Elucidating fungicide complex formation mechanisms with Phytophthora infestans target proteins: In silico insights

The aim of this study was to study of the molecular mechanisms of herbicide binding (pinoxaden, quizalofop- P-tephuryl, prometryn, metribuzin, metsulfuron- methyl) to target proteins (acetyl- CoA carboxylase, acetolactate synthase, cytochrome P450, p-hydroxyphenylpyruvate dioxygenase) of the weed species growing in Siberia: wild oat Avena fatua (L.), wheatgrass Elymus repens (L.), amaranth Amaranthus retro exus (L.), common lamb's quarters Chenopodium album (L.), bristlegrass Setaria viridis (L.). The work was carried out in the Novosibirsk region in 2022-2023. Using bioinformatic methods, virtual three- dimensional complexes of pesticides and their targets were created. A new approach has been proposed to identify the parameters of the cavity of binding sites, which consists in combining the alignment method and machine learning technology. Docking of pesticides with targets was carried out. The calculation of the binding energy indicates a high degree of stability of the ligand- protein complexes. The in silico approach we proposed may be useful for elucidating the behavior of herbicides when bound to weed enzymes. This approach allows a deeper understanding of the mechanism of action and ecotoxicological aspects of the use of pesticides. The studied complexes of herbicides and proteins have a potential high stability due to the low binding energy with receptors, which varies from -5.60 to -13.24 kcal/mol. The binding energy value did not show a direct dependence on the number of amino acid residues of the ligand- binding site. In general, the study supplemented some aspects of the molecular mechanisms of binding herbicides to target proteins of weeds, however, a more in-depth study is required in order to localize critical amino acid points involved in the biological functions of target proteins.

Identification of pesticide targets is of great significance for the development of new pesticides. The new compound GLY-15, containing a pyrimidine heterocycle and a moroxydine skeleton structure, has good anti-TMV activity, but the underlying molecular targets and mechanism of action remain elusive. Here, host malate dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tobacco mosaic virus (TMV) coat protein (CP) were identified as potential targets of GLY-15 using activity-based protein profiling (ABPP) and drug affinity responsive target stability (DARTS), and their interactions with GLY-15 were validated by microscale thermophoresis (MST) and pull-down analysis. Functional analyses demonstrate that MDH silencing significantly reduces TMV accumulation, while transient overexpression of MDH results in elevated viral infection. Meanwhile, yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) analysis uncover that MDH interacts with CP, and their interaction is effectively inhibited by GLY-15. Site-directed mutagenesis identifies E225 as a critical residue for both GLY-15/MDH binding and MDH/CP interaction. Further investigations reveal that GLY-15 functions as an MDH inhibitor and affects its interaction with CP. Meanwhile, we showed that GLY-15 targeting MDH indicates broad antiviral activity against pepper mild mottle virus (PMMoV) and potato virus Y (PVY). This investigation systematically reveals novel insights into the anti-TMV mechanisms of GLY-15, establishing a valuable theoretical basis for antiviral target discovery and plant disease resistance breeding.

Background: NAC, as a unique transcription factor to plants, plays important roles in multiple biological functions, such as regulation of plant growth and development, hormone levels, and responses to various kinds of stresses. However, there is a lack of research of NAC genes in Chinese herbs. Objective: The study aimed to evaluate the potential functions of NAC genes in Scutellaria baicalensis by bioinformatics and expression analysis, and provide evidence of the molecular regulation mechanism involved in flavonoid biosynthesis in S. baicalensis. Methods: The genes of NAC transcription factors in S. baicalensis were obtained from cDNA library and their functions were explored using bioinformatic methods. The NAC genes were screened from the cDNA library of S. baicalensis using BLAST comparison software. Then, the open reading frame (ORF) finder online tool was used to predict the full-length ORFs of NAC genes and their protein characteristics were explored by bioinformatic methods. The expression of NAC genes was then detected by quantitative polymerase chain reaction in different parts of S. baicalensis and different leaves treated by gibberellin GA3 treatment. Results: Six genes of NAC transcription factors were cloned, two of which had complete ORFs. NAC genes cloned in this study were mainly expressed in the flowers of S. baicalensis. The expression levels of NAC2, NAC3, NAC4, NAC5, NAC6 were increased firstly and then decreased gradually after 100 μM GA3 treatment. Meanwhile, some NACs and PAL2 in S. baicalensis showed strong correlation. Conclusion: This study suggested that NACs cloned in this study were mainly regulated the flavonoid biosynthesis in the flowers of S. baicalensis; NAC6 in S. baicalensis might be involved in the regulation of PAL2 transcription and affected the accumulation of flavonoids in the root of S. baicalensis. Our results provided a basis for further understanding the molecular regulation mechanism of flavonoid biosynthesis in S. baicalensis.

Febrifugine, a natural alkaloid, exhibits specific anti-phytophthora activity; however, its mode of action is unclear. In this study, halofuginone, a synthetic derivative of febrifugine, showed significantly higher anti-phytophthora activities than those of febrifugine and the commercial drug metalaxyl against Phytophthora sojae, Phytophthora capsici, and Phytophthora infestans with effective concentration for 50% inhibition (EC50) values of 0.665, 0.673, and 0.178 μg/mL, respectively. Proline could alleviate the growth inhibition of halofuginone on P. capsici, implying that halofuginone might target prolyl-tRNA synthetase (PcPRS). The anti-phytophthora mechanism of halofuginone was then investigated by molecular docking, fluorescence titration, and enzymatic inhibition assays. The results revealed that halofuginone could bind to PcPRS and shared a similar binding site with the substrate proline. Point mutations at Glu316 and Arg345 led to 24.5 and 16.1% decreases in the enzymatic activity of PcPRS but 816.742- and 459.557-fold increases in the resistance to halofuginone, respectively. The results further confirmed that halofuginone was a competitive inhibitor of proline against PcPRS, and Glu316 and Arg345 played important roles in the binding of halofuginone and proline. Taken together, the results indicated that halofuginone is an alternative anti-phytophthora drug candidate and that PcPRS represents a potential target for the development of new pesticides.

Potato late blight is a devastating oomycete disease, which causes great economic losses to agricultural production. In this study, we analyzed the role and potential regulatory mechanism of <italic>StEFR1</italic> in regulating late blight resistance by the relative expression pattern and functional verification combined with the bioinformatics methods. Evolutionary analysis showed that the sequence similarity between StEFR1 and AtEFR was 53.9%. After inoculated with <italic>Phytophthora infestans</italic> for 3 days and treatment with elf18 for 3 hours, the relative expression level of <italic>StEFR1</italic> in isolated leaves of <italic>Atlantic</italic> was upregulated to 1.87 times and 2.31 times compared with the control, respectively. The late blight resistance significantly increased after the overexpression of <italic>StEFR1</italic> in the isolated leaves of <italic>Atlantic</italic> by <italic>agrobacterium</italic> infiltration method. Compared with the control, the area of leaf lesion size decreased and the activity of leaf cells increased. And the marker genes of PTI, SA, and JA signaling pathways in overexpressed leaves were significantly up-regulated to varied degrees, while the relative expression levels of ET related-genes did not change significantly. In conclusion, <italic>StEFR1</italic> was involved in the PTI resistance and regulated the relative expression levels of SA and JA hormone signaling related genes, suggesting that <italic>StEFR1 </italic>positively regulated the potato late blight. This study lays a foundation for further revealing the molecular mechanism of <italic>StEFR1</italic> in regulating the immune response and provides important reference for the molecular breeding of late blight.

The article describes fungicide-free ecologically tolerant technologies for the protection of potato and tomato against Phytophthora infestans (Mont.) de Bary, agent of late blight of these crops. It has been proved that potato cultivars with field resistance (Lugovskoy) provide a protection level comparable to the effectiveness of modern fungicides. Plant extracts from Southern plants can be used as biopesticides in order to additionally suppress the development of late blight. For example, extracts of four species from Yemen (fam. Asteraceae: Psiadia arabica Jaup and Spach; fam. Lamiaceae: Lawsonia inermis L ; Dodonea viscos L ; Euryops arabicus Steud. ex Jaub and Spach) were capable of suppressing late blight development on potato. On the contrary, plant growth regulators, Novosil (active compound triterpene acids), Larixin (active compound dihydroquerticin), and Terpenol (active compound triterpene acids), in spite of the periods of high efficiency against late blight, do not provide satisfying effect since these substances could sometimes even promote a pathogen development. Also, vaccination of tomato with attenuated strain of tobacco mosaic virus (TMV) could improve viability and immune status of plants under normal conditions, and in the case of stress situation it promoted weakness of plants and P. infestans infection. In Eastern Europe, fungicide-free technologies for the protection of potato and tomato against late blight, corresponding to the ecological criterion, should be additionally elaborated and tested for potato and tomato cropping.

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Gibberellin 2-oxidase (GA2ox) is a key enzyme regulating the metabolism of gibberellic acid (GAs) in plants. Identifying GA2ox genes in poplar and analyzing their functions in regulating plant growth and development can provide technical support for breeding new poplar varieties. In this study, bioinformatics methods were used to identify and analyze GA2ox genes in hybrid 'Poplar 741'. A total of 34 GA2ox genes were identified, which were distributed on 7 pairs of chromosomes of hybrid 'Poplar 741'. The tissue expression pattern and GA3-induced expression pattern of PthGA2ox19 were analyzed by quantitative real-time PCR (qRT-PCR). It was found that PthGA2ox19 was highly expressed in stems and its expression level was significantly increased under GA3 induction. The overexpression vector of PthGA2ox19 was constructed and transformed into poplar by Agrobacterium transformation method. It was found that the expression level of PthGA2ox19 in transgenic lines was significantly higher than that in wild-type plants, and the phenotypes showed shorter plant height, thinner stem, shorter internodes and smaller leaves. The development of vascular tissues in transgenic plants was analyzed by paraffin section and microscope observation. The analysis of vascular tissue development showed that the vascular bundles of transgenic plants developed abnormally, the diameter of vessels decreased, and the thickness of xylem and phloem became thinner. In this study, 34 GA2ox genes of hybrid 'Poplar 741' were identified. The overexpression of PthGA2ox19 inhibited the growth and vascular tissue development of poplar, indicating that poplar GA2ox is involved in the regulation of plant growth and development, which can provide a new way for poplar plant type breeding.

Diamondback moths, &lt;italic&gt;Plutella xylostella&lt;/italic&gt; (Linnaeus) are one of the major pests of cruciferous plants such as cabbage and cauliflower (&lt;italic&gt;Brassica oleracea&lt;/italic&gt;) in India. These insect show resistance against commonly used pest management practices such as, chemical insecticides and bacterial pathogens, including &lt;italic&gt;Bacillus thuringiensis&lt;/italic&gt; (Bt). To overcome the resistance problem, develop new pesticides that can act through specific drug targets. To identify such drug targets specific to this insect, data mining and annotation of Expressed Sequence Tags (ESTs) were performed in this study. Expressed Sequence Tags (ESTs, 37,915) of the insect obtained from GenBank were clustered and consensus sequences (4224) were constructed using mining tool (CAP3). Out of it 256 sequences were functionally annotated using three Gene Ontologies (GO) terms, molecular function, biological process and cellular component using similarity search methods (BLASTOGO). By mapping the candidate genes to KEGG Pathway, 38 insect metabolic pathways, inclusive of xenobiotic metabolism by Cytochrome P450 were generated. One of the mapped candidate gene codes for aldehyde dehydrogenase enzyme, which is potentially involved in xenobiotic detoxification of synthetic insecticides, and play a role in the development of resistance to pesticides. Data mining and functional annotation helped to narrow down the choices for potential drug targets, which could aid in the development of new pesticides to overcome resistance in Diamondback moths. This methodology can be extended to other agriculturally important pests to identify the drug targets using EST data.

Chenopodium quinoa is a plant rich in a variety of nutrients, with strong resistance to cold, drought, salt, and barren conditions. BHLH transcription factors are one of the largest transcription factor families in plants, and they have a basic/helix-loop-helix structure. BHLH transcription factors are involved in the regulation of plant growth and development and play a role in stress resistance. Besides, the bHLH family also participates in various signal transduction and anabolic processes, such as light signal transduction and hormone synthesis. In view of the important roles of the bHLH family in plant growth, development, anabolism, signaling, and resistance to stress, the Chenopodium quinoa bHLH (CqbHLH) family was identified in the genome by using bioinformatics methods. Moreover, the structure, chromosomal assignment, replication, and collinearity of all CqbHLH genes were analyzed. The results of this study have laid the foundation for identifying CqbHLH genes that play a key role in the regulation of plant growth and development and in stress resistance.

BackgroundAP2/ERF transcription factors are involved in the regulation of plant growth, development, and stress responses. Our research objective was to characterize novel apple (Malus × domestica Borkh.) genes encoding AP2/ERF transcription factors involved in regulation of plant growth, development, and stress response. The transcriptional level of apple AP2/ERF genes in different tissues and under various biotic and abiotic stress was determined to provide valuable insights into the function of AP2/ERF transcription factors in apple.MethodsThirty full-length cDNA sequences of apple AP2/ERF genes were isolated from ‘Zihong Fuji’ apple (Malus × domestica cv. Zihong Fuji) via homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. Expression levels of apple AP2/ERF genes were detected in 16 different tissues using a known array. Expression patterns of apple AP2/ERF genes were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq with existing data, and the expression of apple AP2/ERF genes was analyzed under NaCl and mannitol treatments using qRT-PCR.ResultsThe sequencing results produced 30 cDNAs (designated as MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF31-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2). Phylogenetic analysis revealed that MdERF11/16, MdERF33/35, MdERF34/39, and MdERF18/23 belonged to groups A-2, A-4, A-5, and A-6 of the DREB subfamily, respectively; MdERF31, MdERF19, MdERF4/25/28/32, MdERF24, MdERF5/6/27, and MdERF3/7/8/17/22/26 belonged to groups B-1, B-2, B-3, B-4, B-5, and B-6 of the ERF subfamily, respectively; MdAP2D60 and MdAP2D62/63/64/65 belonged to the AP2 subfamily; and MdRAV2 belonged to the RAV subfamily. Array results indicated that 30 apple AP2/ERF genes were expressed in all examined tissues to different degrees. RNA-seq results using previously reported data showed that many members of the apple ERF and DREB subfamilies were induced by Alternaria alternate apple pathotype (AAAP) infection. Under salt treatment, many members in the apple ERF and DREB subfamilies were transcriptionally up or down-regulated. Under mannitol treatment, many members of the apple ERF, DREB, and AP2 subfamilies were induced at the transcriptional level. Taken together, the results indicated that the cloned apple AP2/ERF genes were expressed in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol treatment, which suggested they may be involved in regulating growth, development, and stress response in apple.

The long‐term viability of pesticides to control pests, diseases, and weeds is always threatened by resistance. Consequently, there is a need to understand the causes of resistance and the factors that influence its spread, so that the problem can be managed effectively. Chemical pesticides bind to specific sites in their target proteins, and small changes can disrupt this binding. High levels of resistance are often conferred by just a single amino acid change, and the same change causes resistance to a pesticide group in different species, and even phyla. Surprisingly, these changes are generally in highly conserved regions of the target protein. Other mechanisms, such as detoxification, increased efflux associated with multidrug resistance (MDR), overexpression of the target protein and altered metabolic pathways all contribute to resistance, but generally these mechanisms confer lower levels of resistance than target site changes.Biochemistry provides ways to generate resistance, but individuals possessing these mechanisms must not suffer fitness penalties as a consequence. Bioassay remains the main way to monitor resistance, although it is often resource intensive, and it provides little information on the mechanism conferring resistance. Biochemical and DNA methods offer ways to overcome these difficulties, and they are increasingly used to monitor resistance where mechanisms are known.Combining all this information allows assessment of resistance risk and implementation of strategies to manage the problem. Predictive modeling can be useful, but there is always an empirical component in developing anti‐resistance strategies. A corner stone of all anti‐resistance strategies involves using pesticides with different modes of action, and integrating this with nonchemical control measures. Examples of successful anti‐resistance strategies are few, and effective control often rests with the development of new pesticides with novel modes of action.

Heterochronic Genes and the Nature of Developmental Time

The article observes peculiarities of the development of Alternaria solani and Phytophthora infestans potato pathogens for the use of chemical and biological agents and plant growth regulators (PGR) under laboratory conditions. All fungicides of chemical origin — Consento 450 SC, Acrobat MC, Centurion, Anthracol 70 WP, for efficiency were equivalent and showed high efficacy (100%) in laboratory conditions in terms of limiting the development of pathogens of leaf diseases potatoes. The use of biological preparations reduced the development of pathogens of fungi, indicating their effectiveness. The best effect was shown by Phytosporin — M, n — on the 14th day of the experiment, the development of diseases (diameter of the colony on the nutrient medium) was 71.1 and 19.3 mm, which is higher than control at 22.8 and 27.9 mm, respectively. Among PPPs, the most effective for the development of Phytophthora infestans was Gumysol, And in relation to Alternaria solani — Gumysol, Rh. And Poteitin.

Introduction. Methylethylpyridinol (MEPD or Emoxypine) is widely used in practice to treat various eye diseases, exhibiting angioprotective, antiplatelet, antioxidant effects. The molecular mechanisms for the implementation of these and other pharmacological effects of MEPD are not entirely obvious.Aim. To conduct a chemoreactome analysis of MEPD aimed at identifying the molecular mechanisms of the drug action. Materials and methods. The pharmacological/biological properties of MEPD were assessed using methods for chemoinformatic analysis of molecules developed in the scientific school of RAS academicians Yu.I. Zhuravlev and K.V. The analysis procedure is based on the latest machine learning technologies developed in the theory of topological and metric analysis of feature descriptions in application to chemographs.Results and discussion. The results of chemoproteomic profiling of MEPD showed anti-inflammatory, antihypoxic, antioxidant, vasoprotective and vasorelaxant effects of the drug. By inhibiting arachidonate-5-lipoxygenase, leukotriene A4 hydrolase, leu kotriene LTB4 and prostanoid receptors, inhibiting the production of superoxide anions and leukotriene, MEPD helps to reduce local inflammation in eye tissues. Evaluations of the vasodynamic and neuroprotective effects of MEPD in vitro, obtained as a result of chemoreactome analysis, indicated neurotrophic, neuroprotective and vasodilatory effects of MEPD. The neurotrophic effect in vitro in cultured sensory neurons and neuroprotective activity, assessed by the protective effect against L-homocysteine, exceeded the activity of control molecules. The analysis demonstrated the vasodilatory activity of MEPD and the reduction of intraocular pressure in vivo.Conclusions. Based on the results of the chemoreactome analysis, new molecular mechanisms of the anti-inflammatory action of MEPD in eye tissues were proposed, carried out through the inhibition of certain target proteins. The antioxidant properties of MEPD can be associated with both specific interactions with proteome proteins (e.g., activation of antioxidant proteins) and with the direct action of the molecule on reactive oxygen species.