Lyase Activity Research Articles

Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichiacoli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

Abstract Mo026: ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart

Background: Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. Methods: We utilized MyH6-Cas9 mice (male and female) and CRISPR/Cas9 gene editing to generate a cardiac-specific Acly knockdown (AclyKD) mouse model. To assess how the loss of Acly impacts cardiac energy substrate metabolism, we conducted positron emission tomography in vivo and ex vivo stable isotope tracer labelling using working heart preparation. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics. Experimental data were integrated into computational modelling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Results: Loss of ACL expression causes a left-ventricular cardiomyopathy by histology and echocardiography. FDG-PET/CT imaging in male and female mice demonstrated an increased glucose uptake and utilization in AclyKD mice, suggesting a substrate switch from fatty acid oxidation towards glucose. These findings were corroborated by stable isotope tracing, demonstrating that AclyKD suppresses fatty acid oxidation while enhancing lipid remodelling. AclyKD also reduced the efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. Computational flux analysis and integrative multi-omics analysis indicate that ACL activity supports lipid biosynthesis in the heart. Blocking mitochondrial isocitrate dehydrogenase or alpha-ketoglutarate dehydrogenase sensitized cardiomyocytes to ACL inhibition, and overexpressing cytosolic isocitrate dehydrogenase was sufficient to prevent citrate accumulation. Conclusions: Our work demonstrates a previously undescribed function of ACL as a regulator of cardiac energy substrate metabolism and lipid modulation.

A duplexed high-throughput mass spectrometry assay for bifunctional POLB polymerase and lyase activity

Polymerase β (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2.

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.

Optimized medium composition in Physalis alkekengi callus culture altered nitric oxide level for inducing antioxidant enzyme activities and secondary metabolites

Physalis alkekengi L. is a valuable medicinal plant from the Solanaceae family and has multiple therapeutic applications. This study aimed to develop an optimized protocol for callogenesis in P. alkekengi to obtain friable calluses with high biomass. The effect of different concentrations of picloram, casein hydrolysate (CH), basal media (Murashige and Skoog (MS) and Gamborg (B5)), and static magnetic field (SMF) were investigated on the callus induction and growth, signaling molecules, and enzymatic and non-enzymatic antioxidants. Results showed that CH (200 mgL−1) and SMF4 mT for 90 min increased callus induction and fresh weight in P. alkekengi, while different concentrations of picloram reduced callogenesis. Hypocotyl explants showed various callogenesis and metabolic responses depending on the basal medium type. The 2B5 medium supplied with CH 200 (mgL−1) induced friable and cream calluses with high biomass (0.62 g) compared to the MS medium (control). The maximum activity of superoxide dismutase and catalase activities was identified in the 2B5 medium and peroxidase in the 2MS medium. The highest total phenolic (129.44 µg g−1DW) content and phenylalanine-ammonia lyase activity were obtained in the 2MS medium, and total withanolides (49.86 µg g-1DW) and DPPH radical scavenging activity were observed in the 2B5 medium. The 2MS medium boosted the hydrogen peroxide and nitric oxide levels, while their contents alleviated in the 2B5 medium, although these parameters were higher than the control. The findings of this study suggest that an effective protocol for successful callogenesis in P. alkekengi and the nutrient composition of culture medium by affecting the level of signaling molecules can control the antioxidant defense system and callus growth.

Volatiles emitted by Pseudomonas aurantiaca ST-TJ4 trigger systemic plant resistance to Verticillium dahliae

Verticillium dahliae is among the most devastating fungal pathogens, causing significant economic harm to agriculture and forestry. To address this problem, researchers have focused on eliciting systemic resistance in host plants through utilizing volatile organic compounds (VOCs) produced by biological control agents. Herein, we meticulously measured the quantity of V. dahliae pathogens in plants via RTqPCR, as well as the levels of defensive enzymes and pathogenesis-related (PR) proteins within plants. Finally, the efficacy of VOCs in controlling Verticillium wilt in cotton was evaluated. Following treatment with Pseudomonas aurantiaca ST-TJ4, the expression of specific VdEF1-α genes in cotton decreased significantly. The incidence and disease indices also decreased following VOC treatment. In cotton, the salicylic acid (SA) signal was strongly activated 24 h posttreatment; then, hydrogen peroxide (H2O2) levels increased at 48 h, and peroxidase (POD) and catalase (CAT) activities increased to varying degrees at different time points. The malondialdehyde (MDA) content and electrolyte leakage in cotton treated with VOCs were lower than those in the control group, and the expression levels of chitinase (CHI) and PR genes (PR10 and PR17), increased at various time points under the ST-TJ4 treatment. The activity of phenylalanine ammonia lyase (PAL) enzymes in cotton treated with VOCs was approximately 1.26 times greater than that in control plants at 24 h，while the contents of phenols and flavonoids increased significantly in the later stage. Additionally, 2-undecanone and 1-nonanol can induce a response in plants that enhances disease resistance. Collectively, these findings strongly suggest that VOCs from ST-TJ4 act as elicitors of plant defence and are valuable natural products for controlling Verticillium wilt.

Cyclic variable temperature conditioning-maintained storage quality of sweet potatoes by enhancing antioxidant activity

The study investigated the effect of cyclic variable temperature conditioning (CVTC) on the quality parameters of sweet potatoes and their relationship with the antioxidant system (phenols synthesis and reactive oxygen species metabolism). Results showed that CVTC maintained the color and normal respiratory intensity of sweet potatoes and reduced the accumulation of malondialdehyde (MDA) during storage. In detail, total phenols content (TPC) and total flavonoids content (TFC) in CVTC group were significantly higher than those in control (CK), cold storage (CS), and high temperature conditioning (HTC) groups at 8 d, with increases of 17.58% and 85.12% from day 0, respectively. Additionally, phenylalanine ammonia lyase (PAL) and hydroxycinnamoyl transferase (HCT) activities in CVTC group were significantly higher than those in CK, CS, and HTC groups at 8 d. These results showed that CVTC activated the synthesis of phenols by activating the key enzymes activities, resulting in a significant increase in total phenols and flavonoids contents. Besides, CVTC improved the superoxide anion scavenging capacity of sweet potatoes and promoted the decomposition of hydrogen peroxide (H2O2). Meanwhile, superoxide dismutase (SOD) and catalase (CAT) activities in CVTC group were significantly higher than those in CK, CS, and HTC groups in the late storage period. In conclusion, CVTC enhanced the antioxidant activity of sweet potatoes by regulating the phenols synthesis and reactive oxygen species metabolism to maintain their postharvest quality.

Bioprospecting of ornamental and weed plants for hydroxynitrile lyase activity and characterization of novel hydroxynitrile lyases (HNLs) of Euphorbia mili and Cascabela thevetia

Bioprospecting of ornamental and weed plants for hydroxynitrile lyase activity and characterization of novel hydroxynitrile lyases (HNLs) of Euphorbia mili and Cascabela thevetia

Spraying zinc oxide nanoparticles on pistachios (Pistacia vera L.) for preservation in cold storage

ABSTRACT The ability of zinc oxide nanoparticles to extend the shelf life of fresh pistachios in polyethylene packages in cold storage was evaluated. Independent variables were the type and concentration of the disinfectant (0.5%, 1%, 1.5%, and 2% nano-ZnO and 2% peracetic acid) and storage time (at harvest, 25, 50 and 75 days after harvest). Aflatoxin was not detected in the kernels of the disinfectant treatments after storage. A decrease occurred in firmness, chlorophylls and carotenoids in hulls and kernels over time. Phenolics and flavonoids of kernels decreased, parallel to a decrease in weight. Nano-ZnO (2%) prevented this reduction well. In hulls and kernels, nano-ZnO (2%) revealed the highest anthocyanin. Antioxidant activity in kernels remained stable or even increased in response to ZnO (1.5%) on day 75. All ZnO treatments prevented ion leakage while limiting hydrogen peroxide and malondialdehyde production in hulls. Peracetic acid reduced polyphenol oxidase activity on 75th day. ZnO (1% and 1.5%) and peracetic acid increased phenylalanine ammonia lyase activity. On 75th day, ZnO-treated samples had higher peroxidase activity. All ZnO treatments, especially 1.5% and 2%, prevented the loss of quality in hulls, shells, taste, and aroma. Nano-ZnO (2%) can be recommended for fresh pistachio preservation in cold storage.

Discovery and characterization of a novel poly-mannuronate preferred alginate lyase: The first member of a new polysaccharide lyase family

Alginate is one of the most important marine colloidal polysaccharides, and its oligosaccharides have been proven to possess diverse biological functions. Alginate lyases could specifically degrade alginate and therefore serve as desirable tools for the research and development of alginate. In this report, a novel catalytic domain, which demonstrated no significant sequence similarity with all previously defined functional domains, was verified to exhibit a random endo-acting lyase activity to alginate. The action pattern analysis revealed that the heterologously expressed protein, named Aly44A, preferred to degrade polyM. Its minimum substrates and the minimum products were identified as unsaturated alginate trisaccharides and disaccharides, respectively. Based on the sequence novelty of Aly44A and its homologs, a new polysaccharide lyase family (PL44) was proposed. The discovery of the novel enzyme and polysaccharide lyase family provided a new entrance for the gene-mining and acquiring of alginate lyases, and would facilitate to the utilization of alginate and its oligosaccharides.

Drought-induced cell wall degradation in the base of pedicel is associated with accelerated cotton square shedding

Drought significantly impacts cotton square (flower buds with bracts) shedding, directly affecting yield. To address the internal physiological mechanisms of drought affecting cotton square shedding, a polyethylene glycol-simulated drought study was conducted with Dexiamian 1 and Yuzaomian 9110 to investigate cell wall degradation changes in the base of pedicel where the detachment of cotton square takes place, and its relationship with cotton square shedding. Results revealed significant decreases in cellulose, hemicellulose, and pectin contents in the base of square pedicel, leading to cell wall degradation and consequent square shedding. Furthermore, drought stress exacerbated the hydrolysis of cellulose and pectin in the base of pedicel, although not hemicellulose, resulting in more noticeable alterations in the morphology and structure of the base of pedicel, such as more significant degradation in the epidermis, cortex, and phloem. Regarding the cellulose hydrolysis, drought mainly increased the expression of genes β-glucosidase (GhBG1) and endoglucanase (GhEG1), and the activity of β-glucosidase and endoglucanase in the base of pedicel, promoting the conversion of cellulose to cellobiose, and eventually glucose. Regarding the pectin hydrolysis, drought significantly enhanced the expression of the gene pectin methylase (GhPE1), thereby accelerating pectin hydrolysis to generate polygalacturonic acid. Additionally, drought increased the expression of genes pectin lyase (GhPL1) and polygalacturonase (GhPG1), as well as the activity of pectin lyase, which further accelerated the hydrolysis of polygalacturonic acid into galacturonic acid. These findings suggest that drought mainly promotes cellulose and pectin hydrolysis in the base of pedicel, hastening cell wall degradation and final cotton square shedding.

ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart.

Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.

Identification and Characterization of Pectate Lyase as a Novel Allergen in Artemisia sieversiana Pollen

Introduction: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. Methods: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen’s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. Results: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients’ serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients’ basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. Conclusion: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.

Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

(i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Interação fungicida-requeima na síntese de compostos fenólicos e atividade de enzimas de defesa em tomateiro

Due to the significant impact of late blight (LB) (Phytophthora infestans [Mont.]) on tomato (Solanum lycopersicum L.), we investigated the interaction between fungicides and this disease to understand how some plant defense mechanisms are affected over time. Following a randomized design, we evaluated the synthesis of phenolic compounds (PHE) and the activity of phenylalanine ammonium lyase (PAL), peroxidases (POX) and superoxide dismutase (SOD). The experiment involved the application of fosetyl-Al and fluoxastrobin (fungicides with dual modes of action) on healthy and infected tomato plants. LB severity was assessed weekly and leaf samples were collected at various intervals for biochemical analysis. The Kruskal-Wallis test (α = 0.05) analyzed main effects of infection, fungicide, and time on response variables, followed by Bonferroni post hoc for significant group differences and regression models to evaluate variable effects over time. The application of fungicides had no effect on enzymatic activity or PHE accumulation. While PAL and SOD activities were not significantly affected by infection, POX activity was significantly higher in healthy plants (4793.8 U g-1 fresh weight) compared to infected plants (1858.1 U g-1 fresh weight). A complex interaction between PHE accumulation in relation to LB severity and time was observed, with a notable increase in PHE levels at 50 days after transplant when disease severity was between 25 and 50%. Future studies should consider including a broader range of genotypes and isolates of P. infestans, a more extensive set of biochemical responses, and evaluations of the overexpression of genes related to plant defense.

MdVQ17 negatively regulates apple resistance to Glomerella leaf spot by promoting MdWRKY17-mediated salicylic acid degradation and pectin lyase activity.

Glomerella leaf spot (GLS) is a fungal disease caused by Colletotrichum fructicola, which severely restricts the yield and quality of apples. Valine-glutamine (VQ) proteins are transcriptional regulators involved in the regulation of plant growth and stress responses. However, little is known about the role of VQ proteins in the biotic stress response in apple. Here, a VQ gene, MdVQ17, that was highly induced by C. fructicola infection was identified. Overexpression of MdVQ17 in apple increased susceptibility to C. fructicola and significantly reduced the salicylic acid content and β-1,3-glucanase and chitinase activities. Based on yeast two-hybrid screening, MdWRKY17, which promotes susceptibility to C. fructicola, was identified as an MdVQ17-interacting protein. Co-expression of MdVQ17 can promote the binding and transcriptional activation activity of MdWRKY17 on the promoter of Downy Mildew Resistant 6 (MdDMR6), thereby promoting MdWRKY17-mediated salicylic acid degradation. Based on DNA affinity purification sequencing, the pectin lyase-encoding gene MdPL-like was identified as a direct target of MdWRKY17. MdWRKY17 can directly bind to the promoter of MdPL-like and activate its transcription, and the binding and activation of MdWRKY17 on the MdPL-like promoter were significantly enhanced by MdVQ17 co-expression. Functional identification showed that MdPL-like promoted pectin lyase activity and susceptibility to C. fructicola. In sum, these results demonstrate that the MdVQ17-MdWRKY17 module mediates the response to C. fructicola infection by regulating salicylic acid accumulation and pectin lyase activity. Our findings provide novel insights into the mechanisms by which the VQ-WRKY complex modulates plant pathogen defense responses.

The inhibitory effects of monolauroyl-galactosylglycerol against spores of Bacillus cereus and its potential application in dairy

Bacillus cereus is a typical spore-forming strain that can transform into dormant spores to resist unfavorable growth conditions. Among the microorganisms that cause spoilage of dairy products, B. cereus accounts for a large proportion. In this study, we evaluated the inhibitory activity of monolauroyl-galactosylglycerol (MLGG) on B. cereus spores and explored its inhibitory mechanism. The minimum inhibitory spore concentration (MISC) and the minimum sporicidal concentration (MSC) of MLGG against B. cereus spores were 0.313 mg/mL and 2.5 mg/mL, respectively. Scanning electron microscopy was used to observe the changes in spore morphology after MLGG treatment. It was found that low concentrations of MLGG caused irregular spore shape, with a few spores slightly ruptured and high concentrations of MLGG increased the degree of spore rupture, resulting in significant leakage of spore contents. By measuring the effect of MLGG on spore structure, it could be determined that MLGG damaged the coat of the spores and weakened the resistance of the spores, but did not affect the activity of cortical lyases. Therefore, spores could not resume growth even with the addition of exogenous lytic enzymes. After propidium iodide (PI) staining of the spores, flow cytometry analysis revealed that the positive area gradually increased, indicating that the permeability of the inner membrane of the spores was disrupted, leading to dipicolinic acid (DPA) release. The amount of DPA release was proportional to the MLGG concentration, and the growth rate of the spores decreased under hypertonic conditions. These findings indicated that MLGG inactivated spores primarily by damaging the inner membrane of the spores.

Fungicides, bio-controllers and resistance inducers affect bioactive compounds and oxidative metabolism in bell pepper plants inoculated with Phytophthora capsici

ABSTRACT The rational use of chemical fungicides in chilli pepper (Capsicum annuum L.) for controlling Phytophthora capsici Leo. should include molecules that promote induced systemic resistance in the plant. The study objective was to assess how the application of fungicides, bio-controllers and resistance inducers affects bioactive compounds production and oxidative metabolism in pepper plants when inoculated with P. capsici zoospores. Statistical analyses show significant differences in total flavonoids, total phenols and salicylic acid concentrations in plants treated with Trichoderma harzianum, Acibenzolar-S-Methyl (ASM) and phosphonic acid. In addition, the application of ASM and Bacillus subtilis showed the highest values of antioxidant capacity. On the other hand, ASM application favoured increases in superoxide dismutase and phenyl ammonium lyase activity with values of 4.42 ± 0.016 (units · min−1 · g−1) and 3.90 ± 0.22 U · g−1, respectively. However, hydrogen peroxide production was similar to that of phosphonic acid treatments. Similarly, plants treated with B. subtilis and Metalaxyl-M showed increases in peroxidase enzyme activity with values ranging from 2.86 ± 0.05 to 4.52 ± 0.02 (μmol · [mg protein]−1 · min−1). Likewise, phosphonic acid-treated plants exhibited high catalase enzyme activity with values ranging from 2.38 ± 0.032 μmol H2O2 · min−1 · g−1 to 4.30 ± 0.024 μmol H2O2 · min−1 · g−1. The complementary use of products with biological and resistance-inducing functions could help to mitigate the environmental impacts that occur due to the use of chemical fungicides for controlling P. capsici on chilli pepper.

Genetic Diversity and Population Structure of Bursaphelenchus xylophilus in Guangdong, Guangxi, and Jiangsu Provinces in China

This study aimed to investigate the genetic structures of pine wood nematodes (PWNs, Bursaphelenchus xylophilus (Steiner &amp; Buhrer) Nickle)), in Guangdong (GD), Guangxi (GX), and Jiangsu (JS) Provinces (the major PWN dispersal centers). Furthermore, we also explored potential migration routes among the different provinces in order to provide insights into the epidemic source of PWNs in the three provinces in China. We re-sequenced a total of 241 PWNs collected from the above provinces using next-generation sequencing to obtain raw genomic data. Bioinformatics analysis was used to identify the SNPs, genetic structures, and selective sweeps of the PWNs. The results indicate that the PWNs from these three provinces can be classified into five groups (A, B, C, D, and E), among which the genetic variations are significant. All PWN strains from JS were exclusively found in Group A. The PWNs in Groups B and C were composed of strains from GD and GX, while Groups D and E comprised only GD strains. Introgression analysis identified two possible pathways: (1) from Group A to Group B-GX and (2) from Group E to Group D. Selective sweep analysis showed that in Groups B and C, the candidate genes of Group B were mainly related to pectin lyase activity.

Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.