全基因组分析揭示了花生AhCN34基因参与青枯病抗性研究

doi:10.1016/j.jia.2024.03.006

摘要/Abstract

摘要： 花生（Arachis hypogaea L.）青枯病是一种严重威胁花生产量和品质的细菌性土传病害。核苷酸结合富亮氨酸重复序列（NBS-LRR）蛋白是一类植物特异性免疫受体，可识别病原体分泌的效应分子并激活免疫反应以抵抗病原体侵染。然而，在花生中AhCN基因（CN是一类缺乏LRR结构域的NLR基因）的确切功能尚不清楚。本研究通过系统进化发育分析，共鉴定出150个AhCN基因，划分为9个亚家族，发现AhCN基因表现出高度保守的结构特征，并参与植物激素信号转导和防御反应。接种青枯菌后，高抗性花生品种‘H108’在株高、主茎粗和鲜重等生理指标上显著高于感病品种‘H107’，这可能是由于青枯菌在维管束中的增殖和扩散受到抑制造成的。在遭受青枯菌侵染和植物激素处理后，q-PCR验证显示，与H107相比，AhCN34在H108中显著上调。重要的是，在叶片中过表达AhCN34增强了花生对青枯病的抗性。以上结果表明，AhCN34在花生抗病育种中具备一定的潜力，为今后花生高效遗传育种提供理论依据。

Abstract: Peanut (Arachis hypogaea L.) bacterial wilt (BW), caused by Ralstonia solanacearum (RS), is a devastating soil-borne disease that poses a significant threat to peanut yield and quality. Nucleotide-binding leucine-rich repeat (NBS-LRR) proteins are a class of plant-specific immune receptors that recognize pathogen-secreted effector molecules and activate immune responses to resist pathogen infections. However, the precise functions of AhCN genes (CN is a class of NLR genes lacking LRR structural domains) in peanut plants are not fully understood. In this study, a total of 150 AhCN genes were identified and classified into nine subfamilies based on a systematic phylogenetic analysis. The AhCN genes showed highly conserved structural features; promoter cis-elements indicated involvement in plant hormone signaling and defense responses. Following inoculation with RS, the highly resistant peanut variety ‘H108’ significantly outperformed the susceptible variety ‘H107’ in physiological indicators such as plant height, main stem diameter, and fresh weight, likely due to inhibition of bacterial proliferation and diffusion in the stem vascular bundle. AhCN34 was found to be significantly upregulated in H108 compared to H107 during plant infection and in response to treatment with each of three plant hormones. Importantly, AhCN34 overexpression in peanut leaves enhanced their resistance to BW. These findings demonstrate the great potential of AhCN34 for applications in peanut resistance breeding. Our identification and characterization of AhCN genes provide insights into the mechanisms underlying peanut BW resistance and inform future research into genetic methods of improving peanut BW resistance.

Key words: peanut , bacterial wilt , , resistance , , NLR genes , , disease

. 全基因组分析揭示了花生AhCN34基因参与青枯病抗性研究[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.03.006.

Kai Zhao, Yanzhe Li, Zhan Li, Zenghui Cao, Xingli Ma, Rui Ren, Kuopeng Wang, Lin Meng, Yang Yang, Miaomiao Yao, Yang Yang, Xiaoxuan Wang, Jinzhi Wang, Sasa Hu, Yaoyao Li, Qian Ma, Di Cao, Kunkun Zhao, Ding Qiu, Fangping Gong, Zhongfeng Li, Xingguo Zhang, Dongmei Yin. Genome-wide analysis of AhCN genes reveals the AhCN34 involved in bacterial wilt resistance in peanut[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.03.006.

参考文献

Álvarez B, Biosca E G, López M M. 2010. On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 1, 267–279.

Baggs E, Dagdas G, Krasileva K V. 2017. NLR diversity, helpers and integrated domains: Making sense of the NLR IDentity. Current Opinion in Plant Biology, 38, 59–67.

Cai H, Wang W, Rui L, Han L, Luo M, Liu N, Tang D. 2021. The TIR-NBS protein TN13 associates with the CC-NBS-LRR resistance protein RPS5 and contributes to RPS5-triggered immunity in Arabidopsis. Plant Journal, 107, 775–786.

Cao P, Chen J L, Li N N, Zhang S X, Wang R B, Li B J, Liu P Q, An Y Y, Zhang M X. 2023. Seedling Petri-dish inoculation method: A robust, easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions. Journal of Integrative Agriculture, 22, 3709-3719.

Chen T, Yang W, Zhang H, Zhu B, Zeng R, Wang X, Wang S, Wang L, Qi H, Lan Y, Zhang L. 2020. Early detection of bacterial wilt in peanut plants through leaf-level hyperspectral and unmanned aerial vehicle data. Computers and Electronics in Agriculture, 177, 105708.

Chen Y, Ren X, Zhou X, Huang L, Yan L, Lei Y, Liao B, Huang J, Huang S, Wei W, Jiang H. 2014. Dynamics in the resistant and susceptible peanut (Arachis hypogaea L.) root transcriptome on infection with the Ralstonia solanacearum. BMC Genomics, 15, 1–16.

Collier S M, Hamel L P, Moffett P. 2011. Cell death mediated by the N-terminal domains of a unique and highly conserved class of NB-LRR protein. Molecular Plant-Microbe Interactions, 24, 918–931.

Dazy M, Masfaraud J F, Férard J F. 2009. Induction of oxidative stress biomarkers associated with heavy metal stress in Fontinalis antipyretica Hedw. Chemosphere, 75, 297–302.

Deslandes L, Olivier J, Peeters N, Feng D X, Khounlotham M, Boucher C, Somssich I, Genin S, Marco Y. 2003. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proceedings of the National Academy of Sciences of the United States of America, 100, 8024–8029.

Dong Z, Ma C, Tian X, Zhu C, Wang G, Lv Y, Friebe B, Li H, Liu W. 2020. Genome-wide impacts of alien chromatin introgression on wheat gene transcriptions. Scientific Reports, 10, 1–12.

Elsayed T R, Jacquiod S, Nour E H, Sørensen S J, Smalla K. 2020. Biocontrol of bacterial wilt disease through complex interaction between tomato plant, Antagonists, the Indigenous Rhizosphere Microbiota, and Ralstonia solanacearum. Frontiers in Microbiology, 10, 1–15.

FAO (Food and Agriculture Organization). 2021. Online statistical database: Food balance. FAOSTAT. [2022-11-09]. https://www.fao.org/faostat/zh/#data/QCL

Gao H, Narayanan N N, Ellison L, Bhattacharyya M K. 2005. Two classes of highly similar coiled coil-nucleotide binding-leucine rich repeat genes isolated from the Rps1-k locus encode Phytophthora resistance in soybean. Molecular Plant-Microbe Interactions 18, 1035–1045.

Godiard L, Sauviac L, Torii K U, Grenon O, Mangin B, Grimsley N H, Marco Y. 2003. ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt. Plant Journal, 36, 353–365.

Griebel T, Maekawa T, Parker J E. 2014. NOD-like receptor cooperativity in effector-triggered immunity. Trends in Immunology, 35, 562–570.

Gururani M A, Venkatesh J, Upadhyaya C P, Nookaraju A, Pandey S K, Park S W. 2012. Plant disease resistance genes: Current status and future directions. Physiological and Molecular Plant Pathology, 78, 51–65.

Hammond-Kosack K E, Parker J E. 2003. Deciphering plant-pathogen communication: Fresh perspectives for molecular resistance breeding. Current Opinion in Biotechnology, 14, 177-193.

He M, Cui S, Yang X, Mu G, Chen H, Liu L. 2017. Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR. Electronic Journal of Biotechnology, 28, 76–86.

Jiang G, Wei Z, Xu J, Chen H, Zhang Y, She X, Macho A P, Ding W, Liao B. 2017. Bacterial wilt in China: History, current status, and future perspectives. Frontiers in Plant Science, 8, 1–10.

Kourelis J, Sakai T, Adachi H, Kamoun S. 2021. RefPlantNLR is a comprehensive collection of experimentally validated plant disease resistance proteins from the NLR family. PLoS Biology, 19, 1–26.

Lee S K, Song M Y, Seo Y S, Kim H K, Ko S, Cao P J, Suh J P, Yi G, Roh J H, Lee S, An G, Hahn T R, Wang G L, Ronald P, Jeon J S. 2009. Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes. Genetics, 181, 1627–1638.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC_T method. Methods, 25, 402–408.

Lowe-Power T M, Khokhani D, Allen C. 2018. How Ralstonia solanacearum exploits and thrives in the flowing plant xylem environment. Trends in Microbiology, 26, 929–942.

Luo H, Pandey M K, Khan A W, Wu B, Guo J, Ren X, Zhou X, Chen Y, Chen W, Huang L, Liu N, Lei Y, Liao B, Varshney R K. 2019. Next-generation sequencing identified genomic region and diagnostic markers for resistance to bacterial wilt on chromosome B02 in peanut (Arachis hypogaea L .). Plant Biotechnology Journal, 17, 2356-2369.

Macho A P, Zipfel C. 2015. Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system effectors. Current Opinion in Microbiology, 23, 14–22.

Maruta N, Burdett H, Lim B Y J, Hu X, Desa S, Manik M K, Kobe B. 2022. Structural basis of NLR activation and innate immune signalling in plants. Immunogenetics, 74, 5–26.

Mestre P, Baulcombe D C. 2006. Elicitor-mediated oligomerization of the tobacco N disease resistance protein. Plant Cell, 18, 491–501.

Meyers B C, Kozik A, Griego A, Kuang H, Michelmore R W. 2003. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell, 15, 809-834.

Nishimura M T, Anderson R G, Cherkis K A, Law T F, Liu Q L, Machius M, Nimchuk Z L, Yang L, Chung E H, El Kasmi F, Hyunh M, Nishimura E O, Sondek J E, Dangl J L. 2017. TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 114, E2053–E2062.

Parry D A D, Fraser R D B, Squire J M. 2008. Fifty years of coiled-coils and α-helical bundles: A close relationship between sequence and structure. Journal of Structural Biology, 163, 258–269.

Pieterse C M J, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees S C M. 2012. Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Biology, 28, 489–521.

Peng W F, Jiang H F, Ren X P, Lv J W, Zhao X Y, Huang L. 2010. Construction of peanut AFLP genetic map and QTL analysis of bacterial wilt resistance. North China Agricultural Journal, 25, 81-86.

Ren X P, Zhang X J, Liao B S, Lei Y, Huang J Q, Chen Y N, Jiang H F. 2010, Genetic diversity analysis of ICRISAT peanut microcore germplasm resources using SSR markers. Chinese Agricultural Science, 43, 2848-2858.

Rairdan G J, Collier S M, Sacco M A, Baldwin T T, Boettrich T, Moffett P. 2008. The coiled-coil and nucleotide binding domains of the potato Rx disease resistance protein function in pathogen recognition and signaling. Plant Cell, 20, 739–751.

Robert-Seilaniantz A, Grant M, Jones J D G. 2011. Hormone crosstalk in plant disease and defense: More than just JASMONATE-SALICYLATE antagonism. Annual Review of Phytopathology, 49, 317–343.

Schreiber K J, Bentham A, Williams S J, Kobe B, Staskawicz B J. 2016. Multiple domain associations within the Arabidopsis immune receptor RPP1 regulate the activation of programmed cell death. Plos Pathogens, 12, 1–26.

Sheng Y T, Yu X L, Mao T T, Zhang J, Guo X T, Song Z Z, Zhang H X. 2022. Genome sequence data of Leptosphaerulina arachidicola, a causal agent of peanut scorch spot in China. Plant Disease, 106, 748–750.

Takken F L, Albrecht M, Tameling W I L. 2006. Resistance proteins: Molecular switches of plant defence. Current Opinion in Plant Biology, 9, 383–390.

Takken F L W, Goverse A. 2012. How to build a pathogen detector: Structural basis of NB-LRR function. Current Opinion in Plant Biology, 15, 375–384.

Tameling W I L, Baulcombe D C. 2007. Physical association of the NB-LRR resistance protein Rx with a Ran GTPase-activating protein is required for extreme resistance to potato virus X. Plant Cell, 19, 1682-1694.

Wang L, Zhou X, Ren X, Huang L, Luo H, Chen Y, Chen W, Liu N, Liao B, Lei Y, Yan L, Shen J, Jiang H. 2018. A major and stable QTL for bacterial wilt resistance on chromosome B02 identified using a high-density SNP-based genetic linkage map in cultivated peanut Yuanza 9102 derived population. Frontiers in Genetics, 9, 1–13.

Xiao S, Ellwood S, Calis O, Patrick E, Li T, Coleman M, Turner J G. 2001. Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8. Science, 291, 118–120.

Yu G, Xian L, Xue H, Yu W, Rufian J S, Sang Y, Morcillo R J L, Wang Y, Macho A P. 2020. A bacterial effector protein prevents mapk-mediated phosphorylation of sgt1 to suppress plant immunity. Plos Pathogens, 16, 1–30.

Yin J J, Xiong J, Xu L T, Chen X W, Li W T. 2022. Recent advances in plant immunity with cell death: A review. Journal of Integrative Agriculture, 21, 610-620.

Zhang C. 2010. Study on molecular basis of resistence to Ralstonia solanacearum in peanut. MSc thesis, Fujian Agriculture and Forestry University, Fujian, China. (in Chinese).

Zhang C, Chen H, Cai T, Deng Y, Zhuang R, Zhang N, Zeng Y, Zheng Y, Tang R, Pan R, Zhuang W. 2017. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco. Plant Biotechnology Journal, 15, 39–55.

Zhang D D, Guo X J, Wang Y J, Gao T G, Zhu B C. 2017. Novel screening strategy reveals a potent Bacillus antagonist capable of mitigating wheat take-all disease caused by Gaeumannomyces graminis var. tritici. Letters in Applied Microbiology, 65, 512–519.

Zhao K, Ren R, Ma X L, Zhao K K, Qu C X, Cao D, Ma Q, Ma Y Y, Gong F P, Li Z F, Zhang X G, Yin D M. 2022. Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt. The Crop Journal, 10, 809-819.

Zhao Y, Zhang C, Chen H, Yuan M, Nipper R, Prakash C S, Zhuang W, He G. 2016. QTL mapping for bacterial wilt resistance in peanut (Arachis hypogaea L.). Molecular Breeding, 36, 1–11.

Zhou T, Wang Y, Chen J Q, Araki H, Jing Z, Jiang K, Shen J, Tian D. 2004. Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes. Molecular Genetics and Genomics, 271, 402–415.

Zhuang W J, Zhang C, Chen H, Zhuang R R, Chen Y T, Deng Y, Cai T C, Wang S Y, Liu Q Z, Tang R H, Shan S H, Pan R L, Chen L S, Dietz K J. 2019. Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco. Journal of Experimental Botany, 70, 5407–5421.