Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Chonbuk National University

The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, β-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007.

Tenebrio molitor Gram-negative-binding protein 3 (TmGNBP3) is essential for inducing downstream antifungal Tenecin 1 gene expression against infection with Beauveria bassiana JEF-007.

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.

Transformation of Beauveria bassiana to produce EGFP in Tenebrio molitor for use as animal feed additives.

Spraying of entomopathogenic fungi formulated as wettable powders and suspension concentrates based on oils or water has been used for the management of crop pests. However, the use of fungal granules to control paddy field pests has not been fully explored. Herein, several Beauveria bassiana isolates (ERL1170, ERL1578 and ERL836) were produced as granules using a millet-based solid culture. The granules were applied to the rice nursery 3 days before transplanting, and their control efficacy against rice water weevils was determined in paddy fields.

A novel approach: Beauveria bassiana granules applied to nursery soil for management of rice water weevils in paddy fields.

Entomopathogenic fungi are promising pest-control agents but their industrial applicability is limited by their thermosusceptibility. With an aim to increase the thermotolerance of Isaria fumosorosea SFP-198, moisture absorbents were added to dried conidial powder, and the relationship between its water potential and thermotolerance was investigated. Mycotized rice grains were dried at 10℃, 20℃, 30℃, and 40℃ and the drying effect of each temperature for 24, 48, 96, and 140 hr was determined. Drying for 48 hr at 10℃ and 20℃ reduced the moisture content to < 5% without any significant loss of conidial thermotolerance, but drying at 30℃ and 40℃ reduced both moisture content and conidial thermotolerance. To maintain thermotolerance during storage, moisture absorbents, such as calcium chloride, silica gel, magnesium sulfate, white carbon, and sodium sulfate were individually added to previously dried-conidial powder at 10% (w/w). These mixtures was then stored at room temperature for 30 days and subjected to 50℃ for 2 hr. The white carbon mixture had the highest conidial thermotolerance, followed by silica gel, magnesium sulfate, and then the other absorbents. A significant correlation between the water potential and conidial thermotolerance was observed in all conidia-absorbent mixtures tested in this study (r = -0.945). Conidial thermotolerance in wet conditions was evaluated by adding moisturized white carbon (0~20% H2O) to conidia to mimic wet conditions. Notably, the conidia still maintained their thermotolerance under these conditions. Thus, it is evident that conidial thermotolerance can be maintained by drying mycotized rice grains at low temperatures and adding a moisture absorbent, such as white carbon.

Enhancing the Thermotolerance of Entomopathogenic Isaria fumosorosea SFP-198 Conidial Powder by Controlling the Moisture Content Using Drying and Adjuvants.

Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

The bean bug, Riptortus pedestris, is a major agricultural pest that reduces crop quality and value. Chemical pesticides have contributed to pest management, but resistance to these chemicals has significantly limited their use. Alternative strategies with different modes of action, such as entomopathogenic fungi, are therefore of great interest. Herein, we explored how entomopathogenic fungi can potentially be used to control the bean bug and focused on identifying virulence-related genes. Beauveria bassiana (JEF isolates) were assayed against bean bugs under laboratory conditions. One isolate, JEF-007, showed >80 % virulence by both spray and contact exposure methods. Agrobacterium tumefaciens-mediated transformation (AtMT) of JEF-007 generated 249 random transformants, two of which (B1-06 and C1-49) showed significantly reduced virulence against Tenebrio molitor and R. pedestris immatures. Both species were used for rapid screening of virulence-reduced mutants. The two transformants had different morphologies, conidial production, and thermotolerance than the wild type. To determine the localization of the randomly inserted T-DNA, thermal asymmetric interlaced (TAIL) PCR was conducted and analysis of the two clones found multiple T-DNA insertions (two in B1-06 and three in C1-49). Genes encoding complex I intermediate-associated protein 30 (CIA30) and the autophagy protein (Atg22) were possibly disrupted by the T-DNA insertion and might be involved in the virulence. This work provides a strong platform for future functional genetic studies of bean bug-pathogenic B. bassiana. The genes putatively involved in fungal virulence should be experimentally validated by knockdown in future studies.

Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007.

Beauveria bassiana (Bb) is used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Herein, RNA isolated from B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) infected with this strain were firstly subjected to high-throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Due to lack of fungal and host genome information, fungal transcriptome was processed to partially exclude non-infection specific genes and host-flora. Differentially Expressed Gene (DEG) analysis showed that 2381 genes were up-regulated and 2303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by Gene Ontology analysis. Most DEGs were involved in metabolic pathways based on Kyoto Encyclopedia of Genes and Genomes pathway mapping. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. Additionally, transcript levels of several fungal genes were dramatically increased after infection, such as cytotoxic lectin-like protein, bacterial-like toxin, proteins related to cell wall formation, hyphal growth, nutrient uptake, and halogenated compound synthesis. This work provides insight into how entomopathogenic B. bassiana grows in agriculturally harmful bean bug at 6 d post infection.

Up-regulation of carbon metabolism-related glyoxylate cycle and toxin production in Beauveria bassiana JEF-007 during infection of bean bug, Riptortus pedestris (Hemiptera: Alydidae).

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. This is a novel insect positive-sense single-stranded RNA virus, which we named Riptortus pedestris virus-1 (RiPV-1) (GenBank accession no. KU958718). The genome of RiPV-1 consists of 10,554 nucleotides (nt), excluding the poly(A) tail, which contains a single large open reading frame (ORF) of 10,371 nt encoding a polyprotein (3456 aa) and flanked by 71 and 112 nt at the 5' and 3' untranslated regions (UTR), respectively. RiPV-1 genome organization from the 5' end contains a consensus organization of picorna-like RNA virus helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp), in addition to two putative structural proteins located at the 3' region and a poly(A) tail at the 3' end. The viral particles were approximately 30nm in diameter with some dispersal distinctive surface projections. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. RiPV-1 could be found in all stages of lab-reared bean bugs and was detected abundantly in the thorax, abdomen, midgut and fat body, but not in the reproductive organs and muscle. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2-6days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

A novel picorna-like virus, Riptortus pedestris virus-1 (RiPV-1), found in the bean bug, R. pedestris, after fungal infection.

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced <1×10conidia/0.28 cmagar block (wild type: 6.2×10conidia/agar block). Transformant #163 also exhibited different morphology than the wild type, including thicker hyphae with some club-shaped parts. In contrast, the typical morphology of wild type B. bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing.

Se Jin Lee

Department of Agricultural Biology,
College of Agriculture Life Sciences,
Department of Agricultural Biology, College of Agriculture Life Sciences

Chonbuk National University

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Se Jin Lee

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Agricultural Biology,College of Agriculture Life Sciences,Department of Agricultural Biology, College of Agriculture Life Sciences

Chonbuk National University

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Department of Agricultural Biology,
College of Agriculture Life Sciences,
Department of Agricultural Biology, College of Agriculture Life Sciences