Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.

Trehalose in pine wood nematode participates in DJ3 formation and confers resistance to low-temperature stress

Background Recently, pine wood nematode (PWN, Bursaphelenchus xylophilus) has been found in the extreme cold area of northeast China. The third-stage dispersal juvenile (DJ3) of PWN, which is a long-lived stress-resistant stage, plays an important role in the process of PWN spreading to low-temperature areas, as this stage can survive under unfavorable conditions. Results Weighted correlation network analysis (WGCNA) was used to analyze the expression patterns of 15,889 genes included in 21 RNA-Seq results of PWN at DJ3 and the other 6 different stages, and a total of 12 coexpression modules were obtained. Among them, the magenta module has the highest correlation with DJ3, which included a total of 652 genes. KEGG enrichment analysis showed that most of the genes in the magenta module were involved in metabolic processes, which were related to autophagy and longevity regulation. These pathways included starch and sucrose metabolism, which contains trehalose metabolism. To explore the function of trehalose in DJ3 formation and survival under − 20 °C, a trehalose-6-phosphate synthase encoding gene (Bx-tps), a trehalose-6-phosphate phosphatase encoding gene (Bx-tpp) and 7 trehalase encoding genes (Bx-tres) were identified and investigated. The expression of these 9 genes was related to the formation of DJ3. A treatment under − 20 °C induced the accumulation of trehalose. The survival rate of DJ3 at -20 °C reduced after silencing of any of these trehalose metabolism genes. Further analysis suggested that two trehalose synthesis genes were highly correlated with DJ3 and might be involved in autophagy by regulating with energy conversion related genes. Conclusions The above results indicated that trehalose metabolism promotes DJ3 formation and helps DJ3 survive at -20 °C. Although trehalose accumulation is favorable for DJ3 to cope with low-temperature stress, multiple trehalose metabolism genes need to work together. There may be a multi-path regulated physiological process involving trehalose synthesis genes under low-temperature stress resistance. This physiological process may regulate the formation and maintenance of DJ3 through autophagy and energy conversion.

Parallel evolution of trehalose production machinery in anhydrobiotic animals via recurrent gene loss and horizontal transfer

Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose biosynthesis genes remain unidentified in the large majority of metazoan phyla, including vertebrates. To uncover the evolutionary history of trehalose production machinery in metazoans, we scrutinized the available genome resources and identified bifunctional trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase (TPS–TPP) genes in various taxa. The scan included our newly sequenced genome assembly of a desiccation-tolerant tardigrade Paramacrobiotus sp. TYO, revealing that this species retains TPS–TPP genes activated upon desiccation. Phylogenetic analyses identified a monophyletic group of the many of the metazoan TPS–TPP genes, namely ‘pan-metazoan’ genes, that were acquired in the early ancestors of metazoans. Furthermore, coordination of our results with the previous horizontal gene transfer studies illuminated that the two tardigrade lineages, nematodes and bdelloid rotifers, all of which include desiccation-tolerant species, independently acquired the TPS–TPP homologues via horizontal transfer accompanied with loss of the ‘pan-metazoan’ genes. Our results indicate that the parallel evolution of trehalose synthesis via recurrent loss and horizontal transfer of the biosynthesis genes resulted in the acquisition and/or augmentation of anhydrobiotic lives in animals.

Regulation of a Trehalose-Specific Facilitated Transporter (TRET) by Insulin and Adipokinetic Hormone in Rhodnius prolixus, a Vector of Chagas Disease

Using the blood-sucking kissing bug, Rhodnius prolixus as an experimental model, we have studied the involvement of insulin-like peptides (ILPs) and adipokinetic hormone (AKH) signaling in carbohydrate metabolism, focusing on the regulation of the trehalose-specific facilitated transporter (Rhopr-TRET), particularly in the ovaries. We find that trehalose stores in ovaries increase after feeding, synchronously with the beginning of vitellogenesis, but that the transcript expression of enzymes involved in trehalose synthesis show no changes between unfed and blood-fed animals. However, an eightfold increase in Rhopr-TRET transcript expression is observed in the ovaries post-blood meal. In vivo and ex vivo assays using exogenous insulins and Rhopr-AKH, reveal that Rhopr-TRET is up-regulated in ovaries by both peptide families. In accordance with these results, when ILP and AKH signaling cascades are impaired using RNA interference, Rhopr-TRET transcript is down-regulated. In addition, trehalose injection induces an up-regulation of Rhopr-TRET transcript expression and suggests an activation of insulin signaling. Overall, the results support the hypothesis of a direct trehalose uptake by ovaries from the hemolymph through Rhopr-TRET, regulated by ILP and/or AKH. We also show that Rhopr-TRET may work cooperatively with AKH signaling to support the release of trehalose from the ovaries into the hemolymph during the unfed (starved) condition. In conclusion, the results indicate that in females of R. prolixus, trehalose metabolism and its hormonal regulation by ILP and AKH play critical roles in adapting to different nutritional conditions and physiological states.

Comparative Genome Analysis of Carbohydrate-Active Enzymes and Virulence Factors in Lichen-Associated Variovorax Sp. PAMC28711

Background: The genus Variovorax sp. PAMC28711 is a cold-adapted microorganism, isolated from Antarctica lichen Himantormia. The complete genomes of six Variovorax species were analyzed and compared along with the strain PAMC28711. The genomic information was collected from NCBI as well as PATRIC databases. Likewise, CAZyme annotation (dbCAN2 Meta server) was performed in order to predict the CAZyme family responsible for trehalose synthesis and trehalose degradation enzymes. The trehalose metabolic pathway was analyzed via the KEGG database. Bioinformatics tools such as OrthoANI software were used to analyze similar genes in different strains under the same genus. Likewise, MEGA X was used for evolutionary and conserved genes.Results: The complete genome of genus V. sp. PAMC28711 was found to comprise CAZyme families GH (10), GT (9), CB (1), AA5 (1), and CE (1). The three trehalose synthetic pathways (OtsA/OtsB, TS, and TreY/TreZ) and trehalose degradation pathway (TreF) were identified only in V. sp. PAMC28711 among the different strains of Variovorax studied, whereas one to two pathways of trehalose biosynthesis, but not trehalose degradation pathways are involved in other Variovorax strains. The strain PAMC28711 comprises of cytoplasmic trehalase (TreF) as a trehalose degrading enzyme that belongs to the CAZyme family GH37, which is not identified in other strains of Variovorax.Conclusions: To date, although the genus V. sp. PAMC28711 has not been reported to exhibit CAZyme activities such as trehalase, and no microorganism expressed different virulence factors, the results based on PATRIC database showed that the strain carried a few virulence genes. Further, this study provides additional information regarding trehalase as one of the factors facilitating bacterial survival under extreme environments and this enzyme has showed potential application in biotechnology fields.

Unique genomic traits for cold adaptation in Naganishia vishniacii, a polyextremophile yeast isolated from Antarctica

Cold environments impose challenges to organisms. Polyextremophile microorganisms can survive in these conditions thanks to an array of counteracting mechanisms. Naganishia vishniacii, a yeast species hitherto only isolated from McMurdo Dry Valleys, Antarctica, is an example of a polyextremophile. Here we present the first draft genomic sequence of N. vishniacii. Using comparative genomics, we unraveled unique characteristics of cold associated adaptations. From a total of 6183 predicted coding sequences, we identified 336 genes absent in sister species, encoding solute transfers and chaperones, among others. Among genes shared by N. vishniacii and its closest related species we found orthologs encompassing possible evidence of positive selection (dN/dS > 1). Genes associated with photoprotection were found in agreement with high solar irradiation exposure. Also genes coding for desaturases and genomic features associated with cold tolerance (i.e. trehalose synthesis and lipid metabolism) were explored. Finally, biases in amino acid usage (namely enrichment of glutamine and a trend reduction of proline) were observed, possibly conferring increased protein flexibility. To the best of our knowledge, such a combination of mechanisms for cold tolerance has not been previously reported in fungi, making N. vishniacii a unique model for the study of the genetic basis and evolution of cold adaptation strategies.

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background In Saccharomyces cerevisiae, alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear. Results RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members. Conclusions The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background: In Saccharomyces cerevisiae, alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear.Results: RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose.Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members.Conclusions: The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.

MAL62 overexpression enhances uridine diphosphoglucose-dependent trehalose synthesis and glycerol metabolism for cryoprotection of baker’s yeast in lean dough

Background: In Saccharomyces cerevisiae, the alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. Overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear.Results: RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased transcription of uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in intracellular glycerol concentration which was consistent with the elevated transcription of glycerol synthesis pathways. Conclusions: The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol plays a secondary role. These findings shed new light to the mechanism of yeast response to freezing in lean bread dough and can help the improvement of industrial yeast strains.