The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ-WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ-WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37-MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC-NBS-LRR resistance gene MdRPM1. DAP-seq, Y1H, EMSA, and RT-qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37-MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ-WRKY complex in plant pathogen defence responses.

Colorimetric aggregation assay for kanamycin using gold nanoparticles modified with hairpin DNA probes and hybridization chain reaction-assisted amplification.

The authors describe a colorimetric method for determination of kanamycin by using gold nanoparticles (AuNPs) as the element of signal-conversion and by applying hybridization chain reaction-assisted signal amplification. The assay is carried out by monitoring the absorbance change and color change adding salt to the reaction solution containing kanamycin (analyte), hairpin DNA probe, and AuNPs. Three hairpin DNA probes with sticky ends were absorbed on the AuNPs via their sticky ends. Cating with DNA prevents them from salt-induced aggregation (which leads to a color change from red to blue) in the complete absence of kanamycin. In contrast, in the presence of kanamycin, the aptamer hairpin DNA probe binds kanamycin, and the newly exposed section of DNA triggers a cascade of hybridization chain reactions with formation of numerous dsDNAs. On addition of salt, the AuNPs form blue aggregates due to the repulsion between dsDNA and AuNPs. Under optimal conditions, the ration of absorbance at 520 and 630 nm drops with the kanamycin concentration in the range from 1 to 40 µM, and the limit of detection is 0.68 µM. The assay can selectively distinguish kanamycin from other antibiotics. The method was applied to kanamycin detection in (spiked) milk samples and gave excellent recoveries. Graphical abstract Schematic presentation of colorimetric method for kanamycin detection using gold nanoparticles modified with hairpin DNA probes and hybridization chain reaction-assisted amplification.

Identification of Conidiogenesis-Associated Genes in Colletotrichum gloeosporioides by Agrobacterium tumefaciens-Mediated Transformation.

The Colletotrichum gloeosporioides is one of the most significant pathogens leading to huge economic losses. To infect plants and cause disease dissemination, the fungus elaborates to produce asexual spores called conidia, which are long-lived and highly resistant to environmental stresses. Here, we report a large-scale, systematic genome-wide screening of conidiogenesis-associated genes via conidiation assays, and high-efficiency TAIL-PCRs. Of 10,210 independent transformants tested, 59 mutants exhibited significant variation in conidial production. The T-DNA right flanking sequences of 11 conidiation-related transformants were further identified, and the obtained sequences were aligned to the genome sequence to uncover the novel loci of sporogenesis. When considering together, this study provided a large number of conidial production-variation mutants and the conidiation-related genes, which will be a valuable resource for characterizing the molecular mechanisms of conidial formation in the fungus.

[Genome-wide identification and expression analysis of the WRKY gene family in peach].

The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles.

Comparative study on secondary metabolites from different citrus varieties in the production area of Zhejiang.

To investigate the distribution pattern of bioactive components and their correlations between citrus varieties, we thoroughly analyzed secondary metabolites (including flavonoids, phenolic acids, carotenoids, and limonoids) in the peel and pulp of 11 citrus varieties from the production area of Zhejiang. Citrus peels accumulated metabolites far more than the pulp, and the accumulation varied significantly between species. Flavonoids were the most abundant compounds, followed by phenolic acids, with carotenoids and limonoids being far less abundant than the first two, but limonoids were more abundant than carotenoids. Hesperidin was the main flavonoid in most varieties, but cocktail grapefruit and Changshanhuyou contained naringin, with Ponkan having the most abundant polymethoxylated flavones (PMFs). The major components of phenolic acids, carotenoids, and limonoids were ferulic acid, ß-cryptoxanthin, and limonin, respectively. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) indicated that these components were mostly correlated with each other, and these citrus varieties could be categorized into four groups by pulp and three groups by peel. The obtained results filled the data gap for secondary metabolites from local citrus and could provide data references for citrus resource utilization, selection and breeding of superior varieties, and other research.

Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells.

BACKGROUND: Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary. METHODS: Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression. RESULTS: Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined. CONCLUSIONS: In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.

A gold nanoparticle-based visual aptasensor for rapid detection of acetamiprid residues in agricultural products using a smartphone.

Based on the colorimetric analysis of gold nanoparticles and a smartphone readable strategy, a stable, sensitive, and visual method was established for rapid detection of acetamiprid residues in agricultural products. By optimizing the key parameters, the detection process only took 40 minutes with good specificity. The acetamiprid aptamer can help AuNPs to resist salt-induced aggregation. Conversely, in the presence of acetamiprid, the anti-protection is weakened and the AuNPs aggregated with the color change of the solution. The photographs of the solution are recorded by the smartphone and analyzed through image processing. In the range from 25 to 300 µM the method can realize a quantitative analysis of acetamiprid, and the detection limit is about 3.81 µM. Excellent recoveries are taken in samples of cucumber, cabbage, and river water, ranging from 96.78% to 129.95%. These results show no significant difference from the results obtained by the microplate reader. What's more, the method employs a smartphone to read without the assistance of professional equipment, which greatly reduces the cost of detection, and shows a promising application prospect for on-site rapid detection of acetamiprid.

Characterization of the genetic and regulatory networks associated with sugar and acid metabolism in apples via an integrated strategy.

Although sugars and acids have a substantial influence on the taste of apple fruits, the genetic and regulatory networks underlying their metabolism in fruit remain insufficiently determined. To fully decipher the genetic basis of the accumulation of sugars and acids in apple fruits, we adopted an integrated strategy that included time-course RNA-seq, QTL mapping, and whole-genome sequencing to examine two typical cultivars ('HanFu' and 'Huahong') characterized by distinctive flavors. Whole-genome sequencing revealed substantial genetic variation between the two cultivars, thereby providing an indication of the genetic basis of the distinct phenotypes. Constructed co-expression networks yielded information regarding the intra-relationships among the accumulation of different types of metabolites, and also revealed key regulatory nodes associated with the accumulation of sugars and acids, including the genes MdEF2, MdPILS5, and MdGUN8. Additionally, on the basis of QTL mapping using a high-density genetic map, we identified a series of QTLs and functional genes underlying vital traits, including sugar and acid contents. Collectively, our methodology and observations will provide an important reference for further studies focusing on the flavor of apples.

Synthesis of ficin-protected AuNCs in a droplet-based microreactor for sensing serum ferric ions.

A droplet-based microfluidic synthesis approach for preparation of ficin capped gold nano clusters (AuNCs) was developed. Well dispersed AuNCs could be procured within 8 min. Upon excitation wavelength at 340 nm, the resultant AuNCs exhibited a strong blue fluorescence with the maximum emission at 450 nm. Due to the aggregation-induced "turn-off" fluorescence mechanism, the synthesized AuNCs as a fluorescent probe displayed high sensitivity and good selectivity for sensing ferric ions. The relative fluorescence intensity versus ferric ions concentration yielded a good linear calibration in the range of 10.0-1000.0 µM (R2 = 0.998) and the limit of detection was 4.1 µM. Moreover, the possible mechanism for abated fluorescence intensity of AuNCs by adding ferric ions was discussed briefly. Further, the as-prepared fluorescent AuNCs was successfully applied for the detection of serum ferric ions. The results indicated that the droplet-based microfluidic synthesis system could provide a new way for the rapid preparation of AuNCs with good polydispersity and have potential as the sensing probes for the analysis of ferric ions in real biological samples.