Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

Transcriptional Analysis of Cotton Bollworm Strains with Different Genetic Mechanisms of Resistance and Their Response to Bacillus thuringiensis Cry1Ac Toxin.

Transgenic crops producing Bacillus thuringiensis (Bt) insecticidal proteins are grown widely for pest control, but the evolution of resistance in target pests could reduce their efficacy. Mutations in genes encoding cadherin, ABC transporter or tetraspanin were linked with resistance to Cry1Ac in several lepidopteran insects, including the cotton bollworm (Helicoverpa armigera), a worldwide agricultural pest. However, the detailed molecular mechanisms by which these mutations confer insect resistance to Cry1Ac remain largely unknown. In this study, we analyzed the midgut transcriptomes of a susceptible SCD strain and three SCD-derived Cry1Ac-resistant strains of H. armigera (SCD-r1, with a naturally occurring deletion mutation of cadherin; SCD-KI, with a knock-in T92C point mutation in tetraspanin; and C2/3-KO, with both ABCC2 and ABCC3 knocked out). Evaluation of midgut transcript profiles of the four strains without Cry1Ac exposure identified many constitutively differentially expressed genes (DEGs) in the resistant SCD-r1 (n = 1355), SCD-KI (n = 1254) and C2/3-KO (n = 2055) strains. Analysis of DEGs in the midguts of each strain after Cry1Ac exposure revealed similar patterns of response to Cry1Ac in the SCD and SCD-r1 strains, but unique responses in the SCD-KI and C2/3-KO strains. Expression of midgut epithelium healing and defense-related genes was strongly induced by Cry1Ac intoxication in the SCD and SCD-r1 strains, while immune-related pattern recognition receptor and effector genes were highly expressed in the SCD-KI strain after Cry1Ac exposure. This study advances our knowledge of the transcriptomic basis for insect resistance to Bt toxins and provides a valuable resource for further molecular characterization of insect response to Cry1Ac toxin in H. armigera and other pest species.

Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2.

Gestational diabetes mellitus (GDM) is a situation where glucose intolerance is found in pregnant women without a previous diagnosis of diabetes. The role of Kruppel-like factor 9 (KLF9) has not been investigated in GDM, which constituted the aim of our study. HTR8/SVneo cells were induced by high glucose (HG) and pregnant mice were treated with streptozocin (STZ) to establish GDM model in vitro and in vivo, respectively. The expression level of KLF9 was detected by real-time PCR, immunohistochemical staining, and Western blot. Cell viability, apoptosis, inflammation, and oxidative stress were investigated by cell counting kit-8 (CCK-8), TUNEL, enzyme-linked immunosorbent assay (ELISA) and oxidative stress detection kits, respectively. The interaction of KLF9 with dimethylarginine dimethylaminohydrolase 2 (DDAH2) was predicted by bioinformatic tools and confirmed by luciferase reporter assay and chromatin immunoprecipitation (ChIP). The expression of KLF9 was increased in the placental tissues of GDM patients and HG-induced HTR8/SVneo cells. Silencing of KLF9 increased cell viability, reduced cell apoptosis, and suppressed inflammation and oxidative stress in HG-induced HTR8/SVneo cells. KLF9 could bind to DDAH2 promoter and negatively regulate DDAH2 expression. Inhibition of DDAH2 partly weakened the effects of KLF9 silencing on cell apoptosis, inflammation, and oxidative stress. The suppressive effects of KLF9 silencing on blood glucose and insulin concentration in vivo were also abolished by DDAH2 knockdown. In conclusion, we provided evidence that interference of KLF9 could hinder the development of GDM by alleviating cell apoptosis, inflammation, and oxidative stress through upregulating DDAH2, which might instruct the targeting therapies against GDM.Abbreviations: KLF9: Kruppel-like factor 9; DDAH2: dimethylarginine dimethylaminohydrolase 2 ; GDM: gestational diabetes mellitus; ELISA: enzyme-linked immunosorbent assay; CCK-8: cell counting kit-8; ChIP: chromatin immunoprecipitation; sh: short hairpin; HG: high glucose; PBS: phosphate-buffered saline; DAPI: 4, 6-diamidino-2-phenylindole; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; wt: wild-type; mut: mutant.

Proteomic analysis of a clavata-like phenotype mutant in Brassica napus.

Rapeseed is one of important oil crops in China. Better understanding of the regulation network of main agronomic traits of rapeseed could improve the yielding of rapeseed. In this study, we obtained an influrescence mutant that showed a fusion phenotype, similar with the Arabidopsis clavata-like phenotype, so we named the mutant as Bnclavata-like (Bnclv-like). Phenotype analysis illustrated that abnormal development of the inflorescence meristem (IM) led to the fused-inflorescence phenotype. At the stage of protein abundance, major regulators in metabolic processes, ROS metabolism, and cytoskeleton formation were seen to be altered in this mutant. These results not only revealed the relationship between biological processes and inflorescence meristem development, but also suggest bioengineering strategies for the improved breeding and production of Brassica napus.

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy.

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.

A Review of Plant Vacuoles: Formation, Located Proteins, and Functions.

Vacuoles, cellular membrane-bound organelles, are the largest compartments of cells, occupying up to 90% of the volume of plant cells. Vacuoles are formed by the biosynthetic and endocytotic pathways. In plants, the vacuole is crucial for growth and development and has a variety of functions, including storage and transport, intracellular environmental stability, and response to injury. Depending on the cell type and growth conditions, the size of vacuoles is highly dynamic. Different types of cell vacuoles store different substances, such as alkaloids, protein enzymes, inorganic salts, sugars, etc., and play important roles in multiple signaling pathways. Here, we summarize vacuole formation, types, vacuole-located proteins, and functions.

Overexpression of salt-induced protein (salT) delays leaf senescence in rice.

Senescence, a highly programmed process, largely determines yield and quality of crops. However, knowledge about the onset and progression of leaf senescence in crop plants is still limited. Here, we report that salt-induced protein (salT), a new gene, may be involved in leaf senescence. Overexpressing salT could prolong the duration of leaves with higher concentrations of chlorophyll compared with the wild type. Moreover, overexpression of salT could delay the senescence of rice leaves though the inhibition of senescence associated genes (SAGs). Overall, the characterization of salT suggested that it is a new gene affecting the leaf senescence induced by natural and dark conditions.

Timed Knickkopf function is essential for wing cuticle formation in Drosophila melanogaster.

The insect cuticle is an extracellular matrix that consists of the polysaccharide chitin, proteins, lipids and organic molecules that are arranged in distinct horizontal layers. In Drosophila melanogaster, these layers are not formed sequentially, but, at least partially, at the same time. Timing of the underlying molecular mechanisms is conceivably crucial for cuticle formation. To study this issue, we determined the time period during which the function of Knickkopf (Knk), a key factor of chitin organization, is required for wing cuticle differentiation in D. melanogaster. Although knk is expressed throughout metamorphosis, we demonstrate that its expression 30 h prior and 48 h after pupariation is essential for correct wing cuticle formation. In other words, expression beyond this period is futile. Importantly, manipulation of Knk expression during this time causes wing bending suggesting an effect of Knk amounts on the physical properties of the wing cuticle. Manipulation of Knk expression also interferes with the structure and function of the cuticle surface. First, we show that the shape of surface nano-structures depends on the expression levels of knk. Second, we find that cuticle impermeability is compromised in wings with reduced knk expression. In summary, despite the extended supply of Knk during metamorphosis, controlled amounts of Knk are important for correct wing cuticle differentiation and function in a concise period of time.