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Integrative Zoology 2023年第3期18卷 543-551页

作者： Lin TIAN Xixuan LI Yaqi YING Lewen WANG Yanting QIAO Dawei WANG Ying SONG Ning LI Xiaohui LIU state key laboratory for biology of plant diseases and insect pests Key Laboratory of Weed and Rodent Biology and ManagementInstitute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina

The bicoid-related transcription factor 2(Pitx2)plays a crucial role in the development of many organs and tissues by affecting the mitotic cell cycle.Postnatal testis development is related to mitosis and meiosis in mul-tiple cell types,but the role of Pitx2 gene in seasonal inhibition of testicular development remains unknown in rodents.We analyzed PITX2 protein and Pitx2 mRNA expression features using both laboratory and wild male Rattus norvegicus caraco.In postnatal testicle of laboratory colony,we found that PITX2 was expressed in Leydig cells,pachytene spermatocytes,round spermatids,and elongating spermatids rather than spermatogonia and lep-totene/zygotene spermatocytes.Pitx2b expression signiﬁcantly increased along with the occurrence of pachytene spermatocytes and round spermatids,while decreased along with the processes of elongated spermatids.In wild male rats with similar testes weight,a signiﬁcantly suppressed Pitx2b expression occurred with an active meiotic stage in the inhibited testes in autumn and winter,compared with the normally developing testes in spring and summer.These results indicate that Pitx2b expression suppression plays a crucial role in the seasonal inhibition of testis development.

关键词： Pitx2 postnatal testis development Rattus norvegicus seasonal inhibition

Geminiviral C2 proteins inhibit active autophagy to facilitate virus infection by impairing the interaction of ATG7 and ATG8

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Journal of Integrative plant biology 2023年第5期65卷 1328-1343页

作者： Buwei Cao Linhao Ge Mingzhen Zhang Fangfang Li Xueping Zhou state key laboratory for biology of plant diseases and insect pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China state key laboratory of Rice biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Autophagy is a conserved intracellular degradation process that plays an active role in plant response to virus infections.Here we report that geminiviruses counteract activated autophagymediated antiviral defense in plant cells through the C2 proteins they encode.We found that,in Nicotiana benthamiana plants,tomato leaf curl Yunnan virus(TLCYnV)infection upregulated the transcription levels of autophagy-related genes(ATGs).Overexpression of NbATG5,NbATG7,or NbATG8a in N.benthamiana plants decreased TLCYnV accumulation and attenuated viral symptoms.Interestingly,transgenic overexpression of NbATG7 promoted the growth of N.benthamiana plants and enhanced plant resistance to TLCYnV.We further revealed that the C2 protein encoded by TLCYnV directly interacted with the ubiquitinactivating domain of ATG7.This interaction competitively disrupted the ATG7–ATG8 binding in N.benthamiana and Solanum lycopersicum plants,thereby inhibiting autophagy activity.Furthermore,we uncovered that the C2-mediated autophagy inhibition mechanism was conserved in three other geminiviruses.In summary,we discovered a novel counter-defensive strategy employed by geminiviruses that enlists their C2 proteins as disrupters of ATG7–ATG8 interactions to defeat antiviral autophagy.

关键词： autophagy C2 counterdefense geminivirus plant defense the ATG7–ATG8 interaction

Pepino mosaic virus antagonizes plant m^(6)A modification by promoting the autophagic degradation of the m^(6)A writer HAKAI

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aBIOTECH 2023年第2期4卷 83-96页

作者： Hao He Linhao Ge Zhaolei Li Xueping Zhou Fangfang Li state key laboratory for biology of plant diseases and insect pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing100193China state key laboratory of Rice biology Institute of BiotechnologyZhejiang UniversityHangzhou310058China

Autophagy plays an active anti-viral role in plants.Increasing evidence suggests that viruses can inhibit or manipulate autophagy,thereby winning the arms race between plants and viruses.Here,we demonstrate that overexpression of an m^(6)A writer from Solanum lycopersicum,SlHAKAI,could negatively regulate pepino mosaic virus(PepMV)infection,inhibit viral RNA and protein accumulations by affecting viral m^(6)A levels in tomato plants and vice versa.The PepMV-encoded RNA-dependent RNA polymerase(RdRP)directly interacts with SlHAKAI and reduces its protein accumulation.The RdRP-mediated decreased protein accumulation of SlHAKAI is sensitive to the autophagy inhibitor 3-methyladenine and is compromised by knocking down a core autophagy gene.Furthermore,PepMV RdRP could interact with an essential autophagy-related protein,SlBeclin1.RdRP,SlHAKAI,and SlBeclin1 interaction complexes form bright granules in the cytoplasm.Silencing of Beclin1 in Nicotiana benthamiana plants abolishes the RdRP-mediated degradation of SlHAKAI,indicating the requirement of Beclin1 in this process.This study uncovers that the PepMV RdRP exploits the autophagy pathway by interacting with SlBeclin1 to promote the autophagic degradation of the SlHAKAI protein,thereby inhibiting the m^(6)A modification-mediated plant defense responses.

关键词： Pepino mosaic virus HAKAI Autophagy Beclin1

Silencing of odorant binding protein gene AlinOBP4 by RNAi induces declining electrophysiological responses of Adelphocoris lineolatus to six semiochemicals

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insect Science 2017年第5期24卷 789-797页

作者： Xue-Ying Zhang Xiao-Qiang Zhu Shao-Hua Gu Yan-Le Zhou Song-Ying Wang Yong-Jun Zhang Yu-Yuan Guo state key laboratory for biology of plant diseases and insect pests Institute of Plant Protection Chinese Academy of AgriculturalSciences Beijing China

Odorant binding proteins （OBPs） are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction （RT-PCR） analysis, quantitative real-time PCR （qPCR） test and electroantennogram （EAG） assay. The combination of RT-PCR and qPCR analyses revealed that the levels of mes- senger RNA transcript were significantly reduced ~95% in AlinOBP4-siRNA-treated A. lineolatus males and ~75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tride- canal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, （E）-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identifi- cation of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.

关键词： Adelphocoris lineolatus electrophysiological responses odorant bindingprotein AlinOBP4 RNA interference

Relationship between copulation and cold hardiness in Ophraella communa(Coleoptera: Chrysomelidae)

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Journal of Integrative Agriculture 2019年第4期18卷 900-906页

作者： ZHAO Chen-chen YUE Lei WANG Yao GUO Jian-ying ZHOU Zhong-shi WAN Fang-hao state key laboratory for biology of plant diseases and insect pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

Ophraella communa(Coleoptera: Chrysomelidae), the ragweed leaf beetle, is a biological control agent of the invasive common ragweed, Ambrosia artemisiifolia(Asterales: Asteraceae). Adults can survive cold conditions that occur during winter. The adults mate before entering overwintering. Understanding the connection between copulation and overwintering will be useful for determining O. communa seasonality. Determining the relationship between overwintering and copulation required comparison of mated and unmated beetles at mean lethal temperature(LTemp_(50)) exposures for 2 h. Cold-related physiological indices, including the water ratio, super cooling point(SCP), cryoprotectant levels, and energy reserve levels, were also measured. Mating treatment decreased the LTemp_(50) of both sexes by reducing their mean SCP and water ratios. Although the changes of cryoprotectant levels in mated adults were not precisely consistent in between the genders, they increased greatly in both males and females. Body sugar may play a role in copulation and may also elevate cold hardiness in O. communa.

关键词： Ophraella communa mating status cold toleranee hibernation super cooling point energy reserves

Alanine-substituted mutant on Gly^(373) and Asn^(375) of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera

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Journal of Integrative Agriculture 2019年第5期18卷 1064-1071页

作者： LIU Yu-xiao ZHOU Zi-shan LIANG Ge-mei SONG Fu-ping ZHANG Jie state key laboratory for biology of plant diseases and insect pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.

关键词： Bacillus thuringiensis Cry1Ai Domain Ⅱ-loop2 Helicoverpa armigera binding affinity

More stressful event does not always depress subsequent life performance

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Journal of Integrative Agriculture 2019年第10期18卷 2321-2329页

作者： CHEN Ying-ying ZHANG Wei MA Gang MA Chun-sen state key laboratory for biology of plant diseases and insect pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

Climate change has led to a substantial increase in intensity and duration of heat waves worldwide.Predicting the ecological impacts of hot events should incorporate both immediate and potential carry-over effects in different intensities of heat waves. Previous studies suggested that higher heat dose in early life stage of insect generally decreased immediate survival and depressed adult reproduction through carry-over effects, or unchanged adult performance through recovery effects. However, our previous study showed a different pattern, in which longer heat exposures in larval stage did not always decrease but sometimes increase the subsequent adult maturation success in the diamondback moth. We speculated that it might be another important pattern in the carry-over effects vs. heat dose, and conducted experiments using a global pest, Plutella xylostella. Our present results suggested that heat exposures in early life stage reduced the immediate survival and produced general declines with significant zigzag fluctuating patterns in subsequent body size and reproduction as exposure durations increased. The similar patterns were also validated in other insect taxa and other stresses by reanalyzing the experiment data from literatures. The finding highlights the importance for differentiating the biological effects and consequences of changes in heat dose at fine scales;daily exposure hours of a hot day should be considered to predict population dynamic under climate change.

关键词： climate change Plutella xylostella extreme temperature reproduction carry-over effect

Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda

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insect Science 2023年第3期30卷 625-636页

作者： Wei-Kang Han Feng-Xian Tang Hao-Li Gao Yan Wang Na Yu Jian-Jun Jiang Ze-Wen Liu key laboratory of Integrated Management of Crop diseases and pests(Ministry of Education) College of Plant ProtectionNanjing Agricultural UniversityNanjingChina plant protection Research institute Guangxi Academy of Agricultural ScienceGuangxi Key Laboratory of Biology for Crop Diseases and Insect PestsNanningChina

The CRISPR/Cas9 system has been successfully applied in dozens of diverse species;although the screening of successful CRISPR/Cas9 editing events remains particularly laborious,especially for those that occur at relatively low frequency.Recently,a co-CRISPR strategy was proved to enrich the desired CRISPR events.Here,the co-CRISPR strategy was developed in the Fall armyworm,Spodoptera frugiperda,with kynurenine 3-monooxygenase gene(kmo)as a marker.The kmo mosaics induced by single-guide RNAs(sgRNAs)/Cas9 displayed the darker green color phenotype in larvae,compared with wild type(brown),and mosaic-eye adults were significantly acquired from the mosaic larvae group.In the kmo knockout strain,no significant difference was observed in larval development and adult reproduction.Acetylcholinesterase 2(ace2)and Wntl were selected as target genes to construct the co-CRISPR strategy using kmo marker.By coinjection of kmo and ace2 sgRNAs,the mutant efficiency of ace2 was significantly increased in the kmo mosaic(larvae or adults)groups.Similarly,more malformed pupae with Wntl mutations were observed in the darker green larvae group.Taken together,these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm.Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker,the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals.The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.

关键词： co-CRISPR kmo mutation enrichment Spodoptera frugiperda Wnt1

Harnessing nanobodies to expand the recognition spectrumof plant NLRs for diverse pathogens

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aBIOTECH 2023年第3期4卷 272-276页

作者： Ruyi Wang Guo-Liang Wang Yuese Ning state key laboratory for biology of plant diseases and insect pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Department of plant Pathology The Ohio State UniversityColumbusOH 43210USA

The strategy to expand the recognition spectrum of plant nucleotide-binding domain leucine-richrepeat (NLR) proteins by modifying their recognition sequences is generally limited and oftenunsuccessful. Kourelis et al. introduced a groundbreaking approach for generating a customizedimmune receptor, called Pikobody. This method involves integrating a nanobody domain of a fluorescent protein (FP) into a plant NLR. Their research demonstrates that the resulting Pikobody successfully initiates an immune response against diverse pathogens when exposed to the corresponding FP.

关键词： NLR Pik Nanobody Fluorescent protein Immunity

m^(6)A modification of plant virus enables host recognition by NMD factors in plants

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Science China(Life Sciences) 2024年第1期67卷 161-174页

作者： Hao He Linhao Ge Yalin Chen Siwen Zhao Zhaolei Li Xueping Zhou Fangfang Li state key laboratory for biology of plant diseases and insect pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China state key laboratory of Rice biology Institute of BiotechnologyZhejiang UniversityHangzhou 310029China

N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defense mechanism in mammals and plants,but it is largely unknown how m^(6)Aregulates viral infection in plants.Here we report the dynamic changes and functional anatomy of m^(6)Ain Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus(PepMV)infection.m^(6)Amodification in the PepMV RNA genome is conserved in these two species.Overexpression of the m^(6)Awriters,mRNA adenosine methylase A(MTA),and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m^(6)Amodifications,whereas deficiency of these writers decreases the viral RNA m^(6)Alevels but enhances virus infection.Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m^(6)Areaders are involved in anti-viral immunity.Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay(NMD)-related proteins,including NbUPF3 and NbSMG7,but not with NbUPF1.m^(6)Amodification-mediated restriction to PepMV infection is dependent on NMD-related factors.These findings provide new insights into the functionality of m^(6)Aanti-viral activity and reveal a distinct immune response that NMD factors recognize the m^(6)Areaders-viral m^(6)ARNA complex for viral RNA degradation to limit virus infection in plants.

关键词： m^(6)A Pepino mosaic virus plant defense m^(6)A readers NMD factors