黑尾叶蝉PGRP6负调控共生菌抑制水稻矮缩病毒的经卵传播

doi:10.3864/j.issn.0578-1752.2025.05.007

摘要/Abstract

摘要：

【目的】水稻矮缩病毒（rice dwarf virus，RDV）引起的水稻矮缩病是危害中国南方水稻生产的重要病害。结合前人的研究基础，利用分子生物学等手段，解析肽聚糖识别蛋白6（PGRP6）参与调控媒介昆虫黑尾叶蝉（Nephotettix cincticeps）体内共生菌携带RDV经卵垂直传播的机制，为水稻病毒病的生物防治提供理论依据。【方法】利用酵母双杂交技术筛选与经卵传播相关蛋白（prp、Nasuia porin、P8）均互作的PGRP，并通过GST pull-down进一步验证其互作关系；利用实时荧光定量PCR技术测定相关基因在无毒和带毒虫体内的表达差异，并分析干扰PGRP6、prp后对共生菌增殖的影响，同时通过Western blot检测对昆虫体内共生菌膜蛋白积累的影响；分析抑制PGRP6表达对叶蝉存活率、子代带毒率、带毒子代的病毒核酸水平的影响，并利用荧光原位杂交（FISH）和免疫荧光标记观察叶蝉卵巢内prp、PGRP6、Nasuia及RDV的分布情况；通过抑菌圈和肽聚糖降解实验验证PGRP6的功能。【结果】黑尾叶蝉的PGRP6与prp、Nasuia porin、P8均存在互作；PGRP6和prp在RDV侵染的黑尾叶蝉中的表达量均显著升高；抑制PGRP6表达可促进共生菌Nasuia和Sulcia的增殖，而抑制prp降低了共生菌的增殖，其功能与PGRP6相反，当两者同时被干扰后对共生菌的增殖无显著影响；dsPGRP6处理导致黑尾叶蝉存活率显著降低，子代带毒率增加，子代昆虫体内的病毒量明显高于对照组；免疫荧光标记发现PGRP6与prp、Nasuia、RDV均在卵巢组织中分布并发生共定位，prp和Nasuia是包裹的位置关系；PGPR6存在抑菌功能，可发挥抑制共生菌增殖的作用。【结论】PGRP6通过抑制黑尾叶蝉体内共生菌的增殖维持卵巢组织内的稳态，其与prp的功能相拮抗，其既确保了黑尾叶蝉的生命活力，又调控了共生菌携带RDV的垂直传播。

关键词: 水稻矮缩病毒, 黑尾叶蝉, 肽聚糖识别蛋白, 富脯氨酸蛋白, 共生菌, 卵巢, 经卵传播, 抑菌

Abstract:

【Objective】Rice dwarf virus (RDV) causes rice dwarf disease, which is a significant threat to rice production in southern China. This study builds on prior research and employs molecular biology techniques to investigate the role of PGRP6 in regulating the vertical transmission of RDV from symbiotic bacteria within the vector insect Nephotettix cincticeps, thereby establishing a foundation for biological control strategies against rice viral diseases.【Method】Yeast two-hybrid technology was used to screen peptidoglycan recognition proteins (PGRPs) that interact with three proteins (prp, Nasuia porin, and P8) related to transovarial transmission, and the interaction was further confirmed by GST pull-down assay. Real-time fluorescence quantitative PCR technology was employed to assess the differential expression of related genes in nonviruliferous and viruliferous insects, as well as the changes in transcription levels of symbiotic bacteria following interference with PGRP6 and prp. Western blot experiments further confirmed the impact of RNAi treatment on symbiotic bacterial membrane proteins in insects. Following the reduction of PGRP6 expression by RNAi, variations were observed in the survival rate of N. cincticeps, the virus infection rate of the offspring, and the viral nucleic acid level of the infected offspring. Fluorescence in situ hybridization (FISH) and fluorescent immunolabeling techniques were employed to visualize the distribution of prp, PGRP6, Nasuia, and RDV. The functionality of the PGRP6 protein was assessed through inhibition zone assays and experiments on peptidoglycan degradation.【Result】PGRP6 interacted with prp, Nasuia porin, and P8. The expression levels of PGRP6 and prp were significantly elevated in RDV-infected N. cincticeps. Suppression of PGRP6 expression could facilitate the proliferation of symbiotic bacteria Nasuia and Sulcia, while inhibition of prp reduced the proliferation of symbiotic bacteria, acting oppositely to PGRP6. When both were interfered simultaneously, there was no significant effect on the proliferation of symbiotic bacteria. Treatment with dsPGRP6 significantly decreased the survival rate of N. cincticeps and enhanced the transmission of RDV to their offspring, resulting in a substantially higher viral load in the progeny compared to the control group. Immunofluorescence analysis revealed that PGRP6 co-localized with prp, Nasuia, and RDV within ovarian tissue, where prp and Nasuia exhibited a wrapping relationship. Ultimately, PGPR6 was identified as possessing antibacterial properties that inhibited the proliferation of symbiotic bacteria.【Conclusion】PGRP6 maintains the homeostasis of ovarian tissue in N. cincticeps by inhibiting the proliferation of symbiotic bacteria, which functionally antagonizes prp. This mechanism not only ensures the vitality of N. cincticeps but also regulates the transovarial transmission of RDV associated with bacterial symbionts.

Key words: rice dwarf virus (RDV), Nephotettix cincticeps, peptidoglycan recognition protein (PGRP), proline-rich protein (prp), symbiotic bacterium, ovary, transovarial transmission, antibacterial

徐元元, 贾东升, 宾羽, 魏太云. 黑尾叶蝉PGRP6负调控共生菌抑制水稻矮缩病毒的经卵传播[J]. 中国农业科学, 2025, 58(5): 907-917.

XU YuanYuan, JIA DongSheng, BIN Yu, WEI TaiYun. PGRP6 Negatively Regulates Symbiotic Bacteria to Prevent the Transovarial Transmission of RDV in Nephotettix cincticeps[J]. Scientia Agricultura Sinica, 2025, 58(5): 907-917.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.05.007

https://www.chinaagrisci.com/CN/Y2025/V58/I5/907

图/表 5

表1

本研究所用引物序列"

引物名称Primer name	序列Sequence (5′-3′)
AD-PGRP3 F	ccagattacgctcatATGGGTTTAGGTATTGATGAAAATATAAATACCTATTACAAAGT
AD-PGRP3 R	agctcgagctcgatgTTACGCAGCTTGTCTAAGATTCGGGT
AD-PGRP4 F	ccagattacgctcatATGGGGTTGATGGATAAACCACC
AD-PGRP4 R	agctcgagctcgatgCTAGTAATCACCATATTTCCTTCGGTATTCTTCT
AD-PGRP6 F	ccagattacgctcatATGGGATTGTGGTCTTCAAAAGAGG
AD-PGRP6 R	agctcgagctcgatgCTACTCATGCCCTAGAATGTCGC
AD-PGRP7 F	ccagattacgctcatATGTCCGCACCAGAACCAG
AD-PGRP7 R	agctcgagctcgatgTTAAAATAATTTTGTCTCCCCACGGAAAGACTTAACTG
AD-PGRP11 F	ccagattacgctcatATGGCAAGCCCCGTAGTG
AD-PGRP11 R	agctcgagctcgatgTTAAAACAAGTCGACTCTTTCAAAGTTCTGT
AD-PGRP-12 F	ccagattacgctcatATGGGGGACATACCAGATGCT
AD-PGRP-12 R	agctcgagctcgatgTTATTGTTTAGGCGTGATCTGTTGATTTGTTCG
AD-PGRP14 F	ccagattacgctcatATGGGGAAAGATAAGTACAACCCATTTAGG
AD-PGRP14 R	agctcgagctcgatgTTATGAATAGTAAGGGTGGCGAGTGG
AD-PGRP18 F	ccagattacgctcatATGAAACCTATGCATAAACCAATACAAATAGCAATGTTTCT
AD-PGRP18 R	agctcgagctcgatgTCAGTCCCACGGAGGCTCGG
BD-prp F	agaggaggacctgcatatggATGCGTTATCATAAGGACTT
BD-prp R	ttatgcggccgctgcTAGTTATGCGGCCGCTGCTTAAAAGTCTTCAAGCATTG
BD-Nasuia_porin F	gaggacctgcatatgATGTCAAATTCAATTTATTTTTTTTTTATAG
BD-Nasuia_porin R	ttatgcggccgctgcTTAATCTAATAATTTTAAAAATTTTTTATAATAAG
BD-RDV P8 F	gaggacctgcatatgATGTCACGCCAGATGTGGT
BD-RDV P8 R	ttatgcggccgctgcCTAATTTGGTCTATAGTATCTTCCAAATACGGCG
Q-EF1 F	CAGTGAGAGCCGTTTTGAG
Q-EF1 R	AGGGCATCTTGTCAGAGGGC
Q-prp F	ATGTAACCACTCGTGCACCC
Q-prp R	CAACATTTCCGTGTCGCACT
Q-PGRP6 F	AGCGACATTCTAGGGCATGA
Q-PGRP6 R	ACAGCAGTCAACGTAAAGGC
Q-Nasuia 16S F	GGGGAAAACCTCGCGTTATA
Q-Nasuia 16S R	CCACTGCTGCCTCTCGTAAG
Q-Sulcia 16S F	GGGGACTCTAATAAGACTGC
Q-Sulcia 16S R	CTGAGATCGGCTTTCTGGAT
PGEX-RDV-P8 F	aaatcggatctggttATGTCACGCCAGATGTGGTTAG
PGEX-RDV-P8 R	gtcagtcacgatgcgCTAATTTGGTCTATAGTATCTTCCA
PGEX-Nasuia_porin F	aaatcggatctggCGTGGATCCCCGATGTCAAATTCAATTTATTT TTTTTTTATAG
PGEX-Nasuia_porin R	gtcagtcacgatgcgGATGAATTCCGGTTAATCTAATAATTTTAAAAATTT TTTATAATAAG
PGEX-PGRP6 F	aaatcggatctggttATGGGATTGTGGTCTTCAAAAGAGG
PGEX-PGRP6 R	gtcagtcacgatgcgCTCATGCCCTAGAATGTCGC
PET-prp F	gatataccatgggcgATGGTGAAAGTTGGAACCTCTTACG
PET-prp R	gagcggccgcaagctGAAAAGCTTTGGTTTGGGAGCG
PET-PGRP6 F	ggtggacagcaaatgATGGGATTGTGGTCTTCAAAAGAGG
PET-PGRP6 R	gtgctcgagtgcggcCTACTCATGCCCTAGAATGTCGCT
T7-GFP-F	attctctagaagcttaatacgactcactatagggATGAGTAAAGGAGAAGAACTT
T7-GFP-R	attctctagaagcttaatacgactcactatagggTTATTTGTATAGTTCATCCATG
T7-PGRP6 F	attctctagaagcttaatacgactcactatagggTCTTTTCTGAGCGACGGTGT
T7-PGRP6 R	attctctagaagcttaatacgactcactatagggACTGTTCCTGGCTGTATTGC
T7-prp F	attctctagaagcttaatacgactcactatagggACCTCTTACGTGCCGATCAA
T7-prp R	attctctagaagcttaatacgactcactatagggTTTGGTTTGGGAGCGCTTTT

表1

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