Fungal Transformation Research Articles

Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi

Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.

Fungal transformation of 17α-ethinylestradiol in the presence of various concentrations of sodium chloride

17α-Ethinylestradiol (EE2) is a commonly used synthetic estrogen, which is resistant to degradation and accumulates in all parts of the environment. In this work the capability of thirty eight fungal strains to remove this xenobiotic from culture medium was checked. Nine out of the tested strains were able to completely remove EE2 (at initial concentration 10 mg l−1) from the culture medium during 3 d of incubation. The most efficient were two Aspergillus strains (Aspergillus fumigatus IM 6510 and Aspergillus versicolor IM 2161) which also possess the ability to eliminate the xenoestrogen in the presence of NaCl. A. versicolor seems to be less vulnerable to salinity and the transformation process was completed after 3 d of incubation in all tested NaCl concentrations. This strain transformed EE2 and formed its metabolites detected during liquid chromatography–(electrospray) triple quadrupole–mass spectrometry (LC–(ESI)MS–MS) analysis. The quantitative analyses revealed that the EE2 subsequent decrease in samples with or without salinity was accompanied by an increase in the concentrations of estrone (E1) and estradiol (E2). The presence of NaCl slightly inhibited E2 to E1 transformation during the first 24 h of incubation but at the end of the experiment the amount of nontoxic E1 prevailed in all tested samples. The results of this study suggest that Aspergillus sp. possesses an ability to utilize EE2 in the presence of NaCl.

A uninucleate Rhizoctonia sp. from maize plant with ITS heterogeneity and hypersensitive to abiotic stresses

Plant-pathogenic Rhizoctonia spp. are soil-borne fungi with world-wide distribution. Most Rhizoctonia spp. are multinuclear and heterokaryotic fungi, which makes it difficult to build a stable transformation system. In this study, one uninucleate Rhizoctonia isolate, JN, was recovered from a field maize plant. Single nuclear status was observed under fluorescent microscopy after the hyphae were stained with Hoechst33258 solution. The optimal growth conditions of JN mycelia were 28 °C at pH7.0 on potato dextrose agar (PDA) media. The mycelial color of JN cultured on PDA turned from white to light brown with aging. Analysis of the internal transcribed spacer (ITS) sequence of the ribosomal DNA (rDNA) showed that there were two types of ITS sequences within the single isolate and the nucleotide identity of the two sequences is only 95 %. JN is more sensitive to SDS, PEG4000, Sorbitol, hydrogen peroxide (H2O2) and sodium chloride (NaCl) than a multinucleate Rhizoctonia solani, XN5, cultured on PDA. Given the single nucleus, JN has great potential to be used in fungal transformation.

Fluorescence Assisted Selection of Transformants (FAST): Using flow cytometry to select fungal transformants

The availability of drug resistance markers for fungal transformation is often a limiting factor in both fungal genetics research and industrial applications. We describe a new technique using flow cytometry to select fungal transformants using well-known fluorescent proteins as markers for transformation. This new technique, Fluorescence-Assisted Selection of Transformants (FAST), was used for a transformation of Fusarium oxysporum with GFP as a marker targeted at a specific site on chromosome 14. The resulting strain was then transformed again with a gene replacement construct containing both RFP and a gene for drug resistance as markers. By directly comparing FAST with drug resistance selection we show that both methods yield comparable numbers of gene deletion mutants.

PFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

The Role of Cutinase and its Impact on Pathogenicity of Colletotrichum truncatum

The phytopathogenic fungus, Colletotrichum truncatum infects and colonises chili fruit through direct penetration of the cuticle. The cutinase gene of C. truncatum (CtCut1), a cutin degrading enzyme was identified, cloned and shown to be essential in breaching the cuticle of chili fruit. The expression of CtCut1 gene was studied through RNA-mediated gene silencing and its impact on fungal pathogenicity was demonstrated. The vector, pAA1 encoding a hairpin RNA of GFP and CtCut1 was constructed and transformed into C. truncatum pathotype F8-3B (virulent strain). F8-3B-pAA1 transformants exhibited reduced patterns of infection with one isolate having a 45.8% reduction in cutinase activity (reduction in CtCut1 transcript) in comparison to the wild type. Importantly, CtCut1-deficient strains were unable to infect detached chili and soybean hosts as efficiently as the wild type. There was a delay in the infection period by the transformants. Nevertheless, artificial wounding of the cuticle enabled these F8-3B-pAA1 transformants to infect and colonise host tissues, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggested that the defect in pathogenicity was likely due to a failure in penetration of the host cutin. Knowledge of the plant-fungal interactions arose from the development of a fungal transformation system for C. truncatum and implementation of RNAi technology. This technology thus provides an alternative genetic tool for studies of gene function, particularly of essential pathogenicity genes.

Transformation of vanadinite [Pb5 (VO4 )3 Cl] by fungi.

Saprotrophic fungi were investigated for their bioweathering effects on the vanadium- and lead-containing insoluble apatite group mineral, vanadinite [Pb5 (VO4 )3 Cl]. Despite the insolubility of vanadinite, fungi exerted both biochemical and biophysical effects on the mineral including etching, penetration and formation of new biominerals. Lead oxalate was precipitated by Aspergillus niger during bioleaching of natural and synthetic vanadinite. Some calcium oxalate monohydrate (whewellite) was formed with natural vanadinite because of the presence of associated ankerite [Ca(Fe(2+) ,Mg)(CO3 )2 ]. Aspergillus niger also precipitated lead oxalate during growth in the presence of lead carbonate, vanadium(V) oxide and ammonium metavanadate, while abiotic tests confirmed the efficacy of oxalic acid in solubilizing vanadinite and precipitating lead as oxalate. Geochemical modelling confirmed the complexity of vanadium speciation, and the significant effect of oxalate. Oxalate-vanadium complexes markedly reduced the vanadinite stability field, with cationic lead(II) and lead oxalate also occurring. In all treatments and geochemical simulations, no other lead vanadate, or vanadium minerals were detected. This research highlights the importance of oxalate in vanadinite bioweathering and suggests a general fungal transformation of lead-containing apatite group minerals (e.g. vanadinite, pyromorphite, mimetite) by this mechanism. The findings are also relevant to remedial treatments for lead/vanadium contamination, and novel approaches for vanadium recovery.

Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach.

BackgroundAspergillus sojae has been an important filamentous fungus in Biotechnology due to its use in diverse fermentative processes for the production of various food products. Furthermore, this fungus is a common expression system for the production of enzymes and other metabolites. The availability of molecular genetic tools to explore its biology is thus of big interest. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system for A. sojae was developed and its applicability evaluated.ResultsThe donor plasmid named pRM-eGFP was constructed for ATMT of A. sojae. This plasmid contains the ble and egfp genes in its transfer DNA element (T-DNA) to confer phleomycin resistance and express the enhanced green fluorescent protein (EGFP) in A. sojae, respectively. Agrobacterium tumefaciens (LBA4404) harboring the donor plasmid and A. sojae (ATCC 20235) were co-cultured under diverse conditions to achieve ATMT. The maximum number of transformed fungi was obtained after three days of co-culturing at 28°C, and selection with 50 μg/ml phleomycin. Polymerase chain reaction (PCR), fluorescence microscopy and Western Blot analysis for EGFP expression confirmed successful genomic integration of the T-DNA element in A. sojae. The T-DNA was mitotically stable in approximately 40% of the fungal transformants after four generations of sub-culturing under phleomycin pressure.ConclusionWe successfully established a new ATMT protocol for A. sojae. This transformation system should enable further protein expression studies on this filamentous fungus.

Establishment of invasive and non-invasive reporter systems to investigate American elm–Ophiostoma novo-ulmi interactions

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal–plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant–pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.

Microbial diversity of a Mediterranean soil and its changes after biotransformed dry olive residue amendment.

The Mediterranean basin has been identified as a biodiversity hotspot, about whose soil microbial diversity little is known. Intensive land use and aggressive management practices are degrading the soil, with a consequent loss of fertility. The use of organic amendments such as dry olive residue (DOR), a waste produced by a two-phase olive-oil extraction system, has been proposed as an effective way to improve soil properties. However, before its application to soil, DOR needs a pre-treatment, such as by a ligninolytic fungal transformation, e.g. Coriolopsis floccosa. The present study aimed to describe the bacterial and fungal diversity in a Mediterranean soil and to assess the impact of raw DOR (DOR) and C. floccosa-transformed DOR (CORDOR) on function and phylogeny of soil microbial communities after 0, 30 and 60 days. Pyrosequencing of the 16S rRNA gene demonstrated that bacterial diversity was dominated by the phyla Proteobacteria, Acidobacteria, and Actinobacteria, while 28S-rRNA gene data revealed that Ascomycota and Basidiomycota accounted for the majority of phyla in the fungal community. A Biolog EcoPlate experiment showed that DOR and CORDOR amendments decreased functional diversity and altered microbial functional structures. These changes in soil functionality occurred in parallel with those in phylogenetic bacterial and fungal community structures. Some bacterial and fungal groups increased while others decreased depending on the relative abundance of beneficial and toxic substances incorporated with each amendment. In general, DOR was observed to be more disruptive than CORDOR.

Fungal transformation of metallic lead to pyromorphite in liquid medium

Many approaches have been proposed to reduce the toxicity of hazardous substances such as lead in the environment. Several techniques using microorganisms rely on metal removal from solution by non-specific biosorption. However, immobilization of metals through formation of biominerals mediated by metabolic processes offers another solution but which has been given limited attention. In this work, we have investigated lead biomineralization by Paecilomyces javanicus, a fungus isolated from a lead-contaminated soil, in a liquid medium. P. javanicus was able to grow in the presence of metallic lead, supplied as lead shot, and secondary lead minerals were deposited on the lead surfaces as revealed by scanning electron microscopy. Energy dispersive X-ray analysis and X-ray powder diffraction revealed that pyromorphite was formed in the presence of the fungus, but not in abiotic controls. Our results clearly demonstrate that fungal activities can play an important role in lead biocorrosion and biomineralization in an aqueous environment. These findings are relevant to bioremediation approaches for liquid wastes contaminated with lead, or other metals, and also to the immobilization and biorecovery of rare or valuable elements. They also provide further understanding of microbial roles in environmental lead cycling.

Fungal transformation and T-cell proliferation inhibitory activity of melengestrol acetate and its metabolite

Biotransformation of melengestrol acetate (MGA, 17α-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione) (1) was investigated for the first time by using fungal cultures. Incubation of compound 1 with Cunninghamella blakesleeana yielded a new major metabolite, 17α-acetoxy-11β-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2). The metabolite 2 was purified by using HPLC, followed by characterization through 1H- and 13C-NMR and other spectroscopic techniques. Single crystal X-ray diffraction analysis was used to deduce the three dimensional structures of melengestrol acetate (1) and metabolite 2 for the first time. T-cell proliferation assay was employed to evaluate the immunosuppressant effect of compounds 1 and 2 with IC50=0.5±0.07 and 0.6±0.08μg/mL, respectively. The results indicated that these compounds possess sixfold potent T-cell proliferation inhibitory activity as compared to the standard prednisolone (IC50<3.1μg/mL). Both compounds were found to be non-toxic in a 3T3 (mouse fibroblast) cell-based cytotoxicity assay. This discovery of potent anti-inflammatory activity of compounds 1 and 2 can lead the way to develop new immunosuppressant compounds for clinical application.

Sulfonylurea resistance reconstitution as a novel strategy for ILV2-specific integration in Magnaporthe oryzae

A sulfonylurea-resistant allele of the ILV2 gene encoding an acetolactate synthase from the rice-blast fungus Magnaporthe oryzae has been extensively used in fungal transformation as a dominant selectable marker that confers resistance to chlorimuron ethyl. We devised a novel strategy for site-specific integration of foreign DNA via sulfonylurea resistance reconstitution (SRR) by replacing the native ILV2 with the sulfonylurea-resistant ILV2SUR variant. In contrast to random ectopic integration, SRR-based targeted incorporation at a defined locus eliminates position/orientation effects, unnecessary mutations and/or variation in gene expression. Independent transformants derived from the same SRR construct showed consistent and reproducible fluorescent signal in M. oryzae. Furthermore, the high frequency (>95%) of ILV2-specific targeted integration via SRR circumvents the need for a deficiency in non-homologous end joining (NHEJ) pathway in the recipient strain. Unlike the split-marker technique, which is particularly suitable for targeted gene replacement, the SRR strategy should prove useful for promoter analyses, gene tagging and/or complementation analyses in filamentous fungi.

When the boundaries between physics and biology blur: A promising future for fungi as producers of valuable recombinant proteins: Reply to comments on: “Physical methods for genetic transformation of fungi and yeast”

We appreciate the commentaries to our review [1], received from Cruz Hernandez et al. [2], Ortiz-Vazquez [3],Arellano-Carbajal [4], Castano [5],Gold[6], and Moosavi-Nejad and Hosseini [7], who express interesting points ofview, enriching the discussion with their expertise.Fungi constitute a kingdom that can be found all around our planet since prehistoric times. Cruz Hernandez andcolleagues [2] comment on interesting historical aspects of fungi in humans’ life and emphasize that nowadays fungiplay a crucial role in basic scientific research, applied sciences, industry and economy. The impressive economicestimate of the total global market for fungal and yeast products, referred to by these authors, supports this. There arestill many areas for improvement in the field of fungal transformation and many groups around the world are activelyworking toward this goal.

Restriction Enzyme-Mediated Integration 방법으로 확보한 Fusarium oxysporum 형질전환체의 후자리산 생성능 분석

후자린산(FA)는 Fusarium 속 균이 생성하는 독소로서 다른 곰팡이독소보다 독성은 낮으나 다른 독소와 중복 오염시 전체 독성을 증진시키는 것으로 알려졌다. 현재까지 FA 생합성 관련 효소나 유전자가 Fusarium oxysporum에서 밝혀지지 않았기 때문에 본 연구에서는 관련 생합성 유전자의 발굴을 위해 제한효소를 통한 무작위 삽입 형질전환방법인 REMI를 이용하여 FA 생성 F. oxysporum 균주의 생합성유전자의 결손을 시도하였다. F. oxysporum 균주 2주를 대상으로 REMI를 시도한 결과, 평균 3.2주 (<TEX>$1{\mu}g$</TEX> DNA 당)의 효율로 7,100주 이상의 형질전환체를 육성하였다. FA 미생성 형질전환체를 스크리닝 하기 위해 FA가 함유된 배양액에서 다양한 식물종자의 발아여부를 조사한 결과, 11종의 종자 중 가장 감수성인 배추종자를 선발하였다. 각 형질전환체는 Czapek-Dox broth에서 3주간 배양한 후 배양여액을 배추종자의 발아여부 검정에 사용하였다. 검정결과 총 5,000여 주의 REMI 형질전환체 중 53주의 배양여액에서 종자가 발아하지 않아, 이들을 FA 미(저) 생성 추정 형질전환체로 선발하였다. 이중 26주의 FA 생성량을 HPLC로 분석한 결과, 2주의 형질전환체에서 모균주 생성량의 1% 이하의 FA가 검출되었다. 이 중 형질전환체 1주로부터 REMI 벡터 삽입 부위 게놈 DNA의 염기서열(252 bp)을 확보하였으며, 이 부위는 F. fujikuroi의 미동정 게놈부위와 93% 유사성이 있음을 확인하였다. 이 부위의 FA생성 관련성 증명을 위해서는 추후 연구가 필요하다. Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/<TEX>${\mu}g$</TEX> DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at <TEX>$25^{\circ}C$</TEX> then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

Fungal transformation of methyltestosterone by the soil ascomycete Acremonium strictum to some hydroxy derivatives of 17-methylsteroid

The ascomycete Acremonium strictum was used for the biotransformation of methyltestosterone (1), a pharmaceutical steroid substance, into some steroid derivatives (6β-hydroxy-17α-methyltestosterone (2), 6β,12β-dihydroxy-17α-methyltestosterone (3), 7β-hydroxy-17α-methyltestosterone (4), 6β,17β-dihydroxy-17α-methylandrosta-1,4-dien-3-one (5), and 3,17β-dihydroxy-17α-methylestra-1,3,5(10)-triene (6). The fermentation was carried out in Sabouraud-dextrose broth (SDB) supplemented with 1 mM of the substrate, and the temperature and aeration rate were adjusted to 30°C and 150 rpm, respectively. The biotransformation characteristics observed were hydoxylations at C-6β, C-7β, and C-12β, 1,2-dehydrogenation, and ring A aromatization. The best fermentation conditions, such as temperature, substrate concentration, pH, incubation period, and aeration, were found to be 25°C, 1 mM, pH 6.5, 6 days, and 150 rpm, respectively, for maximum biotransformation of 1.

Fungal transformation of diterpenes by Aspergillus phoenix

Microbial transformation is an interesting tool used to increase the variety of chemical structures to be applied in the search for novel bioactive compounds [1]. In present work, the biotransformation of kaurenoic acid (1) and copalic acid (4), two diterpenes isolated in high amounts from Brazilian plants (Mikania glomerata and Copaifera langsdorffii), were performed using submerged liquid culture of Aspergillus phoenix (3,76 X 108 esporos/mL). The microorganism was grown by a two-stage fermentation procedure. Substrate were added as a dimethylsulfoxide solution (0.1 g/L) and incubated for 7 days. The cultures were filtered and the aqueous layers were extracted with ethyl acetate to furnish the extracts codified as AC (obtained from 1) and Aco (obtained from 4). Chemical and NMR studies of AC and Aco allowed us to isolate and to identify four hydroxylated derivatives. Compounds 2 and 3 were isolated from AC, while 5 and 6 from Aco. Both antimicrobial and antitumor activities of such compounds will be investigated by our research group.

Identification of catalase as an early up-regulated gene in Beauveria bassiana and its role in entomopathogenic fungal virulence

The ability of entomopathogenic fungi to infect insects is a complex process involving differential expression of numerous genes some of which are up-regulated when the fungus is in contact with or exposed to insect cuticles. In this report, we identified a set of differentially expressed genes in the entomopathogenic fungus Beauveria bassiana BCC2659 in response to Spodoptera exigua larvae. PCR-select suppression subtractive hybridization (PCR-SSH) was used to identify genes differentially expressed during the initial aspects of the fungal-insect interaction, i.e. up to a 2h post-infection model. Ten fungal genes identified by PCR-SHH were confirmed to be up-regulated by semi-quantitative RT-PCR. Of these genes, a catalase (catE7), implicated in stress resistance, was chosen for further characterization in order to probe its role in B. bassiana pathogenesis and to determine whether over-expression would result in a more virulent strain. To investigate this, a transgenic B. bassiana strain, overexpressing CatE7 was constructed. Fungal transformant lines with extra catE7 copies (Bb::BbcatE7) showed ∼2-fold higher catalase activity than the wild type. Bb::BbcatE7 strains germinated faster than the wild-type parent and exhibited significantly higher virulence against S. exigua larvae. Although the Bb::BbcatE7 strains were no better than wild type in terms of vegetative growth in the presence of exogenous H2O2 concentrations, conidial germination rates were higher in the Bb::BbcatE7 strain in the presence of H2O2. These results suggest that responses mediated by catalases play an important role in the fungal-insect infection process and the manipulation of catalase expression can lead to more effective fungal strains for insect control.

Transformation ofBeauveria bassianato produce EGFP inTenebrio molitorfor use as animal feed additives

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 5days 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.

Bumblebee Venom Serine Protease Increases Fungal Insecticidal Virulence by Inducing Insect Melanization

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.