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Journal of Integrative Agriculture  2025, Vol. 24 Issue (1): 196-208    DOI: 10.1016/j.jia.2023.09.028
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response

Ke Yao1, Menghan Zhang1, Jianjun Xu2, Deliang Peng1, Wenkun Huang1, Ling’an Kong1, 3, Shiming Liu1, Guangkuo Li2#, Huan Peng1, 2#

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

2 Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences/Scientific Observing and Experimental Station of Korla, Ministry of Agriculture and Rural Affairs, Urumqi 830013, China

3 Zhongyuan Research Center, Chinese Academy of Agricultural Sciences, Xinxiang 453000, China

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摘要  

甜菜孢囊线虫(Heterodera schachtii)是重要的植物病原线虫,曾给世界甜菜生产具有毁灭性的破坏2015年在我国新疆首次发现了甜菜孢囊线虫的危害,对我甜菜生产安全造成严重威胁,迫切需要开展相关研究。本研究基于甜菜孢囊线虫的基因组和转录组数据,筛选能够抑制寄主免疫反应的潜在效应蛋白,为解析甜菜孢囊线虫的致病分子机制提供理论依据。采用SignalP 5.0TMHMM2.0生物信息学分析方法,从甜菜孢囊线虫基因组筛选出有信号肽无跨膜结构域的潜在效应子序列,在此基础上,筛选出在植物寄生线虫保守存在,但与自由生活线虫无同源性的蛋白(e<1.0 e-5且覆盖率≥30.0%)的序列,根据甜菜孢囊线虫各发育阶段的基因表达量差异,随后筛选出在甜菜孢囊线虫二龄幼虫和寄生阶段上调表达的序列(差异倍数log2>2且e≤0.01。最后过滤掉功能已知的植物线虫效应蛋白序列(e<1.0 e-10),剩余序列作为潜在的效应蛋白进行下一步分析。将上述候选效应蛋白基因序列构建到植物表达载体pYBA1132中,采用本氏烟草瞬时表达系统,对候选效应蛋白是否能激发或抑制BaxGPA2/RBP1诱导的细胞死亡进行筛选。采用qRT-PCR技术检测候选效应蛋白异源表达植物免疫相关基因PR1PR2WRKY12PI1转录的影响。采用原位杂交方法分析候选效应蛋白在甜菜孢囊线虫中的表达定位。采用生物信息学的方法从甜菜孢囊线虫基因组中筛选51新的候选效应蛋白。利用烟草瞬时表达系统,对37成功克隆的候选效应蛋白基因进行功能验证,筛选出13个能够抑制Bax引起的细胞死亡的候选效应蛋白,其中9个效应蛋白能够抑制GPA2/RBP1诱导的坏死反应qRT-PCR分析发现,13候选效应蛋白基因的瞬时表达能引起寄主植物PR1PR2WRKY12PI1防御基因表达量显著下调。原位杂交结果表明,13候选效应蛋白的表达位点均位于甜菜孢囊线虫的食道腺细胞。本研究从甜菜孢囊线虫基因组中筛选到了13个能够抑制植物免疫应答的新效应蛋白,它们均可以通过抑制寄主抗性相关基因的表达,促进线虫寄生。上述研究结果将为进一步研究甜菜孢囊线虫与寄主的互作提供理论依据。



Abstract  

The sugar beet cyst nematode (Heterodera schachtii) is one of the most destructive pathogens in sugar beet production, which causes serious economic losses every year.  Few molecular details of effectors of Hschachtii parasitism are known.  We analyzed the genome and transcriptome data of Hschachtii and identified multiple potential predicted proteins.  After filtering out predicted proteins with high homology to other plant-parasitic nematodes, we performed functional validation of the remaining effector proteins.  37 putative effectors of Hschachtii were screened based on the Nicotiana benthamiana system for identifying the effectors that inhibit plant immune response, eventually determines 13 candidate effectors could inhibit cell death caused by Bax.  Among the 13 effectors, nine have the ability to inhibit GPA2/RBP1-induced cell death.  All 13 effector-triggered immunity (ETI) suppressor genes were analyzed by qRT-PCR and confirmed to result in a significant downregulation of one or more defense genes during infection compared to empty vector.  For in situ hybridization, 13 effectors were specifically expressed and located in esophageal gland cells.  These data and functional analysis set the stage for further studies on the interaction of Hschachtii with host and Hschachtii parasitic control.

Keywords:  sugar beet cyst nematode       Heterodera schachtii       effector-triggered immunity        hypersensitive response       effector  
Received: 06 June 2023   Accepted: 11 August 2023
Fund: 
This research was supported by the Open Fund of the Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Ministry of Agriculture and Rural Affairs of China (KFJJ202101), the National Key R&D Program of China (2021YFD1400100), the National Natural Science Foundation of China (31972247), the Tianchi Talent Introduction Program in Xinjiang Uygur Autonomous Region, China and the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences. 
About author:  Ke Yao, E-mail: 17794539779@163.com; #Correspondences Huan Peng, Tel/Fax: +86-10-62815576, E-mail: penghuan@caas.cn; Guangkuo Li, Tel: +86-991-4524641, E-mail: lgk0808@163.com

Cite this article: 

Ke Yao, Menghan Zhang, Jianjun Xu, Deliang Peng, Wenkun Huang, Ling’an Kong, Shiming Liu, Guangkuo Li, Huan Peng. 2025. Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response. Journal of Integrative Agriculture, 24(1): 196-208.

Abad P, Gouzy J, Aury J M, Castagnone-Sereno P, Danchin E G, Deleury E, Perfus-Barbeoch L, Anthouard V, Artiguenave F, Blok V C, Caillaud M C, Coutinho P M, Dasilva C, Luca F D, Deau F, Esquibet M, Flutre T, Goldstone J V, Hamamouch N, Hewezi T, et al. 2008. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognitaNature Biotechnology26, 909–915.

Abramovitch R B, Kim Y J, Chen S, Dickman M B, Martin G B. 2003. Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. EMBO Journal22, 60–69.

Ali S, Magne M, Chen S, Côté O, Stare B G, Obradovic N, Jamshaid L, Wang X, Bélair G, Moffett P. 2015. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses. PLoS ONE10, 5819–5829.

Almagro A, José J, Tsirigos K D, Sønderby C K, Petersen T N, Winther O, Brunak S. 2019. Signalp 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology37, 420–423.

de Boer J M, Yan Y, Smant G, Davis E L, Baum T J. 1998. In-situ hybridization to messenger RNA in Heterodera glycinesJournal of Nematology30, 309–312.

Chen C L, Chen Y P, Jian H, Yang D, Dai Y R, Pan L L, Shi F W, Yang S S, Liu Q. 2018. Large-scale identification and characterization of Heterodera avenae putative effectors suppressing or inducing cell death in Nicotiana benthamianaFrontiers in Plant Science8, 2062.

Chen C L, Liu S S, Liu Q, Niu J H, Liu P, Zhao J L, Jian H. 2015. An annexin-like protein from the cereal cyst nematode Heterodera avenae suppresses plant defense. PLoS ONE10, e0122256.

Cotton J A, Lilley C J, Jones L M, Kikuchi T, Reid A J, Thorpe P, Tsai I J, Beasley H, Blok V, Cock P J. 2014. The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode. Genome Biology15, R43.

Dangl J L, Horvath D M, Staskawicz B J. 2013. Pivoting the plant immune system from dissection to deployment. Science341, 746–751.

Davis E L, Hussey R S, Baum T J. 2004. Getting to the roots of parasitism by nematodes. Trends in Parasitology, 20, 134–141.

Davis E L, Hussey R S, Baum T J. 2009. Parasitism genes: What they reveal about parasitism. In: Berg R H, Taylor C G, eds., Cell Biology of Plant Nematode Parasitism. Springer, Berlin, Heidelberg, Germany. pp. 15–44.

Davis E L, Hussey R S, Baum T J, Bakker J, Schots A, Rosso M N, Abad P. 2000. Nematode parasitism genes. Annual Review of Phytopathology38, 365–396.

Davis E L, Hussey R S, Mitchum M G, Baum T J. 2008. Parasitism proteins in nematode–plant interactions. Current Opinion in Plant Biology11, 360–366.

Dong S M, Zhang Z G, Zheng X B, Wang Y C. 2008. Mammalian pro-apoptotic bax gene enhances tobacco resistance to pathogens. Plant Cell Reports27, 1559–1569.

Eves-van den Akker S, Laetsch D R, Thorpe P, Lilley C J, Danchin E G, Da Rocha M, Rancurel C, Holroyd N E, Cotton J A, Szitenberg A. 2016. The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence. Genome Biology17, 124.

Fierst J L, Willis J H, Thomas C G, Wang W, Reynolds R M, Ahearne T E, Cutter A D, Phillips P C. 2015. Reproductive mode and the evolution of genome size and structure in Caenorhabditis nematodes. PLoS Genetics11, e1005323.

Glazebrook J. 2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology43, 205–227.

Gonzalez T L, Liang Y, Nguyen B N, Staskawicz B J, Loqué D, Hammond M C. 2015. Tight regulation of plant immune responses by combining promoter and suicide exon elements. Nucleic Acids Research43, 7152–7161.

Habash S S, Radakovic Z S, Vankova R, Siddique S, Dobrev P, Gleason C. 2017a. Heterodera schachtii tyrosinase-like protein-a novel nematode effector modulating plant hormone homeostasis. Scientific Reports7, 6874.

Habash S S, Sobczak M, Siddique S, Voigt B, Elashry A, Grundler F M W. 2017b. Identification and characterization of a putative protein disulfide isomerase (HsPDI) as an alleged effector of Heterodera schachtiiScientific Reports7, 13536.

Haegeman A, Bauters L, Kyndt T, Rahman M M, Gheysen G. 2013. Identification of candidate effector genes in the transcriptome of the rice root knot nematode Meloidogyne graminicola. Molecular Plant Pathology14, 379–390.

Haegeman A, Mantelin S, Jones J T, Gheysen G. 2012. Functional roles of effectors of plant-parasitic nematodes. Gene492, 19–31.

Hewezi T, Baum T J. 2013. Manipulation of plant cells by cyst and root-knot nematode effectors. Molecular Plant-Microbe Interactions26, 9–16.

Hogenhout S A, Van der Hoorn R A, Terauchi R, Kamoun S. 2009. Emerging concepts in effector biology of plant-associated organisms. Molecular Plant-Microbe Interactions22, 115–122.

Hussey R S. 1989. Disease-inducing secretions of plant-parasitic nematodes. Annual Review of Phytopathology27, 123–141.

Jones J, Gheysen G, Fenoll C. 2011. Genomics and molecular genetics of plant-nematode interactions. Current Nematode Threats to World Agriculture2, 21–43.

Jones J T, Haegeman A, Danchin E G, Gaur H S, Helder J, Jones M G, Kikuchi T, Manzanilla-López R, Palomares-Rius J E, Wesemael W M, Perry R N. 2013. Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology14, 946–961.

Jones M G K. 1981. Host cell responses to endoparasitic nematode attack: Structure and function of giant cells and syncytia. Annals of Applied Biology97, 353–372.

Juvale P S, Baum T J. 2018. “Cyst-ained” research into Heterodera parasitism. PLoS Pathogens14, e1006791.

Kanzaki N, Tsai I J, Tanaka R, Hunt V L, Liu D, Tsuyama K, Maeda Y. 2018. Biology and genome of a newly discovered sibling species of Caenorhabditis elegansNature Communications9, 3216.

Kawai-Yamada M, Jin L, Yoshinaga K, Hirata A, Uchimiya H. 2001. Mammalian Bax-induced plant cell death can be down-regulated by overexpression of Arabidopsis Bax Inhibitor-1 (AtBI-1). Proceedings of the National Academy of Sciences of the United States of America98, 12295–12300.

Kikuchi T, Cotton J A, Dalzell J J, Hasegawa K, Kanzaki N, McVeigh P, Takanashi T, Tsai I J, Assefa S A, Cock P J. 2011. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilusPLoS Pathogens7, e1002219.

Krogh A, Larsson B, Heijne G V, Sonnhammer E L L. 2001. Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes-sciencedirect. Journal of Molecular Biology305, 567–580.

Kumar M, Gantasala N P, Roychowdhury T, Thakur P K, Banakar P, Shukla R N, Jones M G, Rao U. 2014. De novo transcriptome sequencing and analysis of the cereal cyst nematode, Heterodera avenaePLoS ONE, 9, e96311.

Lunt D H, Kumar S, Koutsovoulos G, Blaxter M L. 2014. The complex hybrid origins of the root knot nematodes revealed through comparative genomics. PeerJ2, e356.

Luo S J, Liu S M, Kong L A, Peng H, Huang W K, Jian H, Peng D L. 2019. Two venom allergen-like proteins, HaVAP1 and HaVAP2, are involved in the parasitism of Heterodera avenaeMolecular Plant Pathology20, 471–484.

Masonbrink R, Maier T R, Muppirala U, Seetharam A S, Lord E, Juvale P S, Schmutz J, Johnson N T, Korkin D, Mitchum M G, Mimee B, den Akker S E, Hudson M, Severin A J, Baum T J. 2019. The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes. BMC Genomics20, 119.

Mattevi A, Fraaije M W, Mozzarelli A. 1997. Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: The shape of the active-site cavity controls substrate specificity. Structure5, 907–920.

Mei Y, Wright K M, Haegeman A, Bauters L, Diaz-Granados A, Goverse A, Gheysen G, Jones J T, Mantelin S. 2018. The Globodera pallida SPRYSEC effector GpSPRY-414-2 that suppresses plant defenses targets a regulatory component of the dynamic microtubule network. Frontiers in Plant Science9, 1019.

Mitchum M G. 2016. Soybean resistance to the soybean cyst nematode Heterodera glycines: An update. Phytopathology106, 1444–1450.

Mitchum M G, Hussey R S, Baum T J. 2013. Nematode effector proteins: An emerging paradigm of parasitism. New Phytologist199, 879–894.

Muller J. 1999. The economic importance of Heterodera schachtii in Europe. Helminthologia36, 205–213.

Noon J B, Hewezi T, Maier T R. 2015. Eighteen new candidate effectors of the phytonematode Heterodera glycines produced specifically in the secretory esophageal gland cells during parasitism. Phytopathology, 105, 1362–1372.

Nurnberger T, Brunner F, Kemmerling B, Piater L. 2004. Innate immunity in plants and animals: striking similarities and obvious differences. Immunological Reviews198, 249–266.

Opperman C H, Bird D M, Williamson V M, Rokhsar D S, Burke M, Cohn J, Cromer J, Diener S, Gajan J, Graham S. 2008. Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proceedings of the National Academy of Sciences of the United States of America105, 14802–14807.

Peng H, Liu H, Gao L, Jiang R, Li G K, Gao H F, Wu W, Wang J, Zhang Y, Huang W K, Kong L A, Peng D L. 2021. Identification of Heterodera schachtii on sugar beet in Xinjiang Uygur Autonomous Region of China. Journal of Integrative Agriculture21, 1694–1702.

Pogorelko G, Wang J, Juvale P S, Mitchum M G, Baum T J. 2020. Screening soybean cyst nematode effectors for their ability to suppress plant immunity. Molecular Plant Pathology21, 1240–1247.

Rosso M N, Hussey R S, Davis E L, Smant G, Baum T J, Abad P, Mitchum M G. 2012. Nematode effector proteins: Targets and functions in plant parasitism. In: Martin F, Kamoun S, eds., In: Effectors in Plant-Microbe Interactions. Springer, Heidelberg, Germany. pp. 327–354.

Sacco M A, Koropacka K, Grenier E, Jaubert M J, Blanchard A, Goverse A, Smant G, Moffett P. 2009. The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death. PLoS Pathogens, 5, e1000564.

Sato K, Kadota Y, Shirasu K. 2019. Plant immune responses to parasitic nematodes. Frontiers in Plant Science10, 1165.

Siddique S, Grundler F M W. 2018. Parasitic nematodes manipulate plant development to establish feeding sites. Current Opinion in Microbiology46, 102–108.

Siddique S, Radakovic Z S, Hiltl C, Pellegrin C, Baum T J, Beasley H, Bent A F, Chitambo O, Chopra D, Danchin E G J, Grenier E, Habash S S, Hasan M S, Helder J, Hewezi T, Holbein J, Holterman M, Janakowski S, Koutsovoulos G D, Kranse O P, et al. 2022. The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B5. Nature Communications13, 6190.

Stevens L, Félix M A, Beltran T, Braendle C, Caurcel C, Fausett S, Fitch D, Frézal L, Gosse C, Kaur T, Kiontke K, Newton M D, Noble L M, Richaud A, Rockman M V, Sudhaus W, Blaxter M. 2019. Comparative genomics of ten new Caenorhabditis species. Cold Spring Harbor Laboratory2, 217–236.

Szakasits D, Heinen P, Wieczorek K, Hofmann J, Wagner F, Kreil D P, Sykacek P, Grundler F M, Bohlmann H. 2009. The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant Journal57, 771–784.

Tarek H, Howe P J, Maier T R. 2010. Arabidopsis spermidine synthase is targeted by an effector protein of the cyst nematode Heterodera schachtiiPlant Physiology152, 968–984.

Tefft P M, Bone L W. 1984. Zinc-mediated hatching of eggs of soybean cyst nematode. Heterodera glycinesJournal of Chemical Ecology10, 361–372.

The C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: A platform for investigating biology. Science, 282, 2012–2018.

Verma A, Lin M, Smith D. 2022. A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase. Molecular Plant Pathology23, 1765–1782.

Vieira P, Gleason C. 2019. Plant-parasitic nematode effectors-insights into their diversity and new tools for their identification. Current Opinion in Plant Biology50, 37–43.

Wang G F, Peng D L, Gao B L, Huang W K, Kong L A, Long H B, Peng H, Jian H. 2014. Comparative transcriptome analysis of two races of Heterodera glycines at different developmental stages. PLoS ONE9, e91634.

Wyss U. 1992. Observations on the feeding behaviour of Heterodera schachtii throughout development including events during moulting. Fundamental and Applied Nematology15, 7589.

Xing Q K, Cao Y, Peng J B, Zhang W, Wu J H, Zhou Y Y, Li X H, Yan J Y. 2022. A putative effector LtCSEP1 from Lasiodiplodia theobromae inhibits BAX-triggered cell death and suppresses immunity responses in Nicotiana benthamianaPlants (Basel), 11, 1462.

Yang S S, Dai Y R, Chen Y P, Yang J, Liu Q, Jian H. 2019. A novel G16B09-like effector from Heterodera avenae suppresses plant defenses and promotes parasitism. Frontiers in Plant Science10, 66.

Zhao J, Li L, Liu Q, Liu P, Li S, Yang D, Chen Y, Pagnotta S, Favery B, Abad P, Jian H. 2019. A MIF-like effector suppresses plant immunity and facilitates nematode parasitism by interacting with plant annexins. Journal of Experimental Botany70, 5943–5958.

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