immunity. CaHT1-CaPYR1 module may determine CaNAC2c's tendency toward heat tolerance or immunity by affecting its interaction partner selection. Additionally, CaPti1-CaERF3 module positively regulates bacterial wilt resistance by activating SA-dependent CaPR1 and dehydration tolerance-related genes, and inducing stomatal closure in ABA signaling-dependent manner [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Whether direct or indirect regulatory relationships exist between CaHT1-CaPYR1 module and CaPti1-CaERF3 module (for example, whether CaHT1 affects CaPti1 or CaERF3 activity or coupling with ABA signaling through phosphorylation) will become important directions for future research [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In-depth analysis of how CaHT1-CaPYR1 module integrates ABA, JA, and SA signaling will help construct more comprehensive pepper complex stress response regulatory network models, providing theoretical basis for breeding broad-spectrum resistant pepper varieties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003e校对报告\u003c/h2\u003e \u003cp\u003e当前使用的样式是 [Plant and Soil]\u003c/p\u003e \u003cp\u003e当前文档包含的题录共0条\u003c/p\u003e \u003cp\u003e有0条题录存在必填字段内容缺失的问题\u003c/p\u003e \u003cp\u003e所有题录的数据正常\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Fujian Provincial Natural Science Foundation [Grant Number: 2024J01311873].\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003e \u003cb\u003eXiang Zheng\u003c/b\u003e: Conceptualization, Methodology, Investigation, Data curation, Writing \u0026ndash; original draft. \u003cb\u003eHuifang Shi\u003c/b\u003e: Investigation, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cb\u003eLingxian Yi\u003c/b\u003e: Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cb\u003eDaojin Yu\u003c/b\u003e: Conceptualization, Supervision, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research was supported by the Fujian Provincial Natural Science Foundation (Project No.: 2024J01311873). We thank Shi Huifang for assistance in completing partial experimental work. We also thank Lecturer Yi Lingxian and Professor Yu Daojin for guidance in research design and valuable suggestions during manuscript preparation. We appreciate all laboratory members for helpful discussions and technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHussain A, Li X, Weng Y, Liu Z, Ashraf MF, Noman A, Yang S, Ifnan M, Qiu S, Yang Y, Guan D, He S (2018) CaWRKY22 plays a positive regulatory role in pepper response to \u003cem\u003eRalstonia solanacearum\u003c/em\u003e by forming a network with CaWRKY6, CaWRKY27, CaWRKY40 and CaWRKY58. Int J Mol Sci 19:1426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu A, Wu J, Lei Y, Cai Y, Wang S, Liu Z, Guan D, He S (2018) Putative GSK3/SHAGGY-like kinase CaSK23 in pepper acts as a negative regulator in pepper response to \u003cem\u003eRalstonia solanacearum\u003c/em\u003e invasion. 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Plant Physiol 186:2169\u0026ndash;2189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi L, Li X, Weng Y, Cai H, Liu K, Xie B, Hassan A, Guan D, He S, Liu Z (2022) CaPti1-CaERF3 module positively regulates sweet pepper resistance to bacterial wilt by enhancing immunity and dehydration tolerance. J Plant Biol Cell Mol Biol 111:250\u0026ndash;268\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Capsicum annuum, High temperature and high humidity, Protein kinase, Bacterial wilt, CaHT1, CaPYR1","lastPublishedDoi":"10.21203/rs.3.rs-9298395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9298395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cdiv id=\"ASec1\" class=\"AbstractSection\"\u003e \u003cdiv class=\"Heading\"\u003eAims\u003c/div\u003e \u003cp\u003ePepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e) is susceptible to bacterial wilt caused by \u003cem\u003eRalstonia solanacearum\u003c/em\u003e under high temperature and high humidity (HTHH) conditions, leading to severe yield losses. Our previous studies revealed that the abscisic acid (ABA) receptor PYR1 negatively regulates bacterial wilt resistance under normal temperature but exhibits positive regulation under HTHH conditions. We discovered that PYR1 interacts with HT1, a member of the AGC protein kinase C family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"ASec2\" class=\"AbstractSection\"\u003e \u003cdiv class=\"Heading\"\u003eMethods\u003c/div\u003e \u003cp\u003eTo investigate the function of CaHT1 and its interaction with CaPYR1, we performed subcellular localization, bimolecular fluorescence complementation (BiFC), pull-down assays, virus-induced gene silencing (VIGS), transient and stable overexpression, qRT-PCR, and disease index evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"ASec3\" class=\"AbstractSection\"\u003e \u003cdiv class=\"Heading\"\u003eResults\u003c/div\u003e \u003cp\u003eWe found that CaHT1 localizes to the plasma membrane and nucleus and physically interacts with CaPYR1. Silencing CaHT1 increased pepper susceptibility to R. solanacearum, whereas stable overexpression of CaHT1 in both pepper and tobacco enhanced disease resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"ASec4\" class=\"AbstractSection\"\u003e \u003cdiv class=\"Heading\"\u003eConclusions\u003c/div\u003e \u003cp\u003eThis study demonstrates that CaHT1 positively regulates pepper resistance to bacterial wilt under both normal and HTHH conditions, indicating that HT1 achieves its positive regulatory role through protein interaction with PYR1. These findings hold significant implications for elucidating pepper disease resistance mechanisms and genetic improvement.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"CaHT1 Regulates Pepper Immune Response to Ralstonia solanacearum and Interacts with CaPYR1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 14:28:09","doi":"10.21203/rs.3.rs-9298395/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-15T17:44:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-12T03:16:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-12T02:07:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-10T02:37:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd446275-78c3-487c-b510-88fc7d986400","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T14:28:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 14:28:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9298395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9298395","identity":"rs-9298395","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}