RNA silencing for broad spectrum virus resistance in plants
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A brief outline of the antiviral strategies using RNA silencing pathways with special emphasis on artificial miRNA for broad spectrum virus resistance in plants
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RNA silencing for broad spectrum virus resistance in plants
- 2. Plant virus diseases are key limiting factors causing significant yield loss In India, Green Revolution promoted intensive agricultural practices & reduced varietal diversity Result was emergence of viral diseases in the cultivated crops Introduction
- 3. Crop Disease Yield loss (%) Virus Virus group Cassava Mosaic 18-25 Indian Cassava Mosaic virus Begomovirus Cotton Leaf curl 68-71* Cotton leaf curl virus Begomovirus Groundnut Bud necrosis >80 Groundnut bud necrosis virus Tospovirus Mungbean Yellow mosaic 21-70 Mungbean yellow mosaic virus Begomovirus Blackgram Soybean Pigeonpea Sterility mosaic >80* Pigeonpea sterility mosaic virus Tenuivirus Potato Mosaic 85 Potato virus Y Potyvirus Rice Rice tungro 10 Rice tungro Badna and rice tungro spherical viruses Badnavirus & Waika virus Sunflower Necrosis 12-17 Sunflower necrosis virus Ilaravirus Tomato Leaf Curl 40-100 Tomato leaf curl virus Begomovirus * In epidemic years Dasgupta et al. (2003), Current Scince Viral diseases of crops of India
- 4. Tobacco Mosaic Virus Cassava Mosaic Virus Mungbean yellow mosaic virus Groundnut bud necrosis virus Papaya Ring Spot Virus Dasgupta et al. (2003), Current Scince Viral diseases of crops of India
- 5. Strategies for the management of viral diseases normally include: Control of vector population using insecticides Use of virus-free propagating material Appropriate cultural practices and Use of resistant cultivars But each of the above methods has its own drawback RNA silencing technology provides an impressive tool for engineering resistance to plant viruses Strategies of management
- 6. A collective term Includes several RNA-based inhibition of gene expression at Transcription mRNA stability Translational Discovered in plants as a mechanism of invading nucleic acids •Transgenes •Viruses Those processes share three biochemical features: Formation of dsRNA Processing of dsRNA to small dsRNAs with staggered ends Inhibitory action of a selected sRNA strand (partially or fully complementary RNA or DNA) Act through small (20–26 nt) RNA molecules RNA silencing Central dogma Image source: http://bsj.berkeley.edu/
- 7. sRNAs are produced by RNase III-type enzymes called Dicers Domains: C- terminal dsRNA binding: binds the dsRNA N- terminal RNA helicase: processes long substrates RNase III: cut RNA to produce 2 nt 3’-overhang PAZ (Piwi/Argonaute/Zwille): binds RNA end Small RNA, Dicers and Argonautes: biochemical core of RNA silencing RISC contains: A member of the Argonaute (Ago) protein family Agos: cleave the ‘passenger strand’ An sRNA binding PAZ domain A PIWI domain that provides endonucleolytic activity RISC Complex Image source:https://en.wikiversity.org/wiki/RNA_interference
- 8. (1) Silencing by antisense RNA (2) Co-suppression (3) Silencing by inverted-repeat (IR) sequences (4) MicroRNA (miRNA) pathway (5) Sense-PTGS pathway (6) Transitive silencing or RNA amplification Pathways of RNA silencing for virus resistance
- 9. Inhibits gene expression by o Production of complementary RNA o Targeted mRNA is prevented from translating a viable protein Flavr Savr tomato was produced by antisense RNA (by inhibiting Polygalactouronase enzyme) (1) Silencing by antisense RNA Successful example in viruses: An antisense of Pro gene of PVY induced immunity in potato (Waterhouse et al. 1998) Formation of antisense RNA blocks translation Image source: http://www.scq.ubc.ca/antisense-rna/
- 10. First observed in petunia Attempted to overexpress chalcone synthase (anthrocyanin pigment gene) in petunia, Caused the loss of pigment (Jorgenson et al., 1990) Relies on the “sense RNA over-abundance” resulting in removal of all homologous RNA irrespective of their source Called co-suppression because suppressed expression of both endogenous gene and transgene Wild type Variegated flower (2) Co-suppression Image source:www.cell.com
- 11. Widely used research tool but least understood process High dsRNA levels produced promote the activities of different dicers and RISCs Two classes of siRNA involved: 21nt & 24nt siRNA Two distinct Dicer-like enzymes processes the ds transcripts- DCL3 & DCL4 DCL4 is preffered for production of 21 nt siRNA (3) Silencing by inverted-repeat (IR) sequences Brodersen et al., 2006
- 12. miRNAs are 20-25 nt, non-coding, endogenous small RNAs First discovered in C. elegans Acts through PTGS in a sequence specific manner AmiRNA-mediated viral resistance employed for Turnip yellow mosaic virus , Turnip mosaic virus (Niu et al., 2006) Plum pox virus (Simón-Mateo & García, 2006) Cucumber mosaic virus (Qu et al., 2007; Duan et al., 2008) Potato virus Y , and Potato virus X (Ai et al. 2011) (4) MicroRNA (miRNA) pathway
- 13. (1) Silencing by antisense RNA This pathway is elicited by RNA with aberrant feature Normally leads to degradation through 5’-3’ exonuclease XRN4 Lack of XRN4 triggers convertion into dsRNA (by SGS3, RDR6, SDE3 & WEX) dsRNA is then processed by DCL4 to 21nt siRNA & methylated by HEN1 Two sets of reactions: transitivity or sequence specific target degradation (5)Sense-PTGS pathway Brodersen et al., 2006
- 14. First described in virus-infected tobacco Two distinct siRNA population: Primary (21nt & 24nt) & secondary (21nt) Production of 5’ siRNAs : RDR6/SGS3/SDE3-dependent complementary strand synthesis (primed by primary siRNAs) Production of 3’ siRNA: dsRNA synthesis as S-PTGS (6) Transitive silencing or RNA amplification Brodersen et al., 2006
- 15. Widely accepted for viral defense Initiates a silencing trigger in uninfected systemic tissues Movement of silencing signal is either symplastic or apoplastic Defense is attributed to action of RNA dependent RNA polymerase (RDR) RDR1 too has some roles Transitive silencing in plants
- 16. Case Study:
- 17. Artificial miRNA (amiRNA) technology uses different pre- miRNAs as backbones and has been applied in plants Replacement of several nucleotides in a miRNA sequence does not affect transcription and maturation Viruses targeted: Potato virus Y (PVY) and Tobacco etch virus (TEV) Background of the study
- 18. Designing of a single amiRNA construct to induce resistance to Potato Virus Y (PVYN ) and Tobacco Etch Virus (TEV-SD1) in transgenic plants To determine the most optimal target sequence for amiRNA-mediated viral resistance Objectives
- 19. The amiRNA target sites were identified by comparing PVYN genomes with TEV-SD1 genomes using the DNAMAN 5.2.2 software The potential amiRNA target site was identified using the miRNA Target Finder program & potential amiRNAs were screened based on the amiRNA criteria To eliminate the possibility of targeting the endogenous mRNAs of the host plants, the selected amiRNA sites were placed as inputs in the TIGR tobacco mRNA databases in the miRU program AmiRNA design and construction of expression vector
- 20. Structure of pre-miR319a (176 bp), which is presented as a hairpin The amiRNA or amiRNA* sequences were used to replace miR319 and miR319* sequences by PCR in the pre-miRNA and cloned into the binary vector pROKII AmiRNA design and construction of expression vector Viral amiRNA
- 21. AmiRNA target sequence with high similarity between PVYN and TEV-SD1
- 22. RE sites Target sites The primers used to construct the artificial miRNA in the study
- 23. Special amiRNAs were present in the pRamiRNAs-infiltrated and transgenic leaves AmiRNAs, could not be detected in the control plants Nine amiRNA vectors could be effectively detected and used to generate amiRNAs Accumulation of amiRNAs in infiltrated leaves CK: N. benthamiana plants that were infiltrated with empty vector In1to In9: N. benthamiana plants infiltrated with A. tumefaciens contained binary vectors pRamiRNA1 to pRamiRNA9 Tr1 to Tr9: transgenic plants with amiRNA expression vectors pRamiRNA1 to pRamiRNA9 WT: wild type tobacco NC89 Northern blot Detection of amiRNA expression in a transient expression system and in transgenic plants
- 24. One T1 plant from each line (Random selection) 4-5 leaf stage, two fully expanded upper leaves Screened for symptoms Viral resistance assay of the transgenic plants Inoculation with PVY or TEV Susceptible -ELISA: positive -Symptoms of infection (vein-clearing and mosaic) Resistant -ELISA: negative -Symptomless After 2/3 weeks
- 25. Outcome: Nine PVYN -infected transgenic groups exhibited various degrees of viral resistance from 39% to 57% The resistant plant ratios of pRamiRNA2, pRamiRNA5, pRamiRNA6, pRamiRNA8 & pRamiRNA9 were >50% The highest percentage (57%) of resistant plants was observed in the pRmiRNA9 group TEV-SD1 infected transgenic groups showed resistance from 32% to 52% The resistant plant ratios of pRamiRNA5 and pRamiRNA9 were >50% highest percentage (52%) of resistant plants was observed in the pRamiRNA5 group AmiRNA5 and amiRNA9, which presented the greatest resistance to PVYN and TEV-SD1 (against NIb &CP gene) The ratio of TEV resistance was lower than that of PVY resistance miRNA activity was affected by the mismatched bases between the amiRNA and the target gene Viral resistance assay of transgenic plants
- 26. Viral resistance assay of transgenic plants Line No. of plants infected (PVYN) No. of plants resistant (PVYN) PVYN resistant ratio (%) Total no. of plants infected with TEV-SD1 No. of plants resistant to TEV-SD1 TEV-SD1 resistant ratio (%) Decline in resistance rate (%) T1-amiRNA1 128 52 40.48 ± 5.77 128 42 32.83 ± 2.75 18.9 T1-amiRNA2 96 48 50.38 ±1.48 106 49 46.23 ± 1.56 8.24 T1-amiRNA3 104 41 39.40 ± 2.26 102 33 32.31 ± 1.04 17.99 T1-amiRNA4 83 41 49.97 ± 8.77 94 41 43.61 ± 1.61 12.73 T1-amiRNA5 106 55 52.43 ± 5.62 121 63 52.07 ± 2.61 0.69 T1-amiRNA6 90 46 51.03 ± 5.19 93 43 46.19 ± 3.34 9.48 T1-amiRNA7 109 49 44.93 ± 0.93 112 50 46.52 ± 1.61 −3.54 T1-amiRNA8 100 52 52.05 ± 3.92 101 47 46.52 ± 2.25 10.62 T1-amiRNA9 90 51 56.71 ± 0.44 94 48 51.11 ± 6.54 9.87 WT 30 0 0 30 0 0 --
- 27. Transgene transcripts accumulated in resistant and susceptible transgenic plants The transcript accumulation in resistant plants was lower than susceptible plants No accumulation of target gene transcripts was observed in WT plants Accumulation of transcripts in virus- inoculated plants A–I: transgenic lines with amiRNA expression vectors pRamiRNA1 to pRamiRNA9 WT: wild type plants S: susceptible plant R: resistant plant rRNA: ribosomal RNA Measurement of the silencing effect at the RNA level Northern blot
- 28. Additionally, the viruses were detected in the virus- inoculated plants with the virus-specific probe (CI, NIa, NIb, CP) There was significant viral RNA accumulation in the susceptible plants In the resistant transgenic plants virus was undetectable This result indicated that, the viral resistance was mediated by RNA silencing (via PTGS) Measurement of the silencing effect at the RNA level Northern blot Accumulation of viral RNA in plants A: viral RNA extracted from pRamiRNA1 & pRamiRNA2 transgenic plants; probe used: CI gene B: viral RNA extracted from pRamiRNA3 transgenic plants; NIa ; C: viral RNA extracted from pRamiRNA9 transgenic plants; CP D: viral RNA extracted from pRamiRNA4 to pRamiRNA8 transgenic plants; NIb
- 29. The amiRNAs were extracted from both resistant and susceptible plants Northern blot analysis: accumulation of amiRNA in resistant plants was higher than that in susceptible plants This observation suggested that the accumulation level of amiRNA could indicate susceptibility to viral infection Accumulation of amiRNAs in resistant & susceptible transgenic plants A-I: amiRNAs extracted from pRamiRNA9 transgenic plants Correlation of resistance with accumulation of amiRNA expression
- 30. small RNAs from the non-inoculated and inoculated T2 transgenic plants were extracted (pRamiRNA5 and pRamiRNA9) DIG-labeled sequence of amiRNAs detect the amiRNA DIG-labeled flanking sequence of virus detect the siRNA from virus Northern Blot pRamiRNA5 (amiRNA probe) (Virus upstream probe) (Virus downstream probe) infected Non-infected infected Non-infected pRamiRNA9 Northern Blot Source of small RNA: virus or amiRNA??
- 31. The amiRNA and siRNA hybridization signals were detected in inoculated transgenic plants Only the amiRNA signal was observed in non-inoculated transgenic plants The silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway amiRNA and the siRNA collectively mediated the degradation of viral RNA Results: Source of small RNA: virus or amiRNA??
- 32. Lines used : pRamiRNA5 and pRamiRNA9 An exogenous gene was integrated in tobacco genome and the copy no.s in the two plants were different There was one copy in T1-M5-3, T1- M5-5, T1-M9-3 and T1-M9-4 transgenic plants Seeds from these lines cultivated on a culture medium that contained kanamycin (100 mg/L) After 30 d, the ratio of resistant to Integration and copies of the exogenous genes in T1 & T2 transgenic plants A: T1transgenic plants of pRamiRNA5 (T1-M5) B: T2 transgenic plants of pRamiRNA5 (T2-M5-3, T2-M5-5) C: T1transgenic plants of pRamiRNA9 (T1-M9) D: T2 transgenic plants of pRamiRNA9 (T2-M9-3, T2-M9-4) P: amiRNA expression vectors pRamiRNA5 or pRamiRNA9 Genetic stability of transgenic and viral- resistant plants A B C D
- 33. Kanamycin and viral resistance analysis of T2 transgenic plants T2-M5-3, T2-M5-5: T2 transgenic lines of pRmiRNA5 T2-M9-3, T2-M9-4: T2 transgenic plant lines of pRmiRNA9 Genetic stability of transgenic and viral- resistant plants Result was consistent with Mendel’s laws of inheritance
- 34. viral resistance assay: Resistance ranged from 32% to 52% (with 1-3 mismatched bases) Transgenic that can express active amiRNAs can mediate viral resistance by RNA silencing AmiRNA that targeted CP gene induced highest resistance (57%), Middle segment of CI gene: max.resistance (50%); 5’end of CI gene: only 40% resistance In this study, the accumulation of amiRNA in resistant plants was higher than that in susceptible plants the accumulation of sRNAs may be not the unique determinant of the efficiency of resistance Summary
Editor's Notes
- Some such diseases, which are especially relevant to India, along with their yield losses, are listed in next slide. A majority of plant viruses have a single-stranded positive- sense RNA as the genome. However, some of the most important viruses in tropical countries like India have single-stranded and double-stranded DNA genomes and RNA genomes of ambisence polarity, i.e. genes oriented in both directions. Rapid advances in the techniques of molecular biology have resulted in the cloning and sequence analysis of the genomic components of a number of plant viruses
- 2nd line too long Some such diseases, which are especially relevant to India, along with their yield losses, are listed in next slide. A majority of plant viruses have a single-stranded positive- sense RNA as the genome. However, some of the most important viruses in tropical countries like India have single-stranded and double-stranded DNA genomes and RNA genomes of ambisence polarity, i.e. genes oriented in both directions. Rapid advances in the techniques of molecular biology have resulted in the cloning and sequence analysis of the genomic components of a number of plant viruses
- The paradigm of modern molecular biology, ‘DNA makes RNA makes protein’, predicts a role for RNA as a carrier of information, but not as a regulatory molecule. small RNA biology has significantly improved in recent years, and it is now clear that there are several cellular silencing pathways in addition to those involved in defense. Endogenous silencing pathways have important roles in gene regulation at the transcriptional, RNA stability and translational levels
- Although several mechanisms can generate dsRNA, the sRNA processing and effector steps have a common biochemical core.
- actually tryied to darken flower color
- First Reported in rice using a transgene with a single transcript containing tandem copies of sense & antisense GUS gene that produces dsRNA following transcription (check). An IR transgene construct typically employed for RNAi in plants, produces ds transcripts with perfectly complementary arms. 2 distinct DCL enzymes process dsRNA transcripts. DCL3 probably produces siRNA of 24nt which can direct DNA or histone modifications at homologous loci & which appear to be not required for RNA cleavage. DCL4 probably is preffered enzyme for 21nt siRNA production. One siRNA strand incorporates into AGO-1 loaded RISC to guide endonucleolytic cleavage of homologous RNA, leading to its degradation. Both siRNA species undergo HEN1-mediated methylation.
- The pathway is elicited by RNAs with aberrant features, there might be other triggers too. Aberrations could be lack of poly A tail or 5’ capping. Tha latter would normally lead to RNA degradation through activity of 5’-3’ exonuclease XRN4. Lack of XRN4 would promote accumulation of uncapped mRNAs, thereby trigger their conversion into dsRNA by combined avtion of RDR6, SGS3,SDE3 & WEX. The resultant dsRNA is processed by DCL4 producing 21nt siRNA and methylated by HEN1. then AGO1-loaded RISC to sequence sp. Cleavage of target mRNA. There is a relation of this pathway with transitivity as the 21nt siRNA can be used as primers by RDR6 to reinforce the production of dsRNA from ss templates.
- Transitivity is the ‘transition’ of primary siRNAs to secondary siRNA. a dsRNA source of primary siRNA promotes production of seconadary siRNA both 5’ and 3’ of the initially targeted interval of a transcript. Production of 5’ siRNAs (i) can be explained by RDR6/SGS3/SDE3-dependent complementary strand synthesis that is primed by one of the primary siRNAs. Production of 3’ siRNA (ii) cannot be explained by a primed reaction, and it is possible that RNA fragments resulting from primary siRNA-directed transcript cleavage are recognized as abberant, thereby initiating dsRNA synthesis as S-PTGS. The 5’ and 3’ reactions are not mutually exclusive as siRNA production in (ii) could prime further dsRNA synthesis according to the scheme depicted in (i). DCL4 is shown as putatively involved in 5’ and 3’ secondary siRNA biogenesis. Unlike primary siRNA (21nt and 24nt), seconadary siRNA are exclusively of the 21 nt size class. It is unclear that 24nt primary siRNA can trigger transitive RNA silencing.
- AmiRNA technology aims to produce antiviral plants (after the first point) As a major genus of plant viruses, Potyvirus includes 200 species that infect plants that belong to different families such as Solanaceae, Chenopodiaceae, Leguminosae, andCucurbitaceae, thereby causing great economic loss. two classical species of Potyvirus and contain genomes with a positive-sense single-stranded RNA; these RNAs exhibit high similarities (before the third point)
- PVYN genomes (GenBank No. EU 182576) with TEV-SD1 genomes (GenBank No. EF470242) , host plants were Nicotiana benthamiana and Nicotiana tobacum vat. NC89. The pre-miR319a sequence of Arabidopsis thaliana was used as the backbone to construct amiRNA expression vectors. The natural miR319 and miR319* sequences were replaced with a viral amiRNA sequence by oligonucleotide-directed mutagenesis. To facilitate cloning, we designed the amplification primers, which included a selected amiRNA sequence and a cleavage site (BamHI or KpnI) ( Table 2). The BamHI-KpnI fragment ( Fig. 1) was digested and inserted in the plant binary vector pROKII
- Viral inocula (diluted by phosphate buffer, pH 7.4 in 1:10 ratio) Control used: NC89
- Make the fonts bigger While starting this slide, Statistical analysis showed that the… at last These results further indicated that amiRNA was effective
- Label the lines
- (since high levels of amiRNAs with high viral resistance) six plants from each line to determine the genetic stability and the viral resistance of T2 progeny plants for Southern blot other seeds were cultivated on a kanamycin-free culture medium.
- Reduce texts in red. Yellow fonts; not required?? After the 2nd point, different resistance levels were observed when amiRNAs targeted different viral functional genes or were allocated at different positions in the same functional gene.