Background Viral diseases continue to pose a major threat to the world’s commercial crops. The in-depth exploration and efficient utilization of resistance proteins have become crucial strategies for their control. However, current delivery methods for introducing foreign DNA suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. The nanocarriers provides a convenient channel for the DNA delivery and functional utilization of disease-resistant proteins. Results In this research, we identified a cysteine-rich venom protein (NbCRVP) in Nicotiana benthamiana for the first time. Virus-induced gene silencing and transient overexpression clarified that NbCRVP could inhibit the infection of tobacco mosaic virus, potato virus Y, and cucumber mosaic virus, making it a broad-spectrum antiviral protein. Yeast two-hybrid assay, co-immunoprecipitation, and bimolecular fluorescence complementation revealed that calcium-dependent lipid-binding (CaLB domain) family protein (NbCalB) interacted with NbCRVP to assist NbCRVP playing a stronger antiviral effect. Here, we demonstrated for the first time the efficient co-delivery of DNA expressing NbCRVP and NbCalB into plants using poly(amidoamine) (PAMAM) nanocarriers, achieving stronger broad-spectrum antiviral effects. Conclusions Our work presents a tool for species-independent transfer of two interacting protein DNA into plant cells in a specific ratio for enhanced antiviral effect without transgenic integration, which further demonstrated new strategies for nanocarrier-mediated DNA delivery of disease-resistant proteins. Graphical abstract

Based on the theory of air convection formed by candle burning and 3D model of Abstractflat-plate flow at high angle of attack, the relevant theoretical analysis, experimental exploration and numerical simulation analysis of the design topic u201CCandle Powered Turbineu201D at the 2022 International Young Physicists' Tournament are carried out in a targeted manner. Combined with the model of air convection formed by candle burning, and the three-dimensional model of flat-plate flow at high angle of attack is generalized and applied, the theoretical formula of the torque of the paper spiral in the flow field generated by candles is obtained. The influence of the width and the axial inclination angle of the paper tape on the torque of the paper spiral is explored through experiments, and the optimized parameters are obtained. CFD numerical simulation analysis and experimental research are carried out on the optimized device. The simulation results are in good agreement with the experimental results, which confirms the accuracy of the torque theory proposed in this paper for the motion of paper spirals in the air flow field formed by candle combustion, The natural convection formed by fuel combustion is used as the power source in the rotating device, which has potential application value for the utilization of surplus energy. Moreover, the establishment of the 3D model of flat plate flow at high angle of attack provides a simple calculation method for the total pressure calculation of the flow around a three-dimensional flat plate at a low speed at a high angle of attack with Reynolds number in the range of 104~106.

Tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) are two major economic crops in China. Tobacco mosaic virus (TMV; genus Tobamovirus) is the most prevalent virus infecting both crops. Currently, some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus. Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases. However, stable attenuated TMV mutants are scarce. In this study, we found that the substitutions in the replicase p126, arginine at position 196 (R196) with aspartic acid (D), glutamic acid at position 614 (E614) with glycine (G), serine at position 643 (S643) with phenylalanine (F), or D at position 730 (D730) with S, significantly reduced the virulence and replication of TMV. However, only the mutation of S643 to F reduced the RNA silencing suppression activity of TMV p126. A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants. Furthermore, our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants. This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

Nanoparticles (NPs) derived from RNA interference (RNAi) are considered a potentially revolutionary technique in the field of plant protection in the future. However, the application of NPs in RNAi is hindered by the conflict between the high cost of RNA production and the large quantity of materials required for field application. This study aimed to evaluate the antiviral efficacy of commercially available nanomaterials, such as chitosan quaternary ammonium salt (CQAS), amine functionalized silica nano powder (ASNP), and carbon quantum dots (CQD), that carried double-stranded RNA (dsRNA) via various delivery methods, including infiltration, spraying, and root soaking. ASNP-dsRNA NPs are recommended for root soaking, which is considered the most effective method of antiviral compound application. The most effective antiviral compound tested was CQAS-dsRNA NPs delivered by root soaking. Using fluorescence, FITC-CQAS-dsCP-Cy3, and CQD-dsCP-Cy3 NPs demonstrated the uptake and transport pathways of dsRNA NPs in plants when applied to plants in different modes. The duration of protection with NPs applied in various modes was then compared, providing references for evaluating the retention period of various types of NPs. All three types of NPs effectively silenced genes in plants and afforded at least 14 days of protection against viral infection. Particularly, CQD-dsRNA NPs could protect systemic leaves for 21 days following spraying.

IntroductionKac is a model for all acylation modification studies. Kac plays a critical role in eukaryotes and prokaryotes. It is mainly involved in six major biological functions: gene expression, signal transduction, cell development, protein conversion, metabolism, and metabolite transport.MethodWe investigated and compared the acetylation modification of proteins in healthy and tomato spot wilt virus (TSWV)-infected Nicotiana benthamiana leaves.ResultWe identified 3,418 acetylated lysine sites on 1962 proteins acetylation of proteins in the TSWV-infected and control groups were compared; it was observed that 408 sites on 294 proteins were upregulated and 284 sites on 219 proteins (involved in pentose phosphate, photosynthesis, and carbon fixation in photosynthesis) were downregulated after the infection. Overall, 35 conserved motifs were identified, of which xxxkxxxxx_K_ Rxxxxxxxxx represented 1,334 (31.63%) enrichment motifs and was the most common combination. Bioinformatic analysis revealed that most of the proteins with Kac sites were located in the chloroplast and cytoplasm. They were involved in biological processes, such as cellular and metabolic processes.DiscussionIn conclusion, our results revealed that Kac may participate in the regulation of TSWV infection in N. benthamiana.

Oxathiapiprolin (Otp) is the first successful oxysterol-binding protein (OSBP) inhibitor in oomycete control. It is regarded as a significant milestone in the history of fungicide discoveries and has vast application prospects. There is little available information on the ecotoxicity of Otp to aquatic organisms. In this study, we evaluated the toxic effects of Otp in the Chlorella vulgaris (C. vulgaris). The results revealed the acute toxicity of Otp to C. vulgaris, with a 96-h median effective concentration for growth inhibition of 0.74 mg/L. When algal cells were exposed to 0.5 and 1.5 mg/L Otp, their chlorophyll and carotenoid contents dropped dramatically. As suggested by the significant increase in reactive oxygen species (ROS) and malondialdehyde (MDA) levels and the remarkable changes in the activity of a series of antioxidant enzymes, Otp induces production of ROS, resulting in oxidative damage. In addition, Otp can damage cell structures and could destroy membrane integrity. Finally, the changes in endogenous substances indicated that Otp can perturb energy metabolism and photosynthesis in C. vulgaris cells. The experimental results suggest that Otp can have toxic effects on algal cells by disturbing photosynthesis and causing oxidative damage and abnormal energy metabolism in C. vulgaris cells.

Tobacco (Nicotiana tabacum) is an important economic crop and widely cultivated in rural areas in south of China. A previously uncharacterized disease was observed on field-grown tobacco during 2020 and 2021 around Tongren city, Guizhou province of China (27°59'25.73" N, 108°7'2.43" E). The disease mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate was between 20%-100%. Symptoms first began as yellow-brown necrotic spots on leaves, then merged into larger irregular necrotic spots surrounded by chlorotic halos. Similar lesions were also found on the stems. Ten symptomatic leaf and stem samples were collected from the different infected plants for pathogen isolation. The small pieces of discolored tissues were surface-disinfected with 2% sodium hypochlorite for 3 min and 75% ethanol for 30 s, rinsed three times with sterile water, and blotted on sterile filter paper, placed on potato dextrose agar thenincubated at 28°C in the dark for 3-4 days. The obtained isolates were purified through single-spore culture. Colonies were initially white and fluffy in appearance, later turning gray. Hyphae were smooth, branched, septa, transparent or light brown. Spores were solitary, oblate or nearly spherical, dark brown to black, smooth, 14.3 to 16.1μm × 11.8 to 15.2 μm in diameter. DNA of fungal isolates were extracted using Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), the internal transcribed spacer (ITS) of the ribosomal DNA, β-tubulin (TUB2) gene and translation elongation factor 1-alpha (TEF1-α) were amplified with primers ITS1/ITS4, βt2a/βt2b and EF1-1728F/EF1-986R, respectively. The resulting ITS, TUB2 and TEF1-α sequences were deposited at GenBank, NCBI under accessions MZ882151, MZ927749, MZ927747, respectively. The sequence identity of ITS, TUB2 and TEF1-α with those of Nigrospora oryzae strains HBN (KU254608), HGUP191068 (MZ724102) and LC7307 (KY019409) were 99.64%, 99.29% and 99.65%, respectively. Based on morphological features and phylogenetic analysis, the pathogen was identified as N. oryzae (Wang et al. 2017). Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of tobacco plants grown at 28°C with 60% humidity in greenhouse. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as N. oryzae based on morphological, molecular and phylogenetic analysis, which were fulfilling Koch's postulates. This pathogen was recently identified from watermelon and kiwifruit in the Guizhou (Far and Rossman, 2021). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Nicotiana tabacum in China.

Recombinant alginate lyase was used to degrade alginic acid to prepare unsaturated alginate oligosaccharides (AOs), which were applied to directly reduce silver nitrate to obtain AO-coated silver nanoparticles (AOs@AgNPs), and as a stabilizer as well. First, alginate lyase aly-SJ02 from the marine bacterium Pseudoalteromonas sp. SM0524 was heterologously expressed in Yarrowia lipolytica. Next, the AOs produced through enzymatic degradation were used as reducing and stabilizing agents with silver nitrate to prepare AOs@AgNPs, and the particle size, stability, and other basic data were measured. Finally, based on the enhancement of plant resistance by AOs and the broad-spectrum antiviral bactericidal effect, three kinds of AgNPs had desirable passivation, prevention, and treatment effects on tobacco mosaic virus (TMV). The best control effect was observed for AOs@AgNPs, and it has been verified that AOs@AgNPs have a strong inhibitory effect at both the gene transcription and protein expression levels of TMV. AOs@AgNPs were far better than the AgNPs prepared by using alginate polysaccharides.

Tobacco black shank caused by Phytophthora nicotianae is a devastating disease that causes huge losses to tobacco production across the world. Investigating the regulatory mechanism of tobacco resistance to P. nicotianae is of great importance for tobacco resistance breeding. The jasmonate (JA) signaling pathway plays a pivotal role in modulating plant pathogen resistance, but the mechanism underlying JA-mediated tobacco resistance to P. nicotianae remains largely unclear. This work explored the P. nicotianae responses of common tobacco cultivar TN90 using plants with RNAi-mediated silencing of NtCOI1 (encoding the perception protein of JA signal), and identified genes involved in this process by comparative transcriptome analyses. Interestingly, the majority of the differentially expressed bHLH transcription factor genes, whose homologs are correlated with JA-signaling, encode AtBPE-like regulators and were up-regulated in NtCOI1-RI plants, implying a negative role in regulating tobacco response to P. nicotianae. A subsequent study on NtbHLH49, a member of this group, showed that it’s negatively regulated by JA treatment or P. nicotianae infection, and its protein was localized to the nucleus. Furthermore, overexpression of NtbHLH49 decreased tobacco resistance to P. nicotianae, while knockdown of its expression increased the resistance. Manipulation of NtbHLH49 expression also altered the expression of a set of pathogen resistance genes. This study identified a set of genes correlated with JA-mediated tobacco response to P. nicotianae, and revealed the function of AtBPE-like regulator NtbHLH49 in regulating tobacco resistance to this pathogen, providing insights into the JA-mediated tobacco responses to P. nicotianae.

Background Lysine 2-hydroxyisobutyrylation (Khib) is a novel and conserved post-translational modification (PTM). Frankliniella occidentalis are economically important agricultural pests globally and also notorious for vectoring destructive plant viruses. To better study the disease transmission mechanism of F. occidentalis , it is necessary to conduct in-depth analysis of it. So far, no Khib modification of insects has been reported. Results In this study, a proteome-wide analysis of Khib modifications in F. occidentalis was analyzed for the first time through the combination of high performance liquid chromatography fractionation technology and 2-hydroxyisobutyrylated peptide enrichment and other advanced technologies, 4093 Khib sites were identified on 1125 modified proteins. Bioinformatics and functional enrichment analyses showed that Khib-modified proteins were significantly enriched in many cell compartments and pathways, especially related to various cellular components and biological processes, and were more concentrated in ribosomes and proteasome subunits, involved in energy metabolism, protein synthesis and degradation, compared to the other nine species including Japonica rice, Homo sapiens , P. patens , Botrytis , Ustilaginoidea virens , Saccharomyces cerevisiae , T. gondii , C. albicans , and F. oxysporum . And Khib sites on virus-interacting insect proteins were discovered for the first time, such as cyclophilin and endoCP-GN. Conclusions After three repeated experiments, we found a total of 4093 Khib sites on 1125 proteins. These modified proteins are mainly concentrated in ribosomes and proteasome subunits, and are widely involved in a variety of critical biological activities and metabolic processes of F. occidentalis . In addition, for the first time, Khib modification sites are found on the proteome of F. occidentalis , and these sites could be acted as for the virus interaction, including cyclophilin and endoCP-GN. The global map of 2-hydroxyisobutyrylation in thrips is an invaluable resource to better understand the biological processes of thrips and provide new means for disease control and mitigation of pest damage to crops.

SummaryThe potato virus Y (PVY) is a plant virus that causes massive crop losses globally, especially in Solanaceae crops. A strain of the plant growth‐promoting rhizobacterium (PGPR), Serratia marcescens‐S3 was found to inhibit PVY replication in Nicotiana benthamiana. However, there have been no in‐depth studies demonstrating the underlying mechanism. In the current study, we found that ubiquitination of NbHsc70‐2 is an important way for Serratia marcescens‐S3 to trigger induced systemic resistance (ISR). After the treatment with S. marcescens‐S3, the protein level of NbHsc70‐2 reduced significantly. Inhibiting of ubiquitination increased the accumulation of NbHsc70‐2 in plants and reduced S. marcescens‐S3‐mediated resistance to PVY. Furthermore, transgenic engineered Nicotiana benthamiana NbHsc70‐2KO and NbHsc70‐2USM were constructed using CRISPR‐Cas9‐mediated NbHsc70‐2 knock‐out and ubiquitination respectively. S. marcescens‐S3 significantly reduced the inhibition of NbHsc70‐2 protein accumulation in NbHsc70‐2KO and NbHsc70‐2USM. The virulence of PVY was stronger in NbHsc70‐2USM than the wild‐type plants. These results showed that S. marcescens‐S3 increases the ubiquitination of NbHsc70‐2 to inhibit the recruitment of molecular chaperone NbHsc70‐2 to reduce its replication and infection of PVY.

Western flower thrips (Frankliniella occidentalis) are among the most important pests globally that transmit destructive plant viruses and infest multiple commercial crops. Lysine lactylation (Klac) is a recently discovered novel post-translational modification (PTM). We used liquid chromatography-mass spectrometry to identify the global lactylated proteome of F. occidentalis, and further enriched the identified lactylated proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). In the present study, we identified 1,458 Klac sites in 469 proteins from F. occidentalis. Bioinformatics analysis showed that Klac was widely distributed in F. occidentalis proteins, and these Klac modified proteins participated in multiple biological processes. GO and KEGG enrichment analysis revealed that Klac proteins were significantly enriched in multiple cellular compartments and metabolic pathways, such as the ribosome and carbon metabolism pathways. Two Klac proteins were found to be involved in the regulation of the TSWV (Tomato spotted wilt virus) transmission in F. occidentalis. This study provides a systematic report and a rich dataset of lactylation in F. occidentalis proteome for potential studies on the Klac protein of this notorious pest.

Lysine acetylation (Kac), a reversible PTM, plays an essential role in various biological processes, including those involving metabolic pathways, pathogen resistance, and transcription, in both prokaryotes and eukaryotes. TMV, the major factor that causes the poor quality of Solanaceae crops worldwide, directly alters many metabolic processes in tobacco. However, the extent and function of Kac during TMV infection have not been determined. The validation test to detect Kac level and viral expression after TMV infection and Nicotinamide (NAM) treatment clarified that acetylation was involved in TMV infection. Furthermore, we comprehensively analyzed the changes in the proteome and acetylome of TMV-infected tobacco (Nicotiana benthamiana) seedlings via LC-MS/MS in conjunction with highly sensitive immune-affinity purification. In total, 2082 lysine-acetylated sites on 1319 proteins differentially expressed in response to TMV infection were identified. Extensive bioinformatic studies disclosed changes in acetylation of proteins engaged in cellular metabolism and biological processes. The vital influence of Kac in fatty acid degradation and alpha-linolenic acid metabolism was also revealed in TMV-infected seedlings. This study first revealed Kac information in N. benthamiana under TMV infection and expanded upon the existing landscape of acetylation in pathogen infection.

Background The annual economic loss caused by plant viruses exceeds 10 billion dollars due to the lack of ideal control measures. Quercetin is a flavonol compound that exerts a control effect on plant virus diseases, but its poor solubility and stability limit the control efficiency. Fortunately, the development of nanopesticides has led to new ideas. Results In this study, 117 nm quercetin nanoliposomes with excellent stability were prepared from biomaterials, and few surfactants and stabilizers were added to optimize the formula. Nbhsp70er-1 and Nbhsp70c-A were found to be the target genes of quercetin, through abiotic and biotic stress, and the nanoliposomes improved the inhibitory effect at the gene and protein levels by 33.6 and 42%, respectively. Finally, the results of field experiment showed that the control efficiency was 38% higher than that of the conventional quercetin formulation and higher than those of other antiviral agents. Conclusion This research innovatively reports the combination of biological antiviral agents and nanotechnology to control plant virus diseases, and it significantly improved the control efficiency and reduced the use of traditional chemical pesticides. Graphical Abstract

Our previous study identified that Bacillus siamensis strain LZ88 has significant antagonistic activity against a variety of pathogenic fungi, especially against Alternaria alternata, which causes tobacco brown spot. In the present study, the non-volatile compounds and volatile organic compounds (VOCs) produced by LZ88 were identified and tested for their antifungal activities. The non-volatile compounds had significant inhibitory effects on both mycelial growth and spore germination of A. alternata, with IC50 values of 24.25 mu g/mL and 29.04 mu g/ mL, respectively. HPLC-DAD-ESI-MS/MS analysis revealed that the non-volatile compounds which showed antifungal activities were iturins and macrolactin. HS-SPME-GC-MS identified ten VOCs from strain LZ88, including 8 ketones and 2 acids. Among the VOCs tested, 2-methylbutanoic acid and 3-methylbutanoic acid exhibited significant antifungal activity against A. alternata. Both 2-methylbutanoic acid and 3-methylbutanoic acid could inhibit mycelium growth of A. alternata with IC50 values of 83.10 mg/mL and 104.19 mg/mL, whereas inhibited spore germination with IC50 values of 139.63 mg/mL and 88.07 mg/mL, respectively. Furthermore, when exposed to non-volatile compounds or VOCs, the hyphae and spores of A. alternata were deformed and collapsed, and consequent hyphae without any attached conidia appeared ruptured, shrunken, and twisted. Additionally, the genetic mechanisms were analyzed by whole genome sequencing, and the gene clusters responsible for antibacterial metabolites were mined. These results indicate that strain LZ88 possessed a remarkable potential as alternative agrochemicals toward A. alternata.

为提高粘质沙雷氏菌S3菌株的灵菌红素产率,本研究对粘质沙雷氏菌S3发酵过程中重要的发酵条件参数和关键组分进行逐项优化,并验证了发酵液灵菌红素提取物对本氏烟草病毒的抑制效果.结果表明,最佳培养基配方为:丙三醇0.5％,蛋白胨1.5％,氯化钠0.5％,氯化钾0.25％;最佳发酵条件为:接种量为10％,装液量为60％～70％,pH恒定为6.0,培养温度为28℃,振摇速度160 r/min,发酵周期为60 h.在该发酵条件下,既有利于粘质沙雷氏菌菌体生长,又能够使灵菌红素的产量达到最大化.按照该优化条件进行发酵后,其灵菌红素提取物溶液喷施于接种TMV、CMV和PVY的本氏烟,烟叶中TMV、CMV和PVY病毒量分别为空白对照的12.61％、29.41％和17.69％,对病毒的抑制效果优于氨基寡糖素.试验所得的发酵制备条件能够提高灵菌红素产量,提取物具备显著抗病毒特性,具有产业化开发潜力和经济应用价值.

Tobacco brown spot disease, caused by Alternaria alternata, is responsible for causing significant losses in tobacco production in China. The use of biocontrol microorganisms to prevent and control this disease represent an important, environment-friendly, management strategy. In the present study, 17 bacterial strains with antimicrobial activity were isolated from 40 rhizosphere soil samples and evaluated for antifungal activity to identify an effective biocontrol agent for the control of tobacco brown spot disease in tobacco. Among the isolated strains, Bacillus siamensis, strain LZ88, exhibited the strongest antifungal activity with an inhibition rate of 81.96%. We confirmed that strain LZ88 controlled both mycelial growth and spore production in A. alternata by secreting protein-like antifungal metabolites and through the release of volatile organic compounds. Strain LZ88 also had the ability to induce plant basal immunity as evidenced by increased expression of the defense-related enzymes, peroxidase (POD) and polyphenol oxidase (PPO), in tobacco leaves. Results of greenhouse experiments indicated that LZ88 could effectively reduce the occurrence and degree of severity of tobacco brown spot disease. To the best of our knowledge, this is the first report regarding the use of B. siamensis to control brown spot disease in tobacco.

为明确Rubisco酶小亚基(Rubisco small subunit,RbcS)在烟草抵抗主要病毒侵染胁迫中的作用,以本氏烟为研究材料,利用qRT-PCR、Western blot和瞬时表达体系对烟草RbcS基因在TMV、CMV和PVY侵染中的调控作用进行系统分析.结果表明,3种病毒侵染本氏烟均引起NbRbcS在mRNA水平和蛋白水平上下调,瞬时过表达NbRbcS,病毒拷贝数均显著降低.而沉默NbRbcS,TMV、CMV和PVY拷贝数分别达到阴性对照的11.12、9.57和17.76倍.对PVY引起的斑驳叶片中深绿色和浅绿色区域分别进行qRT-PCR分析发现,在浅叶区NbRbcS mRNA的表达量只有深叶区的49.10％,但PVY的拷贝数却达到了深叶区的5.18倍.以上研究表明,RbcS大量表达可增强烟草对病毒的防御能力,烟草RbcS基因可作为潜在的抗病毒基因利用.

[目的]马铃薯Y病毒(potato virus Y,PVY)是危害我国烟草生产的最重要病毒之一,NAC转录因子与植物的抗病、抗逆密切相关,本论文克隆NbNAC062进行生物信息学分析,并研究其在PVY侵染过程中的作用,为烟草抗病毒药剂的开发提供靶标.[方法]以本氏烟(Nicotiana benthamiana)为材料克隆NbNAC062,利用MEGA、UniProt、SMART、TMHMM Server 2.0、Sol Genomics Network、PlantCARE等技术进行生物信息学分析;利用激光共聚焦与实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)明确PVY侵染前后NbNAC062蛋白定位及mRNA表达量变化;基于病毒介导的基因沉默(virus-induced gene silencing,VIGS)和过表达技术,构建pTRV::NbNAC062沉默载体与pEarleyGate100::RFP::NbNAC062过表达载体,采用qRT-PCR和Western blot检测NbNAC062在本氏烟中沉默与过表达后,PVY的积累量变化及未折叠蛋白应答(unfolded protein response,UPR)相关基因BiP的表达差异.[结果]NbNAC062编码646个氨基酸,N端28—179 aa为NAC结构域,129—185 aa为DNA结合区域,C末端621—643 aa为疏水跨膜结构,系统进化树与蛋白序列分析表明本氏烟NbNAC062与渐狭叶烟草NaNAC062亲缘关系最近.NbNAC062启动子中包含脱落酸、茉莉酸甲酯、水杨酸以及逆境响应相关的多种顺式作用元件.PVY侵染激活NbNAC062从细胞膜转移至细胞核,且诱导NbNAC062上调表达.PVY侵染本氏烟5、7 d,处理组NbNAC062 mRNA水平分别为对照组的2.52、1.95倍;PVY侵染3 d,BiP mRNA表达量为对照组的2.39倍,PVY侵染7 d,BiP表达量极显著低于对照组,下调表达56.77％.本氏烟沉默NbNAC062并接种PVY,接种后3、5、7 d,与对照组相比,沉默组PVY CP mRNA上调表达,分别为对照组的2.12、2.41、1.38倍,BiP mRNA表达量则下调,分别下调28.19％、58.11％、10.77％,接种后5、7 d沉默组PVY CP蛋白含量亦显著高于对照组.过表达NbNAC062并接种PVY,接种后24、48、72 h,与对照组相比,过表达组PVY CP mRNA分别下调22.60％、34.51％、36.21％,接种48、72 h,BiP mRNA上调表达,分别为对照组的1.56、1.35倍,过表达组PVY CP蛋白含量亦低于对照组.[结论]NbNAC062属于NAC类膜结合转录因子,可被PVY侵染激活转移至细胞核,可能通过调控UPR相关基因BiP的表达,促进细胞生存,抑制PVY早期侵染.

[目的]筛选高效靶向降解烟草花叶病毒(tobacco mosaic virus,TMV)的dsRNA,实现其大量制备,并探究其作用机制.[方法]以TMV编码的CP、MP、RdRP功能基因为靶序列,体外转录合成相应的dsRNA,浸润本氏烟(Nicotiana benthamiana),24 h后接种TMV,于接毒后2、3 d取样提取总RNA和蛋白质,以CP基因mRNA水平和蛋白水平为指标,结合TMV病毒生物学症状,综合评价各dsRNA对TMV的抑制效果.同时结合侵染性克隆TMV-30B在本氏烟烟株上的荧光表达现象和TMV在三生烟(Nicot iana tabacum var.Samsun NN)上的过敏性坏死反应(hypersensitive necrosis reaction),通过比较TMV基因组上6个靶序列相对应的dsRNA,筛选出高效抑制TMV的dsRNA片段.为了获取大量的dsRNA,将dsRNA对应的基因片段插入到原核表达载体L4440的双T7启动子之间,转化至RNase Ⅲ缺陷型大肠杆菌(Escherichia coli)HT115(DE3)中,并对原核表达制备的dsRNA喷施烟草后生成的siRNA进行深度测序,比较外源施用dsRNA后,对TMV侵染的small RNA表达特征和富集带的影响.[结果]筛选出高效影响TMVCP基因表达的dsRNA RdRP1461-1774,并构建了可诱导形成目的dsRNA的原核表达载体L4440-dsRdRP1461-1774,可在DE3中大量制备RdRP1461-1774的dsRNA,菌液中提取的dsRNA喷施于烟草上对TMV的防治效果显著.TMV-30B侵染本氏烟时荧光数量减少,并能够延长叶片萎蔫时间,在三生烟上施用时叶片枯斑数量明显减少.小RNA测序结果显示TMV侵染引起的RNAi过程中正义链和反义链以大致相等的频率产生siRNA,而外源性dsRNA的浸润会引起靶向区域siRNA的富集,siRNA反义链累积量骤增,对应的正义链累积量骤减,外源dsRNA的施用能够引起siRNA表达丰度的变化.[结论]通过比较dsRNA介导植物靶向抗TMV侵染的效果来筛选抗烟草花叶病毒的dsRNA序列,最终选定TMV RdRP基因上一段长313 bp的高效作用片段,该片段dsRNA能够高效与靶基因结合,降低染病植株烟草花叶病毒的表达量.同时构建了RdRP1461-1774基因的dsRNA原核表达系统,实现其低成本的高效量产,为后续dsRNA在植物病毒方面的防治应用打下了基础.