RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

Identification of LmUAP1 as a 20‐hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust,Locusta migratoria

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N‐acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20‐hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection‐based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late‐response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi‐based pest control.     

Knockdown of glycogen phosphorylase and glycogen synthase influences expression of chitin synthesis genes of rice brown planthopper Nilaparvata lugens

Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in the glycogen synthesis pathway, which catalyze trehalose and glucose transformation in insects. GS and GP can be regulated by trehalose metabolism, which plays an important role in insect growth. However, it is not known whether these genes can be targeted for pest control through regulation of chitin metabolism. We studied the function of Nilaparvata lugens GS and GP (NLGS and NLGP, respectively) using RNA interference, and reported that trehalose and the chitin biosynthesis pathways are regulated by GP and GS, especially TPS3, TRE1-1, and G6PI1, which decreased following knockdown of these two genes. The expression levels of TPS1, TPS2, and several chitin synthesis pathway family genes were significantly increased following dsNlGP injection. Additionally, despite there being no apparent change to the chitin content, an abnormal molting phenotype and wing deformity appeared, and close to 25% insects died. These results demonstrate that silencing of NLGP or NLGS can lead to molting deformities and an elevated mortality rate through the regulation of chitin pathway genes and chitinase genes. NLGP may play a key role in chitin synthesis due to the number of genes regulated, and higher deformity and mortality rates resulting from its knockdown.     

Cuticular protein LmTwdl1 is involved in molt development of the migratory locust

The cuticle, an essential structure for insects, is produced from cuticular proteins and chitin via a series of biochemical reactions. Tweedle genes are important members of the cuticular protein family and have four conserved motifs binding to chitin. Tweedle family genes have been found to play a profound effect on cuticle development. Here, we report that the cuticular protein gene LmTwdl1 of Locusta migratoria belongs to the Tweedle family. In situ hybridization showed that LmTwdl1 is localized to epidermal cells of the cuticle. The expression patterns of LmTwdl1 showed low expression in the cuticle during the early and middle stages of the fifth‐instar nymphs; in contrast, its expression rapidly increased in the late stages of fifth‐instar nymphs. We performed RNA interference to examine the function of LmTwdl1 in locusts. Silencing of LmTwdl1 resulted in high mortality during the molting process before the next stage. Also, the epicuticle of nymphs failed to molt, tended to be thinner and the arrangement of chitin in the procuticle appeared to be disordered compare to the control group. These results demonstrate that LmTwdl1 plays a critical role in molting, which contributes to a better understanding of the distinct functions of the Tweedle family in locusts.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

Identification of genes encoding N‐glycan processing β‐N‐acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems

Glycoproteins produced by non‐engineered insects or insect cell lines characteristically bear truncated, paucimannose N‐glycans in place of the complex N‐glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N‐glycan processing β‐N‐acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N‐glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing β‐N‐acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing β‐N‐acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing β‐N‐acetylglucosaminidases than degradative or chitinolytic β‐N‐acetylglucosaminidases. In addition, over‐expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing β‐N‐acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus‐insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Insect group II chitinase OfChtII promotes chitin degradation during larva–pupa molting

The insect group II chitinase (ChtII, also known as Cht10) is a unique chitinase with multiple catalytic and chitin-binding domains. It has been proven genetically to be an essential chitinase for molting. However, ChtII's role in chitin degradation during insect development remains poorly understood. Obtaining this knowledge is the key to fully understanding the chitin degradation system in insects. Here, we investigated the role of OfChtII during the molting of Ostrinia furnacalis, a model lepidopteran pest insect. OfChtII was expressed earlier than OfChtI (OfCht5) and OfChi-h, at both the gene and protein levels during larva–pupa molting as evidenced by quantitative polymerase chain reaction and western blot analyses. A truncated OfChtII, OfChtII-B4C1, was recombinantly expressed in Pichia pastoris cells and purified to homogeneity. The recombinant OfChtII-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface as evidenced by scanning electron microscopy. It synergized with OfChtI and OfChi-h when hydrolyzing insoluble α-chitin. These findings suggested an important role for ChtII during insect molting and also provided a strategy for the coordinated degradation of cuticular chitin during insect molting by ChtII, ChtI and Chi-h.     

The chitin‐binding capability of Cy‐AMP1 from cycad is essential to antifungal activity

Antimicrobial peptides are important components of the host innate immune responses by exerting broad‐spectrum microbicidal activity against pathogenic microbes. Cy‐AMP1 found in the cycad (Cycas revoluta) seeds has chitin‐binding ability, and the chitin‐binding domain was conserved in knottin‐type and hevein‐type antimicrobial peptides. The recombinant Cy‐AMP1 was expressed in Escherichia coli and purified to study the role of chitin‐binding domain. The mutants of Cy‐AMP1 lost chitin‐binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy‐AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin‐binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy‐AMP1. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.