Investigating a virus’s host range and cross-infection is important for better understanding the epidemiology and emergence of viruses. Previously, our research group discovered a natural infection of a plant RNA virus, cumber mosaic virus (genus Cucumovirus, family Bromoviridae), in a plant pathogenic basidiomycetous fungus, Rhizoctonia solani, isolated from a potato plant grown in the field. Here, we further extended the study to investigate whether similar cross-infection of plant viruses occurs widely in plant-associated fungi in natural conditions. Various vegetable plants such as spinach, leaf mustard, radish, celery, and other vegetables that showed typical virus-like diseases were collected from the fields in Shandong Province, China. High-throughput sequencing revealed that at least 11 known RNA viruses belonging to different genera, including Potyvirus, Fabavirus, Polerovirus, Waikavirus, and Cucumovirus, along with novel virus candidates belonging to other virus genera, infected or associated with the collected vegetable plants, and most of the leaf samples contained multiple plant viruses. A large number of filamentous fungal strains were isolated from the vegetable leaf samples and subjected to screening for the presence of plant viruses. RT-PCR and Sanger sequencing of the PCR products revealed that among the 169 fungal strains tested, around 50% were carrying plant viruses, and many of the strains harbored multiple plant viruses. The plant viruses detected in the fungal isolates were diverse (10 virus species) and not limited to particular virus genera. However, after prolonged maintenance of the fungal culture in the laboratory, many of the fungal strains have lost the virus. Sequencing of the fungal DNA indicated that most of the fungal strains harboring plant viruses were related to plant pathogenic and/or endophytic fungi belonging to the genera Alternaria, Lecanicillium, and Sarocladium. These observations suggest that the nonpersistent acquisition of plant viruses by fungi may commonly occur in nature. Our findings highlight a possible role for fungi in the life cycle, spread, and evolution of plant viruses.

Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5′ cap structure, and 3′ polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

Cucumber mosaic virus (CMV), the type species of the genus Cucumovirus in the family Bromoviridae, possesses a tripartite, positive-sense RNA genome. CMV is mainly transmitted by more than 80 species of aphids (Gallitelli, 2000) and infects more than 1000 species including a lot of vegetables such as cucumber, tomato and pepper (Roossinck, 2001). Different satellite RNAs (satRNA) associated with CMV have been identified and most of them attenuate symptoms induced by CMV (Simon et al., 2004). Although CMV was reported to infect cabbage in Bejing city of China, the sequences and function of its associated satRNA was undefined. In summer of 2016, celery cabbage samples with severe mosaic symptoms were collected from Tai'an city in Shandong province of China to detect potential CMV and its associated satRNA. CMV was detected from these samples using RT-PCR and western blot. To identify the potential existence of CMV in these samples, total RNAs were extracted from cabbage leaves using Trizol reagent, and then RT-PCR was performed using primers CM-R3-119-F (5-AGGCATGGCTTTCCAAGGTAC-3) and CM-R3-768-R (5-CTGACGGTTTCGTTTGCTCAGC-3), which define a fragment between positions 119-768 of CMV RNA3 (AY429432) . A PCR product of 650 bp was amplified and inserted into pMD18-T (Takara) followed by sequencing two individual clones using universal primers M13F . Through the BLAST analysis on NCBI website, sequences of the 650 bp PCR product shared highest similarity with CMV RNA3. This partial CMV RNA3 sequence from cabbage in Tai'an city was submitted to GenBank obtaining accession number KY646100 as TA-ca isolate. It shared 94.15% identity with RNA3 (D10538) of CMV Fny isolate and 96.92 % identity with RNA3 (AY429432) of CMV Ca isolate . In addition, western blot was performed to detect the coat protein (CP) of CMV. CMV CP with molecular weight of 24 kD can be detected from corresponding diseased cabbage leaves using monoclonal antibody of CMV CP (Du et al., 2013) as specific antibody at 1:8000 dilution. With the same RNA samples, RT-PCR of satCMV was performed using degenerate primers satCMV-int-5F (5-GCGGTGTGGGWTAMCTCC-3, W=A/T, M=C/A) and satCMV-int-3R (5-GCYTCTCCCTGYGTGCG-3, Y=T/C), which were designed based on the alignment of 173 reported satCMV in GenBank. About 220 bp PCR product was detected. The purified fragment was cloned into pMD18-T (Takara) and sequenced using universal primers M13F for two individual clones. Through the BLAST analysis on NCBI website, sequences of 223 bp PCR product shared highest similarity with satCMV. This partial satCMV sequence from cabbage in Tai'an city was submitted to GenBank obtaining accession number KY747451 as TA-ca isolate. It shared 95.52% identity with satCMV China-5 isolate (AJ236905) and 95.07 % identity with satCMV FFQT1-3 isolate (HQ283395). To our knowledge, this is the first report of CMV naturally infecting celery cabbage in Shandong province of China. In addition, this is the first report of CMV-associated satRNA naturally infecting celery cabbage in China. Since satCMV can adjust symptoms caused by CMV, it is necessary to clone the full length genome of satCMV TA-ca isolate and test its effect on symptoms caused by CMV in the near future. It will provide substantial material and data for management of disease caused by CMV.

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a widely used technique for RNA structure analysis using N-methylisatoic anhydride (NMIA) treatment that has been proven to be applicable to different types of RNA templates. In this study, we performed the structural analysis of the viral satellite RNA of cucumber mosaic virus TA-Tb (satCMV TA-Tb) using the SHAPE method. In the preliminary experiment, we optimized the protocol of SHAPE method for analyzing satCMV TA-Tb by determining a suitable quantity of template RNA. This optimization effectively reduced the appearance of a large number of intense bands in the NMIA-untreated sample lane, along with a strong overall background signal that prevented the clear elucidation of the RNA structure. SHAPE analysis indicated the presence of non-structured, single-stranded flexible regions throughout satCMV TA-Tb with prominent flexible stretches located around nucleotide positions 145 to 200. The positions of these flexible regions were particularly consistent with a secondary structure of satCMV TA-Tb predicted by mfold software v.2.3, which consisted of five 5′- and 3′-proximal stem-loops and one internal large multi-branched stem-loop. Sequence alignment and secondary RNA structure prediction of other satCMV sequences that are phylogenetically the same group with satCMV TA-Tb also suggested the presence of 5′- and 3′-proximal stem-loop structures. Our data provide the structural basis for elucidating the mechanism by which satCMV TA-Tb regulates the pathogenicity and replication of its helper virus.