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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2022, Vol. 48 Issue (5): 594-604    DOI: 10.3785/j.issn.1008-9209.2021.11.291
Plant protection     
Cyclic nucleotide-gated ion channel gene CNGC3 positively regulates immunity against Sclerotinia sclerotiorum in Arabidopsis
Mengjiao LIU1(),Hang YI1,Xinzhong CAI1,2()
1.Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University/Zhejiang Provincial Key Laboratory of Crop Pathogen and Insect Biology, Hangzhou 310058, China
2.Hainan Institute, Zhejiang University, Sanya 572025, Hainan, China
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Abstract  

Functions and mechanisms of cyclic nucleotide-gated ion channel (CNGC) in plant immunity against necrotrophic pathogens remain largely unknown. In this study, we explored the functions of AtCNGC3 in immunity against Sclerotinia sclerotiorum, a typical necrotrophic pathogen, through identifying subcellular localization of its expression product, defining its expression patterns and genetically analyzing its function in immunity. The results showed that AtCNGC3 protein was localized in plasma membrane. AtCNGC3 was highly expressed in all analyzed tissues of Arabidopsis thaliana without showing any tissue specificity. Sclerotinia sclerotiorum inoculation strongly and constantly induced the expression of AtCNGC3 gene. The Atcngc3 mutant plants were more susceptible to S. sclerotiorum, while AtCNGC3 overexpression (AtCNGC3-OE) plants exhibited enhanced resistance to S. sclerotiorum, indicating that AtCNGC3 positively regulates the resistance to S. sclerotiorum. Seedlings soaking with Arabidopsis thaliana plant elicitor peptide 1 (AtPep1) could rapidly induce AtCNGC3 gene expression. AtPep1-elicited reactive oxygen species (ROS) burst was weakened in the Atcngc3 mutant seedlings while enhanced in the AtCNGC3-OE plants, demonstrating that AtCNGC3 positively regulates AtPep1-induced ROS burst. Collectively, our results reveal that AtCNGC3 positively regulates immunity against the necrotrophic fungal pathogen S. sclerotiorum in Arabidopsis, thereby providing some insights into the functions of CNGC in plant immunity.

Key words： cyclic nucleotide-gated ion channels      Sclerotinia sclerotiorum      disease resistance      plant immunity      reactive oxygen species     
Received: 29 November 2021      Published: 02 November 2022
CLC:  S 432  
Corresponding Authors: Xinzhong CAI     E-mail: 13777350366@163.com;xzhcai@zju.edu.cn
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Mengjiao LIU
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Cite this article:

Mengjiao LIU,Hang YI,Xinzhong CAI. Cyclic nucleotide-gated ion channel gene CNGC3 positively regulates immunity against Sclerotinia sclerotiorum in Arabidopsis. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(5): 594-604.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.11.291     OR     https://www.zjujournals.com/agr/Y2022/V48/I5/594


环核苷酸门控离子通道基因CNGC3正调控拟南芥抗核盘菌免疫

环核苷酸门控离子通道（cyclic nucleotide-gated ion channel, CNGC）对死体营养型病原物的免疫调控功能及机制尚知之甚少。在本研究中，通过基因产物亚细胞定位、基因表达模式以及免疫调控功能的遗传学分析，探究了AtCNGC3基因对典型死体营养型病原物核盘菌（Sclerotinia sclerotiorum）的免疫调控功能。结果表明：AtCNGC3蛋白定位于质膜上。AtCNGC3基因在拟南芥各组织中均有表达，没有组织特异性。核盘菌接种强烈且持续诱导AtCNGC3基因的表达。Atcngc3突变体植株对核盘菌更易感，而AtCNGC3基因超表达（AtCNGC3 overexpression, AtCNGC3-OE）植株表现出更强的核盘菌抗性，说明AtCNGC3正调控拟南芥对核盘菌的抗性。用植物激发子肽AtPep1浸泡处理拟南芥幼苗能迅速诱导AtCNGC3基因表达，AtPep1诱导的活性氧（reactive oxygen species, ROS）迸发在Atcngc3突变体植株中减弱，而在AtCNGC3-OE植株中增强，说明AtCNGC3正调控AtPep1诱导的ROS迸发。综上所述，本研究结果揭示了AtCNGC3正调控拟南芥对死体营养型病原真菌（核盘菌）的免疫，增进了对CNGC免疫调控功能的认识。


关键词： 环核苷酸门控离子通道,  核盘菌,  抗病性,  植物免疫,  活性氧 

基因

Gene

实验目的

Purpose of experiment

引物名称

Primer name

引物序列（5′→3′）

Primer sequence (5′→3′)

AtCNGC3亚细胞定位分析CNGC3-1300-FagctttcgcgagctcggtaccATGATGAATCCCCAAAGAAACAA
CNGC3-1300-RgcccttgctcaccatggtaccGGTTTCATCCATAGGAAACTCAG
AtCNGC3突变体验证cngc3-LPCTGTTGTGGCTTTAGCCTTTG
cngc3-RPCACTCGTCTTCAAGTTTTGGC
LBb1.3ATTTTGCCGATTTCGGAAC
AtCNGC3基因表达检测CNGC3-qPCR-FATGTTCAGGTTTTATTCAGT
CNGC3-qPCR-RTCCTGAATCATCGTTCTGAA
AtACTIN8基因表达检测ACTIN8-qPCR-FCGAGGCTCCTCTTAACCCAAA
ACTIN8-qPCR-RGGCACAGTGTGAGACACACCA
Sclerotinia ITS菌量检测Scl-qPCR-FGGATCTCTTGGTTCTGGCAT
Scl-qPCR-RGCAATGTGCGTTCAAAGATT
AtCNGC3超表达植株构建CNGC3-1305-FggacagcccagatcaactagtATGATGAATCCCCAAAGAAACAA
CNGC3-1305-RgcccttgctcaccatggatccGGTTTCATCCATAGGAAACTCAGG
Table 1 PCR primers used in this study
Fig. 1 Subcellular localization of AtCNGC3Red and green fluorescences are elicited from the cell membrane marker protein RFP (PIP2A-DsRed) and fusion protein AtCNGC3-GFP, respectively; while yellow fluorescence is the result of overlap of the red and green fluorescences.
Fig. 2 Expression of AtCNGC3 in plant tissues
Fig. 3 Expression response of AtCNGC3 to Sclerotinia sclerotiorum infectionMock: Inoculated with empty PDA plugs; Ss: Sclerotinia sclerotiorum. Single asterisk (*) and triple asterisks (***) indicate significant differences and highly significant differences between Ss inoculation and mock at each time-point at the 0.05 and 0.001 probability levels, respectively.
Fig. 4 Verification of Atcngc3 mutant plantsM: DL2000 DNA marker; WT: Wild type (the same as below).
Fig. 5 Inoculation analysis for resistance of Atcngc3 loss-of-function mutation plants to S. sclerotiorumA. Phenotype of plants after inoculation with S. sclerotiorum; B. Statistical analysis results of lesion area; C. Detection of relative biomass of S. sclerotiorum. Triple asterisks (***) indicate highly significant differences as compared with the WT at the 0.001 probability level (the same as Fig. 6).
Fig. 6 Verification of AtCNGC3 overexpression plantsA. Semi-quantitative PCR assay; B. qRT-PCR analysis.
Fig. 7 Inoculation analysis for resistance of AtCNGC3 overexpression plants to S. sclerotiorumA. Phenotype of plants after inoculation with S. sclerotiorum; B. Statistical analysis results of lesion area; C. Detection of relative biomass of S. sclerotiorum. Single asterisk (*) and triple asterisks (***) indicate significant differences and highly significant differences as compared with the WT at the 0.05 and 0.001 probability levels, respectively.
Fig. 8 Expression response of AtCNGC3 to AtPep1 and its role in AtPep1-induced ROS burstA. Expression response of AtCNGC3 to AtPep1 (200 nmol/L); B. Detection of ROS production in Atcngc3 and AtCNGC3-OE plants induced by AtPep1 (200 nmol/L). Single asterisk (*) indicates significant differences as compared with the H2O treatment at the 0.05 probability level.
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