Roles Of An ARF Homologous Gene, BbarfA, In Pathogenesis Of The Insect Fungal Pathogen Beauveria Bassiana

Beauveria bassiana is a well-known fungal pathogen infecting a variety of insect pests, and is actively being developed as a tool with which to control various insects, including invasive and agricultural pests as well as vectors of human pathogens. Insect fungal pathogens invade their host insects by direct penetraction of the host cuticle. Typically, the invasion process involves in adherence of conidia to the target insect cuticle, germination across the surface, differentation of infection structures known as appressoria which then produce penetration pegs and penetrate into the host cuticle, propagation inside the insect hemocoel, and so on. Therefore, appressoria differentation is vitally important for penetration of the cuticle. And thus, clarification of the mechanism involved in appressorium differentaton is crucial for understanding the fungal invasion and pathogenesis.It is demonstrated that Bbmpkl signal pathway is necessary for regulation of approssorium differentation in B. bassiana. The Bbmpkl disruption strain completely lost the ability to form approssoria. In order to understand the mechanism of approssorium differentiation regulated by Bbmpkl, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Characterization of these differently expressed genes is essential for understand of the regulatory methanism of appressorium differantation and pathogensis by Bbmpkl. In this study, a target clone, A929(GenBank:GR979555.1), of which transcription level was significantly reduced in the Bbmpkl mutant, was selected and characterized. The role of the gene was investigated in appressorium differentation and pathogenesis by employing transcription pattern analysis, RNAi, overexpression, and overexpression of gene mutated at some crucial sites for the function.The major findings obtained are as follows:1. Sequence analysis of BbarfASequence analysis indicated that the complete cDNA of A929clone contained a552-bp open reading frame that encoded a183-amino-acid protein with a predicted molecular mass of 20.9kDa and a deduced pI of6.77. The putative protein showed high homologous to Metarhizium anisopliae Arfl, Magnaporthe oryzae Arf, Saccharomyces cerevisiae Arfl and Aspergillus nidulans ArfA with identities of92-98%, and designated BbarfA (Beauveria-bassiana ADP-Ribosylation Factor A). Phylogenetic analysis showed that BbarfA belongs to ARF1family. The putative amino sequence of BbarfA contained several conserved domains of ARF, such as a14-alkylated site at the2nd glycine, a GTP binding domain at the site between24th-30th amino acid and a GTP hydrolysis domain at the site between67th-7181amono acid.2. Expression pattern of BbarfAReal-time RT-PCR analysis indicated that transcription level of BbarfA was significantly higher in B. bassiana cultured in medium with insect hemocytes as sole nutrition than those cultured in CZM, PDA and SDY media. During conidial germination, transcriprion level was relative high in mature conidia, obviously increased during spore spherical growth (at4h post inoculation), and decreased after emergence of germ tube (8h after inoculation). These results suggested that BbarfA may be involved in pathgenesis and spore spherical growth during conidia germination.3. Effect of BbarfA on the fungal development and pathogenicity.In order to further invesigate roles of BbarfA, transformant strains including BbarfA knock-down (RNAi), overexpression of BbarfA, BbarfAD26G (mutation at GDP binding site) and BbarfAQ711(mutation at GTP hydrolysis site) were obtained.BbarfA influence conidial germination. No obvious difference was observed in colony grwoth and conidiation in the wild-type, RNAi transformant (RNAi), overexpression of BbarfA (OE-arfA) and overexpression of BbarfAD26G (OE-arfAD26G) strains. It was noted that alteration of BbarfA expression patterns (RNAi or overexpression) and overexpression of BbarfAQ711(mutation at the GTP hydrolysis site) caused a distinct change in conidial germination. Knock-down of BbarfA (RNAi) obviously accelerated conidial germination. The median germination time of the RNAi strain (GT50=9.32h) was ca.0.91h shorter than the wild-type strain (GT50=10.23h). However, overexpression BbarfA (OE-arfA) and BbarfAQ711(OE-arfAQ711) resulted in a delay in conidial germination, with ca.1.02h and0.3h of GT50longer than that of the wild-type strain, respectively. Conidial germination was not influenced by overexpression of BbarfAD26G (OE-arfAD26G). The result suggested that BbarfA is involved in conidial germination.BbarfA is involved in the sensitivity of B. bassiana to H2O2. There was no obvious difference in sensitivities to osmotic and high temperature stresses in the all transformant and wild-type strain. But all the transformant strains (RNAi, OE-arfA, OE-arfAQ711and OE-arfAD26G) showed more sensitives to H2O2than the wild-type strain, suggesting that BbarfA is involved in response to H2O2To understand the effect of alteration of BbarfA transcription or expression in B. bassiana on response to H2O2., transcription patterns of several antioxidant genes was evaluated under normal conidition and H2O2stress in all the transformants and wild-type strain. The result showed that transcription levels of superoxide dismutase (Sod-3,4), peroxidase (Pod-3) and thioredoxin (Trx) genes were significantly decreased in all transformants under H2O2stress, comapred to that of the wild-type strain. Transcription patterns of some antioxidant genes including catalase (Cat-1,2,4), superoxide dismutase (Sod-1,2) and peroxidase(Pod-1,2)-encoding genes were altered in the all transformants as compared to those in the wild-type cells. These results suggested that alteration of BbarfA transcription or expression in B. bassiana might interfere with expression patterns of antioxidant genes.BbarfA is required for virulence. No obvious difference was observed in appressorium differentation between all the transformants and the wild-type strain. However, insect bioassays using both topical inoculation and intrahemoceol injection revealed that the virulence of B. bassiana was distinctly affected by alteration of BbarfA transcription or expression. Knock-down of BbarfA (RNAi) caused a distinct increase in virulence, while overexpression BbarfA (OE-arfA) and BbarfAQ711(OE-arfAQ711) significantly resulted in a decrease in virulence in the fungal species. The mean lethal time (LT50) for RNAi were shortened by17.1h and10.7h using topically inoculation and intrahemoceol injection, as compared to the wild-type, respectively. Howeve, overexpression BbarfA (OE-arfA) and BbarfAQ711(OE-arfAQ711) stranis showed a reduction in LT50in both assays, with11.6h-and8.9h-more, and3.5h-and2.9h-more than those of the wild-type strain, respectively. But no obvious change was noted in virulence between overexpression BbarfAD26G (OE-arfAD26G) and the wild-type strain. The result suggested that BbarfA is involved in virulence in B.bassiana.