STAR Protocols
·
2021
·
IF 0
Yunjing Wang,Qian Gong,Fan Huang,Linfang He,Yule Liu
Summary Wounding evokes transient increases in cytosolic calcium (Ca 2+ ) concentration. Visualizing real-time Ca 2+ flux provides new insights into Ca 2+ -signaling pathways. Here, we outline a protocol to detect insect feeding-induced Ca 2+ flux elevation in Nicotiana benthamiana leaves based on the GCaMP3 reporter system by Leica fluorescence stereo microscopes (LFSM). LFSM combines super-fast manual screening with high-end imaging capabilities. Through this protocol, we can clearly observe the calcium flow after aphid’s piercing-sucking. Additionally, we describe a protocol to quantify Ca 2+ level using LFSM. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .
研究领域:
植物生物学
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分子生物学
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昆虫学
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DOI
PubMed
MOLECULAR PLANT PATHOLOGY
·
2021
·Vol. 22
·期 12
·1668-1687
·
IF 5.663
Feng Zhu,Qi-Ping Zhang,Yan-Ping Che,Peng-Xiang Zhu,Qin-Qin Zhang,Zhao-Lin Ji
Systemic acquired resistance (SAR) is induced by pathogens and confers protection against a broad range of pathogens. Several SAR signals have been characterized, but the nature of the other unknown signalling by small metabolites in SAR remains unclear. Glutathione (GSH) has long been implicated in the defence reaction against biotic stress. However, the mechanism that GSH increases plant tolerance against virus infection is not entirely known. Here, a combination of a chemical, virus-induced gene-silencing-based genetics approach, and transgenic technology was undertaken to investigate the role of GSH in plant viral resistance in Nicotiana benthamiana . Tobacco mosaic virus (TMV) infection results in increasing the expression of GSH biosynthesis genes NbECS and NbGS , and GSH content. Silencing of NbECS or NbGS accelerated oxidative damage, increased accumulation of reactive oxygen species (ROS), compromised plant resistance to TMV, and suppressed the salicylic acid (SA)-mediated signalling pathway. Application of GSH or l -2-oxothiazolidine-4-carboxylic acid (a GSH activator) alleviated oxidative damage, decreased accumulation of ROS, elevated plant local and systemic resistance, enhanced the SA-mediated signalling pathway, and increased the expression of ROS scavenging-related genes. However, treatment with buthionine sulfoximine (a GSH inhibitor) accelerated oxidative damage, elevated ROS accumulation, compromised plant systemic resistance, suppressed the SA-mediated signalling pathway, and reduced the expression of ROS-regulating genes. Overexpression of NbECS reduced oxidative damage, decreased accumulation of ROS, increased resistance to TMV, activated the SA-mediated signalling pathway, and increased the expression of the ROS scavenging-related genes. We present molecular evidence suggesting GSH is essential for both local and systemic resistance of N . benthamiana to TMV through a differential modulation of SA and ROS.
研究领域:
植物病理学
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分子生物学
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植物科学
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产品关联
DOI
PubMed
高影响因子
PHYTOCHEMISTRY
·
2019
·
IF 2.905
Fei Luo,Yi Ling,De-Sen Li,Ting Tang,Yan-Chun Liu,Yan Liu,Sheng-Hong Li
Cedrol is an extremely versatile sesquiterpene alcohol that was approved by the Food and Drug Administration of the United States as a flavoring agent or adjuvant and has been commonly used as a flavoring ingredient in cosmetics, foods and medicine. Furthermore, cedrol possesses a wide range of pharmacological properties including sedative, anti-inflammatory and cytotoxic activities. Commercial production of cedrol relies on fractional distillation of cedar wood oils , followed by recrystallization, and little has been reported about its biosynthesis and aspects of synthetic biology . Here, we report the cloning and functional characterization of a cedrol synthase gene ( Lc-CedS ) from the transcriptome of the glandular trichomes of a woody Lamiaceae plant Leucosceptrum canum . The recombinant Lc-CedS protein catalyzed the in vitro conversion of farnesyl diphosphate into the single product cedrol, suggesting that Lc-CedS is a high-fidelity terpene synthase. Co-expression of Lc-CedS , a farnesyl diphosphate synthase gene and seven genes of the mevalonate (MVA) pathway responsible for converting acetyl-CoA into farnesyl diphosphate in Escherichia coli afforded 363 μg/L cedrol as the sole product under shaking flask conditions. Transient expression of Lc-CedS in Nicotiana benthamiana also resulted in a single product cedrol with a production level of 3.6 μg/g fresh weight. The sole production of cedrol by introducing of Lc-CedS in engineered E. coli and N. benthamiana suggests now alternative production systems using synthetic biology approaches that would better address sufficient supply of cedrol.
研究领域:
植物生物化学
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合成生物学
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代谢工程
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DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2021
·Vol. 19
·期 1
·1-14
·
IF 10.435
Wang Yuhui,Xie Zhuomi,Wang Xiuhua,Peng Xin,Zheng Jianping
Background Fluorescent carbon-dots (CDs) with multifaceted advantages have provided hope for improvement of crop growth. Near infrared (NIR) CDs would be more competitive and promising than short-wavelength emissive CDs, which are not directly utilized by chloroplast. The molecular targets and underlying mechanism of these stimulative effects are rarely mentioned. Results NIR-CDs with good mono-dispersity and hydrophily were easily prepared by a one-step microwave-assisted carbonization manner, which showed obvious UV absorptive and far-red emissive properties. The chloroplast-CDs complexes could accelerate the electron transfer from photosystem II (PS II) to photosystem I (PS I). NIR-CDs exhibited a concentration-dependent promotion effect on N. benthamiana growth by strengthening photosynthesis. We firstly demonstrated that potential mechanisms behind the photosynthesis-stimulating activity might be related to up-regulated expression of the photosynthesis and chloroplast synthesis related genes, among which PsbP and PsiK genes are the key regulators. Conclusion These results illustrated that NIR-CDs showed great potential in the applications to increase crop yields through ultraviolet light harvesting and elevated photosynthesis efficiency. This work would provide a theoretical basis for the understanding and applications of the luminescent nanomaterials (not limited to CDs) in the sunlight conversion-related sustainable agriculture. Graphic abstract
研究领域:
农业
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植物生理学
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分子生物学
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纳米材料科学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Zilin Wu,Min Huang,Junmei Jiang,Chun Zhang,Gefei Hao,Moxian Chen,Qing X. Li,Mengao Jia,Junfeng Liu,Xiangyang Li
Ningnanmycin has been proven to effectively inhibit infection by potato virus Y (PVY), although its underlying mechanisms remain unclear. In this study, we report a novel finding that ningnanmycin affects PVY replication. Two approaches were employed: studies using PVY movement-deficient mutants suggest that ningnanmycin, at a concentration of 500 μg/mL, inhibits PVY replication. Ningnanmycin interacts with the PVY-encoded coat protein (CP) with a binding constant of 1.34 μmol/L, and key amino acids Glu168 and Thr206 are involved in this interaction. Additionally, ningnanmycin induces the expression of antiviral response genes in Nicotiana benthamiana, including PRXIIB, PRXIIE, PUB4, and PER42. Furthermore, studies revealed that the overexpression of PUB4 in N. benthamiana confers resistance to PVY infection. These findings highlight the mechanisms by which ningnanmycin activates the PUB4 gene and suppresses CP assembly, thereby inhibiting PVY in N. benthamiana. This study represents an important step toward elucidating the molecular mechanism underlying the antiviral activity of ningnanmycin.
研究领域:
植物病理学
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病毒学
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分子生物学
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植物生物技术
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产品关联
DOI
PubMed
高影响因子
Phytopathology Research
·
2022
·Vol. 4
·期 1
·1-12
·
IF 3.955
Jiang Long,Xiang Shunyu,Lv Xing,Wang Xiaoyan,Li Fengwei,Liu Weina,Liu Changyun,Ran Mao,Huang Jin,Xu Xiaohong,Ma Xiaozhou,Jin Yabo,Sun Xianchao
Silver (Ag)-containing agents or materials are widely used today in plant protection for their antimicrobial activity. In view of the superior inhibitory ability of biosynthesized (aldehyde-modified sodium alginate based) silver nanoparticles (AgNPs) against plant pathogenic fungi in our previous research, here we explored the antagonistic effect of biosynthesized AgNPs on plant pathogenic bacteria and the underlying mechanism. We selected Pseudomonas syringae pv. tabaci , the causal agent of tobacco wildfire disease, as the target and found that 1.2 μg/mL biosynthesized AgNPs completely inhibited the growth of P. syringae pv. tabaci in vitro and in vivo by partly destroying the cell membrane structure of the pathogen, resulting in cytoplasmic leakage. Moreover, Nicotiana benthamiana treated with 1.2 μg/mL biosynthesized AgNPs exhibited a significant upregulation of nonexpressor of pathogenesis-related genes 1 ( NPR1 ) and pathogenesis-related gene 2 ( PR2 ), the typical markers of the salicylic acid (SA)-mediated defense system, and an increase in peroxidase (POD) and polyphenol oxidase (PPO) activities as well as the production of reactive oxygen species (ROS). Furthermore, biosynthesized AgNPs treatment increased the chlorophyll content and dry weight of N. benthamiana . Overall, we demonstrated that biosynthesized AgNPs at a low concentration have high inhibitory effect on the pathogen causing tobacco wildfire disease by destroying bacterial cell membrane and inducing defense resistance in host plant. These results lay a theoretical foundation for further application of biosynthesized AgNPs in the control of plant bacterial diseases.
研究领域:
植物病理学
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纳米技术
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分子植物-微生物互作
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DOI
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2025
·Vol. 26
·期 15
·7170
·
IF 4.9
Maria Alievna Abdullina,Jiarui Li,Feifan Liu,Xinyi Luo,Anastasia Igorevna Barsukova,Svetlana Vladimirovna Trusova
Plant subtilases, as hydrolytic enzymes, contribute to certain plant stress response pathways by cleaving precursor proteins into active peptides or through other less well-characterized mechanisms. Phytaspases represent a specific subgroup of subtilases, and their participation in rapid stress responses, particularly to herbivory attacks and drought, is already well established, in contrast to their poorly understood role in long-term responses. This study investigated the involvement of phytaspase NbSBT1.9-2 in the long-term stress responses ofNicotiana benthamiana. Plants were subjected to either mild to severe mechanical wounding or drought stress, followed by the detection of phytaspase activity and gene expression in the leaf tissue. The results revealed a distinct involvement of phytaspase in the wounding response, showing increased activity and upregulated expression correlated with the extent and recurrence of wounding. In contrast, no significant change in phytaspase activity was observed in the leaves under drought, alongside salinity and heat stress conditions. Consequently, phytaspase association with the long-term response to mechanical injury was demonstrated usingN. benthamianaas a model organism.
研究领域:
植物生物学
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胁迫生理学
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分子植物学
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酶学
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产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2017
·Vol. 7
·期 34
·21028-21035
·
IF 3.108
Yuyu Liu,Xiaohui Wang,Ting Mo,Yaru Yan,Yuelin Song,Yunfang Zhao,Jun Li,Shepo Shi,Xiao Liu,Pengfei Tu
Recently, owing to the important pharmaceutical properties of malonylated glycosides, their chemoenzymatic synthesis using malonyltransferase has received significant attention. In the present study, a new malonyltransferase, NbMaT1, was identified from Nicotiana benthamiana. Extensive enzymatic assays revealed its significant substrate tolerance based on HPLC-UV and HR-MS analyses. Moreover, 16 of the tested glycosides including flavone glycosides, flavonol glycosides, dihydroflavone glycosides, isoflavone glycosides, coumarin glycosides, and phenylethyl chromone glycosides with various sugar moieties (such as glucose, xylose, and galactose) substituted at different positions of their skeleton could be accepted by NbMaT1 to conduct the corresponding malonylation. Among these, enzymatic malonylation of phenylethyl chromone glycosides as well as xylosides and galactosides has rarely been reported earlier. Furthermore, one-pot synthesis using the known malonyl-CoA synthetase MatB and NbMaT1 as well as an unnatural fusion protein MatB–NbMaT1 was designed, which allowed malonic acid to be directly used in the malonylation reaction without the addition of expensive malonyl-CoA. Moreover, a remarkably improved conversion rate was observed for all the tested substrates, with both commercial and industrial application values. The malonylated product of the bioactive flavonoid diglycoside icariin was prepared and NMR spectroscopy revealed that the malonyl group was specifically transferred onto the 6-OH group of the glucose moiety. NbMaT1 was expected to be a universal and effective tool for chemoenzymatic synthesis of diverse bioactive-malonylated glycoside derivatives for drug discovery.
研究领域:
生物化学
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药理学
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植物生物技术
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酶学
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Chen Wang,Yunlong Yan,Min Huang,Guangming Ma,Li Wang,Xin Xie,Wei Xue,Xiangyang Li
Cucumber mosaic virus (CMV) 2b protein plays a key role in the process of CMV infecting plants and symptom formation and is a potential molecular target for the control of this important plant virus. The exploitation of antiviral compounds is one of the strategies with the highest input: output ratio in plant protection. In this study, the CMV 2b recombinant protein was cloned, purified, and identified as the target protein by mass spectrometry. Subsequently, we carried out preliminary functional screening of the LP series of myricetin derivatives designed and synthesized in our laboratory and commercial antiviral compounds by microscale thermophoresis (MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV 2b, with dissociation constant (Kd) values of 1.39, 0.88, 1.52, and 1.77 μM, respectively. Among the commercially available antiviral compounds, ningnanmycin (NNM) was the most active, with a Kd value of 4.09 μM. Then, the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments, with a Kd of 1.19 μM. Among the commercial compounds, NNM had the strongest binding force with CMV 2b, with a Kd of 4.62 μM. Through the screening of commercial compounds and LP series compounds by MST and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to test the in vivo impact of LP11 on CMV. Specifically, the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact on the regulation of CMV pathogenic gene expression, symptoms, and virus titer. The results showed that LP11 had a strong ability to inhibit CMV infection of tobacco at the transcriptional and translational levels. By mutating the CMV 2b protein, the 15th amino acid leucine and the 18th amino acid methionine at the N-terminal region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening and developing anti-CMV agents.
研究领域:
植物病毒学
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植物化学
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分子生物学
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植物病理学
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DOI
PubMed
高影响因子
JOURNAL OF EXPERIMENTAL BOTANY
·
2023
·
IF 6.9
He Chengyong,Xing Fei,Liang Jiahui,Zhang Zhixiang,Zhan Binhui,Habili Nuredin,Wang Hongqing,Li Shifang
Apple necrotic mosaic virus (ApNMV) is associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defence against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, mitochondrial ATP synthase function in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase oligomycin sensitivity-conferring protein (OSCP) subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the ability of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Taken together, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding to its promoter, leading to the susceptibility of apple and N. benthamiana to ApNMV infection. The current study facilitates a comprehensive understanding of the intricate responses of the host to ApNMV infection.
研究领域:
植物病理学
·
分子生物学
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病毒学
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植物生理学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Chunle Wei,Xiong Yang,Shaojie Shi,Lian Bai,Deyu Hu,Runjiang Song,Baoan Song
3-Hydroxy-2-oxindole motif constitutes a core structure in numerous natural products and imparts notable biological activities. Here, we describe the design and synthesis of four series of novel 3-substituted-3-hydroxy-2-oxindole derivatives containing sulfonamide moiety along with their antiviral activities against potato virus Y (PVY). Compound 10b displayed optimal antiviral activity and superior anti-PVY activity compared with the lead compound and commercial Ningnanmycin in terms of curative and protective effects. Additionally, 10b considerably inhibited PVY systemic infection in Nicotiana benthamiana. Physiological and biochemical analyses revealed that the activities of the four crucial defense-related enzymes increased in the tobacco plant following treatment with 10b. RNA-sequencing analysis revealed that 10b substantially induced the upregulation of 38 differentially expressed genes, which were enriched in the photosynthesis pathway. These findings suggest that 10b is a promising antiviral agrochemical that can effectively control PVY infection and trigger plant host immunity to develop virus resistance. This study provides novel molecular entities and ideas for developing new pesticides.
研究领域:
有机化学
·
药物化学
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植物病理学
·
分子生物学
·
农学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2023
·
IF 6.1
Guili Zhao,Wenjuan Zhang,Xiaodong Fu,Xin Xie,Song Bai,Xiangyang Li
The viral protein genome-linked protein (VPg) of telosma mosaic virus (TeMV) plays an important role in viral reproduction. In this study, the expression conditions of TeMV VPg were explored. A series of novel benzenesulfonamide derivatives were synthesized. The binding sites of the target compounds and TeMV VPg were studied by molecular docking, and the interaction was verified by microscale thermophoresis. The study revealed that the optimal expression conditions for TeMV VPg were in Escherichia coli Rosetta with IPTG concentration of 0.8 mM and induction temperature of 25 °C. Compounds A4, A6, A9, A16, and A17 exhibited excellent binding affinity to TeMV VPg, with Kd values of 0.23, 0.034, 0.19, 0.086, and 0.22 μM, respectively. LYS 121 is the key amino acid site. Compounds A9 inhibited the expression of TeMV VPg in Nicotiana benthamiana. The results suggested that TeMV VPg is a potential antiviral target to screen anti-TeMV compounds.
研究领域:
病毒学
·
药物化学
·
分子生物学
·
植物病理学
✓ 已验证
DOI
PubMed
高影响因子
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