Impact of Three Thiazolidinone Compounds with Piperine Skeletons on Trehalase Activity and Development of Spodoptera frugiperda Larvae.

Trehalases (TREs) are pivotal enzymes involved in insect development and reproduction, making them prime targets for pest control. We investigated the inhibitory effect of three thiazolidinones with piperine skeletons (6a, 7b, and 7e) on TRE activity and assessed their impact on the growth and development of the fall armyworm (FAW), Spodoptera frugiperda. The compounds were injected into FAW larvae, while the control group was treated with 2% DMSO solvent. All three compounds effectively inhibited TRE activity, resulting in a significant extension of the pupal development stage. Moreover, the treated larvae exhibited significantly decreased survival rates and a higher incidence of abnormal phenotypes related to growth and development compared to the control group. These results suggest that these TRE inhibitors affect the molting of larvae by regulating the chitin metabolism pathway, ultimately reducing their survival rates. Consequently, these compounds hold potential as environmentally friendly insecticides.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.

Host trehalose metabolism disruption by validamycin A results in reduced fitness of parasitoid offspring.

The general cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is a worldwide destructive omnivorous pest and the endoparasitoid wasp Meteorus pulchricornis (Hymenoptera: Braconidae) is the dominant endoparasitoid of S. litura larvae. Trehalase is a key enzyme in insect trehalose metabolism and plays an important role in the growth and development of insects. However, the specific function of trehalase in parasitoid and host associations has been less reported. In this study, we obtained two trehalase genes (SlTre1 and SlTre2) from our previously constructed S. litura transcriptome database; they were highly expressed in 3rd instar larvae. SlTre1 was mainly expressed in the midgut, and SlTre2 was expressed highest in the head. SlTre1 and SlTre2 were highly expressed 5 days after parasitization by M. pulchricornis. Treatment with the trehalase inhibitor validamycin A significantly inhibited the expression levels of SlTre1 and SlTre2, and the trehalase activity. Besides, the content of trehalose was increased but the content of glucose was decreased 24 h after validamycin A treatment in parasitized S. litura larvae. In addition, the immune-related genes in phenoloxidase (PO) pathway and fatty acid synthesis-related genes in lipid metabolism were upregulated in parasitized host larvae after validamycin A treatment. Importantly, the emergence rate, proportion of normal adults, and body size of parasitoid offspring was decreased in parasitized S. litura larvae after validamycin A treatment, indicating that validamycin A disrupts the trehalose metabolism of parasitized host and thus reduces the fitness of parasitoid offspring. The present study provides a novel perspective for coordinating the application of biocontrol and antibiotics in agroecosystem.

[Prevalence of trehalase enzymopathy genetic determinants in Siberian and Russian Far East populations].

To date, it has been established that the patient's genotype plays a significant role in the formation of trehalase enzymopathy: the level of enzyme activity decreases when the G→A allele replacement occurs in the rs2276064 locus of the TREH gene. To assess the prevalence of trehalase deficiency, extensive population-based studies are needed. Clinical observations show that the reduced activity of bowel trehalase is more common in the Arctic than in European populations. The aim of this research was to analyze the frequency of the alleles and variants of trehalase gene (rs2276064 TREH) in the indigenous small-numbered populations of Siberia and the Russian Far East. Material and methods. Using the Infinium iSelect HD Custom BeadChip biochip on the iScan platform and real-time polymerase chain reaction on a Bio-Rad CFX96 Touch amplifier, genotyping of 1068 DNA samples was carried out, of which 711 represent 10 ethnic groups of the indigenous people of the North of Siberia and the Far East of the Russian Federation. Two reference groups of Russians (n=311) and Yakuts (n=46) represented the "Caucasoid" and "Mongoloid" poles of the Russian population. Results. The reduced trehalase activity that the heterozygous GA*TREH genotype determines can manifest itself in 19.8-53.7% of indigenous northerners. An additional 1.0 to 19.7% of the population are carriers of the AA*TREH genotype, which is associated with apparent trehalose malabsorption. The carriers may experience nausea, abdominal pain, and other dyspeptic symptoms after eating trehalose containing foods. The total risk of trehalase enzymopathy among the indigenous northerners in the Asian part of the Russian Federation is very high and can reach 60-70%. There is a gradient in the A*TREH allele frequencies in the small-numbered indigenous northern groups of Russia from the west (Khanty, Mansi, Nenets) to the east (peoples of the Far East). Conclusion. The results are consistent with previously reported data on the higher carriage of the A*TREH mutant allele in Mongoloid populations compared to Caucasoid groups. It was hypothesized that, while the initial A*TREH allele prevalence in Mongoloid groups was moderately high, an adaptation to a low-sugar protein-lipid "high-latitude" diet led to a weaker control over the maintenance of the carriage of the ancestral G allele. Trehalose malabsorption requires special attention of specialists in the field of nutrition, gastroenterology, public health, and medical genetics working in high-latitude regions.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Prevalence of genetically determined trehalase deficiency in populations of Siberia and Russian Far East.

In order to be digested, the disaccharide trehalose needs to be cleaved by the trehalase enzyme. There were reports suggesting that trehalase deficiency was more common in high-latitude than in the temperate climate populations. New horizons were opened for the epidemiologic research of trehalase enzymopathy when it became clear that reduced trehalase activity is determined by the A allele of tTREH gene (rs2276064). The aim of this study was to analyze the frequencies of the trehalase gene alleles and genotypes among the indigenous peoples of Siberia and the Russian Far East. We genotyped 567 samples representing the indigenous peoples of Siberia and the Russian Far East and 146 samples representing Eastern Slavs as the reference dataset. We found that the frequencies of the A*TREH alleles increased to the east. The A*TREH allele frequency was 0.03 in the reference group, 0.13-0.26 in the North-West Siberian indigenous populations, 0.29-0.30 in the South Siberia, 0.43 in West Siberia, and 0.46 in the low Amur populations. The highest frequency of the A allele (0.63) was observed in the Chukchi and Koryak populations. From 1 to 5% of European origin individuals are at risk of trehalase enzymopathy. In the indigenous populations, the frequency of the A*TREH allele varies 13% to 63%, whereas the frequency of the AA*TREH genotype from 3% to 39%. Thus, the total risk of trehalase enzymopathy among the homo- and heterozygous carriers of the A*TREH allele in the studied indigenous populations may be as high as 24% to 86%.

VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Knockdown of Two Trehalase Genes by RNA Interference Is Lethal to the White-Backed Planthopper Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

Trehalase (Tre) is a crucial enzyme involved in trehalose metabolism, and it plays pivotal roles in insect development and metamorphosis. However, the biological function of Tre genes in Sogatella furcifera remains unclear. In the present study, two Tre genes-SfTre1 and SfTre2-were cloned and identified based on the S. furcifera transcriptome data. Bioinformatic analysis revealed that the full-length complementary DNA of SfTre1 and SfTre2 genes were 3700 and 2757 bp long, with 1728- and 1902-bp open reading frame encoding 575 and 633 amino acid residues, respectively. Expression analysis indicated that SfTre1 and SfTre2 were expressed at all developmental stages, with the highest expression in day two adults. Furthermore, the highest expression levels of SfTre1 and SfTre2 were observed in the ovary; enriched expression was also noted in head tissues. The knockdown of SfTre1 and SfTre2 via injecting double-stranded RNAs decreased the transcription levels of the corresponding mRNAs and led to various malformed phenotypes and high lethality rates. The results of our present study indicate that SfTre1 and SfTre2 play crucial roles in S. furcifera growth and development, which can provide referable information for Tre genes as a potential target for planthopper control.

In silico analysis and a comparative genomics approach to predict pathogenic trehalase genes in the complete genome of Antarctica Shigella sp. PAMC28760.

Although four Shigella species (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii) have been reported, S. sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world's driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic (treA) and cytoplasmic (treF) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes (treF and treA). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S. sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes (treF and treA) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.

Bioinformatic analyses to uncover genes involved in trehalose metabolism in the polyploid sugarcane.

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Opportunities Lost? Evolutionary Causes and Ecological Consequences of the Absence of Trehalose Digestion in Birds.

AbstractTrehalose is a nonreducing disaccharide that is a primary storage and energy source in prokaryotes, yeasts, fungi, and invertebrates. Vertebrates digest trehalose with the intestinal brush border membrane (BBM) enzyme trehalase. Intestinal trehalase activity is reported to be either very low or absent in several bird species. We assayed trehalase activity in 19 avian species, used proteomic analysis to quantify its abundance in the intestinal BBM, and used analyses of available genomes to detect the presence of the gene that codes for trehalase (Treh). We found no intestinal trehalase activity in birds, trehalase was absent from the proteome of their intestinal BBM, and the gene coding for trehalase was absent in their genomes. Surveys of available transcriptomes support the hypothesis that Treh is absent in birds. The trehalase gene was found in the same conserved syntenic block within the genome of all vertebrates surveyed except birds. Our analysis suggests that Treh was lost in an inversion followed by a reinsertion of a large gene block. This event appears to have taken place after the split between crocodiles and birds and dinosaurs. Birds are unable to digest a common dietary sugar like trehalose because their ancestor lost the trehalase gene. The loss of this gene seems to represent an ecological cost, as insectivorous birds seem to be unable to digest a carbohydrate present in their prey. We also speculate that the paucity of mycophagy in birds is due to the presence of large amounts of this sugar in fungal tissues.

Two trehalase isoforms, produced from a single transcript, regulate drought stress tolerance in Arabidopsis thaliana.

KEY MESSAGE: Alternative translation initiation of the unique Arabidopsis trehalase gene allows for the production of two isoforms with different subcellular localization, providing enzyme access to both intra- and extra-cellular trehalose. The trehalose-hydrolyzing enzyme trehalase mediates drought stress tolerance in Arabidopsis thaliana by controlling ABA-induced stomatal closure. We now report the existence of two trehalase isoforms, produced from a single transcript by alternative translation initiation. The longer full-length N-glycosylated isoform (AtTRE1L) localizes in the plasma membrane with the catalytic domain in the apoplast. The shorter isoform (AtTRE1S) lacks the transmembrane domain and localizes in the cytoplasm and nucleus. The two isoforms can physically interact and this interaction affects localization of AtTRE1S. Consistent with their role in plant drought stress tolerance, both isoforms are activated by AtCPK10, a stress-induced calcium-dependent guard cell protein kinase. Transgenic plants expressing either isoform indicate that both can mediate ABA-induced stomatal closure in response to drought stress but that the short (cytoplasmic/nuclear) isoform, enriched in those conditions, is significantly more effective.

Trehalase is required for sex pheromone biosynthesis in Helicoverpa armigera.

Trehalase (Treh) hydrolyzes trehalose to generate glucose and it plays important role in many physiological processes. Acetyl-CoA, the precursor of sex pheromone biosynthesis in the pheromone gland (PG) of Helicoverpa armigera, originates from glucose during glycolysis. However, the function of Treh in sex pheromone biosynthesis remains elusive. In the present study, H. armigera was used as a model to investigate the function of two Trehs (Treh1 and Treh2) in sex pheromone biosynthesis. Results demonstrated that knockdown of HaTreh1 or HaTreh2 in female PGs led to significant decreases in Z11-16:Ald production, female ability to attract males, and successful mating proportions. Pheromone biosynthesis activating neuropeptide (PBAN) treatment triggered HaTreh1 and HaTreh2 activities in the isolated PGs and Sf9 cells. However, the activities of HaTreh1 and HaTreh2 triggered by PBAN were offset by H-89, the specific inhibitor of protein kinase A (PKA). Furthermore, the H-89 treatment significantly decreased the phosphorylation level of Trhe2, which was induced by PBAN. In addition, sugar feeding (5% sugar) increased the enzyme activities of Treh1 and Treh2. In summary, our findings confirmed that PBAN activates Treh1/2 activities by recruiting cAMP/PKA signalling, promotes glycolysis to ensure the supply of acetyl-CoA, and ultimately facilitates sex pheromone biosynthesis and mating behaviour.

[Cloning, expression and properties of trehalase from Pectobacterium cypripedii].

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Purification and Characterization of Trehalase From Acyrthosiphon pisum, a Target for Pest Control.

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs 179PGGRFRELYYWDTY192 and 479QWDFPNAWPP489, a glycine-rich region 549GGGGEY554, and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with Km and Vmax values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health.

Validamycin A Enhances the Interaction Between Neutral Trehalase and 14-3-3 Protein Bmh1 in Fusarium graminearum.

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 µg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

[Genetically determined trehalase deficiency in various population groups of Russia and neighboring countries].

Due to the low specificity and sensitivity of non-invasive clinical tests trehalose malabsorption remained out of sight of gastroenterologists. Therefore, the specialists regard this disorder as rare. Trehalose became widely used in the food industry as a harmless sucrose substitute, sweetener and stabilizer. After the discovery of the trehalase gene (rs2276064 TREH), it was found that the A*TREH allele is the determinant of the disaccharide absorption disorders, and the allele's carriership may be high in some groups. There is not enough information on the A*TREH frequency in the population of Russia. The aim of the study was to analyze the allele and genotype frequencies of the trehalase gene (rs2276064 TREH) in the main population groups of the Russian Federation and neighboring countries. Methods. DNA samples from 1146 unrelated subjects belonging to 21 population groups of Russia, Azerbaijan, Tajikistan and Mongolia were genotyped by the two following methods: 1) using the Infinium iSelect HD Custom Genotyping BeadChip (Illumina, USA) on the iScan platform; 2) by the real time polymer-chain reaction (PCR) method on the Bio-Rad CFX96 Touch amplifier. Results. It has been found that on the territory of the Russian Federation the frequency of the A*TREH allele increases from the west to the east. The frequencies are lowest in the groups of Russians and Finns of the Northwest (0.01-0.03), up to 0.07 in the populations of Central Russia and the Volga region, and even higher toward the Southern Urals (Bashkirs 0.15), in the Transurals and Southern Siberia (0.19 in the Altai people, 0.30 in the Tuvinians and Mongols). Up to 1% of the population of the European part of the Russian Federation have the AA*TREH genotype (i.e. trehalose intolerance in phenotype), and up to 15% (GA*TREH genotype) have a reduced ability to absorb the disaccharide. In the Asian part of the country (Siberia, Altai, Baikal) the genotypes carriers constitute up to 12 and 46% respectively. Conclusion. Trehalose malabsorbtion is an underappreciated problem of particular practical importance for regions with high concentrations of indigenous population (Yakutia-Sakha, Buryatia, Tyva, etc.). It would be feasible to consider food labelling of trehalose.

Trehalose and glucose levels regulate feeding behavior of the phloem-feeding insect, the pea aphid Acyrthosiphon pisum Harris.

Trehalose serves multifarious roles in growth and development of insects. In this study, we demonstrated that the high trehalose diet increased the glucose content, and high glucose diet increased the glucose content but decreased the trehalose content of Acyrthosiphon pisum. RNA interference (RNAi) of trehalose-6-phosphate synthase gene (ApTPS) decreased while RNAi of trehalase gene (ApTRE) increased the trehalose and glucose contents. In the electrical penetration graph experiment, RNAi of ApTPS increased the percentage of E2 waveform and decreased the percentage of F and G waveforms. The high trehalose and glucose diets increased the percentage of E2 waveform of A. pisum red biotype. The correlation between feeding behavior and sugar contents indicated that the percentage of E1 and E2 waveforms were increased but np, C, F and G waveforms were decreased in low trehalose and glucose contents. The percentage of np, E1 and E2 waveforms were reduced but C, F and G waveforms were elevated in high trehalose and glucose contents. The results suggest that the A. pisum with high trehalose and glucose contents spent less feeding time during non-probing phase and phloem feeding phase, but had an increased feeding time during probing phase, stylet work phase and xylem feeding phase.

Autotransporter-based surface expression and complementation of split TreA fragments utilized for the detection of antibodies against bovine leukemia virus.

Bovine Leukemia Virus (BLV) is an oncogenic virus which is the etiological agent of a neoplastic disease in infected cattle called enzootic bovine leukemia (EBL). The most common and sensitive diagnostic methods for EBL like enzyme-linked immunosorbent assay (ELISA) is time-consuming and requires manual handling which makes it unsuitable as an on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have previously reported the use of E. coli periplasmic trehalase (TreA) in a split enzyme sensor diagnostic technology to detect immunoglobulins and antigen-specific antibodies. In the current study, a more sensitive detection was attempted by bacterial surface display of split TreA fragment by fusion with the autotransporter AIDA-I. The split TreA fragments fused to antigens require antigen-specific antibodies for complementation and to trigger trehalase activity. This surface complementation strategy was used to detect anti-BLV antibodies in clinical serum by incorporating the antigenic BLV capsid protein in the fusion proteins. To validate this assay, a panel of serum samples obtained from BLV positive and negative cattle were tested in comparison with ELISA results. Evaluation of this panel resulted in positive detection of all true positive samples. We further demonstrated that this assay can be enhanced by pre-adsorption of clinical serum samples using E. coli cells to increase the specificity and help reduce nonspecific binding. In conclusion, the p24 antigen specific BLV assay is a potential tool for simple and rapid diagnosis of BLV infection, which is compatible with both lab-based and a more user friendly on-farm format.

Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum.

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.