Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Abstract

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ⌬BbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13,14,18,20,29,49,50,51,52,53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11,19,46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm .nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells ...

A fungal homologue of neuronal calcium sensor-1, Bbcsa1, regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana

“…As a broad-host-range insect pathogen, strains of this fungus have been exploited for use against crop and invasive pests as well as for insects that act as human and animal disease vectors such as mosquitoes and ticks (de Faria & Wraight, 2007;Fan et al, 2012;Kirkland et al, 2004). Aside from its interest as a pest biological control agent, B. bassiana is also an emerging model organism that can be used to examine unique aspects of fungal growth and development, including host-pathogen interactions (Jin et al, 2010;Wanchoo et al, 2009). Infection of insects does not require any specialized mode of entry and begins with attachment of fungal spores to the target hosts.…”

High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

“…As a broad host range insect pathogen, strains of this fungus have been exploited for use against crop and invasive pests as well as for insects that act as human and animal disease vectors such as mosquitoes and ticks (de Faria and Wraight, 2007;Farenhorst et al, 2009;Kirkland et al, 2004). Aside from its interest as a pest biological control agent, B. bassiana is also an emerging model organism that can be used to examine unique aspects of fungal growth and development including host-pathogen interactions (Jin et al, 2010;Lewis et al, 2009;Wanchoo et al, 2009). Infection of insects does not require any specialized mode of entry and begins with attachment of fungal spores to the target hosts.…”

“…Feng, personal communication), are likely to increase molecular investigations of this organism (Cho et al, 2006a(Cho et al, ,b, 2007. A number of targeted gene knockouts have also been constructed in B. bassiana; however, the transformation rate remains low and the protocols cumbersome (Fang et al, 2008;Jin et al, 2010;Xu et al, 2009). The generation of a random-insertion mutagenesis library is an invaluable tool for the molecular dissection of B. bassiana growth and development in either its saprophytic or parasitic life-cycle.…”

A strong promoter, PMagpd, provides a tool for high gene expression in entomopathogenic fungus, Metarhizium acridum

“…However, the wild-type strains tend to have disadvantages of slow speed of kill and relatively poor storage, which drastically limit their applications. To improve the virulence and other characteristics of the wild-type strains, engineered manipulations have been performed during recent years, such as introduction of heterologous virulent genes (Wang and St. Leger 2007) and overexpression of homologous genes (Jin et al 2010).…”

“…The promoter of glyceraldehyde-3-phosphate dehydrogenase gene (gpd), which directs the constitutive highly expressed gene gpd, has been used to drive many genes' expression in fungi. Among these gpd promoters, the PgpdA from Aspergillus nidulans is the most frequently used in fungal transformation, providing high levels of constitutive expression of interest genes or marker genes (Cao et al 2007;Jin et al 2010).…”