Jiejun Peng

Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing

RNA interference is a natural defense against viruses in plants. To date, the only viral siRNAs characterized have been those for positive-sense RNA viruses with one or two genome components. Here, we characterized siRNAs derived from rice stripe virus (RSV), a member of the genus Tenuivirus with four genomic RNAs and an ambisense coding strategy. Deep sequencing of small RNAs from infected rice leaves showed that siRNAs were derived almost equally from virion and complementary RNA strands and were mostly 20–22 nucleotides long. Most viral siRNAs were produced within the coding sequences and 5′ termini of the RSV genome. RSV siRNAs had a higher G and lower C content than the viral genome but a strong A/U bias at the first nucleotide and a U bias at the final one, suggesting preferential targeting of such sequences by rice Dicer-like proteins.

Garlic virus X 11-kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus

The subcellular localization of the 11-kDa protein (p11) encoded by ORF3 of Garlic virus X (GarVX; genus Allexivirus, family Alphaflexiviridae) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells of Nicotiana benthamiana. Moreover, the p11-RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17-kDa protein (p17) of garlic interacting with p11 was identified by yeast two-hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells of N. benthamiana, it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11.

Identification and regulation of host genes related to Rice stripe virus symptom production.

Viral infections cause plant chlorosis, stunting, necrosis or other symptoms. The down-regulation of chloroplast-related genes (ChRGs) is assumed to be responsible for chlorosis. We identified the differentially expressed genes (DEGs) in Rice stripe virus (RSV)-infected Nicotiana benthamiana, and examined the contribution of 75 down-regulated DEGs to RSV symptoms by silencing them one by one using Tobacco rattle virus (TRV)-induced gene silencing. Silencing of 11 of the 75 down-regulated DEGs caused plant chlorosis, and nine of the 11 were ChRGs. Silencing of a down-regulated DEG encoding the eukaryotic translation initiation factor 4A (eIF4A) caused leaf-twisting and stunting that were visible on RSV-infected N. benthamiana. A region of RSV RNA4 was complementary to part of eIF4A mRNA and virus-derived small interfering (vsiRNAs) from that region were present in infected N. benthamiana. When expressed as artificial microRNAs, those vsiRNAs could target NbeIF4A mRNA for regulation. We provide experimental evidence supporting the association of ChRGs with chlorosis and show that eIF4A is involved in RSV symptom development. This is also the first report demonstrating that siRNA derived directly from a plant virus can target a host gene for regulation.

Intron retention and 3′-UTR analysis of Arabidopsis Dicer-like 2 transcripts

Arabidopsis thaliana Dicer-like protein 2 (AtDCL2) plays an essential role in the RNA interference pathway. The function of AtDCL2 and other DCLs has been much studied but little has been done to characterize the DCLs transcripts before they are translated into proteins. Here, we investigated AtDCL2 transcripts and showed that all 21 introns of AtDCL2 except intron 9, 18, 20 and 21 could be retained although spliced sequences usually predominated. Intron 10 was more frequently retained and transient expression assays in Nicotiana benthamiana leaves showed that when AG/C at the 3′ splicing site of the intron was changed to AG/G, the intron was more frequently spliced out. Conversely, a high retention of intron 18 was obtained if the AG/G at the 3′ splicing site was changed to AG/C. These results suggest that the sequence at the 3′ splicing site affects the efficiency of intron splicing. The 3′-UTRs of AtDCL2 had lengths between 54 and 154 nts, and the different 3′-UTRs differentially affected the transcriptional levels of fused GFP expressed transiently in N. benthamiana. Further comparisons and mutation experiments suggested that a putative SBF-1 binding site and an AU-rich element in the 3′-UTR both down-regulated expression of the upstream GFP fused to the 3′-UTR. Conversely, a second poly(A) consensus signal sequence in one 3′-UTR up-regulated gene expression. Our results provide insight into the character of AtDCL2 transcripts and demonstrate the potential complexity of factors that affect the frequency and patterns of alternative splicing.

A virus‐based miRNA suppression (VbMS) system for miRNA loss‐of‐function analysis in plants

MicroRNAs (miRNAs) play key roles in plant development and defense against pathogens. To establish the function of individual miRNAs, gain-of-function analysis is usually done by overexpressing a specific miRNA in transgenic plants and has proved very effective. Loss-of-function analysis by the target mimic method is now also increasingly being used. The mimics expressed in the transgenic plants sequester a specific miRNA and lead to changed phenotypes that elucidate miRNA function. However, it takes time to obtain the transgenic plants. To avoid using transgenic plants, we have developed a virus-based miRNA suppression system (VbMS) based on a Tobacco rattle virus vector. The target mimic sequences of miR156, miR319, or miR164 were introduced into the viral genomic RNA, which was then inoculated to Arabidopsis thaliana plants. The resulting phenotypes were consistent with previous reports from transgenic plants, and the expression of targets of the miRNAs was also increased showing that the activity of the miRNAs had been inhibited. VbMS developed here is validated for loss-of-function analysis of miRNA in plants. Moreover, since only simple agroinfiltration rather than transformation is needed, VbMS is suitable for large-scale approaches to miRNA function analysis in plants.

A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor

Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently.

Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections.

Different Virus-Derived siRNAs Profiles between Leaves and Fruits in Cucumber Green Mottle Mosaic Virus-Infected Lagenaria siceraria Plants

RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defense are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of vsiRNAs from leaves and fruits of Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2058) or 22-nt (3996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5′-terminal and 3′-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5′-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.

Altered accumulation of osa-miR171b contributes to rice stripe virus infection by regulating disease symptoms

Reduction of osa-miR171b contributes to rice stripe virus symptoms by regulating its targets, while overexpression attenuates symptoms, providing direct experimental evidence that a plant miRNA affects the development of virus symptoms.

Identification of novel splice variants of the Arabidopsis DCL2 gene.

In Arabidopsis thaliana, Dicer-like protein 2 (DCL2) cleaves double-stranded virus RNA, playing an essential role in the RNA interference pathway. Here, we describe three alternative splicing (AS) forms of AtDCL2: in one, both intron 8 and intron 10 are retained in the mRNA, in second only intron 8 is retained and in the third no intron is retained, but there is a deletion of 56 nucleotides at the end of exon 10. These splicing forms are present in stems and leaves at different development stages. AS was also detected in DCL2 of Brassica rapa, where intron 9, but not intron 8 or intron 10, was retained suggesting that AS may be a common phenomenon in cruciferous plant DCL2s. The retained introns and sequence deletions detected in AtDCL2 changed the reading frame and produced premature terminal codons. The AS forms appeared to be substrates of nonsense-mediated decay of mRNA.