Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules ...successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.
RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) ...in its vector,
Laodelphax striatellus
, using RNAi, dsRNAs against
L. striatellus
genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication.
L. striatellus
genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in
L. striatellus
. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-
L. striatellus
interactions.
Low thermotolerance of entomopathogenic fungi is a major impediment to long-term storage and effective application of these biopesticides under seasonal high temperatures. The effects of high ...temperatures on the viability of an entomopathogenic fungus, Isaria fumosorosea SFP-198 (KCTC 0499BP), produced on different substrates amended with various additives were explored. Ground corn was found to be superior in producing the most thermotolerant conidia compared to yellow soybean, red kidney bean, and rice in a polyethylene bag production system. Using ground corn mixed with corn oil as a substrate resulted in only 7% reduction in germination compared to ground corn alone (67% reduction) after exposure of conidia to 50°C for 2 h. Corn oil as an additive for ground corn was followed by inorganic salts (KCl and NaCl), carbohydrates (sucrose and dextrin), a sugar alcohol (sorbitol), and plant oils (soybean oil and cotton seed oil) in ability to improve conidial thermotolerance. Unsaturated fatty acids, such as linoleic acid and oleic acid, the main components of corn oil, served as effective additives for conidial thermotolerance in a dosage-dependant manner, possibly explaining the improvement by corn oil. This finding suggests that the corn-corn oil mixture can be used to produce highly thermotolerant SFP-198 conidia and provides the relation of unsaturated fatty acids as substrates with conidial thermotolerance.
Insect killing fungi have high potential in controlling agriculturally harmful pest, but their slow progress and high variation in killing insect are major impediments to successful ...industrialization. The present work describes the use of supernatant from the liquid culture of
Beauveria bassiana
SFB-205 to surmount this problem, particularly efficient production of thermotolerant chitinase, which is one of the major pathogenesis-related enzymes in the supernatant. The chitinase was precipitated using varying mineral precipitants and followed by lyophilization, which was compared with a salting out method using ammonium sulfate in effectiveness. Incorporating of the supernatant fraction of the
Beauveria
preparation with attagel at 0.5% (
w/v
) as a precipitant enabled this treatment to show the greatest chitinase-precipitation efficiency (93.4%), followed up with excellent insecticidal activity against cotton aphids when it was mixed with 0.01% (
v/v
) polyoxyethylene-(3)-isotridecyl ether (TDE-3) as a spreading agent in laboratory conditions. Consequently, lyophilized attagel-mediated precipitation pellet was superior to lyophilized salting out pellet in maintaining chitinase activity against a thermal stress at 50°C. This finding provides that the attagel-mediated precipitation can be exploited to improve the thermotolerance of
B. bassiana
SFB-205 chitinase as a novel strategy for biopesticide production.
Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding
ace1 gene from adult heads of ...the diamondback moth (DBM,
Plutella xylostella). The
ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the
ace1 gene was expressed much higher than the
ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling.
In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.
Abstract
Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects ...which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.
Spodoptera litura is a noctuid moth that is considered an agricultural pest. The larvae feed on a wide range of plants and have been recorded on plants from 40 plant families (mostly dicotyledons). ...It is a major pest of many crops. To better understand Spodoptera litura granulovirus (SpliGV), the nucleotide sequence of the SpliGV DNA genome was determined and analyzed.
The genome of the SpliGV was completely sequenced. The nucleotide sequence of the SpliGV genome was 124,121 bp long with 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 or more nucleotides. The 133 putative ORFs covered 86.3% of the genome. Among these, 31 ORFs were conserved in most completely sequenced baculovirus genomes, 38 were granulovirus (GV)-specific, and 64 were present in some nucleopolyhedroviruses (NPVs) and/or GVs. We proved that 9 of the ORFs were SpliGV specific.
The genome of SpliGV is 124,121 bp in size. One hundred thirty-three ORFs that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. No chitinase or cathepsin genes, which are involved in the liquefaction of the infected host, were found in the SpliGV genome, explaining why SpliGV-infected insects do not degrade in a typical manner. The DNA photolyase gene was first found in the genus Granulovirus. When phylogenic relationships were analyzed, the SpliGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XecnGV), which belong to the Type I-granuloviruses (Type I-GV).
Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a ...fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.
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