Characterization of the belowground microbial community and co-occurrence networks of tobacco plants infected with bacterial wilt disease.

Characterizing the microbial communities associated with soil-borne disease incidence is a key approach in understanding the potential role of microbes in protecting crops from pathogens. In this study, we compared the soil properties and microbial composition of the rhizosphere soil and roots of healthy and bacterial wilt-infected tobacco plants to assess their potential influence on plant health. Our results revealed that the relative abundance of pathogens was higher in diseased plants than in healthy plants. Moreover, compared with healthy plants, there was a significantly higher microbial alpha diversity in the roots and rhizosphere soil of diseased plants. In addition, we detected a lower abundance of certain plant microbiota, including species in the genera Penicillium, Trichoderma, and Burkholderia in the rhizosphere of diseased plants, which were found to be significantly negatively associated with the relative abundance of Ralstonia. Indeed, compared with healthy plants, the co-occurrence networks of diseased plants included a larger number of associations linked to plant health. Furthermore, structural equation modeling revealed that these specific microbes were correlated with disease suppression, thereby implying that they may play important roles in maintaining plant health. In conclusion, our findings provide important insights into the relationships between soil-borne disease incidence and changes in the belowground microbial community. These findings will serve as a basis for further research investigating the use of specific plant-associated genera to inhibit soil-borne diseases.

Soil potentials to resist continuous cropping obstacle: Three field cases.

Continuous cropping has become the most common system in intensive, modern agricultural production; however, obstacles often appear in continuous cropping patterns after a few years of use. There have been several studies about the impacts of continuous cropping on soil microbial, but few about differences between soil experiencing continuous cropping obstacles and those where such obstacles had been resisted. Here, after ten or twenty years of continuous tobacco cropping, we collected soil samples investigating discrepancies in soil property and bacterial community between soils experiencing continuous cropping obstacles and soils where the obstacles were resisted providing insight into preventing and controlling continuous cropping obstacles. Results showed that soil organic matter (SOM), available phosphorus (AP), total nitrogen (TN), nitrate-N (NO3--N), and bacterial diversity of samples where continuous cropping obstacles had been resisted were significantly higher than those where continuous cropping obstacles were present. Besides, SOM, AP, TN, and Ammonium-N (NH4+-N) considerably affected the bacterial community. Among all variables, NH4+-N explained the largest proportion of bacterial community variation. Molecular ecological networks were used to putatively identify keystone taxa, including Acidobacteria Gp1, Acidobacteria Gp2, Acidobacteria Gp16, and WPS-1_genera_incertae_sedis. Their relative abundance significantly changed between the two conditions. Overall, our results indicate that decreases in soil nutrient content and bacterial diversity, and significant changes in some keystone taxa abundances may be important factors leading to increased soil-borne diseases and reduced tobacco production potential or quality. Thus, during agricultural production, we could regulate the stability of the soil-crop-microbial ecological system via crop rotation, intercropping, or the use of specialized bio-fertilizers and soil conditioners to mitigate continuous cropping obstacles.

Chlorogenic acid inhibits forming of diabetes mellitus in rats induced by high-fat high-sucrose and streptozotocin.

To evaluate the inhibitory effect of chlorogenic acid on the forming of type 2 diabetes mellitus (T2DM), using Sprague Dawley (SD) rats, a recognized T2DM model induced by high-fat high-sucrose diet (HFSD) and streptozotocin (STZ). Thirty female SD rats were assigned equally to three groups randomly: normal control with standard commercial (NC), chlorogenic acid treatment with HFSD and chlorogenic acid (90mg/kg, CA), and diabetes model with HFSD (DM). Upon treatment with chlorogenic acid, suppression of the onset of diabetes, reduced serum glucose and insulin concentrations, improved glucose tolerance and increased body weight and visceral fat weight were observed. Serum triglyceride, total cholesterol, low density lipoprotein levels, and kidney and pancreas morphology were significantly ameliorated. Chlorogenic acid also inhibited the mRNA levels of hepatic G-6-Pase and up-regulated the mRNA levels of skeletal muscle GLUT4. Our results indicated that before the onset of diabetes, chlorogenic acid had an inhibitory effect against the forming of T2DM induced by HFSD and STZ through regulating the glucose and lipid metabolism.

The spatial and single-cell analysis reveals remodeled immune microenvironment induced by synthetic oncolytic adenovirus treatment.

Oncolytic viruses are multifaceted tumor killers, which can function as tumor vaccines to boost systemic antitumor immunity. In previous study, we rationally designed a synthetic oncolytic adenovirus (SynOV) harboring a synthetic gene circuit, which can kill tumors in mouse hepatocellular carcinoma (HCC) models. In this study, we demonstrated that SynOV could sense the tumor biomarkers to lyse tumors in a dosage-dependent manner, and killed PD-L1 antibody resistant tumor cells in mouse model. Meanwhile, we observed SynOV could cure liver cancer and partially alleviate the liver cancer with distant metastasis by activating systemic antitumor immunity. To understand its high efficacy, it is essential to explore the cellular and molecular features of the remodeled tumor microenvironment (TME). By combining spatial transcriptome sequencing and single-cell RNA sequencing, we successfully depicted the remodeled TME at single cell resolution. The state transition of immune cells and stromal cells towards an antitumor and normalized status exemplified the overall cancer-suppressive TME after SynOV treatment. Specifically, SynOV treatment increased the proportion of CD8+ T cells, enhanced the cell-cell communication of Cxcl9-Cxcr3, and normalized the Kupffer cells and macrophages in the TME. Furthermore, we observed that SynOV could induce distant responses to reduce tumor burden in metastatic HCC patient in the Phase I clinical trial. In summary, our results suggest that SynOV can trigger systemic antitumor immunity to induce CD8+ T cells and normalize the abundance of immune cells to remodel the TME, which promises a powerful option to treat HCC in the future.

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Soil properties, rhizosphere bacterial community, and plant performance respond differently to fumigation and bioagent treatment in continuous cropping fields.

Continuous cropping barriers lead to huge agriculture production losses, and fumigation and biological agents are developed to alleviate the barriers. However, there is a lack of literature on the differences between strong chemical fumigant treatment and moderate biological agent treatment. In this study, we investigated those differences and attempted to establish the links between soil properties, rhizosphere microbial community, and plant performance in both fumigation- and bioagent-treated fields. The results showed that the fumigation had a stronger effect on both soil functional microbes, i.e., ammonia oxidizers and soil-borne bacterial pathogens, and therefore, led to a significant change in soil properties, higher fertilizer efficiency, lower disease infections, and improved plant growth, compared with untreated control fields. Biological treatment caused less changes to soil properties, rhizosphere bacterial community, and plant physiology. Correlation and modeling analyses revealed that the bioagent effect was mainly direct, whereas fumigation resulted in indirect effects on alleviating cropping barriers. A possible explanation would be the reconstruction of the soil microbial community by the fumigation process, which would subsequently lead to changes in soil characteristics and plant performance, resulting in the effective alleviation of continuous cropping barriers.

Genome mining reveals abiotic stress resistance genes in plant genomes acquired from microbes via HGT.

Colonization by beneficial microbes can enhance plant tolerance to abiotic stresses. However, there are still many unknown fields regarding the beneficial plant-microbe interactions. In this study, we have assessed the amount or impact of horizontal gene transfer (HGT)-derived genes in plants that have potentials to confer abiotic stress resistance. We have identified a total of 235 gene entries in fourteen high-quality plant genomes belonging to phyla Chlorophyta and Streptophyta that confer resistance against a wide range of abiotic pressures acquired from microbes through independent HGTs. These genes encode proteins contributed to toxic metal resistance (e.g., ChrA, CopA, CorA), osmotic and drought stress resistance (e.g., Na+/proline symporter, potassium/proton antiporter), acid resistance (e.g., PcxA, ArcA, YhdG), heat and cold stress resistance (e.g., DnaJ, Hsp20, CspA), oxidative stress resistance (e.g., GST, PoxA, glutaredoxin), DNA damage resistance (e.g., Rad25, Rad51, UvrD), and organic pollutant resistance (e.g., CytP450, laccase, CbbY). Phylogenetic analyses have supported the HGT inferences as the plant lineages are all clustering closely with distant microbial lineages. Deep-learning-based protein structure prediction and analyses, in combination with expression assessment based on codon adaption index (CAI) further corroborated the functionality and expressivity of the HGT genes in plant genomes. A case-study applying fold comparison and molecular dynamics (MD) of the HGT-driven CytP450 gave a more detailed illustration on the resemblance and evolutionary linkage between the plant recipient and microbial donor sequences. Together, the microbe-originated HGT genes identified in plant genomes and their participation in abiotic pressures resistance indicate a more profound impact of HGT on the adaptive evolution of plants.

Soil Bacterial Community in the Multiple Cropping System Increased Grain Yield Within 40 Cultivation Years.

The shortage of land resources restricts the sustainable development of agricultural production. Multiple cropping has been widely used in Southern China, but whether the continuous planting will cause a decline in soil quality and crop yield is unclear. To test whether multiple cropping could increase grain yield, we investigated the farmlands with different cultivation years (10-20 years, 20-40 years, and >40 years). Results showed that tobacco-rice multiple cropping rotation significantly increased soil pH, nitrogen nutrient content, and grain yield, and it increased the richness of the bacterial community. The farmland with 20-40 years of cultivation has the highest soil organic carbon (SOC), ammonium nitrogen, and grain yield, but there is no significant difference in the diversity and structure of the bacterial community in farmlands with different cultivation years. The molecular ecological network indicated that the stability of the bacterial community decreased across the cultivation years, which may result in a decline of farmland yields in multiple cropping system> 40 years. The Acidobacteria members as the keystone taxa (Zi ≥ 2.5 or Pi ≥ 0.62) appeared in the tobacco-rice multiple cropping rotation farmlands, and the highest abundance of Acidobacteria was found in the farmland with the highest SOC and ammonium nitrogen content, suggesting Acidobacteria Gp4, GP7, GP12, and GP17 are important taxa involved in the soil carbon and nitrogen cycle. Therefore, in this study, the multiple cropping systems for 20 years will not reduce the crop production potential, but they cannot last for more than 40 years. This study provides insights for ensuring soil quality and enhancing sustainable agricultural production capacity.

Rational Design of Mini-Cas9 for Transcriptional Activation.

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

Chlorogenic Acid Functions as a Novel Agonist of PPARγ2 during the Differentiation of Mouse 3T3-L1 Preadipocytes.

Rosiglitazone (RG) is a well-known activator of peroxisome proliferator-activated receptor-gamma (PPARγ) and used to treat hyperglycemia and type 2 diabetes; however, its clinical application has been confounded by adverse side effects. Here, we assessed the roles of chlorogenic acid (CGA), a phenolic secondary metabolite found in many fruits and vegetables, on the differentiation and lipolysis of mouse 3T3-L1 preadipocytes. The results showed that CGA promoted differentiation in vitro according to oil red O staining and quantitative polymerase chain reaction assays. As a potential molecular mechanism, CGA downregulated mRNA levels of the adipocyte differentiation-inhibitor gene Pref1 and upregulated those of major adipogenic transcriptional factors (Cebpb and Srebp1). Additionally, CGA upregulated the expression of the differentiation-related transcriptional factor PPARγ2 at both the mRNA and protein levels. However, following CGA intervention, the accumulation of intracellular triacylglycerides following preadipocyte differentiation was significantly lower than that in the RG group. Consistent with this, our data indicated that CGA treatment significantly upregulated the expression of lipogenic pathway-related genes Plin and Srebp1 during the differentiation stage, although the influence of CGA was weaker than that of RG. Notably, CGA upregulated the expression of the lipolysis-related gene Hsl, whereas it did not increase the expression of the lipid synthesis-related gene Dgat1. These results demonstrated that CGA might function as a potential PPARγ agonist similar to RG; however, the impact of CGA on lipolysis in 3T3-L1 preadipocytes differed from that of RG.

[Smart therapeutics based on synthetic gene circuits].

Synthetic biology has an important impact on biology research since its birth. Applying the thought and methods that reference from electrical engineering, synthetic biology uncovers many regulatory mechanisms of life systems, transforms and expands a series of biological components. Therefore, it brings a wide range of biomedical applications, including providing new ideas for disease diagnosis and treatment. This review describes the latest advances in the field of disease diagnosis and therapy based on mammalian cell or bacterial synthetic gene circuits, and provides new ideas for future smart therapy design.

Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells.

Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. Therefore, we provide a valuable split-dCas9 toolkit to engineer complex transcription controls, which may inspire new biomedical applications.