Transcriptomic analysis of Metarhizium robertsii during hemocoel colonization after deletion of a spermine synthase MrSPS

Abstract:

[Background] Spermine plays an important role in plant defense response, animal anti-fatigue and anti-aging fight, and fungal growth and metabolism. However, there have been few reports on spermine function in the entomopathogenic fungi. [Objective] The study explored the molecular mechanism of spermine synthase (MrSPS) from Metarhizium robertsii during insect hemocoel colonization. [Methods] Insect bioassays of Galleria mellonella larvae were performed to examine the virulence of Mrsps deleted M. robertsii (ΔMrsps), and the growth characteristics of ΔMrsps in hemolymph were observed. Hemolymph of G. mellonella larvae injected with ΔMrsps and wild-type (WT) strains for 30 h were collected for transcriptome sequencing, and comparisons were made with genes from M. robertsii and G. mellonella separately. Real-time quantitative PCR was performed for verification of transcriptome sequencing. [Results] Compared to WT and complementation (ΔMrsps-cp) strains, ΔMrsps showed significantly decreased virulence, and the pathogenicity of ΔMrsps reduced markedly with decreasing of the injection concentration. After 36 h of infection, spores from both WT and ΔMrsps germinated normally and began to grow in a yeast-like state, while ΔMrsps biomass was less than that of WT after 60 h. A total of 3 202 genes were detected from M. robertsii, of which 1 769 were up-regulated and 922 were down-regulated in ΔMrsps. Lots of differentially expressed genes (DEGs) were involved in carbohydrate metabolism, and transport, catabolism, translation, and amino acid metabolism pathways. All identified 28 genes related to hemocoel colonization from DEGs were down-regulated in ΔMrsps. Real-time quantitative PCR showed that both collagen-like protein and Colonization of hemocoel 1 genes were decreased during the whole stage of hemocoel colonization in ΔMrsps compared to the conditions in WT and ΔMrsps-cp. A total of 13 249 genes were detected from G. mellonella, of which 4 026 genes had differential expression. KEGG analysis revealed most of DEGs were enriched in endocrine system and immune system. Furthermore, among the 22 genes involved in Toll and Imd signaling pathways, 18 were up-regulated in G. mellonella infected by ΔMrsps, which suggested ΔMrsps was more likely to cause the activation of the insect immune system than WT. [Conclusion] The molecular mechanism of MrSPS function in hemocoel colonization of M. robertsii was explored for the first time, which provided a theoretical basis for further revealing the mechanism of spermine function in fungi.