The coat protein of the ilarvirus prunus necrotic ringspot virus mediates long-distance movement.

The coat protein (CP) of plant viruses generally has multiple functions involving infection, replication, movement and pathogenicity. Functions of the CP of prunus necrotic ringspot virus (PNRSV), the causal agent of several threatening diseases of Prunus fruit trees, are poorly studied. Previously, we identified a novel virus in apple, apple necrotic mosaic virus (ApNMV), which is phylogenetically related to PNRSV and probably associated with apple mosaic disease in China. Full-length cDNA clones of PNRSV and ApNMV were constructed, and both are infectious in cucumber (Cucumis sativus L.), an experimental host. PNRSV exhibited higher systemic infection efficiency with more severe symptoms than ApNMV. Reassortment analysis of genomic RNA segments 1-3 found that RNA3 of PNRSV could enhance the long-distance movement of an ApNMV chimaera in cucumber, indicating the association of RNA3 of PNRSV with viral long-distance movement. Deletion mutagenesis of the PNRSV CP showed that the basic motif from amino acids 38 to 47 was crucial for the CP to maintain the systemic movement of PNRSV. Moreover, we found that arginine residues 41, 43 and 47 codetermine viral long-distance movement. The findings demonstrate that the CP of PNRSV is required for long-distance movement in cucumber, which expands the functions of ilarvirus CPs in systemic infection. For the first time, we identified involvement of Ilarvirus CP protein during long-distance movement.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR.

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

Identification of Silencing Suppressor Protein Encoded by Strawberry Mottle Virus.

Strawberry mottle virus (SMoV) is associated with strawberry decline disease, causing losses to fruit yield and quality. In this study, using a screening system that enables detection of both local and systemic plant host (RNA silencing) defense responses, we found that Pro2Glu and P28, encoded by SMoV RNA2 genome, functioned to suppress local and systemic RNA silencing triggered by single- but not double-stranded GFP RNA. Subcellular localization assay revealed that both Pro2Glu and P28 were localized to nucleus and cytoplasm. The deletion of 11 amino acid residues at the C-terminus destabilized Pro2Glu protein, and the disruption of two conserved GW motifs deprived Pro2Glu of ability to suppress RNA silencing. Additionally, SMoV Pro2Glu and P28 enhanced the accumulation of potato virus X (PVX) in Nicotiana benthamiana 22 days post-infiltration, and P28 exacerbated significantly the symptoms of PVX. Collectively, these data indicate that the genome of SMoV RNA2 encodes two suppressors of RNA silencing. This is the first identification of a stramovirus suppressor of RNA silencing.

The GATA factor HANABA TARANU promotes runner formation by regulating axillary bud initiation and outgrowth in cultivated strawberry.

A runner, as an elongated branch, develops from the axillary bud (AXB) in the leaf axil and is crucial for the clonal propagation of cultivated strawberry (Fragaria × ananassa Duch.). Runner formation occurs in at least two steps: AXB initiation and AXB outgrowth. HANABA TARANU (HAN ) encodes a GATA transcription factor that affects AXB initiation in Arabidopsis and promotes branching in grass species, but the underlying mechanism is largely unknown. Here, the function of a strawberry HAN homolog FaHAN in runner formation was characterized. FaHAN transcripts can be detected in the leaf axils. Overexpression (OE) of FaHAN increased the number of runners, mainly by enhancing AXB outgrowth, in strawberry. The expression of the strawberry homolog of BRANCHED1 , a key inhibitor of AXB outgrowth in many plant species, was significantly downregulated in the AXBs of FaHAN -OE lines, whereas the expression of the strawberry homolog of SHOOT MERISTEMLESS, a marker gene for AXB initiation in Arabidopsis, was upregulated. Moreover, several genes of gibberellin biosynthesis and cytokinin signaling pathways were activated, whereas the auxin response pathway genes were repressed. Further assays indicated that FaHAN could be directly activated by FaNAC2, the overexpression of which in strawberry also increased the number of runners. The silencing of FaNAC2 or FaHAN inhibited AXB initiation and led to a higher proportion of dormant AXBs, confirming their roles in the control of runner formation. Taken together, our results revealed a FaNAC2-FaHAN pathway in the control of runner formation and have provided a means to enhance the vegetative propagation of cultivated strawberry.

Identification and molecular characterization of a novel carlavirus infecting rose plants (Rosa chinensis Jacq.).

In the present study, the genome sequence of a potential novel virus, tentatively named "rose virus C" (RVC), was mined from publically available transcriptomic data from a Rosa chinensis plant. The complete genome sequence of RVC consists of 8,386 nt, excluding a 3' poly(A) tail, and contains five ORFs. Phylogenetic analysis showed that RVC clustered with members of the genus Carlavirus, family Betaflexiviridae. The replicase gene had 48.8-52.1% nt sequence identity to those of other carlaviruses, while the CP gene had 40.4-45.9% nt sequence identity, which is far below the species demarcation cutoff of 72%. The incidence of RVC in rose plants was low (5.4%). Overall, our data suggest that RVC is a novel atypical virus of the genus Carlavirus.

Molecular characterization of rose spring dwarf-associated virus isolated from China rose (Rosa chinensis Jacq.) in China.

China rose (Rosa chinensis Jacq.) is an important ornamental plant grown widely in China. In May 2019, we sampled and analyzed a China rose plant by high-throughput sequencing using small RNAs. A luteovirus, rose spring dwarf-associated virus (RSDaV), was detected in this plant, and its complete nucleotide sequence of 5816 nucleotides was determined. The China rose isolate of RSDaV contains five major open reading frames (ORFs) and three putative small ORFs, typical of members of the genus Luteovirus. It shares 94.4% nt sequence identity with the Californian (USA) isolate of the virus. Genomic analysis revealed a deletion of a single U at nt position 5295, which introduced a frameshift mutation, and an insertion of nine nucleotides (AUAAAUGAU) at position 5706-5714, which did not change the reading frame. The aa sequence in that portion of the protein was 90.5% identical to that of the Californian isolate. This is the first report on the occurrence of RSDaV infecting rose plants in China.

The occurrence of strawberry virus 1 infecting strawberry in Shandong province, China.

Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry associated virus 1 (SaV1) is a putative Cytorhabdovirus isolated from strawberry in Fujian province, China (Ding et al., 2019). Strawberry virus 1 (StrV-1) is another putative Cytorhabdovirus characterized from F. ananassa and F. vesca in Czech Republic (Fránová et al., 2019). The complete genomes of isolates of SaV1 and StrV-1 share 79 to 98% nucleotide (nt) identities. In August 2020, foliar chlorotic spots or streaks were observed in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star) in Yantai, Shandong province, China. To identify the associated viruses, symptomatic leaves from two plants of each cultivar (8 samples) were pooled for high-throughput sequencing (HTS). Total RNA was extracted from the composite sample and used for constructing a cDNA library after ribosomal RNA (rRNA)-depletion. Sequencing was carried out on Illumina Hiseq 4000 (Novogene, China). Raw reads were filtered, trimmed and de novo assembled as described previously (Grabherr et al., 2013; Zhou et al. 2020). The resulting contigs were screened by BLASTn and BLASTx against GenBank database. Subsequent analyses indicated the presence of strawberry vein banding virus, strawberry pallidosis associated virus and strawberry mottle virus in the analyzed sample, which had been reported previously in strawberry (Martin and Tzanetakis, 2013; Shi et al., 2018; Bhagwat et al., 2016). Besides, five contigs ranging from 266 to 6,057 nt were obtained. They shared 87 to 91% nt sequence identity with StrV-1 isolate B (GenBank accession no. MK211271). To confirm StrV-1 infection in the strawberry plants, total RNA was isolated from all eight samples using RNAprep Pure Plant Plus Kit (Tiangen, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers StrVp1 (Forward: 5'-CATTACTGAAGCATTCCGTG-3'/Reverse: 5'-AGATATCACGCACAGTGAC-3'), and StrVp2 (Forward: 5'-TTGCGCGAAGCGGATGTCCG-3'/Reverse: 5'-GGCTGCCAGAGCGTTGGATG-3'), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with the expected sizes were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited in GenBank under accession no. MW419123-124 and MW645247-248. Both protein encoding sequences shared 91 to 92% and 80 to 84% nt identities with the corresponding sequences of StrV-1 isolate B and SaV1, respectively, indicating that the isolates from this study are genetic variants of StrV-1 and distantly related to SaV1. Crude sap was prepared by homogenizing leaf tissues of StrV-1 infected strawberry in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy Nicotiana benthamiana plants. Neither the inoculated leaves nor the systemically infected leaves showed obvious symptoms seven days post inoculation. However, StrV-1 was detected by RT-PCR in all five N. benthamiana plants as described above. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in a 667 m2 greenhouse in Yantai had StrV-1-like symptoms. To the best of our knowledge, this is the first report of the occurrence of StrV-1 infecting strawberry in Shandong province, China. Our findings expand the geographic range and genetic diversity of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.

Molecular characterization and pathogenicity analysis of prunus necrotic ringspot virus isolates from China rose (Rosa chinensis Jacq.).

Prunus necrotic ringspot virus (PNRSV) is a viral pathogen with worldwide distribution, infecting many commercial fruit trees and ornamental plants. So far, the correlation between PNRSV infection and China rose mosaic disease has not been studied. Rose mosaic disease is characterized by severe symptoms, including mosaic, line pattern, and ringspot. Six viruses that were potentially associated with mosaic disease, including PNRSV, were tested in China roses. Only PNRSV was detected in China roses showing mosaic disease, and asymptomatic samples tested negative for this virus. This result was confirmed by small RNA sequencing, but rose leaf rosette-associated virus and rose spring dwarf-associated virus were also identified in both samples with mosaic disease and asymptomatic samples. This implied that PNRSV might be associated with China rose mosaic disease. Full genome sequences of two PNRSV isolates were determined, and the RNA1, 2 and 3 segments were found to be 3,332, 2,594 and 1,951 nucleotides (nt) in length, respectively. The three RNA segments shared 88.7-89.1% nt sequence identity in the 3'UTR, while RNA2 and RNA3 shared 98.2-99.4% identity. The higher variability in RNA1 suggests that it might have been under greater selection pressure. Phylogenetic analysis showed that the two PNRSV isolates clustered in group PV-32. Full-length infectious cDNA clones of PNRSV from China rose were constructed and used to agroinfiltrate cucumber seedlings. The inoculated cucumber leaves showed yellowing, chlorotic spots, necrosis, dwarfing, and decline at 23 to 39 days post-inoculation, demonstrating the virulence of the PNRSV isolate from China rose. These data lay a foundation for determining the molecular mechanism of rose mosaic disease caused by PNRSV.

RNA-Seq Reveals Hawthorn Tree as a New Natural Host for Apple Necrotic Mosaic Virus, Possibly Associated with Hawthorn Mosaic Disease.

Apple mosaic disease is widespread in the major apple-producing areas in China and is frequently associated with the presence of the newly identified Apple necrotic mosaic virus (ApNMV), belonging to subgroup 3 of Ilarvirus genus in the family of Bromoviridae. Mosaic symptoms were also observed in a hawthorn tree. Deep sequencing revealed the hawthorn tree with mosaic symptom was infected by ApNMV, which was confirmed by RT-PCR. The complete nucleotide sequences of RNA1 (3,378 nt), RNA2 (2,778 nt), and RNA3 (1,917 nt) of ApNMV from the hawthorn were obtained, sharing 93.8 to 96.8%, 89.7 to 96.1%, and 89.8 to 94.6% nucleotide identities with those from apples and crabapples, respectively. Two hypervariable regions were found, which showed 59.2 to 85.7% and 64.0 to 89.3% sequence identities at position 142 to 198 aa and at position 780 to 864 aa in the POL protein, respectively, between the hawthorn isolate and other isolates (apple, crabapple). A grafting test demonstrated that ApNMV was easily transmissible from hawthorns to apple trees, with severe chlorosis, yellowing, mosaic, curling, and necrosis. In addition, a total of 11,685 hawthorn trees were surveyed for the incidence of mosaic disease from five provinces in China, and only six were found showing typical mosaic symptoms. A total of 145 individual trees (six symptomatic, 68 asymptomatic, and 71 other symptoms) were tested for the presence or absence of ApNMV by RT-PCR. Among them, six symptomatic, four asymptomatic, and 10 other symptomatic trees tested positive for ApNMV. Taken together, these results demonstrated that the hawthorn tree was identified as a new natural host for ApNMV with a relatively low frequency (13.8%, 20 out of 145) in the main producing areas, and it was likely to be the causal pathogen of hawthorn mosaic disease.