Expression of egfp Gene Based on Agrobacterium tumefaciens-mediated Transformation in Beauveria bassiana ERL836

Yu-Shin Nai,Se Jin Lee,Sihyeon Kim,Ho-Jong Ju,Yeon-HoJe,Jae Su Kim 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

Enhanced green fluoresce protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method in this study. The ERL836 transformants were generated with pCambia-egfp binary vector. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% of the fungal putative transformants were inserted by the T-DNA fragment. Of these transformants, 33.33% (2 transformants) expressed the egfp gene. The egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a reference for foreign protein expression in B. bassiana by using the AtMT method.

The seasonal detection of AcSBV (Apis cerana sacbrood virus) prevalence in Taiwan

Yu-Shin Nai,Chong-Yu Ko,Pei-Shou Hsu,Wen-Shi Tsai,Yue-Wen Chen,Meng-Hao Hsu,I-Hsin Sung 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.1

The epizootic disease caused by Apis cerana sacbrood virus (AcSBV) occurred in Eastern hive bee, A. cerana, since2015 in Taiwan. A large-scale survey of this disease from September and December 2016 in Taiwan was performedincluding symptom check and molecular identification in honey bees of A. cerana hives and several A. mellifera hives, which were co-cultured with A. cerana. Based on the nucleotide sequences of partial VP1, thephylogenetic analysis with those of the known AcSBV isolates revealed that most of AcSBV isolates from Taiwanwere closely relative to SBV-FZ and -JL isolates from China, whereas only one sample (N15-5-1) was in a distinctcluster, which was closely relative to SBV-LN from China too. The AcSBV prevalence was occurring in A. ceranahives in most areas of Taiwan except for those in Hualien and Pingtung Counties in Taiwan. Notably, the AcSBVprevalence rate showed the temporal increase from 47.1% to 69.6% within 4 months. In addition, 37.5% ofAcSBV prevalence rate was found in A. mellifera hives. It showed that A. mellifera was also susceptible to AcSBVinfection. The present results would provide the information on the epidemiology and for prospective research.

Functional Analysis of Two iap Genes (iap2 and iap3) of Lymantria xylina Multiple Nucleopolyhedrovirus (LyxyMNPV)

Yu-Shin Nai,Jae Su Kim,Chung-Hsiung Wang 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.04

Baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation stage of viral infection. However, some iap genes are not involved in the anti-apoptotic activity. To investigate the anti-apoptotic activity of the iap genes of Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV), two ly-iap genes (ly-iap2 and ly-iap3) were cloned from LyxyMNPV. From a 5′ RACE analysis, a late promoter motif (TAAG) was found in the upstream (-15 bp) of ly-iap2, but ly-iap3 only posited an enhancer-like element (CGTGC) in the upstream (-22 bp) of 5′ UTR. Gene expression were detected by RT-PCR; the ly-iap2 and ly-iap3 genes began to express in the host cells (IPLB-LD652Y cell line) infected with LyxyMNPV 6 hours post-infection (p.i.) and reached the peak 72 hours p.i., followed by decline 3 to 5 days p.i. Functional assay of the iap genes were performed by an over-expression method in Sf9 cells. Full-length domains of LY-IAP2, LY-IAP3 and LY-IAP2-BIR could differently inhibit the apoptosis which induced by Drosophila RPR protein (DRPR). Interestingly, LY-IAP2-RZF domain was important for LY-IAP2 to rescue apoptosis, but it might be also involved in the ubiquitin activity leading to the degradation of LY-IAP2 protein. LY-IAP3-RZF might be working as a “helper domain” to inhibit DRPR-induced apoptosis. These results can be used to figure out the roles of the ly-iap genes in the apoptosis of host cells.

Enhanced replication of novel picorna-like virus (RiPV-1) in Beauveria bassiana JEF-007-infected bean bug (Riptortus pedestris)

Yi-Ting Yang,Yu-Shin Nai,Se Jin Lee,Sihyeon Kim,Jae Su Kim 한국응용곤충학회 2016 한국응용곤충학회 학술대회논문집 Vol.2016 No.04

A novel insect-infecting positive sense single-stranded RNA virus, Riptortus pedestris virus-1 (RiPV-1), was found in the Riptortus pedestris transcriptome data by de novo assembly and further confirmed by RACE method. The genome of RiPV-1 consists of 10,554 nucleotides (nt) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 10,371 nt encoding a 3,456 aa polyprotein and flanked by 71 and 112 nt 5' and 3' noncoding regions, respectively. RiPV-1 genome contains the consensus genome organization of picorna-like RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) array in that order from the 5' to the 3' end. From the phylogenetic analysis, RiPV-1 was clustered with unassigned insect RNA viruses, APV and KFV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses. Tissue tropism analysis revealed that RiPV-1 was relatively abundant in the thorax, abdomen, midgut and fat body. Interestingly, RiPV-1 replication was enhanced by Beauveria bassiana JEF-007 infection that was quantified using qRT-PCR. This study identified a novel insect-infecting virus and provided further insight into the relationship between virus, fungus and host.

Characterization of functional genes in Beauveria bassiana: Agrobacterium tumefaciens-mediated random transformation and characterization of transformants

Se Jin Lee,Yu-Shin Nai,Sihyeon Kim,Jae Su Kim 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.04

The entomopathogenic fungus, Beauveria bassiana is widely used in integrated pest management (IPM), however its successful application is often limited by the little effort to explore its functions of unknown genes. In this work, egfp-expression cassette was randomly integrated into B. bassiana using Agrobacterium tumefaciensmediated transformation, and the general features of the mutants with unusual characteristics and the localization of the integrated genes were explored. To construct a transformation vector, egfp-expression cassette including gpdA promoter and trpC terminator was cut from pBARKS1-egfp using SacI and HindIII and integrated into pCAMBIA containing hygromycin B resistant hygR gene, designated as pCAMBIA-egfp. Transformed B. bassiana isolates were grown on quarter strength-Sabouraud dextrose agar containing 150 μg hygromycinB ml-1. Expression of egfp was investigated by RT-PCR and a fluorescent microscope (400×). Through the genome walking of the transformants using adaptor primers and gene specific primers, unique bands were detected on the egfp-expressing transformants, which were sequenced to figure out the flanking regions. This work provides a platform of methodology to figure out unknown functional genes of B .bassiana and possibly suggest an improved strategy to use the entomopathogen in IPM.

곤충병원성 곰팡이의 열안정성 향상을 위한 배지의 최적화

유정선,이세진,김시현,Yu-Shin Nai,주호종,김재수 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

곤충 병원성 곰팡이 Metharizium anisopilae JEF 003, 004와 Beauveria bassiana JEF 006, 007의 대량생산배지 조건에 따른 열안정성을 평가하였다. 첫 번째로 millet 배지에서 배양된 포자의 열안정성 평가를 위하여 50℃ 조건에 0, 30, 60, 90, 120 min으로 포자 현탁액 상태와 grain상태로 노출한 결과 현탁액 상태에서 포자 의 열안정성이 더 많이 감소하는 것을 확인하였다. 다음으로 각기 다른 고체배지 (조: 1~5.×109 conidia/g, 수수: 1~2×109 conidia/g, 기장: 2~3×109 conidia/g) 조건에 서 생산된 포자의 열안정성 확인을 위하여 배양이 완료된 포자를 현탁액과 grain상 태에서 50℃ 조건에 0, 1, 2, 4, 8 hours동안 노출하여 열안정성을 평가하였다. 실험 결과 현탁액 상태보다 grain상태에서 포자의 열안정성이 더 높은 것을 확인하였으 며, 조 배지 조건에서 포자의 열안정성이 가장 높게 향상된 것을 확인하였다. 마지 막으로 포자의 열안정성의 추가 향상을 위하여 배양이 완료된 고체배지 포자에 cotton seed oil, coconut oil, soybean oil, castor oil, olive oil, mineral oil을 넣고, 5 0℃의 온도에 0, 1, 2, 4, 8 hours으로 열안정성을 평가하였다. 결과적으로 cotton seed oil, soybean oil, castor oil, olive oil을 처리한 포자에서 높은 열안정성을 확인 하였다. 따라서 곤충병원성 곰팡이의 열안정성 실험 결과로 확인 된 조를 이용하여 높은 열안전성을 확보할 수 있을 것으로 판단이 되며, 추가적인 열안정성 확보를 위 하여 식물성 오일을 제제에 이용할 수 있을 것으로 전망된다.

Control of Entomopathogenic Fungal Disease in Mealworm Rearing System

Sihyeon Kim,Se Jin Lee,Jeong Seon Yu,Yu-Shin Nai,Jae Su Kim 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

Mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) has high and safe protein contents, which enables it to be animal feed. However, occurrence of entomopathogenic fungi in mealworms is one of the limitations for mass production. In this work, we investigated relationships between abiotic conditions and occurrence of fungal pathogens and established an effective control method using fungicides. In virulence assay, third instar mealworm larvae were sprayed by six entomopathogenic Beauveria bassiana isolates and kept under high relative humidity; B. bassiana ERL1575 isolate had highest virulence. Under normal humidity, ERL1575 conidia showed different virulence between spray (~0% virulence) and digestion (~80% virulence) method. Furthermore, mealworms, which digested conidia, were exposed to various temperature (20-35°C) and humidity (1-3 ml distilled water spray/35 mm diam. dish) conditions for 5 days. All the treatments showed ~90% virulence except 35°C incubations (~20% virulence), but irrespective to the humidity conditions. Forty chemical fungicides were assayed against conidial germination and hyphal growth of ERL1575. Fluazinam and mancozeb showed strong inhibition of conidial germination at standard application dose (SD), 1/2 SD and 1/5 SD; besides, fluazinam showed strong inhibition of hyphal growth. When fluazinam and mancozeb were applied to the fungal conidia-inoculated wheat bran, most of mealworms were alive after 3 days post application. However, high mortality rate (~100%) were observed in the conidia-inoculated wheat bran without any fungicides. In conclusion, this work suggests that B. bassiana isolates could be pathogens at <30°C when they were digested by mealworms, and fluazinam and mancozeb would be used as effective control agents against the pathogen.

Transcriptome analysis of entomopathogenic fungus Beauveria bassiana-infected bean bug (Riptortus pedestris) to identify possible fungal virulence factors

Se Jin Lee,Yi-Ting Yang,Yu-Shin Nai,Jae Su Kim 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.10

The ascomycete fungus Beauveria bassiana is a wide host range entomo- pathogenic fungus, which is commonly used as an environmental friendly biopesticide. However, the molecular mechanisms of host-pathogen interaction of B. bassiana are not well understood. Here, the high throughput next generation sequencing was performed to analyze the transcriptome of B. bassiana JEF-007 infected bean bug (Riptorus pedestris). Differentially expressed gene (DEG) analysis results showed that total 4,684 genes including 2,381 up and 2,303 down regulated genes were identified. Most of the DEGs were classified into single- organism, cellular and metabolism processes by gene ontology (GO) analysis. Metabolism pathway was the most abound category of DEGs via KEGG pathway mapping. Several possible candidates of virulence factors were dramatically expressed after infection, such as cytotoxic lectin, bacterial-like toxin, and proteins related to cell wall, hyphal growth, nutrient uptake and halogenated compounds synthesis. Furthermore, we also found the highest expression of a novel small RNA virus in the infected bean bug, but the relationship between fungal virulence and the RNA virus was under determination. The functional roles of these possible virulence factors are remained unclear, but this work provides a new insight for further fungal studies. Our results reflect systemic impacts of fungal pathogenesis and these findings represent a significant advance in the fungal functional genomics.

Transcriptome analysis of entomopathogenic fungus Beauveria bassiana-infected bean bug (Riptortus pedestris)

Se Jin Lee,Yi-Ting Yang,Yu-Shin Nai,Jae Su Kim 한국응용곤충학회 2016 한국응용곤충학회 학술대회논문집 Vol.2016 No.04

Beauveria bassiana (Bb) is an entomopathogenic fungus with a wide host range, and is commonly used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Here, RNA isolated from a highly virulent strain of B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) (bean bug) infected with this strain were subjected to high throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Differentially expressed gene (DEG) analysis showed that 2,381 genes were up-regulated and 2,303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by gene ontology (GO) analysis. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. This work provides insight into how entomopathogenic B. bassiana occupies agriculturally harmful bean bug at the late stage, which might be essential during fungal infection.

A new strategy using entomopathogenic fungi for the control of tree borer insects

Shin Tae Young,Lee Mi Rong,Kim Jong‐Cheol,Nai Yu‐Shin,Kim Jae Su 한국곤충학회 2022 Entomological Research Vol.52 No.7

In order to establish a strategy for controlling pests living in trees and causing damage, injected entomopathogenic fungal conidia suspension of Beauveria bassiana into a four-year-old Canary Island date palm, Phoenix canariensis. As a result, B. bassiana Bb-egfp#3 expressing green fluorescent protein was dispersed from the injection site to the top and bottom of the tree. To evaluate the insecticidal activity of the Bb-egfp#3 injected into the tree, we released mealworm larvae (Tenebrio molitor) on the ground palm tree sample that was injected with the conidia suspension. As a result, mycosis caused by the injected Bb-egfp#3 in the trees was generated in all cadavers. This study suggests the possibility of establishing a new control strategy against tree pests by injecting an entomopathogenic fungal conidia suspension into trees.