| Chalkbrood is a honeybee disease caused by an entomopathogenic fungus Ascosphaera apis which severely reduces the number of viable offspring in the nest.Although artificial methods using spore inoculation and in vitro larval rearing techniques shown that pathogenicity varies among different strains and mutants,yet,the molecular level mechanisms in pathogenicity is unclear.We sequenced,assembled and annotated the transcriptomes of three REMI(Restricted Enzyme-Mediated Integration)constructed mutants with different pathogenicity: less pathogenic(M1),pathogenic(M4),and nonpathogenic(M7)and wild-type(WT)of A.apis pathogen using mRNA from hyphae and spores of each sample.Illumina sequencing was used to generate a total of 394,910,604 clean reads from 12 samples,that were de novo Trinity-based assembled into 12,989 unigenes with an N50 length of 853 bp and assigned to putative annotations.Among them,9,598 genes were similar to known proteins as determined by a BLASTx search(E-value ≤ 1.0E-05)against NCBI nr UniProt database,7,807 genes were annotated in Interpro database,6,583 genes were classified in to Gene Ontology(GO)database and assigned to 2,521 GO terms that contained three main categories,6,561 genes were similar to known proteins in Pfam database,230 genes were assigned to Cluster of Orthologous Groups(COG)categories.Moreover,2,503 genes were mapped into 314 pathways in KEGG(Kyoto Encyclopedia of Genes and Genomes)database.In addition,functional annotation revealed the presence of 356 fungal specific transcriptional factor(TF)genes grouped in to 32 families.Understanding gene regulation is vital in unravelling the effect of genetic variation on both normal development and mutations.Differentially expressed genes(DEGs)in each mutant compared to wild-type samples were identified to better understand the gene expression regulation during hyphae development and sporulation.Comparative analysis of transcriptomes revealed a total of 172,3,996,and 650 up-regulated DEGs and 4,403,2,845,and 3,016 down-regulated DEGs between M1 vs WT,M4 vs WT,and M7 vs WT,respectively.In this study,numerous genes with a potential role in fungal pathogenicity have been detected down-regulated in mutants compared to wild type.For example,53 hydrolytic enzymes,54 transcriptional factors,and 25 cell wall related genes were more down-regulated in M1 and M7 than in M4 compared to WT.Hydrolytic enzymes are potentially involved in virulence through host invasion and escape process,whereas TFs genes play a vital role in the transcriptional regulation of pathways implicated in virulence;and cell wall related genes known to determine cell shape,shielding the cells from environmental stresses,interact with host cells,with dynamic structures that are crucial for cell morphology and pathogenesis.The expression level of 12 commonly down-regulated genes in all the three mutants were validated with qRT-PCR and the result shown that the genes were consistently expressed.Furthermore,the functions and pathways of DEGs between REMI constructed mutants and wild type samples of A.apis were mapped into GO and KEGG pathway databases,respectively.A total of 811,691,and 660 GO terms were filtered as DEGs in M1,M4 and M7 mutants,respectively,compared to wild type transcriptome.GO and KEGG enrichment analysis of DEGs between mutants and wild type suggesting that several genes are potentially contributing to A.apis pathogenicity.KEGG pathway enrichment analysis revealed that a total of 216 genes enriched into nine pathways were down-regulated in the three mutants compared to wild type.Among these,five pathways known to be involved in fungal pathogenicity were suppressed by random mutagenesis(three in M1 and two in M7).Pathways uniquely down-regulated in M1 in comparison to wild type were: glycine,serine and threonine metabolism(28 genes);and aminoacyl-tRNA biosynthesis(31 genes).In addition,54 genes involved in ribosome biogenesis in eukaryotes were down-regulated in M1 compared to M4.Seventeen genes enriched into fatty acid metabolism were down-regulated in M7 compared to wild type.Furthermore,21 genes involved in basal transcription factors pathway were down-regulated in M7 compared to M4.The down-regulation of pathogenicity related pathways in M1 and M7 than M4 supports their less pathogenicity during in vitro bioassay experiment compared to M4.Additionally,key protein encoding genes associated to pathogenicity were differentially expressed between mutants and wild type,indicating that these genes may play vital roles in pathogenicity.In conclusion,the genes that are differentially expressed in the three mutants play relevant roles in morphogenesis and development,and pathogenesis in other organisms which are worthwhile of further investigations specific to A.apis.The result of this study provides valuable genetic resources for further investigation on transcriptome variation caused by mutation and the functional validation of candidate pathogenicity genes in A.apis. |
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