1) dsRNA-based strategies show potential for managing plant diseases caused by fungi, nematodes, insects, and oomycetes. 2) Direct delivery of dsRNA to crop plants (HIGS) or spray application (SIGS) can induce gene silencing in pathogens. 3) Studies have demonstrated the ability of dsRNA to suppress the fungus Fusarium graminearum, both in transgenic Arabidopsis plants and after foliar application to wheat. 4) However, more evidence is still needed to demonstrate efficient control of fungi and oomycetes under field conditions using dsRNA approaches.

1. Karl-Heinz Kogel
Institute für Phytopathologie
JLU Giessen
Potential for dsRNA-based
management of plant diseases

2. • 11 Bill. people in 2100
• Sustain. criteria: no further land increase for agriculture acceptable
• Most important driver for biodiversity loss / greenhouse gas
• Extreme yield losses without plant protection measures
• Organic farming ~25% less productive than integrated production
• No other option than increase the global yield efficiency
Source: Farming without plant protection:
EPRS European Parlamentary Research Service (PE 634.416 – March 2019)
challenges

3. RNA-based Plant Protection
Fighting against pest and diseases
using non-coding RNAs
• Convincing data in insect control !
• What about fungal pathogens ?
• Nature provides a blue print for HIGS / SIGS strategies:
sRNA effectors are „natural“ !
outline

4. Agricultural application of
double-stranded (ds)RNA
viruses
insects
nematodes
fungi
oomycets
√
√
√
√
√
Recent reviews:
Cai et al. 2018. Curr Opin Microbiol 46:58-64
Andrade & Hunter 2016. InTech pp. 391-409

5. Delivery of dsRNA to suppress pathogens / pests
o Direct delivery via the crop plant (GMO)
( Host-Induced Gene Silencing, HIGS )*
*Nowara et al. (2010) Plant Cell 22:3130
**Koch et al. (2016) PloS Pathogens
o Delivery via spray-application (RNA as fungicide)
( Spray-Induced Gene Silencing, SIGS )*

6. Abdellatef et al. 2014, Plant Biotech. J. 13:849
Aphid target „SHEATH PROTEIN“ (SHP)
www.lastrefuge.co.uk
SHP
Target: Sitobion avenae

7. Aphid generation
2 weeks on
shp-dsRNA plants
7 generations on wt plants
Abdellatef et al. 2014, Plant Biotech. J. 13:849
Transgenerational silencing SHP in shp-dsRNA-
expressing barley plants
HIGS
(GMO)

8. Targeting the mycotoxin-producing fungus
Fusarium graminearum by RNA
Jansen et al. 2005 PNAS 102:16892Necrotrophic growth
Head blight disease
Macroconidia
Axenic culture

9. An „old“ fungicide target is a good target for dsRNA
Cytochrome P450 sterol
14α-demethylase (CYP51)
De Souza et al. 2009
= Target of AZOLE fungicides:
sterol demethylation inhibitors (DMI)
Ergosterol
biosynthesis
Fusarium graminearum
has three CYP51 genes
(Fan et al. 2013)
FgCYP51B:
sterol 14α-demethylation
FgCYP51A:
sterol 14α demethylase
induced on ergosterol
depletion
FgCYP51C:
virulence on wheat ears

10. CYP51-dsRNA targets all
three fungal CYP51 genes
= 791 nt long

11. Control of Fusarium graminearum
on CYP51-dsRNA expressing Arabidopsis
Fusarium
Fusarium
+ dsRNA
Koch et al. 2013, PNAS 110:19324
HIGS
(GMO)

12. dsRNA as fungicide?
Koch et al. 2016, Plos ONE ppat.1005901

13. SIGS-mediated systemic control of Fusarium graminearum
CYP51-dsRNA
Buffer
GFP-dsRNA
systemic leaf area
dsRNA
Spray
INOCULATION
systemic leaf area

14. Uptake of CYP51-dsRNAatto488
CYP51-dsRNA
CYP51-dsRNA-488
Buffer
GFP-dsRNA

15. Fungi amenable to environmental dsRNA
Fungus RNA Treat. Activity Ref.
Fusarium
graminearum
dsRNA
siRNA
foliar,
liquid culture
reduced
virulence
Koch et al.
2016
Fusarium
culmorum
dsRNA liquid culture reduced
growth
Koch et al.
2018
Botrytis
cinerea
dsRNA
siRNA
foliar reduced
virulence
Wang et al.
2016
Sclerotinia
sclerotiorum
dsRNA foliar reduced
virulence
McLoughlin
et al. 2018
Zymoseptoria
tritici
dsRNA axenic
culture
none Kettles et al.
2018
Verticillium
longisporum
dsRNA liquid culture uptake of
dsRNA
Kogel,
unpublished

16. Possible drawbacks of dsRNA approaches
• Emergence of resistant microbes/pests ?
• Yield panelty ?
• Off targets ?
• Immune stimulatory activity of dsRNA ?

17. upper
leaf part
Off-targets of CYP51-dsRNA
*Simulations were run using Si-Fi software (v3.1) for predicting off-targets prediction
(http://labtools.ipk-gatersleben.de).
†Number of 21-mer siRNA sequences with perfect match to the query sequence.
‡Number of 21-mer siRNA sequences with perfect match to the query sequence that fulfill additional criteria for efficient RNAi (See Si-
Fi software).

18. Perspective
• Potential application of dsRNA includes GMO and non-GMO strategies
• The technique has extremely broad application reaches
• However, so far little substantial evidence for (agronomically)
efficient control of fungi and oomycetes
• dsRNA Strategies can be used for target evaluation / validation
• Viruses and insects are extremley amenable to control by
dsRNA/sRNAs

19. Artificial RNAi strategies seem to work
almost everywhere in nature
An evolutionarily highly conserved
mechanism of RNA exchange between
cells, organs and organisms
must exist?

20. MicrobePlant
Natural Cross-Kingdom RNA interference
Exchange of sRNA effectors between
hosts and microbes is a natural phenomenon
sRNA
Weiberg et al. 2013. Science 342:118
Zhang et al. 2016. Nat Plants 2(10):16153
Zanini et al. 2018. Front Plant Sci doi.org/103389/fpls
Kusch et al. 2018. Fungal Biol. 122:1050

21. Bidirectional RNA exchange
Artificial
sRNAs and dsRNAs
(HIGS)
STOP immunity
Endogenous
natural
plant ncRNAs
AGO1
Plant cell
STOP virulence
Fungal
sRNA
DCL1
RNAi
RNAi