The invasion process of the entomopathogenic fungus Ophiocordyceps sinensis into the larvae of ghost moths (Thitarodes xiaojinensis) using a GFP-labeled strain.

Chinese cordyceps is a well-known and valuable traditional Chinese medicine that forms after Ophiocordyceps sinensis parasitizes ghost moth larvae. The low natural infection rate of O. sinensis limits large-scale artificial cultivation of Chinese cordyceps, and the invasion process is unclear. To investigate the temporal and spatial regulation when O. sinensis enters ghost moths, we constructed an O. sinensis transformant that stably expresses green fluorescent protein (GFP). Inoculating Thitarodes xiaojinensis larvae with a high concentration of GFP-labeled O. sinensis, we observed that O. sinensis conidia could adhere to the host cuticle within 2 days, germinate penetration pegs within 4 days, and produce blastospores in the host hemocoel within 6 days. The reconstructed three-dimensional (3D) structures of the invasion sites showed that penetration pegs germinated directly from O. sinensis conidia at the joining site with the larval cuticle. Differentiated appressoria or hyphae along the host epicuticle are not required for O. sinensis to invade ghost moths. Overall, the specific invasion process of O. sinensis into its host is clarified, and we provided a new perspective on the invasion process of entomopathogenic fungi.

Genome Analysis of a Novel Clade b Betabaculovirus Isolated from the Legume Pest Matsumuraeses phaseoli (Lepidoptera: Tortricidae).

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonellagranulovirus (CpGV) and Cryptophlebia leucotretagranulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps.

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.

A New High-Quality Draft Genome Assembly of the Chinese Cordyceps Ophiocordyceps sinensis.

Ophiocordyceps sinensis (Berk.) is an entomopathogenic fungus endemic to the Qinghai-Tibet Plateau. It parasitizes and mummifies the underground ghost moth larvae, then produces a fruiting body. The fungus-insect complex, called Chinese cordyceps or "DongChongXiaCao," is not only a valuable traditional Chinese medicine, but also a major source of income for numerous Himalayan residents. Here, taking advantage of rapid advances in single-molecule sequencing, we assembled a highly contiguous genome assembly of O. sinensis. The assembly of 23 contigs was ∼110.8 Mb with a N50 length of 18.2 Mb. We used RNA-seq and homologous protein sequences to identify 8,916 protein-coding genes in the IOZ07 assembly. Moreover, 63 secondary metabolite gene clusters were identified in the improved assembly. The improved assembly and genome features described in this study will further inform the evolutionary study and resource utilization of Chinese cordyceps.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.