Geetanjali Baruah

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

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 tungroBadna 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

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

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

A collective termIncludes several RNA-based inhibition of gene expression at TranscriptionmRNA stabilityTranslationalDiscovered 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/

sRNAs are produced by RNase III-type enzymes called Dicers

Domains: C- terminal dsRNA binding: binds the dsRNAN- terminal RNA helicase: processes long substrates RNase III: cut RNA to produce 2 nt 3’-overhangPAZ (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

(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

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 translationImage source: http://www.scq.ubc.ca/antisense-rna/

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

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

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

(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

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

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

Unlike animals, transitive silencing is bidirectional (5’ and 3’)

Transitive silencing in plants

Case Study:

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

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

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

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

AmiRNA target sequence with high similarity between PVYN and TEV-SD1

RE sites Target sites

The primers used to construct the artificial miRNA in the study

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 vectorIn1to 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

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

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

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 --

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 plantsA–I: transgenic lines with amiRNA expression vectors pRamiRNA1 to pRamiRNA9WT: wild type plantsS: susceptible plantR: resistant plantrRNA: ribosomal RNA

Measurement of the silencing effect at the RNA level

Northern blot

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 

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 plantsThis observation suggested that the accumulation level of amiRNA could indicate susceptibility to viral infection

Accumulation of amiRNAs in resistant & susceptible transgenic plantsA-I: amiRNAs extracted from pRamiRNA9 transgenic plants

Correlation of resistance with accumulation of amiRNA expression

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-infec

ted

infected

Non-infec

tedpRamiRNA9

Northern Blot

Source of small RNA: virus or amiRNA??

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??

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 non-resistant plants was 3:1

Integration and copies of the exogenous genes in T1 & T2 transgenic plantsA: 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

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

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 silencingAmiRNA 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