Identification of a novel member of the genus Laulavirus (family Phenuiviridae) from the entomopathogenic ascomycete fungus Cordyceps javanica.

The virus family Phenuiviridae (order Hareavirales, comprising segmented negative-sense single stranded RNA viruses) has highly diverse members that are known to infect animals, plants, protozoans, and fungi. In this study, we identified a novel phenuivirus infecting a strain of the entomopathogenic fungus Cordyceps javanica isolated from a small brown plant hopper (Laodelphax striatellus), and this virus was tentatively named "Cordyceps javanica negative-strand RNA virus 1" (CjNRSV1). The CjNRSV1 genome consists of three negative-sense single stranded RNA segments (RNA1-3) with lengths of 7252, 2401, and 1117 nt, respectively. The 3'- and 5'-terminal regions of the RNA1, 2, and 3 segments have identical sequences, and the termini of the RNA segments are complementary to each other, reflecting a common characteristic of viruses in the order Hareavirales. RNA1 encodes a large protein (∼274 kDa) containing a conserved domain for the bunyavirus RNA-dependent RNA polymerase (RdRP) superfamily, with 57-80% identity to the RdRP encoded by phenuiviruses in the genus Laulavirus. RNA2 encodes a protein (∼79 kDa) showing sequence similarity (47-63% identity) to the movement protein (MP, a plant viral cell-to-cell movement protein)-like protein (MP-L) encoded by RNA2 of laulaviruses. RNA3 encodes a protein (∼28 kDa) with a conserved domain of the phenuivirid nucleocapsid protein superfamily. Phylogenetic analysis using the RdRPs of various phenuiviruses and other unclassified phenuiviruses showed CjNRSV1 to be grouped with established members of the genus Laulavirus. Our results suggest that CjNRSV1 is a novel fungus-infecting member of the genus Laulavirus in the family Phenuiviridae.

Identification of a negative-strand RNA virus with natural plant and fungal hosts.

The presence of viruses that spread to both plant and fungal populations in nature has posed intriguingly scientific question. We found a negative-strand RNA virus related to members of the family Phenuiviridae, named Valsa mali negative-strand RNA virus 1 (VmNSRV1), which induced strong hypovirulence and was prevalent in a population of the phytopathogenic fungus of apple Valsa canker (Valsa mali) infecting apple orchards in the Shaanxi Province of China. Intriguingly, VmNSRV1 encodes a protein with a viral cell-to-cell movement function in plant tissue. Mechanical leaf inoculation showed that VmNSRV1 could systemically infect plants. Moreover, VmNSRV1 was detected in 24 out of 139 apple trees tested in orchards in Shaanxi Province. Fungal inoculation experiments showed that VmNSRV1 could be bidirectionally transmitted between apple plants and V. mali, and VmNSRV1 infection in plants reduced the development of fungal lesions on leaves. Additionally, the nucleocapsid protein encoded by VmNSRV1 is associated with and rearranged lipid droplets in both fungal and plant cells. VmNSRV1 represents a virus that has adapted and spread to both plant and fungal hosts and shuttles between these two organisms in nature (phyto-mycovirus) and is potential to be utilized for the biocontrol method against plant fungal diseases. This finding presents further insights into the virus evolution and adaptation encompassing both plant and fungal hosts.

Similar Characteristics of siRNAs of Plant Viruses Which Replicate in Plant and Fungal Hosts.

RNA silencing is a host innate antiviral mechanism which acts via the synthesis of viral-derived small interfering RNAs (vsiRNAs). We have previously reported the infection of phytopathogenic fungi by plant viruses such as cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Furthermore, fungal RNA silencing was shown to suppress plant virus accumulation, but the characteristics of plant vsiRNAs associated with the antiviral response in this nonconventional host remain unknown. Using high-throughput sequencing, we characterized vsiRNA profiles in two plant RNA virus-fungal host pathosystems: CMV infection in phytopathogenic fungus Rhizoctonia solani and TMV infection in phytopathogenic fungus Fusarium graminearum. The relative abundances of CMV and TMV siRNAs in the respective fungal hosts were much lower than those in the respective experimental plant hosts, Nicotiana benthamiana and Nicotiana tabacum. However, CMV and TMV siRNAs in fungi had similar characteristics to those in plants, particularly in their size distributions, proportion of plus and minus senses, and nucleotide preference for the 5' termini of vsiRNAs. The abundance of TMV siRNAs largely decreased in F. graminearum mutants with a deletion in either dicer-like 1 (dcl1) or dcl2 genes which encode key proteins for the production of siRNAs and antiviral responses. However, deletion of both dcl1 and dcl2 restored TMV siRNA accumulation in F. graminearum, indicating the production of dcl-independent siRNAs with no antiviral function in the absence of the dcl1 and dcl2 genes. Our results suggest that fungal RNA silencing recognizes and processes the invading plant RNA virus genome in a similar way as in plants.

Long-Term Preservation of Plant Viruses in Cryopreserved Shoot Tips.

Availability of the methods for long-term virus preservation facilitates easy acquirement of viruses, which are needed in many basic and applied virological studies. Cryopreservation is currently considered an ideal means for long-term preservation of plant germplasm. Recent studies have shown that cryopreservation provided an efficient and reliable method for long-term preservation of plant viruses. Here, we describe the detailed procedures of droplet vitrification for long-term preservation of apple stem grooving virus (ASGV), which represents a type of viruses that can invade meristematic cells of the shoot tips, and potato leafroll virus (PLRV), which is a phloem-limited virus that does not infect the apical meristem. Shoot tip cryopreservation provides an advantageous strategy for the long-term preservation of plant viruses.

Facilitative and synergistic interactions between fungal and plant viruses.

Plants and fungi are closely associated through parasitic or symbiotic relationships in which bidirectional exchanges of cellular contents occur. Recently, a plant virus was shown to be transmitted from a plant to a fungus, but it is unknown whether fungal viruses can also cross host barriers and spread to plants. In this study, we investigated the infectivity of Cryphonectria hypovirus 1 (CHV1, family Hypoviridae), a capsidless, positive-sense (+), single-stranded RNA (ssRNA) fungal virus in a model plant, Nicotiana tabacum CHV1 replicated in mechanically inoculated leaves but did not spread systemically, but coinoculation with an unrelated plant (+)ssRNA virus, tobacco mosaic virus (TMV, family Virgaviridae), or other plant RNA viruses, enabled CHV1 to systemically infect the plant. Likewise, CHV1 systemically infected transgenic plants expressing the TMV movement protein, and coinfection with TMV further enhanced CHV1 accumulation in these plants. Conversely, CHV1 infection increased TMV accumulation when TMV was introduced into a plant pathogenic fungus, Fusarium graminearum In the in planta F. graminearum inoculation experiment, we demonstrated that TMV infection of either the plant or the fungus enabled the horizontal transfer of CHV1 from the fungus to the plant, whereas CHV1 infection enhanced fungal acquisition of TMV. Our results demonstrate two-way facilitative interactions between the plant and fungal viruses that promote cross-kingdom virus infections and suggest the presence of plant-fungal-mediated routes for dissemination of fungal and plant viruses in nature.

Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata.

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Symptomatic plant viroid infections in phytopathogenic fungi.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.