Effects of editing DFR genes on flowers, leaves, and roots of tobacco.

BACKGROUND: DFR is a crucial structural gene in plant flavonoid and polyphenol metabolism, and DFR knockout (DFR-KO) plants may have increased biomass accumulation. It is uncertain whether DFR-KO has comparable effects in tobacco and what the molecular mechanism is. We employed the CRISPR/Cas9 method to generate a knockout homozygous construct and collected samples from various developmental phases for transcriptome and metabolome detection and analysis. RESULTS: DFR-KO turned tobacco blossoms white on homozygous tobacco (Nicotiana tabacum) plants with both NtDFR1 and NtDFR2 knockout. RNA-seq investigation of anthesis leaf (LF), anthesis flower (FF), mature leaf (LM), and mature root (RM) variations in wild-type (CK) and DFR-KO lines revealed 2898, 276, 311, and 101 differentially expressed genes (DEGs), respectively. DFR-KO primarily affected leaves during anthesis. According to KEGG and GSEA studies, DFR-KO lines upregulated photosynthetic pathway carbon fixation and downregulated photosystem I and II genes. DFR-KO may diminish tobacco anthesis leaf photosynthetic light reaction but boost dark reaction carbon fixation. DFR-KO lowered the expression of pathway-related genes in LF, such as oxidative phosphorylation and proteasome, while boosting those in the plant-pathogen interaction and MAPK signaling pathways, indicating that it may increase biological stress resistance. DFR-KO greatly boosted the expression of other structural genes involved in phenylpropanoid production in FF, which may account for metabolite accumulation. The metabolome showed that LF overexpressed 8 flavonoid metabolites and FF downregulated 24 flavone metabolites. In DFR-KO LF, proteasome-related genes downregulated 16 amino acid metabolites and reduced free amino acids. Furthermore, the DEG analysis on LM revealed that the impact of DFR-KO on tobacco growth may progressively diminish with time. CONCLUSION: The broad impact of DFR-KO on different phases and organs of tobacco development was thoroughly and methodically investigated in this research. DFR-KO decreased catabolism and photosynthetic light reactions in leaves during the flowering stage while increasing carbon fixation and disease resistance pathways. However, the impact of DFR-KO on tobacco growth steadily declined as it grew and matured, and transcriptional and metabolic modifications were consistent. This work offers a fresh insight and theoretical foundation for tobacco breeding and the development of gene-edited strains.

The CBL-CIPK network is involved in the physiological crosstalk between plant growth and stress adaptation.

Plants have evolved to deal with different stresses during plant growth, relying on complex interactions or crosstalk between multiple signalling pathways in plant cells. In this sophisticated regulatory network, Ca2+ transients in the cytosol ([Ca2+ ]cyt ) act as major physiological signals to initiate appropriate responses. The CALCINEURIN B-LIKE PROTEIN (CBL)-CBL-INTERACTING PROTEIN KINASE (CIPK) network relays physiological signals characterised by [Ca2+ ]cyt transients during plant development and in response to environmental changes. Many studies are aimed at elucidating the role of the CBL-CIPK network in plant growth and stress responses. This review discusses the involvement of the CBL-CIPK pathways in two levels of crosstalk between plant development and stress adaptation: direct crosstalk through interaction with regulatory proteins, and indirect crosstalk through adaptation of correlated physiological processes that affect both plant development and stress responses. This review thus provides novel insights into the physiological roles of the CBL-CIPK network in plant growth and stress adaptation.

High-throughput creation of Nicotiana tabacum gene-targeted mutants based on CRISPR/Cas9.

KEY MESSAGE: 4382 available sgRNAs targeting 1060 tobacco genes were obtained, and 10,682 targeted mutants were created using high-throughput methods. Four optimization experiments were established to solve problems encountered during genetic transformation.

Biochemical characterization and overexpression of an α-amylase (BmAmy) in silkworm, Bombyx mori.

Silkworm (Bombyx mori) is the only fully domesticated insect. As an economically important insect, nutrition utilization is important for its productivity. Hence, the present study investigated the expression pattern of BmAmy, an α-amylase, in B. mori. BmAmy protein purification and biochemical characterization were performed, and effects of BmAmy overexpression were assessed. Real-time quantitative reverse transcription polymerase chain reaction indicated that BmAmy transcription was positively correlated with the silkworm's food intate. Moreover, enzymatic activity assay results showed that BmAmy had significant α-amylase activity of about 1 mg/min/mg protein. Furthermore, treatment with mulberry amylase inhibitors MnAI1 and MnAI2 resulted to 89.92% and 93.67% inhibition in BmAmy activity, respectively, and the interaction between BmAmy and MnAI was also confirmed by protein docking analysis. A silkworm line that specifically overexpressed BmAmy in the midgut was generated through piggyBac-based transgenic technology, and compared to those of non-transgenic silkworms, the whole cocoon and cocoon shell weights of these transgenic silkworms increased by 10.13% and 18.32%, respectively, in the female group, and by 5.83% and 6.00%, respectively, in the male group. These results suggested that BmAmy may be a suitable target for breeding better silkworm varieties in the future.

Interaction between Brassica yellows virus silencing suppressor P0 and plant SKP1 facilitates stability of P0 in vivo against degradation by proteasome and autophagy pathways.

P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0-SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br ). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br , which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br . Genetic analyses indicated that the P0Br -SKP1 interaction is not directly required for silencing suppression activity of P0Br , but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Molecular characterization of a new recombinant brassica yellows virus infecting tobacco in China.

Brassica yellows virus (BrYV), prevalently distributed throughout mainland China and South Korea while triggering serious diseases in cruciferous crops, is proposed to be a new species in the genus Polerovirus within the family Luteoviridae. There are three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) reported in cabbage and radish. Here, we describe a new BrYV isolate infecting tobacco plants in the field, which was named BrYV-NtabQJ. The complete genome sequence of BrYV-NtabQJ is 5741 nt in length, and 89% of the sequence shares higher sequence identities (about 90%) with different BrYV isolates. However, it possesses a quite divergent region within ORF5, which is more close to Beet western yellows virus (BWYV), Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV). A significant recombination event was then detected among BrYV-NtabQJ, BrYV-B Beijng isolate (BrYV-BBJ) and BWYV Leonurus sibiricus isolate (BWYV-LS). It is proposed that BrYV-NtabQJ might be an interspecific recombinant between BrYV-BBJ and BWYV-LS, and the recombination might result in the successful aphid transmission of BrYV from cruciferous crops to tobacco. And it also poses new challenges for BrYV diagnosis and the vegetable production.

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum.

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384 × 106 Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30 °C and 180 rpm: carbon source, 50 g/L maltose; initial pH 7; and 8 g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3 L. The fermentation broth contained 303 g/L maltose, which produced 122.34 g/L pullulan with an average productivity of 1.0195 g/L/h and 82.32 g/L dry biomass within 120 h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251 g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.

Species Diversity and Functional Prediction of Surface Bacterial Communities on Aging Flue-Cured Tobaccos.

Microbes on aging flue-cured tobaccos (ATFs) improve the aroma and other qualities desirable in products. Understanding the relevant organisms would picture microbial community diversity, metabolic potential, and their applications. However, limited efforts have been made on characterizing the microbial quality and functional profiling. Herein, we present our investigation of the bacterial diversity and predicted potential genetic capability of the bacteria from two AFTs using 16S rRNA gene sequences and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) software. The results show that dominant bacteria from AFT surfaces were classified into 48 genera, 36 families, and 7 phyla. In addition, Bacillus spp. was found prevalent on both ATFs. Furthermore, PICRUSt predictions of bacterial community functions revealed many attractive metabolic capacities in the AFT microbiota, including several involved in the biosynthesis of flavors and fragrances and the degradation of harmful compounds, such as nicotine and nitrite. These results provide insights into the importance of AFT bacteria in determining product qualities and indicate specific microbial species with predicted enzymatic capabilities for the production of high-efficiency flavors, the degradation of undesirable compounds, and the provision of nicotine and nitrite tolerance which suggest fruitful areas of investigation into the manipulation of AFT microbiota for AFT and other product improvements.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco.

Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

CRISPR/Cas9-mediated knockout of NtMYC2a gene involved in resistance to bacterial wilt in tobacco.

MYC2 is a class of bHLH family transcription factors and a major regulatory factor in the JA signaling pathway, and its molecular function in tobacco has not been reported. In this study, CRISPR/Cas9-mediated MYC2 gene NtMYC2a knockout mutants at tobacco was obtained and its agronomic traits, disease resistance, and chemical composition were identified. Comparing with the WT, the leaf width of the KO-NtMYC2a was narrowed, the nornicotine content and mecamylamine content increased significantly and the resistance to Ralstonia solanacearum significantly decreased. The transcriptome sequencing results showed that DEGs related to immunity, signal transduction and growth and development were enriched between KO-NtMYC2a and WT. NtJAR1 and NtCOI1 in KO-NtMYC2a were down-regulated to regulating the JA signaling pathway, result in a significant decrease in tobacco's resistance to R. solanacearum. Our research provides theoretical support for the functional research of MYC2 and the study of the mechanism of tobacco bacterial wilt resistance.

Loss function of NtGA3ox1 delays flowering through impairing gibberellins metabolite synthesis in Nicotiana tabacum.

Flowering time, plays a crucial role in tobacco ecological adaptation besides its substantial influence on tobacco production and leaf quality. Meanwhile, it is sensitive to biotic or abiotic challenges. The plant hormones Gibberellins (GAs), controlling a number of metabolic processes, govern plants growth and development. In this study, we created a late flowering mutant HG14 through knocking out NtGA3ox1 by CRISPR/Cas9. It took around 13.0 and 12.1 days longer to budding and flowering compared to wild type Honghuadajinyuan. Nearly all of the evaluated agronomic characters deteriorated in HG14, showing slower growth and noticeably shorter and narrower leaves. We found that NtGA3ox was more prevalent in flowers through quantitative reverse transcription PCR analysis. Transcriptome profiling detected 4449, 2147, and 4567 differently expressed genes at the budding, flowering, and mature stages, respectively. The KEGG pathway enrichment analysis identified the plant-pathogen interaction, plant hormone signal transduction pathway, and MAPK signaling pathway are the major clusters controlled by NtGA3ox1 throughout the budding and flowering stages. Together with the abovementioned signaling pathway, biosynthesis of monobactam, metabolism of carbon, pentose, starch, and sucrose were enriched at the mature stage. Interestingly, 108 up- and 73 down- regulated DEGs, impairing sugar metabolism, diterpenoid biosynthesis, linoleic and alpha-linolenic acid metabolism pathway, were continuously detected accompanied with the development of HG14. This was further evidenced by the decreasing content of GA metabolites such as GA4 and GA7, routine chemicals, alkaloids, amino acids, and organic acids Therefore, we discovered a novel tobacco flowering time gene NtGA3ox1 and resolved its regulatory network, which will be beneficial to the improvement of tobacco varieties.

Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR.

CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T1 generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T2 mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.

Autotoxins in continuous tobacco cropping soils and their management.

Tobacco belongs to the family Solanaceae, which easily forms continuous cropping obstacles. Continuous cropping exacerbates the accumulation of autotoxins in tobacco rhizospheric soil, affects the normal metabolism and growth of plants, changes soil microecology, and severely reduces the yield and quality of tobacco. In this study, the types and composition of tobacco autotoxins under continuous cropping systems are summarized, and a model is proposed, suggesting that autotoxins can cause toxicity to tobacco plants at the cell level, plant-growth level, and physiological process level, negatively affecting soil microbial life activities, population number, and community structure and disrupting soil microecology. A combined strategy for managing tobacco autotoxicity is proposed based on the breeding of superior varieties, and this approach can be combined with adjustments to cropping systems, the induction of plant immunity, and the optimization of cultivation and biological control measures. Additionally, future research directions are suggested and challenges associated with autotoxicity are provided. This study aims to serve as a reference and provide inspirations needed to develop green and sustainable strategies and alleviate the continuous cropping obstacles of tobacco. It also acts as a reference for resolving continuous cropping challenges in other crops.

Nucleotide sequence of a chickpea chlorotic stunt virus relative that infects pea and faba bean in China.

We determined the genome sequence of a new polerovirus that infects field pea and faba bean in China. Its entire nucleotide sequence (6021 nt) was most closely related (83.3% identity) to that of an Ethiopian isolate of chickpea chlorotic stunt virus (CpCSV-Eth). With the exception of the coat protein (encoded by ORF3), amino acid sequence identities of all gene products of this virus to those of CpCSV-Eth and other poleroviruses were <90%. This suggests that it is a new member of the genus Polerovirus, and the name pea mild chlorosis virus is proposed.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Differential Effects of Ammonium (NH4+) and Potassium (K+) Nutrition on Photoassimilate Partitioning and Growth of Tobacco Seedlings.

Plants utilize carbohydrates as the main energy source, but much focus has been on the impact of N and K on plant growth. Less is known about the combined impact of NH4+ and K+ nutrition on photoassimilate distribution among plant organs, and the resultant effect of such distribution on growth of tobacco seedlings, hence this study. Here, we investigated the synergetic effect of NH4+ and K+ nutrition on photoassimilate distribution, and their resultant effect on growth of tobacco seedlings. Soluble sugar and starch content peaks under moderate NH4+ and moderate K+ (2-2 mM), leading to improved plant growth, as evidenced by the increase in tobacco weight and root activity. Whereas, a drastic reduction in the above indicators was observed in plants under high NH4+ and low K+ (20-0.2 mM), due to low carbohydrate synthesis and poor photoassimilate distribution. A strong positive linear relationship also exists between carbohydrate (soluble sugar and starch) and the activities of these enzymes but not for invertase. Our findings demonstrated that NH4+ and K+-induced ion imbalance influences plant growth and is critical for photoassimilate distribution among organs of tobacco seedlings.

Effect of electronic cigarette and tobacco smoking on the human saliva microbial community.

Increasing evidence demonstrated the oral microbial community profile characteristics affected by conventional cigarettes smoking, but few studies focus on oral microbiome in response to electronic cigarettes (E-cigarettes). This study aimed to investigate the effect of E-cigarettes on the oral microbiome and to describe the difference of oral community profiles between E-cigarette smokers and tobacco smokers. 16S rRNA V4 gene sequencing was performed to investigate the oral microbial profiles of 5 E-cigarette smokers, 14 tobacco smokers, 8 quitting tobacco smokers, and 6 nonsmokers. The Chao1, ACE, and Shannon diversity indexes increased significantly in saliva samples collected from E-cigarette smokers and tobacco smokers compared to the non-smokers, and no significant difference was found in alpha diversity between E-cigarette smokers and tobacco smokers. The main phyla Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria and major genera Neisseria, Streptococcus, Prevotellaceae, Fusobacterium, and Porphyromonas dominated in the smoking groups, while Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria became the dominant phyla along with the genera Corynebacterium, Neisseria, Streptococcus, Actinomyces, and Porphyromonas in the nonsmokers. The differences in the phylum Actinobacteria and genus Corynebacterium contributed to various functional differences between smokers and nonsmokers. The difference on oral microbial and composition between E-cigarettes and common tobacco were associated with increased Prevotellaceae and decreased Neisseria. Additionally, smoking cessation could lead to re-establishment of the oral microbiome to that of nonsmokers. Our data demonstrate that E-cigarette smoking had different effects on the structure and composition of the oral microbial community compared to tobacco smoking. However, the short- and long-term impact of E-cigarette smoking on microbiome composition and function needs further exploration.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.