Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia

Abstract

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Keywords: Animalia; Plantae; cross-kingdom infection; fungal virus; partitivirus.

Fig. 1.

Replication of fungal partitiviruses in N. benthamiana protoplasts. (A) Experimental procedure. See the Materials and Methods section for the details of viruses, original fungal host strains, experimental host organisms, and methodologies utilized. (B and C) Relative quantification of RnPV2 and RnPV11 (B) and RnPV18 and RnPV19 (C) viral RNA by RT-qPCR. Virus contents at different time points posttransfection were quantitatively compared; the value at 0 hpt was arbitrarily assigned as 1. The data are presented as the fold change of viral RNA accumulation. (D and E) Agarose gel electrophoretic analysis of total RNA extracted from transfected N. benthamiana protoplasts with RnPV11 (D) and RnPV18 and RnPV19 (E). Total RNA fractions obtained at different time points were electrophoresed in 1.3% agarose gel using 1× TAE buffer. Only RnPV18 and RnPV11 dsRNA are visible in the gel. Note that the two dsRNA genomic segments (dsRNA1 and dsRNA2) of RnPV11, RnPV18, and RnPV19 comigrate in agarose gel. A size standard (1-kb DNA ladder) was electrophoresed in parallel (lane M). VF refers to the virus-free sample. Mean values and SEs were calculated from three biological replicates, each with three technical replicates, in this and subsequent figures. hpt refers to hours posttransfection.

Fig. 2.

Replication of RnPV18 in carrot calluses and plants (A) Procedure for the regeneration of carrot plants from protoplasts transfected by RnPV18 and RnPV19. (B) Agarose gel electrophoresis of RT-PCR products derived from RnPV18 in different carrot calluses. The numbers circled with red are positive for the RnPV18 infection. (C) RT-PCR analysis of systemic infection of different carrot plant tissues by RnPV18. Two carrot plants (Virus free and Carrot 12-3C), derived from a virus-free callus and RnPV18-infected callus (No. 12), respectively, were subjected to the analysis. L1–L4 denote leaf samples, and R1 denotes a root sample. Total RNA fractions obtained from fungal strain W442 (Rn W442 RNA) and plant or callus line derived from untransfected carrot protoplasts (NTC) served as a positive and a negative control. (D and E) Electron micrographs of RnPV18 particles purified from carrot callus (D) and plant leaves (E). The samples were examined using a Hitachi electron microscope model H-7650. The white arrows point to typical virus-like particles.

Fig. 3.

Replication of RnPV18 and RnPV19 RNA in Sf9 and S2 insect cells. (A and B) Relative quantification of RnPV18 and RnPV19 RNA in Sf9 (A) and S2 (B) cells. RT-qPCR was performed as described in the Fig. 1 legend at different time points posttransfection (0, 24, and 96 hpt). (C and D) Northern blotting of RnPV18 (Upper rows in C and D) and RnPV19 mRNA (Lower rows in C and D) in army worm Sf9 (C) and fruit fly S2 (D) cells. Equal amounts of single-stranded RNA fractions (20 µg) from insect cells were subjected to northern blotting. DIG-labeled probes specific to RnPV18 and RnPV19 RdRP encoding dsRNA1 were used. W97 is a virus-free R. necatrix strain (VF), while W442 is the natural host of RnPV18 and RnPV19, for which a much smaller amount (1 µg) of RNA was probed.

Fig. 4.

Properties of RnPV18- and RnPV19-derived small RNAs in three different host organisms. (A and B) Size distribution of RnPV18 dsRNA1-(A) and RnPV19 dsRNA1-derived siRNAs (B) produced in plant, fungal, and insect cells. Comparison of siRNAs (sense and antisense, 15 to 32 nucleotides) derived from the RdRP-coding dsRNA1 genomic segments in N. benthamiana cells (Top row), insect S2 cells (Middle row), and the original host R. necatrix W442 mycelia (Bottom row) in each panel. N. benthamiana and fruit fly S2 cells were harvested at 3 d posttransfection for high through-put small RNA sequencing, while R. necatrix W442 mycelia were collected after culturing in potato dextrose broth for 1 wk. Read numbers were normalized against one million small RNA reads. (C and D) Hot spots of RnPV18 dsRNA1-(C) and RnPV19 dsRNA1-derived small RNAs (D) produced in N. benthamiana (Nb), fruit fly S2 (S2) and R. necatrix (Rn) cells. Small RNA reads are mapped to their genomes. The vsiRNA reads were not normalized. (C and F) Ratios in percentage of the 5′-terminal nucleotides of RnPV18 dsRNA1-(E) and RnPV19 dsRNA1-derived siRNAs (F) are shown in graph form. See SI Appendix, Fig. S9, for the properties of the CP-coding genomic segment (dsRNA2).