Conceived and designed the experiments: Insect-killing entomopathogenic fungi have high potential for controlling agriculturally harmful pests.
Asexual definition rae dong, 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 ERL 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 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. Insect killing entomopathogenic fungi have high potential in controlling agriculturally harmful pests . Some products have been industrialized as follows: The main active components of these commercial products are conidia asexual spores with high variability in virulence and slow pathogenesis thus having difficulties in the expansion of fungal insecticide market .
So far, some efforts have been given to the expression of pathogenesis-related genes, such as B. These proteins were previously reported expressed in baculovirus expression vector system BEVS with the assessment of insecticidal potentials. Additionally, insect cuticle-degrading fungal own Pr1 protease and fusion protein of Pr1 and chitinase gene were over-expressed
Asexual definition rae dong M.
But much more virulent entomopathogenic fungi need to be developed for efficacious pest management. Melanization was studied as a rapid insect response to challenges of its immune system and as a novel strategy to accelerate host mortality.
When arthropods encounter an immune challenge, they initiate a serine protease cascade that, in turn, leads to the activation of prophenoloxidase proPO -activating factors PPAFs . These factors are activated by cleavage between clip of PPAFs "Asexual definition rae dong" serine protease domains.
This causes the conversion of phenols to diphenol, quinine and, finally, melanin. Injection of purified Bi-VSP induces a lethal melanization response in target insects by modulating innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease which exhibits mammalian fibrinolytic activity. The fibrinolytic activity of Bi-VSP, possibly inhibiting blood coagulation, can facilitate the spread of toxic components throughout the bloodstream.
Blood coagulation disorders are a global and frequently lethal medical disease. When clots are not dissolved, they accumulate in blood vessels and cause thrombosis leading to myocardial infraction and other cardiovascular diseases . It is hypothesized that the fungi-based expression of Bi-VSP induces fast melanization of whole insect bodies, and the transformants possibly have much higher insecticidal potency than the previous achievements.
For the extracellular secretion of Bi-VSP protein, the active domain of the vsp gene was tailed to the signal sequence of B. A selected transformant was injected to yellow spotted longicorn beetle larvae to confirm insect melanization and sprayed on beet armyworms to examine mortality. For integration of the vsp gene into B. The pABeG has phosphinothricin PPT resistant bar and egfp genes, and each gene is expressed under the control of the same gpdA promoter in the same transcriptional direction.
The binary plasmid, linearized by cutting with Hin dIII, was transformed into ERL by the restriction enzyme-mediated integration based on blastospores . The fusion form of PCR product was confirmed by sequencing. Bb signal, shadowed; and Bi VSP, not-shadowed. Transformation of the competent blastospores of wild type B.
White mycelial growth and in 7 days a similar number of conidial production Wt: From an economic standpoint, additional efforts are not necessary to increase BbsVSP- conidial production to the level of the wild type.
Transcription of Bbs-vsp "Asexual definition rae dong" and translation followed by extracellular secretion of VSP was confirmed. Serine proteases are known to have fibrinolytic activity.
Phosphate buffered saline PBS solution was used as a base for all the treatments. To determine the biological activity of the transformant BbsVSP-a fibrin plate assay and a bioassay against yellow spotted longicorn beetle larvae were conducted. In fibrin plate assay in a mm dish, supernatant concentrate fold of BbsVSP- strain made a clear area degradation of fibrin on the fibrin plate but no corresponding clearance with the wild type Figure 2d.
BbsVSP- strain had supernatant dosage-dependent fibrinogen degradation activity when fibrinogen clots were submerged to the supernatant Figure S2. In the BbsVSP- treatment, small dark brown spots were observed 2 days post-injection, followed by complete insect melanization without fungal outgrowth in 7 days, but the wild type-injected larvae turned light pink as the mycosis developed without forming dark spots on the host cuticle until the hosts were covered by a sporulating mass of mycelium Figure S3.
The transformant had significantly faster virulence than the wild type in controlling beet armyworms Figure 3a. Thus, it takes 2. BbsVSP- treated beet armyworms Asexual definition rae dong black in 4 days and no further development was observed Figure 3b. The melanization in beet armyworms began during the early stages of fungal pathogenesis, and soon after fungal germination and hyphal penetration.
However, beet armyworms in the wild type treatment developed to 4th "Asexual definition rae dong" larvae with mycosis in 7 days. The wild type fungus had more time to achieve mechanical penetration and enzymatic degradation for complete mortality. Non-treated control beet armyworms developed to 5th instar in 10 days. BbsVSP- treated beet armyworms turned black in 4 days and no stage development was observed. But beet armyworms in the wild type treatment developed to fourth instars with mycosis in 7 days and completely mycotized in 10 days.
The fungus-based expression of bumblebee serine protease significantly increased the virulence of wide type, which may be compared to the expression of scorpion neurotoxin AaIT  and the over-expression of fungal own Pr1 protease  as mentioned above.
In the BbsVSP- treatment, it took 2. In the of neurotoxin, AaIT59 transformant required 4. Approximately it took 1. Similarly, in the over-expression of Pr1 protease, transformants required 93—96 h wild type: VSP-integrated insect-killing fungi have some advantages in pest management. They control agriculturally harmful insects in a short time compared to the wild type.
Activation of melanization cascade is very sensitive to initiators and proceeds very quickly . Thus, low levels of hyphal penetration may be enough to induce the melanization cascade when VSP-integrated fungus is applied.
From an economic standpoint, VSP-integrated fungi do not need to spend great deal of Asexual definition rae dong for hyphal penetration into the haemocoel. However, in research on the expression of chitinase, vegetative insecticidal protein and insect-specific scorpion toxin expressed in haemocoelhyphal growth and penetration should be fully accomplished for the expression Asexual definition rae dong integrated genes.
Fungal penetration and expression of VSP is a strategically well combined tactic to achieve fast control with high biological performance. Particularly, VSP-integrated fungi can be more useful in controlling pests with a short-term life cycle.
Another merit of VSP-integrated insect-killing fungi is the self-restriction of further reproduction and dispersal in the environments by the genetically modified fungus. Because VSP-mediated insect melanization quickly kills target insects and the fungal pathogen that introduced this toxin, no further development by the fungus should be possible.
The rapid killing of its host insects and the death of treated fungus explains why no mycoses were found in the VSP-integrated fungus treatments. Melanization improves the effectiveness of other immune responses that promote arthropod resistance to microbial infection  and suppresses the infection of parasitoids . Dispersal of genetically modified fungi can be naturally inhibited in the environment so that the registration process may be minimized although fundamental safety tests are required.
But, genetically modified fungi, even those whose reproduction or dispersal is self-limited, may or may not be easy or even possible to register in many countries merely because they are GMOs, regardless of their beneficial properties. It may take more times in the industrialization of this VSP-integrated insect-killing fungus than expectation.
However, in other cases expression of other functional insecticidal proteins such as chitinase, vegetative insecticidal protein, and insect-specific scorpion toxindispersal of genetically modified fungi may be usual events, so it should be carefully controlled in the environment.
VSP-integrated insect-killing fungi inherit any fungal own host spectrum, by which VSP can be expressed in potentially many insects.
Among microbial pest control agents, Bacillus thuringiensis Bt and baculoviruses can be alternatively considered, but host spectrums are mainly limited to lepidopteran pests moths and BEVS is not available in VSP expression as described above. Insect-killing fungi, particularly B. Mass production of entomopathogenic fungi has been effectively developed and is cost-effective .
Thanks to the development of industrialization technology, VSP-integrated fungal spores conidia or VSP proteins can be easily mass-produced and harvested. A great deal of effort has been given to increase the shelf life of fungal spores during distribution and after application. Some studies are necessary to investigate whether any directed genetic modification of a particular fungal strain does or does not affect such critically important properties of the fungus.
In conclusion, for the first time "Asexual definition rae dong" bee venom serine protease Bi-VSP has been successfully expressed in the insect-killing fungus, B. Asexual definition rae dong research highlights the expression of multi-functional Bi-VSP not available in BEVS in a fungal platform that is especially relevant for agricultural fungal application and pharmacological purified proteases fields with much stronger biological activities. These results could significantly increase the economic value of entomopathogenic fungi for at least some specific application.
The wild type strain B. A fungal transformation vector, pABeG expressing egfp gene provided by Dr. The pABeG has phosphinothricin PPT resistant bar and egfp genes, and each gene is expressed under the control of gpdA promoter in the same transcriptional direction.
Active domain of vsp gene was tailed with B. Cultured broth was filtered using 3M filter papers and syringe filters 0. Byung Rae Jin, was derived from mouse and used to detect the expression of VSP secreted from mycelia of transformants. The PVDF membrane was incubated with a 1,fold dilution of the Asexual definition rae dong antibody and a 2,fold dilution of goat anti-rabbit IgG horseradish peroxidase HRP as the second antibody.
The original fibrin plate assay  was slightly modified for use here in measuring the fibrinolytic activity of the supernatants.
An aliquot of 5 ml fibrinogen Sigma-Aldrich, from human plasma 0. After the incubation, solutions in the tubes were completely removed using a pipette and the amount of remaining fibrin was observed. Yellow spotted longicorn beetle Psacothea hilaris for fungal injection and beet armyworm Spodoptera exigua for fungal spray were supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea.
They were reared as previously described  and subjected to bioassays . PBS and siloxane solution served as controls.
Injected larvae were placed in mm diam. Petri dishes were not stacked to keep from excess moisture from forming inside of the dishes. Symptom of melanization and mycosis was observed daily for 10 days. Fungal suspensions were sprayed at 10 ml per dish using a microsprayer, and dishes were covered with lids and sealed Asexual definition rae dong Parafilm. Mortality was assessed by counting the number of live and dead larvae per dish daily for 7 days.
This entire bioassay was repeated twice using different batches of conidial suspensions on different days. Rabe, Ian Rae, Monique Richard (to whom 1 owe special thanks for heip in the uanslation of .