Effects of Exogenous miR3979 on Chemotaxis, Infection and Development of Root Knot Nematode Meloidogyne graminicola in Rice

doi:10.16819/j.1001-7216.2025.240906

Abstract

Abstract: 【Objective】 miR3979-3p is involved in rice responses to multiple biotic and abiotic stresses. Its expression is downregulated in root tissues infected by the root-knot nematode Meloidogyne graminicola. Therefore, it is important to investigate the role of miR3979-3p in the interaction between M. graminicola and rice.【Method】 Rice roots were treated with artificially synthesized double-stranded miR3979 (ds-miR3979) via root soaking. Endogenous miR3979-3p expression levels were quantified using qPCR. Chemotaxis and infectivity of second-stage juveniles (J2s) were assessed through plate inoculation and tissue staining. The effects of ds-miR3979 treatment on root gall formation and nematode development were evaluated using pot inoculation assays.【Result】 Treatment with 100–800 nmol/L ds-miR3979 for 12–24 h had no significant effect on J2 motility. Compared with ddH₂O-treated controls, root soaking with 400 nmol/L ds-miR3979 for 12–24 h significantly upregulated miR3979-3p expression in both roots and leaves of rice seedlings at the seedling emergence and three-leaf stages. Continuous microscopic observation revealed reduced chemotactic movement of J2s toward root tips within 2–8 h, with significantly fewer nematodes aggregating around root tips. Plate inoculation showed a decrease in the number of nematodes penetrating roots at 1–3 days post-inoculation (dpi). Pot inoculation assays demonstrated significant reductions in root gall number, number of females within roots, and proportion of females at 7–21 dpi, along with delayed female development.【Conclusion】 Artificially synthesized ds-miR3979 can be taken up by rice plants, upregulating endogenous miR3979-3p expression, thereby inhibiting M. graminicola infection and female development.

Key words:

text-align:justify, "> small RNA, miR3979-3p, rice, Meloidogyne graminicola, infection, development

摘要： 【目的】miR3979-3p参与水稻响应多种生物和非生物胁迫，在拟禾本科根结线虫(Meloidogyne graminicola)侵染的根组织中下调表达，因此探究miR3979-3p对根结线虫与水稻互作中的作用具有重要意义。【方法】利用人工合成的双链miR3979(ds-miR3979)浸泡水稻根组织，采用qPCR测量水稻内源miR3979-3p的表达水平，通过平板接种和组织染色观察二龄幼虫的趋性和侵染能力，结合盆钵接种评价ds-miR3979处理对水稻根结产生和线虫发育的影响。【结果】100~800 nmol/L ds-miR3979处理12~24 h对二龄幼虫的运动活性无显著影响；与ddH2O处理的对照相比，400 nmol/L ds-miR3979浸泡根系12~24 h显著提高了立针期和三叶期水稻根系和叶片miR3979-3p的表达水平；连续显微观察2~8 h，发现二龄幼虫向水稻根尖移动趋性降低，集聚在根尖周围的线虫数量显著减少；同时，平板接种1~3 d侵入根内的线虫数量减少，盆钵接种7~21 d根系上的根结数目、根内雌虫数量和占比显著下降，且雌虫的发育进程延缓。【结论】人工合成的ds-miR3979可被水稻吸收并上调内源miR3979-3p表达，进而影响根结线虫的侵染和雌虫发育。

关键词: 小RNA, miR3979-3p, 水稻, 拟禾本科根结线虫, 侵染, 发育

YANG Xingzhou, CUI Miaomiao, WEI Lihui, GU Aiguo, LI Dongxia, LE Xiuhu, FENG Hui. Effects of Exogenous miR3979 on Chemotaxis, Infection and Development of Root Knot Nematode Meloidogyne graminicola in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(5): 703-710.

杨行洲, 崔苗苗, 魏利辉, 顾爱国, 李东霞, 乐秀虎, 冯辉. 外源miR3979处理水稻对拟禾本科根结线虫趋性、侵染和发育的影响[J]. 中国水稻科学, 2025, 39(5): 703-710.

References

[1] Bologna N G, Voinnet O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis[J]. Annual Review of Plant Biology, 2014, 65: 473-503. [2] Borges F, Martienssen R A. The expanding world of small RNAs in plants[J]. Nature Reviews Molecular Cell Biology, 2015, 16(12): 727-741. [3] Molnar A, Melnyk C, Baulcombe D C. Silencing signals in plants: A long journey for small RNAs[J]. Genome Biology, 2011, 12(1): 215. [4] Li S, Wang X, Xu W, Liu T, Cai C, Chen L, Clark C B, Ma J. Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts[J]. Nature Plants, 2021, 7(1): 50-59. [5] Betti F, Ladera-Carmona M J, Weits D A, Ferri G, Iacopino S, Novi G, Svezia B, Kunkowska A B, Santaniello A, Piaggesi A, Loreti E, Perata P. Exogenous miRNAs induce post-transcriptional gene silencing in plants[J]. Nature Plants, 2021, 7(10): 1379-1388. [6] Li T, Li H, Zhang Y X, Liu J Y. Identification and analysis of seven H₂O₂-responsive miRNAs and 32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica)[J]. Nucleic Acids Research, 2011, 39(7): 2821-2833. [7] Nguyen D Q, Nguyen N L, Nguyen V T, Tran T H G, Nguyen T H, Nguyen T K L, Nguyen H H. Comparative analysis of microRNA expression profiles in shoot and root tissues of contrasting rice cultivars (Oryza sativa L.) with different salt stress tolerance[J]. PLoS One, 2023, 18(5): e0286140. [8] Bakhshi B, Mohseni Fard E, Nikpay N, Ali Ebrahimi M, Bihamta M R, Mardi M, Salekdeh G H. microRNA signatures of drought signaling in rice root[J]. PLoS One, 2016, 11(6): e0156814. [9] Liu Q, Zhang H. Molecular identification and analysis of arsenite stress-responsive miRNAs in rice[J]. Journal of Agricultural and Food Chemistry, 2012, 60(26): 6524-6536. [10] Sato Y, Nosaka-Takahashi M, Suzuki T, Shimizu-Sato S. Small RNAs in Rice: Molecular Species and Their Functions[M]. Rice Genomics, Genetics and Breeding, Singapore: Springer Nature, 2018: 21-36. [11] Xu D, Mou G, Wang K, Zhou G. MicroRNAs responding to southern rice black-streaked dwarf virus infection and their target genes associated with symptom development in rice[J]. Virus Research, 2014, 190: 60-68. [12] Wu Y, Lü W, Hu L, Rao W, Zeng Y, Zhu L, He Y, He G. Identification and analysis of brown planthopper-responsive microRNAs in resistant and susceptible rice plants[J]. Scientific Reports, 2017, 7(1): 8712. [13] Javed M, Reddy B, Sheoran N, Ganesan P, Kumar A. Unraveling the transcriptional network regulated by miRNAs in blast-resistant and blast-susceptible rice genotypes during Magnaporthe oryzae interaction[J]. Gene, 2023, 886: 147718. [14] Jeong D H, Park S, Zhai J, Gurazada S G R, De Paoli E, Meyers B C, Green P J. Massive analysis of rice small RNAs: Mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage[J]. The Plant Cell, 2011, 23(12): 4185-4207. [15] Mantelin S, Bellafiore S, Kyndt T. Meloidogyne graminicola: A major threat to rice agriculture[J]. Molecular Plant Pathology, 2016, 18: 3-15. [16] Kyndt T, Fernandez D, Gheysen G. Plant-parasitic nematode infections in rice: Molecular and cellular insights[J]. Annual Review of Phytopathology, 2014, 52: 135-153. [17] 刘国坤, 肖顺, 张绍升, 张敦富, 王玉. 拟禾本科根结线虫对水稻根系的侵染特性及其生活史 [J]. 热带作物学报, 2011, 32(4): 743-748. Liu G K, Xiao S, Zhang S S, Zhang D F, Wang Y. Infection characteristics and life cycle of Meloidogyne graminicola on rice roots[J]. Chinese Journal of Tropical Crops, 2011, 32(4): 743-748. (in Chinese with English abstract) [18] Niu Y, Xiao L, de Almeida-Engler J, Gheysen G, Peng D, Xiao X, Huang W, Wang G, Xiao Y. Morphological characterization reveals new insights into giant cell development of Meloidogyne graminicola on rice[J]. Planta, 2022, 255(3): 70. [19] Verstraeten B, Atighi M R, Ruiz-Ferrer V, Escobar C, de Meyer T, Kyndt T. Non-coding RNAs in the interaction between rice and Meloidogyne graminicola[J]. BMC Genomics, 2021, 22(1): 560. [20] Hui F, Zhou C R, Zhu F, Le X H, Jing D D, Daly P, Zhou D M, Wei L H. Resistance analysis of the rice variety Huaidao 5 against root-knot nematode Meloidogyne graminicola[J]. Journal of Integrative Agriculture, 2023, 22(10): 3081-3089. [21] 冯辉, 聂国嫒, 陈曦, 张金凤, 周冬梅, 魏利辉. 拟禾谷根结线虫江苏分离群体形态学和分子鉴定[J]. 江苏农业学报, 2017, 33(4): 794-801. Feng H, Nie G A, Chen X, Zhang J F, Zhou D M, Wei L H. Morphological and molecular identification of Meloidogyne graminicola isolates from Jiangsu Province[J]. Jiangsu Journal of Agricultural Sciences, 2017, 33(4): 794-801. (in Chinese with English abstract) [22] Wang C, Lower S, Williamson V M. Application of Pluronic gel to the study of root-knot nematode behaviour[J]. Nematology, 2009, 11(3): 453-464. [23] Cuperus J T, Fahlgren N, Carrington J C. Evolution and functional diversification of MIRNA genes[J]. The Plant Cell, 2011, 23(2): 431-442. [24] Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative CT method[J]. Nat Protocols, 2008, 3(6): 1101-1108. [25] Koeppe S, Kawchuk L, Kalischuk M. RNA Interference past and future applications in plants[J]. International Journal of Molecular Sciences, 2023, 24(11): 9755. [26] Bilir Ö, Göl D, Hong Y, McDowell J M, Tör M. Small RNA-based plant protection against diseases[J]. Frontiers in Plant Science, 2022, 13: 951097. [27] Dalakouras A, Wassenegger M, Dadami E, Ganopoulos I, Pappas M L, Papadopoulou K. Genetically modified organism-free RNA interference: Exogenous application of RNA molecules in plants[J]. Plant Physiology, 2020, 182(1): 38-50. [28] Niu D, Hamby R, Sanchez J N, Cai Q, Yan Q, Jin H. RNAs: A new frontier in crop protection[J]. Current Opinion in Biotechnology, 2021, 70: 204-212. [29] 王帅, 魏钰洋, 张羲, 谢佳, 胡展, 孙然锋. 根结线虫趋化性研究进展 [J]. 农药学学报, 2022, 24(5): 982-996. DOI: 10.16801/j.issn.1008-7303.2022.0090. Wang S, Wei Y Y, Zhang X, Xie J, Hu Z, Sun R F. Research progress on chemotaxis of root-knot nematodes[J]. Chinese Journal of Pesticide Science, 2022, 24(5): 982-996. DOI: 10.16801/j.issn.1008-7303.2022.0090. (in Chinese with English abstract) [30] Ghorbanzadeh Z, Hamid R, Jacob F, Mirzaei M, Zeinalabedini M, Abdirad S, Atwell B J, Haynes P A, Ghafari M R, Salekdeh G H. MicroRNA profiling of root meristematic zone in contrasting genotypes reveals novel insight into rice response to water deficiency[J]. Journal of Plant Growth Regulation, 2022, 42(6): 3814-3834. [31] 张静文. 普通野生稻(Oryza rufipogon Griff.)干旱胁迫相关的 microRNAs 的鉴定与功能分析[D]. 北京: 中国农业大学，2017. Zhang J W. Identification and functional analysis of drought stress-related microRNAs in Oryza rufipogon Griff.[D]. Beijing: China Agricultural University, 2017. (in Chinese) [32] Chowdhury A T, Hasan M N, Bhuiyan F H, Islam M Q, Nayon M R W, Rahaman M M, Hoque H, Jewel N A, Ashrafuzzaman M, Prodhan S H. Identification, characterization of Apyrase (APY) gene family in rice (Oryza sativa) and analysis of the expression pattern under various stress conditions[J]. PLoS One, 2023, 18(5): e0273592.