The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.

Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

Genetic diversity and population structure of beet necrotic yellow vein virus in China.

Beet necrotic yellow vein virus (BNYVV) is a serious threat to the sugar beet industry worldwide. However, little information is available regarding the genetic diversity and population structure of BNYVV in China. Here, we analyzed multiple sequences from four genomic regions (CP, RNA3, RNA4 and RNA5) of a set of Chinese isolates. Sequence analyses revealed that several isolates were mixed infections of variants with different genotypes and/or different p25 tetrad motifs. In total, 12 distinct p25 tetrads were found in the Chinese BNYVV population, of which four tetrads were newly identified. Phylogenetic analyses based on four genes (CP, RNA3-p25, RNA4-p31 and RNA5-p26) in isolates from around the world revealed the existence of two to four groups, which mostly corresponded to previously reported phylogenetic groups. Two new subgroups and a new group were identified from the Chinese isolates in p25 and p26 trees, respectively. Selection pressure analysis indicated that there was a positive selection pressure on the p25 from the Chinese isolates, but the other three proteins were under a negative selection pressure. There was frequent gene flow between geographically distant populations, which meant that BNYVV populations from different provinces were not geographically differentiated.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Discovery and Characterization of a Novel Carlavirus Infecting Potatoes in China.

A new carlavirus, tentatively named Potato virus H (PVH), was found on potato plants with mild symptoms in Hohhot, Inner Mongolia Autonomous Region, China. PVH was confirmed by genome sequencing, serological reactions, electron microscopy, and host index assays. The PVH particles were filamentous and slightly curved, with a modal length of 570 nm. Complete RNA genomic sequences of two isolates of PVH were determined using reverse transcription-PCR (RT-PCR) and the 5' rapid amplification of cDNA ends (5' RACE) method. Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus, with a positive-sense single-stranded genome of 8410 nt. It shared coat protein (CP) and replicase amino acid sequence identities of 17.9-56.7% with those of reported carlaviruses. Phylogenetic analyses based on the protein-coding sequences of replicase and CP showed that PVH formed a distinct branch, which was related only distantly to other carlaviruses. Western blotting assays showed that PVH was not related serologically to other potato carlaviruses (Potato virus S, Potato virus M, and Potato latent virus). PVH systemically infected Nicotianaglutinosa but not Nicotiana tabacum, Nicotianabenthamiana, or Chenopodiumquinoa, which is in contrast with the other potato carlaviruses. These results support the classification of PVH as a novel species in the genus Carlavirus. Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.

A novel strain of Beet western yellows virus infecting sugar beet with two distinct genotypes differing in the 5'-terminal half of genome.

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ(A) and BWYV-BJ(B) (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ(A) and BWYV-BJ(B) were 5674 and 5626nt in length, respectively. BWYV-BJ(B) was 48nt shorter than BWYV-BJ(A) in the regions 1589-1615 and 1629-1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ(A) and BWYV-BJ(B) shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3-88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3-99.5%). The complete nucleotide sequences of BWYV-BJ(A) and BWYV-BJ(B) were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ(A) and BWYV-BJ(B) shared the highest homology with BWYV-US (nucleotide identity 91.2-93.3, 86.7-89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ(A), BWYV-BJ(B) were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ(A) and BWYV-BJ(B) might be recombinant viruses.

Molecular characterization of two Chinese isolates of Beet western yellows virus infecting sugar beet.

Beet western yellows virus (BWYV) has previously been reported as an agent of sugar beet yellowing disease in China. In this article, the complete genomic RNA sequences of two Chinese BWYV isolates infecting beet from Inner Mongolia (BWYV-IM) and Gansu (BWYV-GS) were determined and compared with three beet poleroviruses (BMYV, BChV and BWYV-US) and other non-beet-infecting poleroviruses. The genomes of the two isolates were 5,668 nt in length, and had almost the same genomic organization and characteristics as BWYV-US. The full length of BWYV-IM shared nucleotide sequence identities of 97.4, 86.6, 64.4 and 70.8% with BWYV-GS, BWYV-US, BChV and BMYV, respectively. Further sequence analysis indicated that the Chinese BWYV isolates were more closely related to BWYV-US; however, the identity of any gene product between the Chinese isolates and BWYV-US was <90%. Therefore, on the basis of genome sequence, we propose that these Chinese isolates are a distinct strain of BWYV that infect sugar beet. In addition, recombinant detection analysis revealed that BWYV-IM might be a recombinant virus.