This document summarizes two case studies on using externally applied dsRNA to manage plant pathogens. The first case study investigated using a β2-tubulin dsRNA to silence the gene in the fungal pathogen Fusarium asiaticum. It was found to reduce fungal growth, virulence, and increase sensitivity to fungicides. The second case study designed dsRNA constructs targeting different regions of Tomato leaf curl virus and Cucumber mosaic virus. Application of these dsRNAs provided protection against the viruses in tomato and nicotiana plants. The hybrid dsRNA provided protection against both a DNA and RNA virus. Both studies demonstrate the potential of using exogenous dsRNA to induce RNAi in plants and

1. “SIGS- externally applied dsRNA mediated management
plant pathogens”
Indian Agricultural Research Institute
Division of Plant Pathology
Speaker - Pankhuri Singhal, 11567
P.hD. first year
Seminar leader - Dr. M.S. Saharan,
Dr. Basavaraj
Chairman - Dr. V.K.Baranwal
Credit Seminar: Pl. Path. 691

2. Plant Protection
options
Cultural methods
Host plant resistance
Chemical control
How well this strategy helps our food crop to
protect itself ?

3. RNA silencing/ RNAi/
Gene quelling (Fungi)
Endogenous cellular
mechanism
Found only in higher
eukaryotes
Initiated by dsRNA or
stem loop RNA structures
(hpRNAs)
(Dalakouras et al., 2019)

4. (Joga et al., 2016)
Transgenic versus non-transgenic RNAi
(Mitter et al. 2017)

5. Mode of entry and movement of dsRNA in plants
(Kim et al., 2019)

6. Diverse areas wherein
dsRNA mediated RNAi
has been exploited:
 Against fungi
 Against viruses/
viroids
 Against insects
 Nematodes
 Mites
 Weeds
 Manage pesticide
resistance
 Against mycotoxins
(Dubrovina et al., 2019)
Via
extracellular
vesicles
RNAi in different eukaryotes

7. Target Origin Of RNA RNA ,Virus
Application
Plant Host Effect Reference
RP gene of
PMMoV, TEV,
and AMV
In vitro synthesized
dsRNA
Mechanical
inoculation
Tobacco,
pepper
Resistance to
PMMoV, TEV,
and AMV
Tenllado
and Díaz-
Ruíz (2001)
RP gene of
PMMoV
Crude extracts of
bacterially
expressed dsRNA
Mechanical
inoculation or
spraying with atomizer
Tobacco Resistance to
PMMoV
Tenllado et
al. (2003)
Viroid-
specific
dsRNAs
In vitro synthesized
dsRNA and siRNA
Mechanical
inoculation
Tomato,
chrysanthem
um, gynura
Resistance to
PSTVd, CEVd
and CChMVd
Carbonell et
al. (2008)
p126 and CP
genes of
TMV
In vitro synthesized
dsRNA
Mechanical
inoculation
Tobacco Resistance to
TMV
Konakalla
(2016)
RP gene of
PMMoV; 2b
supressor of
CMV2b
In vitro transcribed
RP dsRNA, crude
extracts of
bacterially
expressed 2b dsRNA
naked /LDH loaded
Spray inoculation Tobacco,
cowpea
Resistance to
PMMoV and
CMV
Mitter et al.
(2017)
HC-Pro and
CP genes of
ZYMV
In vitro synthesized
dsRNAs
Mechanical
inoculation
cucumber,
watermelon
and squash
Resistance to
ZYMV
Kaldis et al.
(2018)
Exogenous dsRNA for plant viral resistance

8. Target Origin Of
RNA
RNA ,Fungal
Application
Plant Host Effect Reference
CYP51A, CYP51B,
and CYP51C
genes of
Fusarium
graminearum
In vitro
synthesized
CYP3-dsRNA
RNA spraying;
fungal inoculation
48 hpt
Barley Inhibition of
fungal growth,
weaker disease
symptoms
Koch et al.
(2016)
DCL1 and DCL2
genes of Botrytis
cinerea
In vitro
synthesized
dsRNA
RNA dropped onto
the surface of each
plant specimen
Tomato, rose,
strawberry, grape,
lettuce, onion,
same Wang et al.
(2016)
59 target genes of
Sclerotinia
sclerotiorum
In vitro
synthesized
dsRNAs
Foliar RNA
application to the
leaf surface with
Silwet L-77
Oilseed rape,
Arabidopsis
same McLoughlin
et al.
(2018)
Myosin 5 gene of
Fusarium
asiaticum
In vitro
synthesized
dsRNA
RNA spraying;
fungal inoculation
12 hpt
Wheat same Song et al
(2018)
β2Tub gene of
Fusarium
asiaticum
In vitro
synthesized
dsRNA
RNA spraying after
leaf wounding with
quartz sand; fungal
inoculation 12 hpt
Cucumber, soya,
barley, wheat
F. asiaticum, B.
cinerea,
M.oryzae, and
Colletotrichum
truncatum
Gu et al.
(2019)
Exogenous dsRNA for plant fungal resistance

9. Methods of dsRNA production
In vivo
(Bacterially
expressed)
In vitro
One-step PCR, Transcription and
hybridization
Two-step PCR, Transcription and
hybridization
One-step PCR, Transcription and
Replication
Escherichia coli using T7 RNA
Polymerase
Pseudomonas syringae
using Phage phi6 Polymerase
complex
(Voloudakis et al., 2015)

10. CASE STUDY I :
Aim: To determine whether RNAi molecules derived from an essential and
fungus specific-gene could confer resistance to fungal pathogens by
spraying dsRNA on plants.

11. Designing of β2-tubulin dsRNA constructs and
evaluation through PCR and southern blot analysis

12. Selection of efficient RNAi segments by analyzing
the mycelial growth and asexual reproduction
The Faβ2TubRNAi−3 strain
grew extremely slowly with
71% lower and formed fewer
aerial hyphae compared to
the FaNJ003
The conidiation of
Faβ2TubRNAi−1,−2, and −3
was significantly decreased,
especially for the
Faβ2TubRNAi−3
transformant, with shorter
conidia and less septum
number.
Unexpectedly, the conidia
production and characteristic
of Faβ2TubRNAi−4 were
similar with the non
transgenic strain FaNJ003
Effect of the silenced of β-tubulin gene on virulence
toward plants
Virulence of the Faβ2TubRNAi strains was
assayed by seedling inoculation of wheat
cultivar Yumai35 with conidial spores
During the succeeding days, disease symptoms
quickly spread in all the inoculated seeding
except those inoculated with the
Faβ2TubRNAi−3 strain also the lesion length was
significantly reduced in Faβ2TubRNAi-3 strain
followed by 1, 2, and 4 as compared to the
original strain FaNJ003.
These results demonstrated that the third segment of β2-tubulin was the
highest efficient RNAi segment to silence the β2-tubulin gene in F.asiaticum

13. Uptake of dsRNA in plant cells and induction of RNAi
Wheat coleoptiles and barley,
cucumber and soya leaves were
treated with 560-nt GFP dsRNA
(control) or Faβ2Tub−3 dsRNA
Relative fungal biomass on plant
The disease symptoms of leaves
spraying Faβ2Tub−3 dsRNA were
clearly lower than leaves spraying
GFP dsRNA.
The relative fungal biomass on
plants spraying with Faβ2Tub−3
dsRNA were significantly reduced
by 54%–90%, compared with
control groups.

14. The Faβ2Tub−3 dsRNA was added into
fungal cultural medium to detect the
effect of dsRNA on fungal growth and
development
Fusarium spp. mycelia were severely
inhibited with conglobated and multiple-
branching structures.
The infection structure formation of B.
cinerea, was seriously blocked by
Faβ2Tub−3 dsRNA.
Faβ2Tub−3 dsRNA resulted in mycelial
growth inhibition of Magnaporthe oryzae
and Colletotrichum truncatum, as well as
morphological changes in vitro.
The different effects of Faβ2Tub−3 dsRNA
on different fungal species may result from
the off-targets effects or the diverse
functions of β-tubulin in diverse fungi.
The effects of Faβ2Tub−3 dsRNA on hyphal morphology
The Faβ2Tub−3 dsRNA was added into
fungal cultural medium to detect the
effect of dsRNA on fungal growth and
development
Fusarium spp. mycelia were severely
inhibited with conglobated and multiple-
branching structures.
The infection structure formation of B.
cinerea, was seriously blocked by
Faβ2Tub−3 dsRNA.
Faβ2Tub−3 dsRNA resulted in mycelial
growth inhibition of Magnaporthe oryzae
and Colletotrichum truncatum, as well as
morphological changes in vitro.
The different effects of Faβ2Tub−3 dsRNA
on different fungal species may result from
the off-targets effects or the diverse
functions of β-tubulin in diverse fungi.

15. Investigation of MBC sensitivity of the Faβ2Tub dsRNA
The mycelia were severely inhibited in medium containing Faβ2Tub−3 dsRNA
and medium containing Faβ2Tub−3 dsRNA plus MBC, compared with two
control media Moreover, the antifungal activity of Faβ2Tub−3 dsRNA plus MBC
effected longer than single Faβ2Tub−3 dsRNA.
Plant spraying assay: lesion
lengths were shorter by
treatment with Faβ2Tub−3
dsRNA plus MBC than that
by treatment with
Faβ2Tub−3 dsRNA alone.
SNA medium
These results demonstrated that Faβ2Tub−3 dsRNA increased the sensitivity of the
fungus to MBC and effected more effective mixing with MBC.

16. Findings
 Faβ2Tub−3 transformant was effective and stable in reducing conidiation,
conidial length, mycelial growth and pathogenicity.
 The expression of β2-tubulin was decreased 90% in Faβ2TubRNAi−3
transformant.
 Because the β2-tubulin was conserved in fungi, Faβ2Tub−3 dsRNA showed a
multiple-antifungal activity against Fusarium spp., Botrytis cinerea,
Magnaporthe oryzae and Colletotrichum truncatum
 Mixture of Faβ2Tub−3 dsRNA and MBC effected more effective and longer than
the single Faβ2Tub−3 dsRNA.
 In addition, the fungal resistance to MBC was highly reduced by silencing β2-
tubulin

17. CASE STUDY II :
Aim: To design dsRNA constructs from various regions of ToLCV and CMV
and test their efficacy in protection against the cognate viruses by
exogenous application

18. Regions of ToLCV and CMV used for
dsRNA synthesis

19. Evaluation of two-step PCR products and
dsRNA through gel electrophoresis

20. Tomato plants cv. Pusa early dwarf and the
treatments given

21. ToLCV
ToLCV + dsAC1/AC4 (ds1)
ToLCV + dsAV1/AV2 (ds2)
ToLCV + dsAC1/AC4_AV1/A2
ToLCV + ds2b_AV1/A2
ToLCV + ds2b (nonhomologous)
Evaluation of the effect of different treatments of
dsRNA compared to mock in tomato against ToLCV
ToLCV ToLCV +
AC1/AC2
ToLCV +
AV1/AV2
ToLCV +
AC1/AC2_
AV1/AV2
ToLCV +
2b_AV1/AV2
(hybrid construct)
ToLCV +
CMV-2b
18 dpi 41.67 25 16.67 20.83 12.50 33.33
22 dpi 50 29.17 25 25 20.83 41.67
26 dpi 54.17 33.33 25 33.40 25 41.67
30 dpi 70.83 41.67 25 33.40 29.17 58.33
34 dpi 79.17 45.83 33.33 41.67 37.50 62.50
38 dpi 83.33 45.83 33.33 41.67 37.50 79.17

22. Estimation of the life span of dsRNA in tomato
DsAV1/AV2 was exogenously applied on two leaves of each tomato plant. A pool of seven
plants was used for RNA extraction at several time points. Detection in local (treated)
leaves was carried out at 1, 6, 9, 14, 21 dpi, while in non treated (systemic) leaves was
carried out at 1, 6, 9, 14, 21, 41 dpi. Second panel indicate the presence of the
endogenous gene cyclophilin (cyclo, 127 bp)
The applied dsRNA
(dsAV1/AV2) were detected
in local leaves up to 21 dpi
and while in case of
systemic leaves up to 41
dpi.
These results confirm the movement of dsRNA from local to the systemic
tissues in tomato.

23. CMV Infected Healthy
CMV ds2b CMV
ds2b_AV1/AV2
Evaluation of the effect of different treatments of
dsRNA compared to mock in Nicotiana against CMV
CMV CMV + ds2b CMV + ds2b
_AV1/AV2
CMV + ds-
AV1/AV2
8 dpi 30 3.33 13.33 23.33
10 dpi 80 30 33.33 40
12 dpi 90 40 50 83.33
14 dpi 96.67 46.67 56.67 83.33
16 dpi 100 60 66.67 93.33
CMV
CMV + ds2b
CMV + dsAV1/AV2
(non-homologous control)
CMV + ds2b_AV1/AV2

24. Findings
 ds1, 2, 3, 4 provided 45%, 60%, 50% and 55% protection against ToLCV,
respectively
 The slightly higher protection provided by ToLCV15 AC1/AC4_AV1/AV2 dsRNA as
compared to ToLCV-AC1/AC4 could be explained by the presence of AV1/AV2
region
 Detection of dsRNA in local and systemic leaves (for 21 and 41 days post
application) confirmed the systemic movement as well as the persistence of
dsRNA in tomato
 Using a single dsRNA molecule, namely the hybrid construct CMV-2b_ToLCV-
AV1/AV2, protection against both a DNA and a RNA virus was observed

25. Conclusions
Spray-induced gene silencing (SIGS) stimulates host defence mechanism without
the need to develop stably transformed plants.
dsRNA mediated RNAi can also be used to overcome the resistance of pathogens
against fungicides

26. Future Prospects
 Optimization of methods of dsRNA production,
delivery and stability in plants.
 New researches in the field of nanoparticles, RNA
stabilizing agents, materials promoting RNA
adhesion, or protein carriers could enhance
efficiency of exogenous dsRNA treatments.

27. Thank You