Transcriptome profiling reveals insertional mutagenesis suppressed the expression of candidate pathogenicity genes in
Awraris Getachew1,2, Tessema Aynalem Abejew1,2, Jiangli Wu1
1Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture; Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, 100093, Beijing, China.
Abstract:
Chalkbrood disease is caused by Ascosphaera apis which severely affects honeybee brood. Spore inoculation experiments shown pathogenicity varies among different strains and mutants, however, the molecular mechanism of pathogenicity is unclear. We sequenced, assembled and annotated the transcriptomes of wild type (SPE1) and three mutants (SPE2, SPE3 and SPE4) with reduced pathogenicity that were constructed in our previous study. Illumina sequencing generated a total of 394,910,604 clean reads and de novo Trinity-based assembled into 12,989 unigenes, among these, 9,598 genes were successfully annotated to known proteins in UniProt database. A total of 172, 3,996, and 650 genes were up-regulated and 4,403, 2,845, and 3,016 genes were down-regulated between SPE2-SPE1, SPE3-SPE1, and SPE4-SPE1, respectively. Overall, several genes with a potential role in fungal pathogenicity were detected down-regulated in mutants including 100 hydrolytic enzymes, 117 transcriptional factors, and 47 cell wall related genes. KEGG pathway enrichment analysis reveals 216 genes involved in nine pathways were down-regulated in mutants compared to wild type. The down-regulation of more pathways involved in pathogenicity in SPE2 and SPE4 than SPE3 supports their lower pathogenicity during in-vitro bioassay experiment. Expression of 12 down-regulated genes in mutants was validated by quantitative real time PCR. This study provides valuable information on transcriptome variation caused by mutation for further functional validation of candidate pathogenicity genes in A. apis.
Insights
This study reveals key genes and pathways in Ascosphaera apis, the fungus causing chalkbrood disease in honeybees. Mutations reducing pathogenicity led to down-regulation of essential fungal genes, offering insights into disease mechanisms.
Area of Science:
- Mycology
- Insect Pathology
- Molecular Biology
Background:
- Chalkbrood disease, caused by Ascosphaera apis, significantly impacts honeybee populations.
- The molecular basis of A. apis pathogenicity remains largely unknown.
- Previous studies generated A. apis mutants with varying pathogenicity levels.
Purpose of the Study:
- To investigate the molecular mechanisms underlying pathogenicity variations in Ascosphaera apis.
- To identify genes and pathways affected by mutations that reduce fungal pathogenicity.
- To provide a foundation for functional validation of candidate pathogenicity genes.
Main Methods:
- Transcriptome sequencing and de novo assembly of wild-type and mutant A. apis strains.
- Differential gene expression analysis using Illumina sequencing and Trinity assembly.
- KEGG pathway enrichment analysis to identify affected biological processes.
- Quantitative real-time PCR (qRT-PCR) for validating gene expression changes.
Main Results:
- Transcriptome analysis identified thousands of differentially expressed genes between wild-type and mutant strains.
- Mutants with reduced pathogenicity showed down-regulation of genes related to fungal pathogenicity, including hydrolytic enzymes, transcriptional factors, and cell wall components.
- KEGG analysis revealed down-regulation of nine pathways in mutants, with more affected pathways in highly attenuated strains (SPE2, SPE4).
- Expression of 12 down-regulated genes was confirmed by qRT-PCR.
Conclusions:
- Transcriptome variations provide insights into the molecular basis of A. apis pathogenicity.
- Down-regulation of specific genes and pathways correlates with reduced pathogenicity in A. apis mutants.
- This study identifies candidate genes for future functional studies to understand and combat chalkbrood disease.
