Genome characterization of a novel narnavirus infecting the plant-pathogenic fungus Ustilaginoidea virens.

Mycoviruses are viruses that infect fungi and oomycetes. They are widespread in all major groups of plant-pathogenic fungi and oomycetes. To date, only the full genome of dsRNA mycoviruses and the contigs of positive-sense single-stranded RNA (+ssRNA) mycoviruses have been reported in Ustilaginoidea virens, which is the notorious causal agent of rice false smut (RFS). Here, we report the molecular characterization of a novel +ssRNA mycovirus, Ustilaginoidea virens narnavirus 4 (UvNV4), isolated from U. virens strain Uv418. UvNV4 has a genome of 3,131 nucleotides (nt) and possesses an open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RdRp) of 1,017 amino acids (aa) sequence with a molecular mass of 116.6 kDa. BLASTp analysis revealed that the RdRp showed 50.34% aa sequence identity to that of the previously described Zhangzhou Narna tick virus 1. Phylogenetic analysis indicated that UvNV4 is closely related to members of the family Narnaviridae. Taken together, these results clearly demonstrate that UvNV4 is a novel +ssRNA virus infecting U. virens.

Characterization of a novel gammapartitivirus infecting the phytopathogenic fungus Pyricularia oryzae.

In this study, we identified a novel double-strand RNA (dsRNA) mycovirus in Pyricularia oryzae, designated "Magnaporthe oryzae partitivirus 4" (MoPV4). The genome of MoPV4 consists of a dsRNA-1 segment encoding an RNA-dependent RNA polymerase (RdRP) and a dsRNA-2 segment encoding a capsid protein (CP). Phylogenetic analysis indicated that MoPV4 belongs to the genus Gammapartitivirus within family Partitiviridae. The particles of MoPV4 are isometric with a diameter of about 32.4 nm. Three-dimensional structure predictions indicated that the RdRP of MoPV4 forms a classical right-handed conformation, while the CP has a reclining-V shape.

Deficiency of ChPks and ChThr1 Inhibited DHN-Melanin Biosynthesis, Disrupted Cell Wall Integrity and Attenuated Pathogenicity in Colletotrichum higginsianum.

Colletotrichum higginsianum is a major pathogen causing anthracnose in Chinese flowering cabbage (Brassica parachinensis), posing a significant threat to the Chinese flowering cabbage industry. The conidia of C. higginsianum germinate and form melanized infection structures called appressoria, which enable penetration of the host plant's epidermal cells. However, the molecular mechanism underlying melanin biosynthesis in C. higginsianum remains poorly understood. In this study, we identified two enzymes related to DHN-melanin biosynthesis in C. higginsianum: ChPks and ChThr1. Our results demonstrate that the expression levels of genes ChPKS and ChTHR1 were significantly up-regulated during hyphal and appressorial melanization processes. Furthermore, knockout of the gene ChPKS resulted in a blocked DHN-melanin biosynthetic pathway in hyphae and appressoria, leading to increased sensitivity of the ChpksΔ mutant to cell-wall-interfering agents as well as decreased turgor pressure and pathogenicity. It should be noted that although the Chthr1Δ mutant still exhibited melanin accumulation in colonies and appressoria, its sensitivity to cell-wall-interfering agents and turgor pressure decreased compared to wild-type strains; however, complete loss of pathogenicity was not observed. In conclusion, our results indicate that DHN-melanin plays an essential role in both pathogenicity and cell wall integrity in C. higginsianum. Specifically, ChPks is crucial for DHN-melanin biosynthesis while deficiency of ChThr1 does not completely blocked melanin production.

Dual Transcriptome Analysis Reveals That ChATG8 Is Required for Fungal Development, Melanization and Pathogenicity during the Interaction between Colletotrichum higginsianum and Arabidopsis thaliana.

Anthracnose disease of cruciferous plants caused by Colletotrichum higginsianum is a serious fungal disease that affects cruciferous crops such as Chinese cabbage, Chinese flowering cabbage, broccoli, mustard plant, as well as the model plant Arabidopsis thaliana. Dual transcriptome analysis is commonly used to identify the potential mechanisms of interaction between host and pathogen. In order to identify differentially expressed genes (DEGs) in both the pathogen and host, the conidia of wild-type (ChWT) and Chatg8 mutant (Chatg8Δ) strains were inoculated onto leaves of A. thaliana, and the infected leaves of A. thaliana at 8, 22, 40, and 60 h post-inoculation (hpi) were subjected to dual RNA-seq analysis. The results showed that comparison of gene expression between the 'ChWT' and 'Chatg8Δ' samples detected 900 DEGs (306 upregulated and 594 down-regulated) at 8 hpi, 692 DEGs (283 upregulated and 409 down-regulated) at 22 hpi, 496 DEGs (220 upregulated and 276 down-regulated) at 40 hpi, and 3159 DEGs (1544 upregulated and 1615 down-regulated) at 60 hpi. GO and KEGG analyses found that the DEGs were mainly involved in fungal development, biosynthesis of secondary metabolites, plant-fungal interactions, and phytohormone signaling. The regulatory network of key genes annotated in the Pathogen-Host Interactions database (PHI-base) and Plant Resistance Genes database (PRGdb), as well as a number of key genes highly correlated with the 8, 22, 40, and 60 hpi, were identified during the infection. Among the key genes, the most significant enrichment was in the gene encoding the trihydroxynaphthalene reductase (THR1) in the melanin biosynthesis pathway. Both Chatg8Δ and Chthr1Δ strains showed varying degrees of reduction of melanin in appressoria and colonies. The pathogenicity of the Chthr1Δ strain was lost. In addition, six DEGs from C. higginsianum and six DEGs from A. thaliana were selected for real-time quantitative PCR (RT-qPCR) to confirm the RNA-seq results. The information gathered from this study enriches the resources available for research into the role of the gene ChATG8 during the infection of A. thaliana by C. higginsianum, such as potential links between melanin biosynthesis and autophagy, and the response of A. thaliana to different fungal strains, thereby providing a theoretical basis for the breeding of cruciferous green leaf vegetable cultivars with resistance to anthracnose disease.

Metatranscriptomic Analysis Reveals Rich Mycoviral Diversity in Three Major Fungal Pathogens of Rice.

In recent years, three major fungal diseases of rice, i.e., rice blast, rice false smut, and rice-sheath blight, have caused serious worldwide rice-yield reductions and are threatening global food security. Mycoviruses are ubiquitous in almost all major groups of filamentous fungi, oomycetes, and yeasts. To reveal the mycoviral diversity in three major fungal pathogens of rice, we performed a metatranscriptomic analysis of 343 strains, representing the three major fungal pathogens of rice, Pyricularia oryzae, Ustilaginoidea virens, and Rhizoctonia solani, sampled in southern China. The analysis identified 682 contigs representing the partial or complete genomes of 68 mycoviruses, with 42 described for the first time. These mycoviruses showed affinity with eight distinct lineages: Botourmiaviridae, Partitiviridae, Totiviridae, Chrysoviridae, Hypoviridae, Mitoviridae, Narnaviridae, and Polymycoviridae. More than half (36/68, 52.9%) of the viral sequences were predicted to be members of the families Narnaviridae and Botourmiaviridae. The members of the family Polymycoviridae were also identified for the first time in the three major fungal pathogens of rice. These findings are of great significance for understanding the diversity, origin, and evolution of, as well as the relationship between, genome structures and functions of mycoviruses in three major fungal pathogens of rice.

Co-infection of Four Novel Mycoviruses from Three Lineages Confers Hypovirulence on Phytopathogenic Fungus Ustilaginoidea virens.

Rice false smut caused by Ustilaginoidea virens has become one of the most important diseases of rice. Mycoviruses are viruses that can infect fungi with the potential to control fungal diseases. However, little is known about the biocontrol role of hypoviruses in U. virens. In this study, we revealed that the hypovirulence-associated U. virens strain Uv325 was co-infected by four novel mycoviruses from three lineages, designated Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8), Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), and Ustilaginoidea virens narnavirus virus 13 (UvNV13), respectively. The U. virens strain co-infected by four mycoviruses showed slower growth rates, reduced conidial yield, and attenuated pigmentation. We demonstrated that UvRV16 was not only the major factor responsible for the hypovirulent phenotype in U. vriens, but also able to prevent U. virens to accumulate more mycotoxin, thereby weakening the inhibitory effects on rice seed germination and seedling growth. Additionally, we indicated that UvRV16 can disrupt the antiviral response of U. virens by suppressing the transcriptional expression of multiple genes involved in autophagy and RNA silencing. In conclusion, our study provided new insights into the biological control of rice false smut.

A peptide derived from the N-terminal of NS2A for the preparation of ZIKV NS2A recognition polyclonal antibody.

Zika virus non-structural protein NS2A participates in viral replication, organization, and budding, as well as escaping host immunity. NS2A also involved in the induction of microcephaly by ZIKV. However, the above studies were mainly performed through NS2A with a tag due to the lack of available antibodies against NS2A. ZIKV NS2A is a multiplex transmembrane protein, which leads to difficulties in the preparation of its recognition antibodies, thus seriously affecting the study of ZIKV NS2A. In this study, we found that a peptide (GSTDHMDHFSLGVLC) derived from the N-terminal of ZIKV NS2A coupled to KLH induced antibodies recognizing ZIKV NS2A in rabbits. The purified polyclonal antibody recognized ZIKV NS2A in ZIKV-infected cells with high efficiency and specificity, as detected by western blot and immunofluorescence assay. Our study has important implications for the preparation of ZIKV NS2A antibodies and the in-depth study of ZIKV NS2A.

Genome Organizations and Functional Analyses of a Novel Gammapartitivirus from Rhizoctonia solani AG-1 IA Strain D122.

Here, we describe a novel double-stranded (ds) RNA mycovirus designated Rhizoctonia solani dsRNA virus 5 (RsRV5) from strain D122 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. The RsRV5 genome consists of two segments of dsRNA (dsRNA-1, 1894 bp and dsRNA-2, 1755 bp), each possessing a single open reading frame (ORF). Sequence alignments and phylogenetic analyses showed that RsRV5 is a new member of the genus Gammapartitivirus in the family Partitiviridae. Transmission electron microscope (TEM) images revealed that RsRV5 has isometric viral particles with a diameter of approximately 20 nm. The mycovirus RsRV5 was successfully removed from strain D122 by using the protoplast regeneration technique, thus resulting in derivative isogenic RsRV5-cured strain D122-P being obtained. RsRV5-cured strain D122-P possessed the traits of accelerated mycelial growth rate, increased sclerotia production and enhanced pathogenicity to rice leaves compared with wild type RsRV5-infection strain D122. Transcriptome analysis showed that three genes were differentially expressed between two isogenic strains, D122 and D122-P. These findings provided new insights into the molecular mechanism of the interaction between RsRV5 and its host, D122 of R. solani AG-1 IA.