| Appressorium formation and penetration the insect cuticle are the key links of the pathogen pathogenicity of Beauveria bassiana. Previous research found, Bbmpkl MAPK signal pathway is an important way for regulaton of B. bassiana appressourium formation and penetration the insect cuticle. Inhibition of this pathway would impair appressorium formation and file to penetrate the host cuticle. A Subtractive Hybridization Library (SSH) was constructed during appressorium formation with WT transcrips as Tester and Bbmpkl disruption mutand transcripts as the Driver. The results showed that transcription levels of31genes were significantly downregulated or extensively expressed in the wild type strain (Zhang et al.,2010). Investigation of these target genes is crucial for understand of Bbmpkl-regulated appressorium and pathogencity. A570cloning is specific gene expression of appressorium formation tage of wild strains. Bbrpdl shows high homology to many fungal regulatory P domain-contain protein, named Bbrpdl. Recently, Bbrpd1encoding protein has been annotated as a transcriptional regulator in the Genome sequence of B. bassiana ARSEF2860. The upstream sequence of Bbrpdl contains several possible nitrogen regulatory elements, suggesting that this gene Bbrpdl might be related with utilization of nitrogen sources in intection infection. Our previous study demonstrated that Bbrpdl affect conidiation depending on the nitrogen sourcesin in B. bassiana (Wang,2011). However, the details of fungal developmental differentiation and pathogencity mediated by Bbrpds still need to be clearified. In this study, we further investigated the roles of Bbrpdl in the fungal growth, conidiation, and virulence using expression analysis, overexpression of the gene, as well as the gene disruption mutant. The main results are as follows:Analysis of putative amino acid sequence and expression of Bbrpdl.Bioinformatics analysis reveals that the N-end of Bbrpdl encodes24AA signal pe ptides, while a387amino acids anchor B domain (1.8E-118) exist in the C end which b elongs to the TIGR04312secreted protein family. Otherwise, No obvious subcellular lo calization signal or DNA binding domain exists. So we can postulate Bbrpdl may enco des a secrete protein other than transcription factor or regulator.Real-time RT-PCR analysis shows that the expression of Bbrpdl decreases as the conidia matures. With the nutrient consumption increases and time consumes, the expression of Bbrpdl shows a rise trend. Under different nitrogen sources inducing conditions, Expression of Bbrpdl was strongly induced by nitrate nitrogen and nitrite nitrogen. It is speculated that Bbrpdl is related to conidia production, nutrient stress or nitrogen metabolism of Beauveria bassiana.Disruption or overexpression of Bbrpdl affects growth of Beauveria bassiana.The growth rate of⊿Bbrpdl, compared to the WT strain, decreases8.5%,15.6%and10.3%on PDA, SDAY and the basic culture medium CZM (with N03"’-N as the sole nitrogen source), respectively. Meantime, the growth rate of OE-rpdl decreases significantly, with8.1%,5.3%, compared to the wild strain, on PDA and SDAY, respectively. When the NO2-1-N medium acts as sole nitrogen source, both⊿Bbrpdl and OE-rpdl grow slower than the wild strain, reduced by9.4%and6.2%, respectively. Moreover, inactivation of Bbrpdl significantly increases the growth rate, with30.0%,7.0%and11.7%higher compared to the WT while NH4+-N, Cuticle, Peptone medium act as sole nitrogen source, respectively. However the growth rate of OE-rpdl increases with5.9%,4.6%and19.6%, compared to the WT on NH4+-N, cuticle, peptone medium as sole nitrogen source, respectively. Otherwise when cultivated on medium using G1n as unique nitrogen source the growth rate of⊿Bbrpdl boosts8.1%while OE-rpdl decreases7.1%compared to the WT. Our result reveals that Bbrpdl is responsible for vegetative growth of Beauveria bassiana, while strictly regulated by nitrogen source.Interestingly, nitrate reductase activity of OE-rpdl increases significantly when using NO2-1-N as the sole nitrogen source supply. Meanwhile, the nitrate reductase activity of△Bbrpdl descends. Therefore, Bbrpdl, regulated by NO2-1-N, is involved in nitrogen utility. Disruption or overexpression of Bbrpdl is involved in conidiation depending on nitrogen sources.Bbrpdl affected conidia production of B. bassiana responsing to nitrogen sources. Inactivation of Bbrpdl significantly increased conidiation (conidia/mm2), which is increased by0.51,1.32,1.19,0.3and3.84-fold on SDAY, PDA, minimal medium CZM (MM), and MM containing peptone or G1n as sole nitrogen source compared to the wild type strain, respectively. Moreover, OE-rpdl caused a significant reduction in conidiation, with a0.5,0.49,0.77,0.88and1.25fold decrease in conidia/mm2compared to the WT on SDAY and PDA, the basic culture medium CZM (with NO3-1-N as the sole nitrogen source) and peptone and G1n as the only nitrogen source culture, respectively. In the medium with NH4+-N as sole nitrogen source,△Bbrpdl and OE-rpdl conidia yield was higher than the wild strain, Increased by3.95and3fold, respectively. Sporulation of△Bbrpdl and OE-rpdl were significantly lower than the wild strain on NO2-1-N as the sole nitrogen source conditions. This shows that Bbrpdl is dependent on the nitrogen source to effect conidia production of B. bassiana. The expression of five5conidia related genes after growth on various nitrogen sources as sole sources was examined in test strains. The results show that expression level of5conidia related genes differ significantly among different condia yield of the tested strains. In conclude, Bbrpdl which affect the conidiation while response to nitrogen availability may involve in the regulation of sporulation related genes.Disruption or overexpression of Bbrpdl influences conidial germinationConidia viability determination shows that GT50of⊿Bbrpdl strain (GT50=9.4h) is1.8h faster than the wild-type strain (GT50=11.2h). But The GT50of the OE-rpdl strain (GT50=12.2h h) is1h shorter than the wild-type strain (GT50=11.2h). The result suggested that Bbrpdl may involve in conidial germination.Disruption or overexpression of Bbrpdl influences adhesion of conidia.The adhesion determination shows that the hydrophobicity of disruption mutant and over expression strain of Bbrpdl are both higher than the wild type strain. Meanwhile, the hydrophilicity of⊿Bbrpdl and OE-rpdl are weaker than WT strain. Therefore, Bbrpdl may act as an important factor affecting the surface property and virulence of B. bassiana.Disruption or overexpression of Bbrpdl affects virulence of B.bassiana.Two types of insect bioassays using fresh conidia and third-Galleria mellonella larvaes were performed.The first, in which the fungal conidia were topically applied represents the natural route of infection, and the second, in which conidia are injected into the hemoceol of the insect thus by-passing the cuticle. In both assay,△rpdl mutants displayed a increase in the mean lethal time (LT50) it takes to kill as compared to the wild-type strain. Meanwhile, overexpression of Bbprdl caused a decrease in virulence, with a reduction of LT50it takes to kill as compared to the wild-type strain. So, Bbrpdl may act as one of the important factors influencing the virulence. |
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