Post-ingestive stability of a mulberry Kunitz-type protease inhibitor MnKTI-1 in the digestive lumen of silkworm: dual inhibition towards α-amylase and serine protease.

BACKGROUND: Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS: MnKTI-1, a mulberry Kunitz-type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up-regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori, and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI-1 proteins were detected in gut content and frass of specialist B. mori, and exhibited significant post-ingestive stability. Recombinant refolded MnKTI-1 (rMnKTI-1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α-amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI-1 to BmAmy can be reverted by pre-incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION: These findings demonstrate that mulberry MnKTI-1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.

Mulberry-derived miR168a downregulates BmMthl1 to promote physical development and fecundity in silkworms.

Plant-derived miRNAs and their interactions with host organisms are considered important factors in regulating host physiological processes. In this study, we investigated the interaction between the silkworm, an oligophagous insect, and its primary food source, mulberry, to determine whether mulberry-derived miRNAs can penetrate silkworm cells and regulate their functions. Our results demonstrated that miR168a from mulberry leaves enters the silkworm hemolymph and binds to the silkworm Argonaute1 BmAGO1, which is transported via vesicles secreted by silkworm cells to exert its regulatory functions. In vivo and in vitro functional studies revealed that miR168a targets the mRNA of silkworm G protein-coupled receptor, BmMthl1, thereby inhibiting its expression and activating the JNK-FoxO pathway. This activation reduces oxidative stress responses, prolongs the lifespan of silkworms, and improves their reproductive capacity. These findings highlight the challenges of replacing mulberry leaves with alternative protein sources and provide a foundation for developing silkworm germplasms suitable for factory rearing.

COG-imposed Golgi functional integrity determines the onset of dark-induced senescence.

Plant survival depends on dynamic stress-response pathways in changing environments. To uncover pathway components, we screened an ethyl methanesulfonate-mutagenized transgenic line containing a stress-inducible luciferase construct and isolated a constitutive expression mutant. The mutant is the result of an amino acid substitution in the seventh subunit of the hetero-octameric conserved oligomeric Golgi (COG) complex of Arabidopsis thaliana. Complementation studies verified the Golgi localization of cog7, and stress tests established accelerated dark-induced carbon deprivation/senescence of the mutant compared with wild-type plants. Multiomics and biochemical analyses revealed accelerated induction of protein ubiquitination and autophagy, and a counterintuitive increased protein N-glycosylation in senescencing cog7 relative to wild-type. A revertant screen using the overexpressor (FOX)-hunting system established partial, but notable rescue of cog7 phenotypes by COG5 overexpression, and conversely premature senescence in reduced COG5 expressing lines. These findings identify COG-imposed Golgi functional integrity as a main player in ensuring cellular survival under energy-limiting conditions.

Origin and functional differentiation of (E)-ß-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-ß-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-ß-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Uncovering the functional residues of Arabidopsis isoprenoid biosynthesis enzyme HDS.

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.

Evolutionary and functional analysis of mulberry type III polyketide synthases.

BACKGROUND: Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses. RESULTS: Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol. CONCLUSIONS: Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

Biotic stress-induced expression of mulberry cystatins and identification of cystatin exhibiting stability to silkworm gut proteinases.

MAIN CONCLUSION: Biotic stresses induce the expression of mulberry cystatins. MaCPI-4 protein is stable in silkworm digestive fluid and accumulates in gut food debris and frass. Plant cystatins are considered to be involved in defense responses to insect herbivores though little is known about how cystatins from the natural host respond to a specialist herbivory and the following postingestive interaction is also poorly understood. Here, we studied the biotic stress-mediated inductions of cystatins from mulberry tree, and examined the stability of mulberry cystatin proteins in the gut of silkworm, Bombyx mori, a specialist insect feeding on mulberry leaf. First, we cloned and characterized six cystatin genes from a mulberry cultivar, Morus atropurpurea Roxb., named as MaCPI-1 to MaCPI-6. The recombinant MaCPI-1, MaCPI-3 and MaCPI-4 proteins, which showed inhibitory effects against papain in vitro, were produced. Silkworm herbivory as well as methyl jasmonate (MeJA) treatment induced the expression of five mulberry cystatin genes, and the highest inductions were observed from MaCPI-1 and MaCPI-6. Mechanical wounding led to the inductions of four cystatin genes. The differential induction occurred in MaCPI-2. The induced protein changes were detected from three mulberry cystatins comprising MaCPI-1, MaCPI-3 and MaCPI-4. In vivo and in vitro assays showed that MaCPI-1 and MaCPI-3 proteins were susceptible to silkworm digestive fluid and MaCPI-4 had an antidigestive stability, and was detected in silkworm gut and frass. Collectively, our data indicated that biotic stresses resulted in the transcriptional inductions and protein changes of mulberry cystatins (MaCPIs), and identified MaCPI-4 with stability in the gut of its specialist herbivore.

Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli.

Bombyx mori, a lepidopteran insect, is one of the earliest models for pattern recognition of Gram-negative bacteria, which may induce the IMD pathway for production of antibacterial peptides. So far, several recognition proteins have been reported in B. mori. However, the connection between pattern recognition of Gram negative bacteria and activation of BmRelish1, a transcription factor controlled by the IMD pathway remains largely unknown. In the present study, we identify BmCPT1, a cuticle protein bearing a Tweedle domain. Its gene expression is co-regulated by NF-kappaB and juvenile hormone signals. BmCPT1 is induced by Escherichia coli in fat bodies and hemocytes, but is constitutively expressed in the epidermis. In vitro binding assays indicate that BmCPT1 protein recognizes and binds to E. coli peptidoglycan. Post-transcriptionally modified BmCPT1 in the hemolymph binds to E. coli cells through interactions with peptidoglycan recognition protein-5 (BmPGRP5) and lipopolysaccharide binding protein (BmLBP). Transgenic overexpression of BmCPT1 causes the upregulated expression of BmRelish1 and clear induction of two gloverin genes. Therefore, BmCPT1 may work along with BmPGRP-S5 and BmLBP to recognize E. coli in the hemolymph and indirectly activate BmRelish1 to induce antimicrobial peptide synthesis.

Draft genome sequence of the mulberry tree Morus notabilis.

Human utilization of the mulberry-silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species' spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant-herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.

Expression profile of cuticular genes of silkworm, Bombyx mori.

BACKGROUND: Insect cuticle plays essential roles in many physiological functions. During molting and metamorphosis tremendous changes occur in silkworm cuticle where multiple proteins exist and genes encoding them constitute about 1.5% of all Bombyx mori genes. RESULTS: In an effort to determine their expression profiles, a microarray-based investigation was carried out using mRNA collected from larvae to pupae. The results showed that a total of 6676 genes involved in various functions and physiological pathways were activated. The vast majority (93%) of cuticular protein genes were expressed in selected stages with varying expression patterns. There was no correlation between expression patterns and the presence of conserved motifs. Twenty-six RR genes distributed in chromosome 22 were co-expressed at the larval and wandering stages. The 2 kb upstream regions of these genes were further analyzed and three putative elements were identified. CONCLUSIONS: Data from the present study provide, for the first time, a comprehensive expression profile of genes in silkworm epidermal tissues and evidence that putative elements exist to allow massive production of mRNAs from specific cuticular protein genes.

Identification of the chitin-binding proteins from the larval proteins of silkworm, Bombyx mori.

The silkworm is a model organism for Lepidoptera. Its cuticle is composed mainly of chitin and proteins, which plays essential roles in multiple physiological functions. The binding of proteins to chitin plays an important role for cuticle formation. In this research, a chitin-binding assay followed by a proteomics analysis was carried out using the proteins extracted from the 5th instar larval cuticles. As results, twenty-two proteins were identified including nine cuticular proteins, two lysozyme precursors, two proteins with chitin-binding-type 2 domains, and other proteins. A cuticular protein with the RR-1 consensus, BmorCPR56, and a silkworm Tweedle protein, BmorCPT1, were detected in the chitin-binding fraction for the first time and their chitin-binding activities were further confirmed in vitro. The results of this research increase our understanding of the structure of the silkworm larval cuticle.