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The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii.

ABSTRACT: The polyubiquitin gene is a highly conserved open reading frame that encodes different numbers of tandem ubiquitin repeats from different species, which play important roles in different biological processes. Metarhizium robertsii is a fungal entomopathogen that is widely applied in the biological control of pest insects. However, it is unclear whether the polyubiquitin gene is required for fungal development, stress tolerance, and virulence in the entomopathogenic fungus. In the present study, the polyubiquitin gene (MrUBI4, MAA_02160) was functionally characterized via gene deletion in M. robertsii. Compared to the control strains, the MrUBI4 deletion mutant showed delayed conidial germination and significantly decreased conidial yields (39% of the wild-type 14 days post-incubation). Correspondingly, the transcript levels of several genes from the central regulatory pathways associated with conidiation, including brlA, abaA, and wetA, were significantly downregulated, which indicated that MrUBI4 played an important role in asexual sporulation. Deletion of MrUBI4 especially resulted in increased sensitivity to ultraviolet (UV) and heat-shock stress based on conidial germination analysis between mutant and control strains. The significant increase in sensitivity to heat-shock was accompanied with reduced transcript levels of genes related to heat-shock protein (hsp), trehalose, and mannitol accumulation (tps, tpp, nth, and mpd) in the MrUBI4 deletion mutant. Deletion of MrUBI4 has no effect on fungal virulence. Altogether, MrUBI4 is involved in the regulation of conidiation, conidial germination, UV stress, and heat-shock response in M. robertsii.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6627135 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual conidiation, and virulence. However, functions of G proteins in entomopathogenic fungi remain unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gαi, in conidiation, stress resistance, and virulence in Metarhizium robertsii. MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG, flbD, brlA, wetA, phiA, and stuA. Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ΔMrGpa1 and wild-type (WT) strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in tolerance to antifungal drug and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ΔMrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. Further assays showed that MrGpa1 regulated intracellular cyclic AMP (cAMP) levels, but feeding with cAMP could not recover the appressorium formation rate of ΔMrGpa1. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity, and cell wall integrity in M. robertsii. This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.

Project description:Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (?MrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ?MrKu70 was 93% compared to 7% in the wild-type strain. Since ?MrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.

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The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii.

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