Fujiang Wen

Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system

In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage λ-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.

GhWRKY25 , a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana

WRKY transcription factors are involved in various processes, ranging from plant growth to abiotic and biotic stress responses. Group I WRKY members have been rarely reported compared with group II or III members, particularly in cotton (Gossypium hirsutum). In this study, a group I WRKY gene, namely, GhWRKY25, was cloned from cotton and characterized. Expression analysis revealed that GhWRKY25 can be induced or deduced by the treatments of abiotic stresses and multiple defense-related signaling molecules. Overexpression of GhWRKY25 in Nicotiana benthamiana reduced plant tolerance to drought stress but enhanced tolerance to salt stress. Moreover, more MDA and ROS accumulated in transgenic plants after drought treatment with lower activities of SOD, POD, and CAT. Our study further demonstrated that GhWRKY25 overexpression in plants enhanced sensitivity to the fungal pathogen Botrytis cinerea by reducing the expression of SA or ET signaling related genes and inducing the expression of genes involved in the JA signaling pathway. These results indicated that GhWRKY25 plays negative or positive roles in response to abiotic stresses, and the reduced pathogen resistance may be related to the crosstalk of the SA and JA/ET signaling pathways.

Production of transgenic rice new germplasm with strong resistance against two isolations of Rice stripe virus by RNA interference

Rice stripe disease, with the pathogen Rice stripe virus (RSV), is one of the most widespread and severe virus diseases. Cultivating a resistant breed is an essential and efficient method in preventing rice stripe disease. Following RNA interference (RNAi) theory, we constructed three RNAi binary vectors based on coat protein (CP), special-disease protein (SP) and chimeric CP/SP gene sequence. Transgenic lines of rice cv. Yujing6 were generated through Agrobacterium-mediated transformation. We inoculated T1 generation plants from each line derived from CP/SP, CP, and SP transgenic rice plants with two RSV isolates from Shandong Province and Jiangsu Province using viruliferous vector insects. In these resistance assays, chimeric CP/SP RNAi lines showed stronger resistance against two isolates than CP or SP single RNAi lines. Stable integration and expression of RNAi transgenes were confirmed by Southern and northern blot analysis of independent transgenic lines. In the resistant transgenic lines, lower levels of transgene transcripts and specific short interference RNAs were observed relative to the susceptible transgenic plant, which showed that virus resistance was increased by RNAi. Genetic analysis demonstrated that transgene and virus resistance was stably inherited in the T2 progeny plants.

Bacterially Expressed Double-Stranded RNAs against Hot-Spot Sequences of Tobacco Mosaic Virus or Potato Virus Y Genome Have Different Ability to Protect Tobacco from Viral Infection

Posttranscriptional gene silencing, also known as RNA interference, involves degradation of homologous mRNA sequences in organisms. In plants, posttranscriptional gene silencing is part of a defense mechanism against virus infection, and double-stranded RNA is the pivotal factor that induces gene silencing. In this paper, we got seven hairpin RNAs (hpRNAs) constructs against different hot-spot sequences of Tobacco mosaic virus (TMV) or Potato virus Y (PVY) genome. After expression in Escherichia coli HT115, we extracted the seven hpRNAs for the test in tobacco against TMV or PVY infection. The data suggest that different hpRNAs against different hot-spot sequences of TMV or PVY genome had different ability to protect tobacco plants from viral infection. The resistance to TMV conferred by the hpRNA against the TMV movement protein was stronger than other TMV hpRNAs; the resistance to PVY conferred by the hpRNA against the PVY nuclear inclusion b was better than that induced by any other PVY hpRNAs. Northern blotting of siRNA showed that the resistance was indeed an RNA-mediated virus resistance.

Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY

In this study, 16 hairpin RNA (hpRNA) vectors were constructed, each harboring 50 bp viral RNA sequence as the stem. They all targeted the coat protein (CP) gene of Potato virus Y (PVY). Virus resistance assay revealed that hairpin constructs targeting the anterior 200 bp regions of the CP gene were unable to induce virus resistance, while the 12 hpRNA constructs targeting posterior 600 bp regions induced high virus resistance up to 77.78 %. Northern blot analysis revealed that 50 bp-length hpRNA constructs could be transcribed efficiently and processed into siRNAs; however, no correlation between siRNA accumulation and degree of antiviral defense was observed. Results presented here indicated that the middle and 3′ end of the CP cDNA was important for hpRNA-mediated PVY resistance, improving the design of pathogen-derived hpRNA expression cassettes for transgenic plant against viruses.

Conserved Sequences of Replicase Gene-Mediated Resistance to Potyvirus through RNA Silencing

Nuclear inclusion protein b (NIb) genes of three Potato virus Y isolates PVY-SD1 (O strain), PVY-SD4 (N strain), PVY-SD5 (NTN strain), and Tobacco etch virus isolate TEV-SD1 in Shandong Province were cloned and sequenced. Sequence analysis showed that the sequence homology of the entire NIb gene among the four viruses ranged from 65% to 95%. Hairpin RNA (hpRNA) constructs were designed based on five conserved regions derived from PVY-SD1 and introduced into tobacco plants. After asexual propagation, the transgenic plants were analyzed for resistance to PVY-SD1, PVY-SD4, PVY-SD5, and TEV-SD1. We obtained resistance ratios of 26.2%, 22.7%, 36.4%, 20.3%, and 21.7% to PVY-SD1. When inoculated with the PVY-SD5 virus, the transgenic plants had resistance ratios ranging from 2.4% to 15.9%, but no resistance at all to the other viruses, PVY-SD4 and TEV-SD1. No correlation was found between resistance of transgenic plants and the transgene copy numbers. Northern blot and small interfering RNA (siRNA) analysis demonstrated that the resistance was attributable to RNA silencing. Genetic analysis demonstrated that virus resistance was stably inherited in progeny T1 and T2. These results indicate that the siRNA molecules against conserved regions can confer virus resistance but are restricted to viruses with more than 90% sequence homology.

Manipulation of Auxin Response Factor 19 affects seed size in the woody perennial Jatropha curcas

Seed size is a major determinant of seed yield but few is known about the genetics controlling of seed size in plants. Phytohormones cytokinin and brassinosteroid were known to be involved in the regulation of herbaceous plant seed development. Here we identified a homolog of Auxin Response Factor 19 (JcARF19) from a woody plant Jatropha curcas and genetically demonstrated its functions in controlling seed size and seed yield. Through Virus Induced Gene Silencing (VIGS), we found that JcARF19 was a positive upstream modulator in auxin signaling and may control plant organ size in J. curcas. Importantly, transgenic overexpression of JcARF19 significantly increased seed size and seed yield in plants Arabidopsis thaliana and J. curcas, indicating the importance of auxin pathway in seed yield controlling in dicot plants. Transcripts analysis indicated that ectopic expression of JcARF19 in J. curcas upregulated auxin responsive genes encoding essential regulators in cell differentiation and cytoskeletal dynamics of seed development. Our data suggested the potential of improving seed traits by precisely engineering auxin signaling in woody perennial plants.

Virus resistance obtained in transgenic tobacco and rice by RNA interference using promoters with distinct activity

To induce virus resistance in tobacco and rice we constructed hairpin RNA expression system harbouring inverted repeat fragments of coat protein cDNA of Potato virus Y (PVY) or Rice stripe virus (RSV). These structures were driven by three promoters [cauliflower mosaic virus 35S (CaMV 35S), polyubiqutin gene of maize (Ubi), and Pharbitis nil leucine zipper gene (PNZIP)] which have different tissue-specific activity. PVY resistance ratios were 65.18, 24.33 and 83.54 % in transgenic tobacco plants harboring p35S-PVY, pUbi-PVY and pPNZIP-PVY. RSV resistance was 16.21, 28.61 and 29.33 % in transgenic rice plants harboring p35S-RSV, pUbi-RSV and pPNZIP-RSV. Northern blotting and GUS assay demonstrated that virus resistance levels were related to promoter activity. Therefore, choice of the more effective and tissue-specific promoter to reinforce transcription of hpRNAs will favour the cultivation of highly virusresistant transgenic plants.

Viral resistance mediated by shRNA depends on the sequence similarity and mismatched sites between the target sequence and siRNA

Viral resistance can be effectively induced in transgenic plants through their silencing machinery. Thus, we designed nine short hairpin RNAs (shRNA) constructs to target nuclear inclusion protein b (NIb), helper component proteinase (HC-Pro), cylindrical inclusion protein (CI) and viral protein genome linked (VPg) genes of Potato virus Y (PVYN) and Tobacco etch virus (TEV-SD1). The shRNAs were completely complementary to the genes of PVYN, and contained 1–3 nt mismatches to the genes of TEV-SD1. To study the specificity of gene silencing in shRNA-mediated viral resistance, the constructs were introduced into tobacco plants. The results of viral resistance assay revealed that these nine kinds of transgenic tobacco plants can effectively induce viral resistance against both PVYN and TEV-SD1, and the shRNA construct targeting the NIb gene showed higher silencing efficiency. Northern blot and short interfering RNA (siRNA) analyses demonstrated that the viral resistance can be attributed to the degradation of the target RNA through the RNA silencing system. Correlation analysis of siRNA sequence characteristics with its activity suggested that the secondary structure stability of the antisense strand did not influence siRNA activity; 1 to 3 nt 5’ end of the sense strand caused a significant effect on siRNA activity where the first base such as U was favourable for silencing; the base mismatch between the siRNA and the target gene may be more tolerated in the 5’ end.

Different target genes and chimeric-gene hairpin structures affect virus resistance mediated by RNA silencing in transgenic tobacco

Two types of hairpin RNA (hpRNA) constructions were designed using a chimeric gene formed from two genes, the coat protein (CP) gene or the silencing suppressor gene, from the Cucumber mosaic virus (CMV) and the Potato virus Y (PVYN), respectively; one type generated a single hairpin structure, whereas the other formed a two-hairpin structure. Four constructs, pDCPSH (double CP gene fragments, single hairpin), pDCPDH (double CP gene fragments, double hairpins), pHC2bSH (two silencing suppressor gene fragments, single hairpin), and pHC2bDH (two silencing suppressor gene fragments, double hairpins), were individually introduced into tobacco plants. A transcript analysis demonstrates that the small interference RNA (siRNA) processing efficiency was greater with the double-hairpin construct than with the single-hairpin construct, although the expression of their target genes were similar. A viral resistance assay shows that the transgenic tobacco plants effectively resisted a mixed infection of CMV and Potato virus Y (PVYN) and that pDCPDH exhibited the highest silencing efficiency. The accumulation of siRNA in the inoculated transgenic plants expressing different hairpin structures was similar. A genetic analysis reveals that viral resistance in the transgenic plants was stably inherited from the T0 to T1 generation. A transcript analysis and a viral resistance assay indicate that the double-hairpin structure of the same target sequences tended to produce more siRNA before the virus inoculation and thus strengthened RNA-mediated viral resistance.