MdLRR-RLK1-MdGRP1-LIKE module improved biotic resistance in apple

MdLRR-RLK1-MdGRP1-LIKE module improved biotic resistance in apple | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 June 2025 V1 Latest version Share on MdLRR-RLK1-MdGRP1-LIKE module improved biotic resistance in apple Authors : Wenjun Chen , Chao Zhang , Wei Guo , Yi Zhao , Yingying Lei , Cui Chen , Ziwen Wei , Xiaoming Li , Yue Ma , and Hongyan Dai 0000-0003-2025-0026 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175022852.28343178/v1 161 views 136 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Apple is an important economic species, and it always suffered by biotic stress during its growth and development. Fungi and pests are two types of biotic stress that have significant destructive effects on apples. Besides, the LRR-RLKs family play a key role in regulating plant responses to biotic stress. In this study, overexpressing MdLRR-RLK1 enhanced apple resistance to Colletotrichum fructicola and aphids by promoting the expression of resistance genes such as WRKYs , PRs and JA-pathway genes, as well as increasing the content of antioxidant enzymes s and econdary metabolites. Additionally, MdLRR-RLK1 could interact with MdGRP1-LIKE in vivo and in vitro , and MdLRR-RLK1 could phosphorylate MdGRP1-LIKE in vitro . Overexpressing MdGRP1-LIKE enhanced apple resistance to C. fructicola by increasing the expression of resistance genes such as WRKYs and PRs and the content of antioxidant enzymes. However, overexpressing MdGRP1-LIKE did not enhance the apple resistance to aphids. These findings reveal the mechanism of the MdLRR-RLK1-MdGRP1-LIKE module regulated apple resistance to biotic stress. Introduction Apple ( Malus domestica ), a widely cultivated and economically valuable fruit in the World (Wang et al., 2020), is a rich source of nutrients. Regular consumption of apples can reduce the risk of numerous chronic diseases (Boyer and Liu, 2004). However, apple always is susceptible to various stresses especially biotic stresses, leading to significant economic losses. Although some apple varieties with high stress tolerance have been cultivated, their practical application remains challenging due to the slow selection processes and the complexity of genetic backgrounds (Nemeskéri et al., 2009; Martínez-Fortún et al., 2022). Therefore, investigating the molecular mechanisms of resistance to biotic stress is crucial for breeding apple varieties. Biotic stresses, such as insects, viruses, bacteria, and fungi always cause significant damage to plants (Muhammad et al., 2019). These organisms attack plants, reducing their vitality and leading to host plants death (Pandey et al., 2017). Among these biotic stresses, fungi and insects are particularly detrimental to apple plants. Pathogenic fungi typically penetrate plant cells with hyphae, killing host tissues to extract nutrients, thereby causing plant death (Raman et al., 2012). Pests especially aphids are major pests on various crops with high reproductive potential and phenotypic plasticity (Simon and Peccoud, 2018; Dedryver et al., 2010). During their life cycle, most aphids adopt seasonal cyclic mechanisms, producing both winged and wingless biomorphic (Loxdale and Balog, 2018; Nalam et al., 2019). While feeding on the phloem sap of host plants, aphids use their mouthparts to pierce nearly all plant tissues and can inoculate viruses into plants (Brault et al., 2010; Erb and Reymond, 2019). As the most efficient vectors of plant viruses, aphids transmit over 30% of insect-borne viruses (Jayasinghe et al., 2021). Under biotic stress, reactive oxygen species (ROS) including superoxide anions and hydrogen peroxide were significant accumulated (H 2 O 2 ), which could disrupt physiological metabolism of plants (Mishra et al., 2023). Antioxidant enzymes such as POD, CAT, and SOD play crucial roles in eliminating ROS and enhancing plant biotic stress resistance (Sachdev et al., 2021). Study have demonstrated the vital function of antioxidant enzymes in plant defense against biotic stress (Rani and Pratyusha, 2013). Additionally, plants also can produce various secondary metabolites to enhance biotic resistance (Awmack and Leather, 2002). Previous studies demonstrated that tannins, glucosinolates, and organic acids can enhance plant biotic stress resistance (Hopkins et al., 2009; Bang et al., 2021; Valentinuzzi et al., 2021). Consequently, the contents of antioxidant enzymes and secondary metabolites can serve as reliable indicators of plant resistance to biotic stress. Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest RLK family in plants, with over 200 members identified in the Arabidopsis genome (Shiu et al., 2001). LRR-RLKs are composed of an extracellular domain with LRRs, a transmembrane domain, and an intracellular domain containing a kinase domain (Kobe and Kajava, 2001; Torii, 2004). Numerous LRR-RLKs have been shown to perceive plant hormones, regulate growth, and participate in defense responses (Chakraborty et al., 2019). Evidence highlight the critical role of LRR-RLKs in plant defense. In rice, OsLRR-RLK1 positively regulates resistance to pests (Hu et al., 2018). In Arabidopsis, FLS2 and BAK1 are one of LRR-RLKs family to activate immune responses (Sun et al., 2003). Our previous studies demonstrated that overexpressing CpMdLRR-RLK1 enhanced bacterial tolerance in Arabidopsis (Guo et al., 2020). However, the function of MdLRR-RLK1 involved in biotic stress remains poorly understood in apple. In this study, we found that MdLRR-RLK1 expression was significantly induced under biotic stress. We also demonstrated that overexpressing MdLRR-RLK1 enhanced apple resistance to Colletotrichum fructicola and aphids by increasing the contents of secondary metabolite and antioxidant enzyme. Although MdGRP1-LIKE overexpression lines exhibited higher tolerance to C. fructicola , it did not improve tolerance to aphids in apple. Furthermore, MdLRR-RLK1 could phosphorylate MdGRP1-LIKE in vitro . This study elucidated the mechanism of MdLRR-RLK1 and MdGRP1-LIKE increase the contents of antioxidant enzyme to improve apple resistance under C. fructicola stress. MATERIALS AND METHODS Plant materials and growth conditions This study used wild-type apple plants ( Malus domestica ) ‘GL-3’. Before transplantation, the seedlings were cultivated in rooting media for three months to ensure root system development. During transplantation, both rooted GL-3 and transgenic lines were placed in a substrate composed of peat, vermiculite, and perlite at a ratio of 3:2:1. The substrate was covered with film to maintain moisture. After 20 days of cultivation under the film, the film was removed, and cultivation continued for an additional two to five months. The plants were cultivated at a temperature of 25°C under a photoperiod of 12 hours of light and 12 hours of darkness (Dai et al., 2013). Stable genetic transformation To achieve stable genetic transformation, the full-length CDS of MdGRP1-LIKE was inserted into the pRI-101-AN vector with 35s promoter. The constructed vector was subsequently transformed into Agrobacterium tumefaciens strain EHA105. The GL-3 tissue-cultured seedlings were then used for stable genetic transformation following the method described (Lei et al., 2024). RT‒qPCR Total RNA was extracted via the CTAB method (Chang et al., 2007). cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser (Takara RR047A). Quantitative PCR (qPCR) using SYBR Premix Ex Taq (Cwbio UltraSYBR Mixture CW2601H) on an IQ6 qPCR instrument (Applied Biosystems QuantStudio 6 FLEX). Three biological and three technical replicates were performed to calculate the relative expression level. Actin was used as the internal control for apple, and data analysis was conducted via the 2 -ΔΔCt method (Livak and Schmittgen, 2001). The RT-qPCR primers used in this study are detailed in Table S1. Colletotrichum fructicola treatment Colletotrichum fructicola was cultured on fungal medium until spore production. Spores were aseptically collected in a sterile environment, dissolved in sterile water, and adjusted to 10 7 spores/mL using a hemocytometer under a microscope to prepare a spore suspension (Zhang et al., 2019a). The suspension was sprayed on GL-3 and transgenic plants. Subsequently sampled at 0 d, 4 d, and 8 d post-inoculation and observed phenotypes. Aphid treatment Aphids ( Aphis citricolavander Goot) were collected from the field, and 15 healthy individual aphids were selected and inoculated to the young tissues of GL-3 and transgenic plants. The inoculated tissues were covered with mesh bags to maintain a ventilated yet relatively sealed environment. Phenotypes were observed, and samples were collected at specified time intervals post-inoculation. Bioinformatics analysis NCBI (https://www.ncbi.nlm.nih.gov/) was used to search for the amino acid sequences of GRPs related genes for phylogenetic analysis. MEGA7 software was used to construct a phylogenetic tree. Pfam (https://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) were used to perform protein domain analysis. DNAMAN version 9 software was used for visualizing and annotating the relevant domains. Accession numbers The proteins (and their accession numbers) for phylogenetic analysis in NCBI can be found and include the following: MdGRP1-LIKE (XP_008377301), MsGRP1A-like (XP_050106879), PcGRP1A-like (XP_068323854), PbGRP2A (XP_018506897), GmGRP2 (XP_003527212), AhGRP (XP_025610241), JrGRP-like (XP_018857590), PpGRP-like (XP_020426007), VvGRP2A (XP_003631658), TcGRP1A (XP_007042188), RcGRP (XP_024166795), GhGRP1A (XP_016742901), PdGRP (XP_034196860). The genes (and their accession numbers) for RT-qPCR in NCBI can be found and include the following: MdLRR-RLK1 (XM_008395995), MdGRP1-LIKE (XM_008379079), MdWRKY15 (XM_008395555), MdWRKY23 (XM_008383213), MdWRKY33 (XM_070809708), MdCOI1 (XM_008394693), MdEGL1 (XM_008363357), MdLOX3 (XM_008392656), MdPR2 (XM_008366971), MdPR4 (XM_008372483), MdPR5 (XM_029108558), MdPR8 (XM_070820590), MdACT (XM_008382322). Determination of enzyme activity Weighed 0.1 g sample and extracted the enzyme mixture via 0.05 mol/L PBS buffer at 4°C for 3 days. Subsequently, centrifuged the solutions at 4°C and 12,000 rpm for 20 minutes. The supernatant obtained after centrifugation was used as the enzyme mixture. The determination of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activity (Mahmood et al., 2016). Determination of MDA Weighed 1.0 g each sample and pulverized in a 0.1% (w/v) trichloroacetic acid (TCA) solution. Subsequently, centrifuged at 12,000 rpm for 15 minutes. Followed by adding 0.5% thiobarbituric acid (TBA) to the supernatant. The mixture was heated in a 95°C water bath for 50 minutes and then rapidly cooled in an ice bath to halt the reaction. Next, centrifuged the solution again at 12,000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm. The quantification of MDA was performed via the spectrophotometric method on the basis of its molar absorption coefficient (155 mM⁻¹ cm⁻¹) (Cakmak and Horst, 1991). Determination of Titratable Acid 0.01 mol/L sodium hydroxide (NaOH) standard solution and 1% phenolphthalein indicator were prepared. 2 g samples were ground by liquid nitrogen, mixed with 30 mL distilled water in a 50 mL conical flask and 80°C water bath for 30 minutes. After cooling, the mixture was filtered into a 50 mL volumetric flask and diluted to volume with distilled water. A 5-10 mL aliquot of the extract was transferred to a 50 mL conical flask, and 3-5 drops of 1% phenolphthalein indicator were added. The solution was titrated with 0.01 mol/L NaOH until a faint pink color persisted for 30 seconds, indicating the titration endpoint. The volume of NaOH consumed was recorded, and the average of three replicates was calculated. Subsequently calculated the titratable acid content. Determination of Tannin Tannin content was determined using the Folin-Ciocalteau reagent (Charanjit and Harish, 2002). Weighed 2 g sample and homogenized in 80% ethanol at room temperature and centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was retained, and the residue was re-extracted twice with 80% ethanol. The combined supernatants were evaporated to dryness at room temperature. The residue was dissolved in 5 mL of distilled water. A 100 μL aliquot of the extract was diluted to 3 mL with water, and 0.5 mL of Folin-Ciocalteau reagent was added. 2 mL of 20% sodium carbonate was added after 3 minutes, and measured the solution at 650 nm after 60 minutes. not-yet-known not-yet-known not-yet-known unknown Determination of Glucosinolate Glucosinolate content was determined as previously reported (Doorn et al., 1999). Subcellular localization The subcellular localization method (Wang et al., 2019). The coding sequences of MdGRP1-LIKE without stop codons were inserted into the vectors p MdGRP1-LIKE -GFP. Nicotiana benthamiana leaves were then infiltrated with A. tumefaciens strain GV3101. Following a 48-hour dark incubation and a 24-hour normal light incubation, GFP fluorescence was visualized via laser confocal microscopy (TCS SP8, Leica, Germany, with a scanning resolution of 8192 * 8192 pixels). CD3-1007 mCherry serves as a cell membrane marker (Wang et al ., 2019). Yeast library screening and validation GL-3 tissue cultured seedlings were cultivated for 3 months. Samples were taken from the leaves and roots to construct a yeast nuclear membrane dual protein library. The library was developed by Shanghai OE Biotech Co., Ltd. (https://www.oebiotech.com/). The full-length CDS of MdLRR-RLK1 was introduced into the vector pBT3-STE- MdLRR-RLK1 and used for protein screening. The screening library method was provided by Shanghai OE Biotech Co., Ltd. The full-length CDS of MdGRP1-LIKE was introduced into the vector pPR3-N-MdGRP1-LIKE. The screening was performed via SD/-Trp/-Leu/-His/-Ade media with 40 μg/mL X-α-gal and 30 mM 3-AT (QDO/X/3-AT). Yeasts obtained from the screening were sequenced via Sanger sequencing. After the corresponding vector was constructed, the screened genes were validated in QDO/X/3-AT for a period of 3-5 days at 30°C. Bimolecular fluorescence complementation assay (BiFC assay) The bimolecular fluorescence complementation (BiFC) assay (Walter et al., 2004). The full-length coding sequences of MdLRR-RLK1 and MdGRP1-LIKE were subsequently cloned and inserted into the vectors MdLRR-RLK1 -YFP n and MdGRP1-LIKE -YFP c , respectively. The subsequent steps and the use of a cell membrane marker were in accordance with the subcellular localization method. Pull-down assay The pull-down assay followed the protocol described in previous research (Li et al., 2024). The full-length coding sequences of MdLRR-RLK1 and MdGRP1-LIKE were inserted into the vectors MdLRR-RLK1 -His and MdGRP1-LIKE -GST, respectively. Subsequently, these constructs were transformed into competent Escherichia coli BL21 (DE3) cells and induced with IPTG. The bacterial cells were harvested, and the proteins were extracted via Tiachi E. coli Lysis Buffer (ACE Biotechnology, BR0005-2). Protein purification was performed using Ni NTA Resin (TransGenBiotech, DP101-01) for MdLRR-RLK1 -His and GST Resin (TransGenBiotech, DP201-01) for MdGRP1-LIKE -GST. The antibodies used included mouse anti-His-Tag monoclonal antibody (ABclonal, AE003), mouse anti-GST-Tag monoclonal antibody (ABclonal, AE001), and HRP-conjugated goat anti-mouse IgG (H+L) (ABclonal, AS003). In vitro Phosphorylation assay The in vitro phosphorylation assay method (Li et al., 2023). The antibodies used were a pan phospho-serine/threonine rabbit polyclonal antibody (Beyotime, AF5725) and HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208). The recombinant proteins MdLRR-RLK1 -His and MdGRP1-LIKE -GST were overexpressed, and phosphatase inhibitors (phosphate inhibitor cocktail A from Beyotime, product number P1081) were added during bacterial lysis to obtain proteins for Western blot (WB) analysis. Using MdLRR-RLK1-His as the kinase and MdGRP1-LIKE-GST as the substrate, the kinase and substrate were mixed in a 1:5 ratio with kinase assay buffer (25 mM Tris-HCl [pH 7.5], 10 mM MgCl 2 , 1 mM CaCl 2 , 10 mM ATP, and 1 mM DTT) and incubated at 30°C for 2 hours. The reaction was then stopped by heating with 1×SDS loading buffer at 100°C for 5 minutes. The protein mixture was separated on a 12% SDS-PAGE gel. WB detection was performed using an anti-phospho serine/threonine antibody (Beyotime, AF5725). Expression of the MdLRR-RLK1 gene was induced by biotic stress In previous study, ectopic overexpression of CpLRR-RLK enhanced the resistance to bacteria in Arabidopsis (Guo et al., 2020). To further elucidate the MdLRR-RLK function in biotic stress, we inoculated C. fructicola in GL-3 (Supplemental Figure 1a). The results showed that GL-3 leaves gradually darkened and died over time post-inoculation. RT-qPCR analysis revealed that MdLRR-RLK1 expression was significantly induced in GL-3 plants inoculated with C. fructicola compared to controls (Figure 1b). Additionally, GL-3 plants were inoculated with aphids ( Aphis citricolavander Goot) (Supplemental Figure 1b). After 15 days, the apical young tissues of GL-3 were densely infested with aphids (Figure 1c), and MdLRR-RLK1 expression was also significantly induced compared to controls (Figure 1d). These results indicated that MdLRR-RLK1 plays a crucial role in biotic stress. Figure 1. MdLRR-RLK1 is sensitive to biotic stress. (a) Phenotypes of GL-3 tissue-cultured seedlings inoculated with C. fructicola at 0 d, 4 d, and 8 d post-inoculation. H 2 O-treated GL-3 plants served as controls. Scale bar, 1 cm. (b) Expression levels of MdLRR-RLK1 in C. fructicola treatment by RT-qPCR. Error bars represent standard deviation ( n = 3). (c) Phenotypes of GL-3 after aphid inoculation 15 d. Scale bar, 1 cm. (d) Expression levels of MdLRR-RLK1 in aphid treatment by RT-qPCR. Error bars represent standard deviation ( n = 3). Asterisks indicate significant differences as determined by Student’s t -test (**, P < 0.01). MdLRR-RLK1 modulated apple biotic resistance To elucidate the MdLRR-RLK1 function in biotic stress, we inoculated C. fructicola in MdLRR-RLK1 overexpression and RNAi lines (Chen et al., 2024). MdLRR-RLK1 overexpression lines exhibited light disease symptoms after 8 d inoculating, whereas MdLRR-RLK1 RNAi lines exhibited severe leaf darkening and wilting (Figure 2). POD, CAT, SOD, and MDA revealed that overexpressing MdLRR-RLK1 enhanced antioxidant enzyme activity and reduced leaf damage, whereas MdLRR-RLK1 RNAi lines exhibited lower antioxidant activity and higher leaf damage compared to other lines (Figure 2b-d, Supplemental Figure 2a). Additionally, overexpressing MdLRR-RLK1 significantly reduced disease incidence, while silencing MdLRR-RLK1 increased disease incidence (Supplemental Figure 2b). 30 d after aphids inoculating, MdLRR-RLK1 overexpression lines had the fewest aphids on apical young tissues, and MdLRR-RLK1 RNAi lines had the most (Figure 2e and f). Concurrently, POD, CAT, SOD, MDA, and relative electrical conductivity indicated that overexpressing MdLRR-RLK1 significantly enhanced antioxidant enzyme levels and mitigated leaf damage, whereas silencing MdLRR-RLK1 showed the opposite effect (Figure 2g-i, Supplemental Figure 2c). Tannic and glucosinolates act as an important secondary metabolite for plants to resist aphid damage (Bang et al., 2021; Valentinuzzi et al., 2021). Determination of tannins content, glucosinolates content and titratable acids content revealed that overexpressing MdLRR-RLK1 enhanced the content of anti-insect compounds. These results demonstrated MdLRR-RLK1 could modulate biotic resistance in apple. Figure 2. Phenotypes of MdLRR-RLK1 transgenic lines under biotic stress. (a) Phenotypes of tissue-cultured GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 4 d, and 8 d after inoculating with C. fructicola . Scale bar, 1 cm. (b-d) Determination of POD, CAT, and SOD in GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 4 d, and 8 d after inoculating with C. fructicola . Error bars represent standard deviation ( n = 3). (e) Phenotypes of GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 10 d, and 30 d after inoculating with aphids. Scale bar, 1 cm. (f) Number of aphids in GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 10 d, and 30 d after inoculating with aphids. Error bars represent standard deviation ( n = 3). (g-i) Determination of POD, CAT, and SOD in GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 10 d, and 30 d after inoculating with aphids. Error bars represent standard deviation ( n = 3). OE stands for the MdLRR-RLK1 overexpression line. RNAi stands for the MdLRR-RLK1 RNA interference line. not-yet-known not-yet-known not-yet-known unknown MdLRR-RLK1 promoted expression level of resistance genes in apple Given the differential biotic stress resistance phenotypes observed in MdLRR-RLK1 transgenic lines and GL-3, RNA-seq analysis was conducted to investigate the expressions of resistance genes. Heatmap analysis revealed the differential resistance genes between MdLRR-RLK1 overexpression lines and MdLRR-RLK1 RNAi lines (Figure 3a). RT-qPCR demonstrated that overexpressing MdLRR-RLK1 significantly increased the transcriptional levels of resistance genes such as WRKYs (MdWRKY15, MdWRKY23, MdWRKY33 ), PRs (MdPR4, MdPR5, MdPR8 ), and JA-pathway genes (MdCOI, MdEGL1, MdLOX3 ) under C. fructicola stress, whereas silencing MdLRR-RLK1 significantly decreased those resistance gene expressions compared to GL-3 (Figure 3b-j). Similarly, the transcriptional levels of resistance genes under aphid stress were consistent to C. fructicola stress, with higher gene expressions in MdLRR-RLK1 overexpression lines and lower gene expressions in MdLRR-RLK1 RNAi lines (Supplemental Figure 3a-g). These results demonstrated that MdLRR-RLK1 plays a critical role in regulating the apple resistance to biotic stress. Figure 3 Expression of resistance genes in GL-3 and MdLRR-RLK1 transgenic lines under C. fructicola stress. (a) Heat map of resistance genes in GL-3 and MdLRR-RLK1 transgenic lines. Error bars represent standard deviation ( n = 3). (b-j) Expression of MdWRKYs , MdPRs , and JA-pathway genes in GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 4 d, and 8 d after inoculating with C. fructicola . Error bars represent standard deviation ( n = 3). OE stands for the MdLRR-RLK1 overexpression line. RNAi stands for the MdLRR-RLK1 RNA interference line. MdLRR-RLK1 could phosphorylate MdGRP1-LIKE To investigate the mechanism of MdLRR-RLK1 enhanced resistance to biotic stress, the pBT3-STE- MdLRR-RLK vector was constructed for yeast library screening, leading to the identification of MdGRP1-LIKE ( XM_008379079 in NCBI) as the interacting partner. Further yeast two-hybrid assays with the pBT3-STE -MdLRR-RLK and pPR3-N -MdGRP1-LIKE vectors confirmed the interaction between MdLRR-RLK1 and MdGRP1-LIKE in yeast cells (NMY51) (Figure 4a). BiFC assays were performed to verify the interaction. The results indicated MdLRR-RLK1 -YFP n and MdGRP1-LIKE -YFP c could interact in the cell membrane of tobacco cells, whereas negative controls ( MdLRR-RLK1 -YFP n /pYFP c and pYFP n / MdGRP1-LIKE- YFP c ) did not produce YFP signals (Figure 4b). Pull-down assays also demonstrated that MdLRR-RLK1 -His could interact with MdGRP1-LIKE-GST (Figure 4c). These results indicated that MdLRR-RLK1 could interact with MdGRP1-LIKE both in vivo and in vitro . Due to MdLRR-RLK1 serves as a kinase (Chen et al., 2024), we hypothesized that MdGRP1-LIKE could be phosphorylated by MdLRR-RLK1. In vitro phosphorylation assays demonstrated that MdLRR-RLK1-HIS could phosphorylate MdGRP1-LIKE-GST (Figure 4d). Moreover, phosphorylated MdLRR-RLK1-His (phos-MdLRR-RLK1-His) also phosphorylated MdGRP1-LIKE-GST in vitro (Figure 4e). indicating that the phosphorylation of MdGRP1-LIKE by MdLRR-RLK1 was independent of MdLRR-RLK1 phosphorylation status. not-yet-known not-yet-known not-yet-known unknown Figure 4 MdLRR-RLK1 could phosphorylate MdGRP1-LIKE in vitro. (a) Yeast two-hybrid validation. pBT3-STE-MdLRR-RLK1, pPR3-N-MdGRP1-LIKE, and control plasmids were co-transformed into NMY51 competent yeast, subsequently cultured on media for 3-5 days after transformation. (b) BiFC assays. The MdLRR-RLK1 -YFPn, MdGRP1-LIKE -YFPc, and control plasmids were injected into tobacco leaves via A. tumefaciens . Scales bar, 75 μm. (c) Pull-down assay. E. coli containing MdLRR-RLK1 -His, MdGRP1-LIKE -GST, and empty plasmids were induced to obtain the corresponding proteins, and detected via SDS-PAGE. (d and e) MdLRR-RLK1 phosphorylates and degrades MdGRP1-LIKE in vitro . E. coli containing MdLRR-RLK1 -His, MdGRP1-LIKE -GST, and empty plasmids were induced to obtain the corresponding proteins. MdGRP1-LIKE -GST with MdLRR-RLK1-His and MdGRP1-LIKE -GST with phos-MdLRR-RLK1-His were co-incubated for 2 h, 4 h, and 6 h respectively, subsequently detected via SDS-PAGE. OE stands for the MdLRR-RLK1 overexpression line. RNAi stands for the MdLRR-RLK1 RNA interference line. Overexpressing MdGRP1-LIKE enhanced resistance to Colletotrichum fructicola in apple To elucidate the function of MdGRP1-LIKE, a phylogenetic tree was constructed and indicated MdGRP1-LIKE is closely related to RcGRP1 (Figure 5a). Subcellular localization showed MdGRP1-LIKE localized in the cytoplasm (Figure 5b). 4 MdGRP1-LIKE overexpression lines were obtained, and #OE-1 and #OE-2 were selected for further experiments (Supplemental Figure 5a). Additionally, overexpressing MdGRP1-LIKE enhanced apple resistance to C. fructicola compared to GL-3 (Figure 5c). Similarly, MdGRP1-LIKE overexpression lines exhibited higher POD, CAT, and SOD activity, along with lower MDA levels (Supplemental Figure 5b-e). However, overexpressing MdGRP1-LIKE did not enhance apple resistance to aphids (Figure 5e and f). MdGRP1-LIKE overexpression lines exhibited higher POD, CAT, and SOD activity compared to GL-3 after inoculating aphid (Supplemental Figure 5f-h), but there were no significant differences in MDA, titratable acid content, tannin content, or glucosinolate content (Supplemental Figure 5i, Figure 5f-h). Furthermore, resistance-related genes were significantly upregulated in MdGRP1-LIKE overexpression lines after inoculating C. fructicola or aphids (Figure 5j-s). However, the transcription levels of JA-pathway genes ( MdCOI and MdEGL1 ) in MdGRP1-LIKE overexpression lines were significant downregulated compared to GL-3 under biotic stress (Supplemental Figure 6a-d). These results indicate that overexpressing MdGRP1-LIKE enhanced resistance to C. fructicola but does not improve resistance to aphids. Figure 5. Phenotypes of MdGRP1-LIKE transgenic lines under biotic stress. (a) Phylogenetic tree analysis of the MdGRP1-LIKE. Proteins from 13 species were selected to construct a phylogenetic tree, then analyzed using the Neighbor-Joining method. (b) Subcellular localization assay. pMdGRP1-LIKE-GFP and pRI101-GFP were transformed into tobacco leaves using A. tumefaciens GV3101. Scale bar, 75 μm. (c) Phenotypes of tissue-cultured GL-3 and MdLRR-RLK1 transgenic lines at 0 d, 4 d, and 8 d after inoculating with C. fructicola . Scale bar, 1 cm. (d) Incidence rate of GL-3 and MdGRP1-LIKE transgenic lines at 0 d, 4 d, and 8 d after inoculating with C. fructicola . Error bars represent standard deviation ( n ＞ 10). (e) Phenotypes of GL-3 and MdGRP1-LIKE transgenic lines at 0 d and 20 d after inoculating with aphids. Scale bar, 1 cm. (f) Number of aphids in GL-3 and MdGRP1-LIKE transgenic lines at 0 d and 20 d after inoculating with aphids. Error bars represent standard deviation ( n = 3). (g-i) Determination of titratable acids content, tannins content, and glucosinolates content, in GL-3 and MdLRR-RLK1 transgenic lines at 0 d and 20 d after inoculating with aphids. Error bars represent standard deviation ( n = 3). OE stands for the MdGRP1-LIKE overexpression line. Asterisks indicate significant differences as determined by Student’s t -test (*, P < 0.05; **, P < 0.01). Discussion not-yet-known not-yet-known not-yet-known unknown MdLRR-RLK1 regulated apple resistance to biotic stress LRR-RLKs represent the largest subfamily of receptor-like kinases in plants (Fischer et al., 2016). Approximately 226 LRR-RLKs were identified in Arabidopsis (Wu et al., 2016) and 332 in rice (Dufayard et al., 2017). RLK genes are well-characterized as R genes and serve as critical components in plant defense against various biotic stresses (Gómez-Gómez et al., 2000; Tameling et al., 2002). The wheat TaWRKY56 and TaWRKY70 regulated the expression of TaRLK1, thereby influencing the resistance of sharp eyespot (Chen et al., 2016). SISERK1 was involved in the resistance process against potato aphids mediated by Mi-1 in tomatoes (Ma et al., 2016). Our findings also demonstrated that overexpressing MdLRR-RLK1 enhanced resistance to C. fructicola and aphids in apple, whereas silencing MdLRR-RLK1 has the opposite effect (Figure 2). Plant hormones are fundamental to responses and environmental signals, and JA is recognized as a critical regulator of biotic stress responses (Ku et al., 2018; Al-Zahrani et al., 2020). JA-induced FtCYP94C1 may enhance buckwheat resistance to the fungal pathogen Agrocybe agri-HGI3 by accumulating flavonoids (He et al., 2023). Study demonstrated that OsCOI could regulate resistance by inducing various secondary metabolites in rice (Ye et al., 2012; Yan et al., 2018). Additionally, PR4, PR5, and PEROXIDASE (PEROX ) could enhance resistance to Fusarium graminearum in wheat (Ameye et al., 2015). Our results also revealed that overexpressing MdLRR-RLK1 upregulated the expression of resistance genes (WRKYs and PRs ) and JA-pathway genes, thereby enhancing apple resistance to biotic stress (Supplemental Figure 3, Supplemental Figure 4). Secondary metabolites such as POD, CAT, SOD, tannins, glucosinolates, and organic acids were always considered as important substances involved in biotic stress (Cruz and Maria, 2008; Iqbal and Poór, 2025; Tamara et al., 2007; Rao et al., 2021). Previous studies have shown that overexpressing CaLRR-RLK1 increased accumulation of H2O2 and enhanced the resistance to Ralstonia solanacearum in pepper (Capsicum annuum ) (Mou et al., 2019). ZmPEPR2 enhanced plant resistance to Pythium stalk rot by increased content of antioxidant enzymes in maize (Zea mays ), (He et al., 2025). Overexpression of ScLRR-RLK in tobacco enhanced plant resistance to pathogen infection by inducing the expression of reactive oxygen species (ROS) scavenging-related genes. (Wang et al., 2025). Our findings also demonstrate that overexpressing MdLRR-RLK1 increased content of antioxidant enzymes, tannins, glucosinolates, and organic acids under biotic stress (Figure 2, Figure 3, Supplemental Figure 2). MdLRR-RLK1-MdGRP1-LIKE module enhanced resistance to C. fructicola in apple Phosphorylation is a critical post-translational modification under stress conditions in plants (Arsova et al., 2018; Zhang et al., 2020). In eukaryotes, phosphorylation is one of the important post-translational modification methods of proteins. It affects the function of proteins by altering their conformation and stability (Bentem and Hirt, 2009). Moreover, RLKs are a representative type of protein receptor kinases (Nishi et al., 2011; Nishi et al., 2014). BRAK enhanced the resistance to Botrytis cinerea by phosphorylating PSKR1s in tomato (Ding et al., 2024). GbSOBIR1 could phosphorylate GbbHLH171, thereby positively regulating the resistance to Verticillium dahliae in cotton (Zhou et al., 2019). Our study demonstrated that MdLRR-RLK1 could phosphorylate MdGRP1-LIKE in vitro as well (Figure 4). As a part of signaling cascades, RLKs could also be phosphorylated, which may affect their ability to phosphorylate target genes (Jagodzik et al., 2018; Lee et al., 2012b; Komis et al., 2018). Interestingly, we found that the phosphorylation of MdGRP1-LIKE by MdLRR-RLK1 was independent of MdLRR-RLK1 phosphorylation status (Figure 4), and the mechanisms require further investigation. Glycine-rich proteins (GRPs) are characterized by high glycine content (up to 70%) and the presence of glycine-rich motifs composed of repetitive amino acid residues (Mangeon et al., 2009). GRPs have been reported to participate in plant resistance caused by biotic stress. Overexpressing AtGRP7 enhanced plant resistance to tobacco mosaic virus and Pseudomonas syringae (Lee et al., 2012a). Ectopic overexpressing LsGRP1 improved disease tolerance in Arabidopsis (Lin et al., 2020). Overexpressing GhGRPL enhanced cotton resistance to biotic stress (Yu et al., 2024). Our study demonstrated that overexpressing MdGRP1-LIKE significant upregulated resistance-related genes expressions and increased apple resistance to C. fructicola (Figure 5, Supplemental Figure 5). Therefore, we hypothesize that MdLRR-RLK1 can enhance the resistance of apples to C. fructicola by phosphorylating MdGRP1-LIKE. MdGRP1-LIKE did not affect aphid resistance in apple Plants employ diverse signaling pathways to respond to fungi or insect invasion (Hettenhausen et al., 2015). MPK4 enhanced resistance to the bacterial pathogen Pseudomonas syringae DC3000 by regulating ROS. However, silencing MPK4 in tobacco enhances resistance to the tobacco moth by regulating jasmonic acid (JA) signaling (Zhang et al., 2019b). The research found that the transgenic wheat resistant to Blumeria graminis tritici had a larger population of aphids compared to the non-transgenic strain, indicating that plants that are protected from Blumeria graminis tritici may be more favorable for aphids (Burg et al., 2012). These results demonstrated that a single gene may only affect specific resistance to plants. Our study demonstrated that JA-pathway genes were significantly downregulated in MdGRP1-LIKE overexpression lines (Supplemental Figure 6). And overexpressing MdGRP1-LIKE did not increase aphid resistance or secondary metabolites such as tannins, glucosinolates, and titratable acids, suggesting MdGRP1-LIKE may not be involved in apple resistance to aphids (Figure 5). This suggests that MdGRP1-LIKE may enhance apple fungal resistance via other pathway, and the mechanism need further investigate. Therefore, the mechanism of overexpressing MdLRR-RLK1 enhanced aphid resistance in apple still needs further exploration. Thus, we proposed a mechanism of the MdLRR-RLK1 regulates resistance to biotic stress in apple. Under aphid stress, MdLRR-RLK1 was significantly induced, increasing antioxidant enzymes content, secondary metabolites content, and transcriptional levels of resistance genes to enhance plant resistance to aphid stress; under C. fructicola stress, MdLRR-RLK1 was significantly induced and MdGRP1-LIKE was extensively phosphorylated, increasing antioxidant enzyme contents and transcriptional levels of resistance genes to enhance plant resistance to fungi stress (Figure 6). Figure 6. Mechanism of MdLRR-RLK1 modulates biotic resistance in apple. AUTHOR CONTRIBUTIONS HY Dai designed research. WJ Chen, C Zhang and Y Zhao performed research. W Guo, YY Lei, C Chen and ZW Wei analyzed data. XM Li provided the laboratory. Y Ma provided the materials. WJ Chen drafted the manuscript. HY Dai revised the manuscript. CONFLICT OF INTEREST The authors declare no conflicts of interest. not-yet-known not-yet-known not-yet-known unknown ACKNOWLEDGEMENTS We thank Dr. Huixia Shou at Zhejiang University for providing the CD3-1007 vector. We thank Dr Yue Ma at Shenyang Agricultural University for providing the Colletotrichum fructicola. This research was supported by the National Natural Science Foundation of China (Grant No. 32472715 and No. 31972380). Supplementary data The following materials are available in the online version of this article. Supplemental Figure 1. Organism used for stress treatments. Supplemental Figure 2 Physiological indicators of GL-3 and MdLRR-RLK1 transgenic lines under biotic stress. Supplemental Figure 3 Expression of JA-pathway resistance genes in GL-3 and MdLRR-RLK1 transgenic lines under biotic stress. Supplemental Figure 4 Physiological indicators of GL-3 and MdGRP1-LIKE overexpression lines under biotic stress. Supplemental Figure 5 Expression of JA-pathway genes in GL-3 and MdGRP1-LIKE overexpression lines under biotic stress. Table. S1. Primers for the construction of vectors. Table. S2. Primers for RT-qPCR. Data availability The authors confirm that the data supporting the findings of this study are available within the article and its Supporting Information. not-yet-known not-yet-known not-yet-known unknown References Al-Zahrani, W., Bafeel, S.O., El-Zohri, M. 2020. Jasmonates mediate plant defense responses to Spodoptera exigua herbivory in tomato and maize foliage. Plant Signaling and Behavior, 15, e1746898. https://doi.org/10.1080/15592324.2020.1746898 Ameye, M., Audenaert, K., Zutter, N., Steppe, K., Meulebroek, L., Vanhaecke, L., Vleesschauwer, D., Haesaert, G., Smagghe, G. 2015. Priming of wheat with the green leaf volatile Z-3-Hexenyl acetate enhances defense against Fusarium graminearum but boosts deoxynivalenol production. 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Keywords colletotrichum fructicola mdlrr-rlk1 aphid apple signaling Authors Affiliations Wenjun Chen Shenyang Agricultural University College of Horticulture View all articles by this author Chao Zhang Shenyang Agricultural University College of Horticulture View all articles by this author Wei Guo Shenyang Agricultural University College of Horticulture View all articles by this author Yi Zhao Shenyang Agricultural University College of Horticulture View all articles by this author Yingying Lei Shenyang Agricultural University College of Horticulture View all articles by this author Cui Chen Shenyang Agricultural University College of Horticulture View all articles by this author Ziwen Wei Shenyang Agricultural University College of Horticulture View all articles by this author Xiaoming Li Shenyang Agricultural University College of Horticulture View all articles by this author Yue Ma Shenyang Agricultural University View all articles by this author Hongyan Dai 0000-0003-2025-0026 [email protected] Shenyang Agricultural University College of Horticulture View all articles by this author Metrics & Citations Metrics Article Usage 161 views 136 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wenjun Chen, Chao Zhang, Wei Guo, et al. MdLRR-RLK1-MdGRP1-LIKE module improved biotic resistance in apple. Authorea . 18 June 2025. DOI: https://doi.org/10.22541/au.175022852.28343178/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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