Metabolomic and Transcriptomic Analyses Reveal the Molecular Mechanism Underlying the Massive Accumulation of Secondary Metabolites in Fenugreek (Trigonella foenum-graecum L.) Seeds.

Fenugreek (Trigonella foenum-graecum L.) is a traditional medicinal plant for treating human diseases that is widely cultivated in many countries. However, the component and related metabolic pathways are still unclear. To understand the changes in expression of the component and related genes during seed development, this study employed metabolomic and transcriptomic analyses and integrative analysis to explore the metabolites and pathways involved in the growth of fenugreek. The antifungal activity of the fenugreek seeds was also analyzed. A total of 9499 metabolites were identified in the positive ion mode, and 8043 metabolites were identified in the negative ion mode. Among them, the main components were fatty acyls, prenol lipids, steroids, steroid derivatives, flavonoids, and isoflavonoids. Among these enriched pathways, the top 20 pathways were "flavone and flavonol biosynthesis", "isoflavonoid biosynthesis", and "flavonoid biosynthesis". 3,7-Di-O-methylquercetin, flavonoids, pseudobaptigenin, isoflavonoids, methylecgonine, alkaloids, and derivatives were the most significantly upregulated metabolites. There were 38,137 differentially expressed genes (DEGs) identified via transcriptomic analysis. According to the KEGG pathway enrichment analysis, 147 DEGs were significantly enriched in "flavonoid biosynthesis". Ten DEGs of the six key enzymes were found to be involved in three pathways related to flavonoid and alkaloid synthesis in fenugreek. The antifungal activity test revealed the inhibitory effect of the ethanol extract of fenugreek seeds on Alternaria tenuissima (Kunze)Wiltshire and Magnaporthe oryzae. These findings further prove that the use of botanical pesticides in fenugreek fruit has research value.

Variation of gene expression of fatty acid acyl CoA reductase associated with wax secretion of a scale insect, Ericerus pela, and identification of its regulation factors through the accessible chromatin analyses and yeast one-hybrid.

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.

The Complete Mitochondrial Genome of the Chinese White Wax Scale Insect, Ericerus pela Chavannes (Hemiptera: Coccidae), with Novel Gene Arrangement and Truncated tRNA Genes.

The Chinese white wax scale insect, Ericerus pela Chavannes (Hemiptera: Coccidae), is one of the scale insects with great economic value and has been dispersed and reared in China for over one thousand years. Its mitochondrial genome provides essential information for the molecular identification and genetic study of this species. We assembled the complete mitochondrial genome of E. pela based on PacBio sequencing and analyzed its genomic features. The genome was 17,766 bp in length with 13 protein-coding genes, 22 tRNAs, and two rRNA genes. The analysis results showed E. pela had significant gene rearrangements involving tRNAs compared with other Coccoidea species. Furthermore, E. pela's nine tRNAs were identified to have obvious truncated structures. The phylogenetic tree compiled of the species showed a long branch of the Coccoidea lineage, which indicated the high evolutionary rate in this group. Our study revealed the mitochondrial characteristics of E. pela and enriched the mitochondrial genetic information on Coccoidea species. It also determined the occurrence of gene rearrangement for the species in this superfamily.

Two Cladosporium Fungi with Opposite Functions to the Chinese White Wax Scale Insect Have Different Genome Characters.

Insects encounter infection of microorganisms, and they also harbor endosymbiosis to participate in nutrition providing and act as a defender against pathogens. We previously found the Chinese white wax scale insect, Ericerus pela, was infected and killed by Cladosporium sp. (pathogen). We also found it harbored Cladosporium sp. (endogensis). In this study, we cultured these two Cladosporium fungi and sequenced their genome. The results showed Cladosporium sp. (endogensis) has a larger genome size and more genes than Cladosporium sp. (pathogen). Pan-genome analysis showed Cladosporium sp. (endogensis)-specific genes enriched in pathways related to nutrition production, such as amino acid metabolism, carbohydrate metabolism, and energy metabolism. These pathways were absent in that of Cladosporium sp. (pathogen). Gene Ontology analysis showed Cladosporium sp. (pathogen)-specific genes enriched in the biosynthesis of asperfuranone, emericellamide, and fumagillin. These terms were not found in that of Cladosporium sp. (endogensis). Pathogen Host Interactions analysis found Cladosporium sp. (endogensis) had more genes related to loss of pathogenicity and reduced virulence than Cladosporium sp. (pathogen). Cytotoxicity assay indicated Cladosporium sp. (pathogen) had cytotoxicity, while Cladosporium sp. (endogensis) had no cytotoxicity. These characters reflect the adaptation of endosymbiosis to host-restricted lifestyle and the invader of the entomopathogen to the host.

Genomic Analyses of the Fungus Paraconiothyrium sp. Isolated from the Chinese White Wax Scale Insect Reveals Its Symbiotic Character.

The Chinese white wax scale, Ericerus pela, is an insect native to China. It harbors a variety of microbes. The Paraconiothyrium fungus was isolated from E. pela and genome sequenced in this study. A fungal cytotoxicity assay was performed on the Aedes albopictus cell line C6/36. The assembled Paraconiothyrium sp. genome was 39.55 Mb and consisted of 14,174 genes. The coding sequences accounted for 50.75% of the entire genome. Functional pathway analyses showed that Paraconiothyrium sp. possesses complete pathways for the biosynthesis of 20 amino acids, 10 of which E. pela lacks. It also had complementary genes in the vitamin B groups synthesis pathways. Secondary metabolism prediction showed many gene clusters that produce polyketide. Additionally, a large number of genes associated with 'reduced virulence' in the genome were annotated with the Pathogen-Host Interaction database. A total of 651 genes encoding carbohydrate-active enzymes were predicted to be mostly involved in plant polysaccharide degradation. Pan-specific genomic analyses showed that genes unique to Paraconiothyrium sp. were enriched in the pathways related to amino acid metabolism and secondary metabolism. GO annotation analysis yielded similar results. The top COG categories were 'carbohydrate transport and metabolism', 'lipid transport and metabolism', and 'secondary metabolite biosynthesis, transport and catabolism'. Phylogenetic analyses based on gene family and pan genes showed that Paraconiothyrium sp is clustered together with species from the Didymosphaeriaceae family. A multi-locus sequence analysis showed that it converged with the same branch as P. brasiliense and they formed one group with fungi from the Paraconiothyrium genus. To validate the in vitro toxicity of Paraconiothyrium sp., a cytotoxicity assay was performed. The results showed that medium-cultured Paraconiothyrium sp. had no harmful effect on cell viability. No toxins were secreted by the fungus during growth. Our results imply that Paraconiothyrium sp. may establish a symbiotic relationship with the host to supply complementary nutrition to E. pela.