ABSTRACT
Prolonged periods of host-lethal infection by entomopathogenic fungi pose challenges to the development of biological control agents. The obligate entomopathogen C. obscurus, however, rapidly kills aphid hosts, warranting investigation. This study investigated the interaction between C. obscurus and a bean aphid Megoura crassicauda during the incubation period of infection, using transcriptome analysis to map host gene expression profiles. Results indicate C. obscurus-inoculated aphid activation of the wound healing immune responses, alongside suppression of the key molecules involved in Toll signaling, melanization, and metabolism. Furthermore, neuromotor system-related genes were upregulated, paralleling the intoxication observed in a nematode pest treated with C. obscurus-derived CytCo protein. To deepen interaction insights, a His-tag pull-down assay coupled with mass spectrometry analysis was conducted using CytCo as a bait to screen for potential aphid protein interactors. The proteins were identified based on the assembled transcriptome, and eleven transmembrane proteins were predicted to bind to CytCo. Notably, a protein of putatively calcium-transporting ATPase stood out with the highest confidence. This suggests that CytCo plays a vital role in C. obscurus killing aphid hosts, implicating calcium imbalance. In conclusion, C. obscurus effectively inhibits aphid immunity and exhibits neurotoxic potential, expediting the infection process. This finding facilitates our understanding of the complex host-pathogen interactions and opens new avenues for exploring biological pest management strategies in agroforestry.
ABSTRACT
Cytolytic (Cyt)-like genes are present in both pathogenic bacteria and fungi. Bacterial Cyt proteins can destroy insect midgut epithelial cells after ingestion by hosts and some of them have been developed as biopesticides; however, few studies have investigated their functions in fungal pathogens. This study investigated the effects of a Cyt-like protein (CytCo) derived from Conidiobolus obscurus (Entomophthoromycotina) on the hemocytes of the greater wax moth Galleria mellonella larvae. The results showed a significant decline in hemocyte viability after treatment with CytCo in vivo or in vitro. The hemocyte density in the hemolymph was reduced by 65.2% and 50.2% after 12 h in vivo and 6 h in vitro treatments, respectively. Apoptosis/necrosis tests using fluorescence microscopy demonstrated that CytCo-treated hemocytes displayed apoptosis, and many of them also showed necrosis after 6 h in vitro treatment. Based on transcriptome analysis, several genes involved in the programmed cell death signaling pathway were upregulated in the CytCo-treated hemocytes. Meanwhile, the differentially expressed genes related to energy production, signal transduction, transcription regulation, and melanization were upregulated, demonstrating activated immune responses; those putatively related to hemocyte adhesion were downregulated, possibly in response to the reduction of hemocytes in hemolymph. In conclusion, CytCo as a virulence factor, could irreversibly incapacitate host hemocytes, playing an important role in debilitating insect immunity. This novel insecticidal protein holds a potential to develop biopesticide for controlling agroforestry pests.
Subject(s)
Hemocytes , Moths , Animals , Larva , Proteins , Insecta , NecrosisABSTRACT
Fungi in the Entomophthorales order can cause insect disease and epizootics in nature, contributing to biological pest control in agriculture and forestry. Most Entomophthorales have narrow host ranges, limited to the arthropod family level; however, rare genomic information about host-specific fungi has been reported. Conidiation is crucial for entomopathogenic fungi to explore insect resources owing to the important roles of conidia in the infection cycle, such as dispersal, adhesion, germination, and penetration into the host hemocoel. In this study, we analyzed the whole genome sequence of the aphid-obligate pathogen Conidiobolus obscurus strain ARSEF 7217 (Entomophthoromycotina), using Nanopore technology from Biomarker Technologies (Beijing, China). The genome size was 37.6 Mb, and encoded 10,262 predicted genes, wherein 21.3% genes were putatively associated to the pathogen-host interaction. In particular, the serine protease repertoire in C. obscurus exhibited expansions in the trypsin and subtilisin classes, which play vital roles in the fungus' pathogenicity. Differentially expressed transcriptomic patterns were analyzed in three conidiation stages (pre-conidiation, emerging conidiation, and post-conidiation), and 2915 differentially expressed genes were found to be associated with the conidiation process. Furthermore, a weighted gene co-expression network analysis showed that 772 hub genes in conidiation are mainly involved in insect cuticular component degradation, cell wall/membrane biosynthesis, MAPK signaling pathway, and transcription regulation. Our findings of the genomic and transcriptomic features of C. obscurus help reveal the molecular mechanism of the Entomophthorales pathogenicity, which will contribute to improving fungal applications in pest control.
ABSTRACT
Virulence attenuation frequently occurs in in vitro culturing of pathogenic microbes. In this study, we investigated the total putative long noncoding RNAs (lncRNAs) in an aphid-obligate pathogen, Conidiobolus obscurus, and screened the differentially expressed (DE) lncRNAs and protein-coding genes involved in the virulence decline. The virulence was significantly attenuated after eight subculturing events, in which the median lethal concentration of the conidia ejected from mycelial mats relative to the bamboo aphid, Takecallis taiwanus, increased from 36.1 to 126.1 conidia mm-2, four days after inoculation. In total, 1,252 lncRNAs were identified based on the genome-wide transcriptional analysis. By characterizing their molecular structures and expression patterns, we found that the lncRNAs possessed shorter transcripts, lower expression, and fewer exons than did protein-coding genes in C. obscurus. A total of 410 DE genes of 329 protein-coding genes and 81 lncRNAs were identified. The functional enrichment analysis showed the DE genes were enriched in peptidase activity, protein folding, autophagy, and metabolism. Moreover, target prediction analysis of the 81 lncRNAs revealed 3,111 cis-regulated and 23 trans-regulated mRNAs, while 121 DE lncRNA-mRNA pairs were possibly involved in virulence decline. Moreover, the DE lncRNA-regulated target genes mainly encoded small heat shock proteins, secretory proteins, transporters, autophagy proteins, and other stress response-related proteins. This implies that the decline in virulence regulated by lncRNAs was likely associated with the environmental stress response of C. obscurus. Hence, these findings can provide insights into the lncRNA molecules of Entomophthoromycotina, with regards to virulence regulators of entomopathogens.
