| Beauveria bassiana is an important entomopathogenic fungi, which has been widely used for pests control of agriculture, forestry and city, and it is becoming a model strain for the research of host-fungus interaction. However, its insecticide efficacy in the field is often influenced by adverse environmental stresses, such as desiccation, high temperature, and UV radiation, which limit the widespread using of the fungal agent. In addition, fungal pathogens also face stresses to immune responses from host insect. Thus, understanding of fungal pathogen responses to stresses from environment or host insect would facilitate improvement and production of a more efficient biocontrol agent.The fungal cell wall is a barrier that protect cells against adverse environment, and is also involved in the fungal growth, morphogenesis. Thus, fungal cell wall is an important target for investigating the fungal responses to environment stresses and interaction between the fungal pathogen and host insect. The cell wall integrity (CWI) pathway, slt2/mpkl/pkcl pathway, not only plays a vital role in maintaining cell wall structure and composition, but also mediates fungal growth, development and stress responses. MADS-box Rlml, a potential target transcription factor of CWI pathway, has been characterized in Saccharomyces cerevisiae and several other fungal species. It has been shown that role of Rlml homologs is conserved, but also distinctly varied in fungal species.Bbslt2, a MAPK (mitogen-activated protein kinase) of CWI pathway, is vital for cell wall integrity in B. bassiana. However, role of Rlml orthologs, a potential target of Bbslt2, is still unclear. Here, we cloned an Rlml homolog, Bbrlml, in B. bassiana and characterized using gene knock-out, reverse complement and over expression strategies.Follows are the main results:1. Cloning of BbrlmlAccording to conserved amino acids analysis of Rlml orthologs in Saccharomyces cerevisiae, Aspergillus nidulans, Aspergillus fumigatus, an Rlml ortholog encoding gene, designed Bbrlml, was cloned in B. bassiana by BLAST searching the genomic database. Sequence analysis showed that Bbrlml contained a 1965 bp ORF. The putative protein of Bbrlml consisted of 654 amino acid residues with calculated Mr of 70.08 kDa and pI of 5.31. The predicted Bbrlm1 contains MADS-MEF2 domain at sites from 2 to 70 amino acid of N terminal and a domain of unknown function, DUF3827 domain, at the region of 365 aa-516 aa. Phylogenetic analysis indicated that Bbrlml closely clustered with Cordyceps militaris r1mA.2. Function of BbrlmlBbrlml mediated the fungal growth. To investigate roles of Bbrlm1, transformants of gene disruption and overexpression were generated by homologous recombination and constitutive expression strategies. Determination of colony growth indicated that Bbrlml mediated the fungal growth. On the minimal medium (CZM), a distinct growth was detected in gene knock-out mutant (ABbrlml), while an increase was observed in overexpression strain (OE-Bbrlml). On complete medium (1/4 SDY), either ΔBbrlml or OE-Bbrlml displayed a decrease colony growth as compared to the wild-type strain. Colony of ABbrlml appeared compact and thin. In contrast, OE-Bbrlm1 displayed flourishing and thick colony as compared to the wild-type. These results suggested that Bbrlml mediated growth of B. bassiana depend on nutrition.Bbrlml influenced sporulation of B. bassiana. Determination of conidia yield indicated that disruption of Bbrlml caused a significant increase in conidiation on minimal medium, while a distinct reduction in the overexpression strain. On complete medium, no obvious difference in conidiation was detected in the ABbrlml, OE-Bbrlm1 and wild type strains. Either disruption or overexpression of the gene leaded to a decrease in blastospore yield in the complete liquid broth. The results suggested that Bbrlm1 was involved in sporulation in B. bassiana.Bbrlml mediated the cell wall integrity. To understand role of Bbrlml in cell wall integrity, sensitivities of ΔBbrlml and OE-Bbrlml to cell wall synthesis inhibitors and high temperature were evaluated on minimal and complete media. On minimal medium, ABbrlml was more sensitive to high temperature and SDS, but more tolerant to calcofluor white (CFW) than the wild-type strain. OE-Bbrlml displayed an increased in tolerance to CFW and CR, but no effect on SDS and high temperature sensitivities as compared to the wild-type strain. On complete medium, disruption of Bbrlml caused a distinct increase in sensitivity to CFW and SDS, but a decrease in sensitivity to CR, and no effect on high temperature sensitivity. However, Bbrlm1 overexpression resulted in an increase in tolerance to all of the stress conditions. Observation with transmission electron microscopy (TEM) showed that the ΔBbrlml conidia appeared distinctly distorted. No obvious difference in blastospore cell wall was noted between the wild-type and Bbrlml disruption mutant. The OE-Bbrlm1 was also distorted and cell wall became thinner than the wild-type. Although morphology of OE-Bbrlml blastospore was normal, similar to the conidia, cell wall became thinner than the wild type cells. These results suggested that Bbrlm1 regulated cell wall integrity and construction dependent on nutrition.Bbrlml controlled high osmotic stress response. Effect of Bbrlml on osmotic stress response was investigated on minimal and complete media using NaCl and sorbitol as osmotic stressors. On minimal medium, ABbrlm1 was more sensitive to high osmotic stresses (1.2 M sorbitol or 0.6 M NaCl). However, no obvious difference in sensitivity to osmotic stressors was detected between the OE-Bbrlml and the wild-type strains. On complete medium, either ΔBbrlml or OE-Bbrlml strain displayed an increase tolerance to high osmitic stressors (1.0 M NaCl or 1.2 M sorbitol). These results suggested that Bbrlml is involved in osmotic stress response, which was also influenced by varied nutrition.Bbrlml mediated virulence. Two types of insect bioassays using fresh conidia and third-instar larvae of Galleria mellonella were performed. The first, in which the fungal conidia were topically applied represents the natural route of infection, and the second, in which conidia were injected into the hemoceol of the insect thus by-passing the cuticle, was used to examine deficiencies in immune evasion and intra-insect survival. Assay results of topically inoculation showed that disruption and overexpression of Bbrlml caused decrease of 11% and 7% in virulence as compared to the wild-type strain, respectively. Similarly, intrahemoceol injection assay showed that disruption or overexpression of the gene also resulted in reduction of 9% and 13% in virulence, respectively. These results showed that Bbrlm1 rigorously affected virulence in B. bassiana.Bbrlml affected secondary metabolism. Bbrlml was found to be as a pH-dependent regulator of an orange pigment in B. bassiana. The pigment production in the ABbrlml strain increased under the acidic condition, but decreased under the alkaline condition.Bbrlml influenced the acid production. pH value detection indicated that the cultured broth of ΔBbrlml in the complete medium displayed a distinct decrease in pH value as compared to that of the wild-type cells, suggesting the mutant strain produced a amount of acid materials. |
PDF Full Text Request