Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (10): 2165-2180.doi: 10.3864/j.issn.0578-1752.2026.10.008

• PLANT PROTECTION • Previous Articles     Next Articles

Receptor-Like Kinase Gene PbeCRLK2 Positively Regulates the Pyrus betulaefolia Resistance to Pear Valsa Canker

YANG JingHua(), SUN E, LIU XiaoHuan, ZHANG Pan, DAI YingBao, WANG WenHui, DONG Han, ZUO CunWu()   

  1. College of Horticulture, Gansu Agricultural University, Lanzhou 730070
  • Received:2026-01-14 Accepted:2026-03-13 Online:2026-05-16 Published:2026-05-20
  • Contact: ZUO CunWu

RichHTML

1

PDF

8

Abstract:

【Objective】CRLK (calcium/calmodulin-regulated receptor-like kinase) plays an important role in low-temperature stress and starch biosynthesis, but its function in plant immunity remains unclear. This study aimed to investigate the role of PbeCRLK2 in the immune response of Pyrus betulifolia to pear Valsa canker, and to provide theoretical basis and genetic resources for the breeding of pear trees resistant to Valsa canker.【Method】The basic characteristics of PbeCRLK2 were identified through bioinformatics and subcellular localization analysis. The role of PbeCRLK2 in disease resistance was verified using transient expression in fruits and stable expression in suspension cells. Downstream immune pathways were explored by measuring reactive oxygen species (ROS) and the expression of immune-related genes. To further investigate the mechanism by which PbeCRLK2 regulates resistance to pear Valsa canker, transcriptomic analysis and qRT-PCR verification were conducted on PbeCRLK2 overexpressing cells at different time points after treatment with metabolites of V. pyri.【Result】PbeCRLK2 was a conserved cytoplasmic receptor-like kinase localized to the cell membrane. Its expression was significantly induced by metabolites of V. pyri. Both transient and stable expression of PbeCRLK2 significantly inhibited V. pyri infection and enhanced the resistance of P. betulaefolia to pear Valsa canker. Transient expression for 72 h reduced lesion diameter by 11.59% and stable expression by 44.06% on average. PbeCRLK2 enhanced pattern-triggered immunity (PTI) responses by activating WRKY22 expression. It also induced ROS bursts and up-regulated the expression of RBOHD and OXI1. Meanwhile, the expression of the key salicylic acid pathway gene ChiV was significantly up-regulated. All of these phenomena contributed to enhanced P. betulaefolia defense capabilities. GO and KEGG enrichment analyses were conducted separately for up- and down-regulated DEGs from four groups (DC_vs_YC, DT1_vs_YT1, DT2_vs_YT2 and DT3_vs_YT3) in the transcriptome data. DEGs were mainly enriched in pathways such as protein dephosphorylation, chitin catabolic processes, lipid metabolism, secondary metabolite and terpenoid biosynthesis, etc. qRT-PCR results showed that the expression trends of the selected genes were consistent with the transcriptome data, validating the transcriptomic findings.【Conclusion】PbeCRLK2 may be involved in chitin signal transduction. PbeCRLK2 enhances the resistance of P. betulaefolia to Valsa canker through the coordinated activation of PTI responses, ROS burst, and the salicylic acid signaling pathway. At the same time, it may maintain cellular immune homeostasis by dynamically regulating the expression of dephosphorylation-related genes.

Key words: Pyrus betulaefolia, Valsa pyri, PbeCRLK2, disease resistance function, transcriptomic analysis

Table 1

Primers used for gene cloning, vector construction and qRT-PCR"

基因名称Gene name 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
PbeCRLK2-pFGC5941 GAGGCGCGCCATGGTTCATCAAACTGATCTTGTTA GAGGCGCGCCATGGTTCATCAAACTGATCTTGTTA
PbeCRLK2-GFP GAGAACACGGGGGACGAGCTCATGGTTCATCAAA
CTGATCTTGTTA		 CTTGCTCACCATGGTGTCGACTTCAGTTACTCTTTC
GTTCACGCTC
PbeCRLK2-qPCR GTTATCATTGGCGTCTCTGTTGGT TTGTGCGTATAGGTAGAGTGGTAAGA
Actin TTCAGATACTGTTGTGGAGCCTTAC AGTAACTCCAGACATTGTTGCAGAG
LOX1 GCTTATGTGGCTGTAAATGACTCTG GAGGATGCAGAAGTTTGTAAATTGG
ChiV CTACAGCATAAACTACCCCTTCCAG CGTTCCTATACCGGATCCATTAGC
RBOHD TACGTGGAGATCACTCTCGACATC CCGCTTCAGCTCCTGAGAGAC
OXI1 CAACTTGGAAAACTCCGAGAAGTC TTGTCAGTATCCGATAAAACGTCTC
WRKY22 CATATCCAAGGGGATATTACAGATG GTGACTATAAAAATATTCGGGTCGG

Fig. 1

Basic bioinformatic and evolutionary analysis of PbeCRLK2"

Fig. 2

PbeCRLK2 overexpression enhances the resistance of P. betulaefolia to V. pyri"

Fig. 3

Overexpression of PbeCRLK2 enhances VpM resistance and induces ROS burst in P. betulaefolia suspension cells"

Fig. 4

PbeCRLK2 activates the expression of key genes in immune pathways"

Table 2

Statistics and quality inspection of sequencing data"

样本
Sample		 原始数据
Raw data		 过滤后数据
Clean data		 过滤后数据比率
Clean data ratio (%)		 Q20 (%) Q30 (%) GC (%)
YC-1 41866810 41866810 100 98.13 96.73 46.75
YC-2 43720318 43720318 100 98.36 96.97 46.65
YC-3 37260460 37260460 100 98.11 96.70 46.84
YT1-1 40110802 40110802 100 98.06 96.63 46.57
YT1-2 36265408 36265408 100 98.17 96.81 46.52
YT1-3 36308808 36308808 100 98.07 96.61 46.62
YT2-1 45657690 45657690 100 98.47 97.08 46.31
YT2-2 39679760 39679760 100 98.25 96.92 46.35
YT2-3 38180810 38180810 100 98.16 96.78 46.15
YT3-1 41359618 41359618 100 98.30 97.01 46.03
YT3-2 42150220 42150216 100 98.40 96.97 46.09
YT3-3 41439948 41439948 100 98.37 97.12 46.11

Fig. 5

Number of differentially expressed genes (DEGs) (A) and PbeCRLK2 expression trends (B)"

Fig. 6

GO enrichment analysis of DEGs"

Fig. 7

KEGG enrichment analysis of DEGs"

Fig. 8

Analysis of expression patterns of DEGs in key pathways"

Fig. 9

qRT-PCR and RNA-Seq results of DEGs related to disease resistance"

[1]
张绍铃, 谢智华. 我国梨产业发展现状、趋势、存在问题与对策建议. 果树学报, 2019, 36(8): 1067-1072.
ZHANG S L, XIE Z H. Current status, trends, main problems and the suggestions on development of pear industry in China. Journal of Fruit Science, 2019, 36(8): 1067-1072. (in Chinese)
[2]
联合国粮农组织. 联合国粮食及农业组织统计数据库. (2025-12-31)[2026-03-01]. https://www.fao.org/statistics/zh/.
Food and Agriculture Organization. Food and Agriculture Organization Statistical Database. (2025-12-31)[2026-03-01]. https://www.fao.org/statistics/zh/. (in Chinese)
[3]
吴芳, 刘红霞, 侯世星, 温俊宝. 梨树腐烂病在香梨树体上的空间分布特点. 中国农学通报, 2012, 28(10): 277-281.
WU F, LIU H X, HOU S X, WEN J B. Spatial distribution characteristics of Valsa canker on fragrant pear. Chinese Agricultural Science Bulletin, 2012, 28(10): 277-281. (in Chinese)
[4]
王金友, 冯明祥. 新编梨树病虫害防治技术. 北京: 金盾出版社, 2005.
WANG J Y, FENG M X. Newly Compiled Techniques for Disease and Pest Control in Pear Trees. Beijing: The JinDun Publishing House, 2005. (in Chinese)
[5]
王静璞. 梨树主要病害及其防治方法. 现代园艺, 2020, 43(7): 194-195.
WANG J P. Main diseases of pear trees and control methods. Contemporary Horticulture, 2020, 43(7): 194-195. (in Chinese)
[6]
TANG D Z, WANG G X, ZHOU J M. Receptor kinases in plant-pathogen interactions: More than pattern recognition. The Plant Cell, 2017, 29(4): 618-637.

doi: 10.1105/tpc.16.00891 pmid: 28302675
[7]
HARDIE D G. Plant protein serine/threonine kinases: Classification and functions. Annual Review of Plant Physiology and Plant Molecular Biology, 1999, 50: 97-131.

pmid: 15012205
[8]
DING S T, FENG S X, ZHOU S B, ZHAO Z R, LIANG X, WANG J, FU R S, DENG R, ZHANG T, SHAO S J, YU J Q, FOYER C H, SHI K. A novel LRR receptor-like kinase BRAK reciprocally phosphorylates PSKR1 to enhance growth and defense in tomato. The EMBO Journal, 2024, 43(23): 6104-6123.

doi: 10.1038/s44318-024-00278-z
[9]
ZHENG Y, ZHAO D, LU Y, CHEN Z J, LIU Z H, SUN E, YU H Q, MAO X, CAI M R, ZUO C W. The Malectin-like kinase gene MdMDS1 negatively regulates the resistance of Pyrus betulifolia to Valsa canker by promoting the expression of PbePME1. Physiologia Plantarum, 2025, 177(1): e70032.

doi: 10.1111/ppl.v177.1
[10]
LU Y, MAO X, WANG C, ZHENG Y, DUO H, SUN E, YU H Q, CHEN Z J, ZUO C W. Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by wall-associated kinase gene MbWAK1. Physiologia Plantarum, 2024, 176(3): e14330.

doi: 10.1111/ppl.v176.3
[11]
LUO X M, XU N, HUANG J K, GAO F, ZOU H S, BOUDSOCQ M, COAKER G, LIU J. A lectin receptor-like kinase mediates pattern- triggered salicylic acid signaling. Plant Physiology, 2017, 174(4): 2501-2514.

doi: 10.1104/pp.17.00404
[12]
HOOK S S, MEANS A R. Ca2+/CaM-dependent kinases: From activation to function. Annual Review of Pharmacology and Toxicology, 2001, 41: 471-505.

doi: 10.1146/pharmtox.2001.41.issue-1
[13]
YANG S, CAI W W, SHEN L, CAO J S, LIU C L, HU J, GUAN D Y, HE S L. A CaCDPK29-CaWRKY27b module promotes CaWRKY40- mediated thermo-tolerance and immunity to Ralstonia solanacearum in pepper. The New Phytologist, 2022, 233(4): 1843-1863.

doi: 10.1111/nph.v233.4
[14]
WANG J Y, WANG S Z, HU K, YANG J, XIN X Y, ZHOU W Q, FAN J B, CUI F H, MOU B H, ZHANG S Y, WANG G L, SUN W X. The kinase OsCPK4 regulates a buffering mechanism that fine-tunes innate immunity. Plant Physiology, 2018, 176(2): 1835-1849.

doi: 10.1104/pp.17.01024
[15]
SUN C C, CHEN Y M, MA A F, WANG P, SONG Y Y, PAN J X, ZHAO T T, TU Z P, LIANG X X, WANG X D, FAN J, BI G Z, MENG X Z, DOU D L, XU G Y. The kinase CPK5 phosphorylates MICRORCHIDIA1 to promote broad-spectrum disease resistance. The Plant Cell, 2025, 37(3): koaf051.
[16]
WANG J P, MUNYAMPUNDU J P, XU Y P, CAI X Z. Phylogeny of plant calcium and calmodulin-dependent protein kinases (CCaMKs) and functional analyses of tomato CCaMK in disease resistance. Frontiers in Plant Science, 2015, 6: 1075.
[17]
YANG T B, SHAD ALI G, YANG L H, DU L Q, REDDY A S N, POOVAIAH B W. Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants. Plant Signaling and Behavior, 2010, 5(8): 991-994.

doi: 10.4161/psb.5.8.12225
[18]
CHEN Y, SHI H F, YANG G L, LIANG X Y, LIN X L, TAN S P, GUO T, WANG H. OsCRLK2, a receptor-like kinase identified by QTL analysis, is involved in the regulation of rice quality. Rice, 2024, 17(1): 24.

doi: 10.1186/s12284-024-00702-2 pmid: 38587574
[19]
YU H Q, SUN E, MAO X, CHEN Z J, XU T, ZUO L G, JIANG D J, CAO Y N, ZUO C W. Evolutionary and functional analysis reveals the crucial roles of receptor-like proteins in resistance to Valsa canker in Rosaceae. Journal of Experimental Botany, 2023, 74(1): 162-177.

doi: 10.1093/jxb/erac417
[20]
SUN E, YU H Q, CHEN Z J, CAI M R, MAO X, LI Y Y, ZUO C W. Enhanced Valsa canker resistance conferred by expression of MdLecRK-S.4.3 in Pyrus betulifolia is largely suppressed by PbePUB36. Journal of Experimental Botany, 2023, 74(14): 3998-4013.

doi: 10.1093/jxb/erad126
[21]
LIU S H, LI J H, LI N, ZHOU P, LI L L. Genome-wide identification of calcium-dependent protein kinases (CDPKs) in pear (Pyrus bretschneideri Rehd) and characterization of their responses to Venturia nashicola infection. Horticulture, Environment, and Biotechnology, 2022, 63(6): 903-915.

doi: 10.1007/s13580-022-00444-4
[22]
杜成龙. 杜梨PbeLecRK-S.4-PbePIP1-4应答效应子NIS1/2调控腐烂病抗性的机制研究[D]. 兰州: 甘肃农业大学, 2025.
DU C L. Study on the mechanism of PbeLecRK-S.4-PbePIP1-4 response effector NIS1/2 regulating resistance to Valsa canker in Pyrus betulaefolia[D]. Lanzhou: Gansu Agricultural University, 2025. (in Chinese)
[23]
YANG T B, CHAUDHURI S, YANG L H, DU L Q, POOVAIAH B W. A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants. The Journal of Biological Chemistry, 2010, 285(10): 7119-7126.

doi: 10.1074/jbc.M109.035659
[24]
LI P, ZHAO L L, QI F, HTWE N M, LI Q Y, ZHANG D W, LIN F C, SHANG-GUAN K K, LIANG Y. The receptor-like cytoplasmic kinase RIPK regulates broad-spectrum ROS signaling in multiple layers of plant immune system. Molecular Plant, 2021, 14(10): 1652-1667.

doi: 10.1016/j.molp.2021.06.010
[25]
THAPA G, GUNUPURU L R, HEHIR J G, KAHLA A, MULLINS E, DOOHAN F M. A pathogen-responsive leucine rich receptor like kinase contributes to Fusarium resistance in cereals. Frontiers in Plant Science, 2018, 9: 867.

doi: 10.3389/fpls.2018.00867
[26]
JAISWAL N, LIAO C J, MENGESHA B, HAN H, LEE S, SHARON A, ZHOU Y, MENGISTE T. Regulation of plant immunity and growth by tomato receptor-like cytoplasmic kinase TRK1. New Phytologist, 2022, 233(1): 458-478.

doi: 10.1111/nph.v233.1
[27]
LUKAN T, COLL A. Intertwined roles of reactive oxygen species and salicylic acid signaling are crucial for the plant response to biotic stress. International Journal of Molecular Sciences, 2022, 23(10): 5568.

doi: 10.3390/ijms23105568
[28]
PENG Y J, YANG J F, LI X, ZHANG Y L. Salicylic acid: Biosynthesis and signaling. Annual Review of Plant Biology, 2021, 72: 761-791.

doi: 10.1146/annurev-arplant-081320-092855 pmid: 33756096
[29]
UM-E-AIMAN, NISAR N, TSUZUKI T, LOWE A, ROSSITER J T, JAVAID A, POWELL G, WASEEM R, AL-MIJALLI S H, IQBAL M. Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants. International Journal of Biological Macromolecules, 2021, 178: 253-262.

doi: 10.1016/j.ijbiomac.2021.02.164
[30]
WASZCZAK C, CARMODY M, KANGASJÄRVI J. Reactive oxygen species in plant signaling. Annual Review of Plant Biology, 2018, 69: 209-236.

doi: 10.1146/annurev-arplant-042817-040322 pmid: 29489394
[31]
RAMOS R N, ZHANG N, LAUFF D B, VALENZUELA-RIFFO F, FIGUEROA C R, MARTIN G B, POMBO M A, ROSLI H G. Loss-of-function mutations in WRKY22 and WRKY25 impair stomatal-mediated immunity and PTI and ETI responses against Pseudomonas syringae pv . tomato. Plant Molecular Biology, 2023, 112(3): 161-177.

doi: 10.1007/s11103-023-01358-0
[32]
RAMOS R N, MARTIN G B, POMBO M A, ROSLI H G. WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana. Plant Molecular Biology, 2021, 105(1/2): 65-82.

doi: 10.1007/s11103-020-01069-w
[33]
HUSSAIN A, LI X, WENG Y H, LIU Z Q, ASHRAF M F, NOMAN A, YANG S, IFNAN M, QIU S S, YANG Y J, GUAN D, HE S L. CaWRKY22 acts as a positive regulator in pepper response to Ralstonia solanacearum by constituting networks with CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58. International Journal of Molecular Sciences, 2018, 19(5): 1426.

doi: 10.3390/ijms19051426
[34]
LIU F, ZENG M Z, SUN Y J, CHEN Z Y, CHEN Z D, WANG L, CUI J R, ZHANG F S, LV D, CHEN X, XU Y P, DUAN K X, WANG Y, WANG Y C. BAK1 protects the receptor-like kinase BIR2 from SNIPER2a/b-mediated degradation to promote pattern-triggered immunity in Nicotiana benthamiana. The Plant Cell, 2023, 35(9): 3566-3584.

doi: 10.1093/plcell/koad187
[35]
BISHOP J G, DEAN A M, MITCHELL-OLDS T. apid evolution in plant chitinases: Molecular targets of selection in plant-pathogen coevolution. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(10): 5322-5327.
[36]
MIR Z A, ALI S, SINGH A, YADAV P, TYAGI A, CHATURANI G D G, GROVER A. In silico analysis and overexpression of chitinase class IV gene in Brassica juncea improves resistance against Alternaria brassicae. Industrial Crops and Products, 2021, 169: 113555.

doi: 10.1016/j.indcrop.2021.113555
[37]
NAUMANN T A, PRICE N P J. Truncation of Class IV chitinases from Arabidopsis by secreted fungal proteases. Molecular Plant Pathology, 2012, 13(9): 1135-1139.

doi: 10.1111/mpp.2012.13.issue-9
[38]
CHEN Y L, HUANG T C, YOU C H, CHEN Y, CHEN Y, QUE Y X, SU Y C. The function and regulatory network of sugarcane chitinase gene ScChiIV1 in response to pathogen stress. Plant Physiology and Biochemistry, 2025, 221: 109630.

doi: 10.1016/j.plaphy.2025.109630
[39]
KIM D S, KIM N H, HWANG B K. The Capsicum annuum Class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses. Journal of Experimental Botany, 2015, 66(7): 1987-1999.

doi: 10.1093/jxb/erv001
[40]
LU D P, WU S J, GAO X Q, ZHANG Y L, SHAN L B, HE P. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(1): 496-501.
[41]
DIAO Z H, YANG R Q, WANG Y Z, CUI J M, LI J H, WU Q Q, ZHANG Y X, YU X S, GONG B Q, HUANG Y, et al. Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1. Molecular Plant Pathology, 2024, 25(4): e13447.

doi: 10.1111/mpp.v25.4
[42]
蔡敏蕊. 苹果类受体激酶基因MdLYK7调控梨腐烂病的抗性功能研究[D]. 兰州: 甘肃农业大学, 2024.
CAI M R. Study on the resistance function of apple receptor kinase gene MdLYK7 in the regulation of Valsa canker in pear[D]. Lanzhou: Gansu Agricultural University, 2024. (in Chinese)
[43]
GOGGIN F L, SHAH J, GILLASPY G. Editorial: Lipid metabolism and membrane structure in plant biotic interactions. Frontiers in Plant Science, 2022, 13: 1096268.

doi: 10.3389/fpls.2022.1096268
[44]
THOLL D. Biosynthesis and biological functions of terpenoids in plants// Advances in Biochemical Engineering-Biotechnology, 2015, 148: 63-106.

doi: 10.1007/10_2014_295 pmid: 25583224
[1] WANG Fan, LIU ChenWei, LU HongChen, XU RenChao, BIAN XiaoChun. Transcriptome Analysis of Vicia faba Response to Alternaria alternata Infection and Validation of the Disease Resistance Function of VfPR4 [J]. Scientia Agricultura Sinica, 2025, 58(22): 4656-4672.
[2] LI Han, JIANG ShangTao, PENG HaiYing, LI PeiGen, GU ChangYi, ZHANG JinLian, CHEN TingSu, XU YangChun, SHEN QiRong, DONG CaiXia. Effects of Inoculation with Indigenous and Exogenous Arbuscular Mycorrhizal Fungi on Drought Resistance of Pyrus betulaefolia and Its Adaptation Mechanism [J]. Scientia Agricultura Sinica, 2024, 57(1): 159-172.
[3] LI Hui, ZHANG YuFeng, LI XiaoGang, WANG ZhongHua, LIN Jing, CHANG YouHong. Identification of Salt-Tolerant Transcription Factors in the Roots of Pyrus betulaefolia by the Association Analysis of Genome-Wide DNA Methylation and Transcriptome [J]. Scientia Agricultura Sinica, 2023, 56(7): 1377-1390.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd