Project description:A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.
Project description:Amylostereum areolatum is the symbiotic fungus of the Eurasian woodwasp, Sirex noctilio, a globally invasive species. The mutualistic symbiont is associated with the woodwasp, assisting the damage process and providing nutrition for its insect partners. Colonization and growth of A. areolatum have essential impacts on the development and spread of S. noctilio, though the mechanism of interaction between the two has been poorly described. In this study, the first genome of this symbiotic fungus was sequenced, assembled, and annotated. The assembled A. areolatum genome was 57.5 Mb (54.51% GC content) with 15,611 protein-coding genes. We identified 580 carbohydrate-active enzymes (CAZymes), 661 genes associated with pathogen-host interactions, and 318 genes encoding transport proteins in total. The genome annotation revealed 10 terpene/phytoene synthases responsible for terpenoid biosynthesis, which could be classified into three clades. Terpene synthase gene clusters in clade II were conserved well across Russulales. In this cluster, genes encoding mevalonate kinase (MK), EGR12 (COG1557), and nonplant terpene cyclases (cd00687) were the known biosynthesis and regulatory genes. Genome sequence analysis of this fungus would prove the possibility of A. areolatum volatiles affecting the host selection of S. noctilio on a molecular basis. We further clarified that A. areolatum was a strict obligate symbiotic fungus. The wasps might protect the fungus before it was introduced into a suitable host substrate by oviposition, while the fungus would provide S. noctilio with a suitable environment and nutrients for the larval growth. These results would lay a foundation for our understanding of the mechanism of this entomogenous symbiosis.IMPORTANCESirex noctilio (F.), together with Amylostereum areolatum, a wood-decaying symbiotic fungus, causes severe damage to Pinus species worldwide. In China, it causes extensive death of Mongolian pine (Pinus sylvestris var. mongolica). There is an obligate dependency mutualism between the woodwasp and its fungus. Studies have suggested that the fungal growth rate affected the size of the wasps: larger adults emerged from sites with a higher fungus growth rate. This genome is the first reported genome sequence of a woodwasp symbiotic fungus. Genome sequence analysis of this fungus would prove the possibility of A. areolatum volatiles affecting the host selection of S. noctilio on a molecular basis. We further clarified that A. areolatum was a strict obligate symbiotic fungus and that it would provide S. noctilio with a suitable environment and with nutrients for the larval growth. These results would lay a foundation for our understanding of the mechanism of this entomogenous symbiosis.