are big melanized multicellular survival structures and tend to
form aggregates resulting in more difficult and imprecise conidia count.
12. Stored conidia will start to germinate after 1 or 2 days at 4
C.
To conserve them for a longer period, resuspend conidia in
distilled water (centrifuge at 3000 Â g at RT for 10 min and
replace PDB with dH 2 O). Place the conidia solution after
immediate freezing in liquid nitrogen at À80
C.
13. To avoid loss of material due to evaporation and/or wrong
handling, 96-well plates can be sealed with a plastic transparent
sticky foil like the one used for sealing qPCR well plates.
References
1. Baulcombe D (2004) RNA silencing in plants.
Nature 431:7006
2. Castel SE, Martienssen RA (2013) RNA interference in the nucleus: roles for small RNAs in
transcription, epigenetics and beyond. Nat Rev
Genet 14(2):100–112
3. Weiberg A, Wang M, Lin FM, Zhao H,
Zhang Z, Kaloshian I, Jin H (2013) Fungal
small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science
342(6154):118–123
4. Cai Q, He B, Kogel KH, Jin H (2018) Crosskingdom RNA trafficking and environmental
RNAi-nature’s blueprint for modern crop protection strategies. Curr Opin Microbiol
46:58–64
5. Zanini S, S ˇ ec ˇic ´ E, Busche T, Kalinowski J,
Kogel KH (2019) Discovery of interactionrelated sRNAs and their targets in the Brachypodium distachyon and Magnaporthe oryzae
pathosystem. BioRxiv 631945. https://doi.
org/10.1101/631945
6. Nowara D, Gay A, Lacomme C, Shaw J,
Ridout C, Douchkov D, Schweizer P (2010)
HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22(9):3130–3141
7. Koch A, Biedenkopf D, Furch A, Weber L,
Rossbach O, Abdellatef E, Linicus L,
Johannismeier J, Jelonek L, Goesmann A,
Cardoza V, McMillan T, Mentzel T, Kogel
KH (2016) An RNAi-based control of Fusarium graminearum infections through spraying
of long dsRNAs involves a plant passage and is
controlled by the fungal silencing machinery.
PLoS Pathog 12(10):e1005901
8. Koch A, Kumar N, Weber L, Keller H, Imani J,
Kogel KH (2013) Host-induced gene silencing
of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong
resistance to Fusarium species. Proc Natl Acad
Sci U S A 110(48):19324–19329
9. Head GP, Carroll MW, Evans SP, Rule DM,
Willse AR, Clark TL, Meinke LJ (2017) Evaluation of SmartStax and SmartStax PRO maize
against western corn rootworm and northern
corn rootworm: efficacy and resistance management. Pest Manag Sci 73(9):1883–1899
10. McLoughlin AG, Wytinck N, Walker PL, Girard IJ, Rashid KY, Kievit T, Belmonte MF
(2018) Identification and application of exogenous dsRNA confers plant protection against
Sclerotinia sclerotiorum and Botrytis cinerea. Sci
Rep 8(1):7320
11. Zhang DD, Wang XY, Chen JY, Kong ZQ, Gui
YJ, Li N, Bao YM, Dai XF (2016) Identification and characterization of a pathogenicityrelated gene VdCYP1 from Verticillium dahliae. Sci Rep 6:27979
12. Niehl A, Soininen M, Poranen MM, Heinlein
M (2018) Synthetic biology approach for plant
protection using dsRNA. Plant Biotechnol J 16
(9):1679–1687
13. Kettles GJ, Hofinger BJ, Hu P, Bayon C, Rudd
JJ, Balmer D, Courbot M, Hammond-Kosack
KE, Scalliet G, Kanyuka K (2019) sRNA
profiling combined with gene function analysis
reveals a lack of evidence for cross-kingdom
RNAi in the wheat – Zymoseptoria tritici Pathosystem. Front Plant Sci 10:892
14. Koch A, Ho ¨fle L, Timo Werner B, Imani J,
Schmidt A, Jelonek L, Kogel KH (2019)
SIGS vs HIGS: a study on the efficacy of two
dsRNA delivery strategies to silence genes in
infected host and non-host plants. Molecular
Plant Pathology 20 (12):1636–1644
238
Matteo Galli et al.
form aggregates resulting in more difficult and imprecise conidia count.
12. Stored conidia will start to germinate after 1 or 2 days at 4
C.
To conserve them for a longer period, resuspend conidia in
distilled water (centrifuge at 3000 Â g at RT for 10 min and
replace PDB with dH 2 O). Place the conidia solution after
immediate freezing in liquid nitrogen at À80
C.
13. To avoid loss of material due to evaporation and/or wrong
handling, 96-well plates can be sealed with a plastic transparent
sticky foil like the one used for sealing qPCR well plates.
References
1. Baulcombe D (2004) RNA silencing in plants.
Nature 431:7006
2. Castel SE, Martienssen RA (2013) RNA interference in the nucleus: roles for small RNAs in
transcription, epigenetics and beyond. Nat Rev
Genet 14(2):100–112
3. Weiberg A, Wang M, Lin FM, Zhao H,
Zhang Z, Kaloshian I, Jin H (2013) Fungal
small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science
342(6154):118–123
4. Cai Q, He B, Kogel KH, Jin H (2018) Crosskingdom RNA trafficking and environmental
RNAi-nature’s blueprint for modern crop protection strategies. Curr Opin Microbiol
46:58–64
5. Zanini S, S ˇ ec ˇic ´ E, Busche T, Kalinowski J,
Kogel KH (2019) Discovery of interactionrelated sRNAs and their targets in the Brachypodium distachyon and Magnaporthe oryzae
pathosystem. BioRxiv 631945. https://doi.
org/10.1101/631945
6. Nowara D, Gay A, Lacomme C, Shaw J,
Ridout C, Douchkov D, Schweizer P (2010)
HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22(9):3130–3141
7. Koch A, Biedenkopf D, Furch A, Weber L,
Rossbach O, Abdellatef E, Linicus L,
Johannismeier J, Jelonek L, Goesmann A,
Cardoza V, McMillan T, Mentzel T, Kogel
KH (2016) An RNAi-based control of Fusarium graminearum infections through spraying
of long dsRNAs involves a plant passage and is
controlled by the fungal silencing machinery.
PLoS Pathog 12(10):e1005901
8. Koch A, Kumar N, Weber L, Keller H, Imani J,
Kogel KH (2013) Host-induced gene silencing
of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong
resistance to Fusarium species. Proc Natl Acad
Sci U S A 110(48):19324–19329
9. Head GP, Carroll MW, Evans SP, Rule DM,
Willse AR, Clark TL, Meinke LJ (2017) Evaluation of SmartStax and SmartStax PRO maize
against western corn rootworm and northern
corn rootworm: efficacy and resistance management. Pest Manag Sci 73(9):1883–1899
10. McLoughlin AG, Wytinck N, Walker PL, Girard IJ, Rashid KY, Kievit T, Belmonte MF
(2018) Identification and application of exogenous dsRNA confers plant protection against
Sclerotinia sclerotiorum and Botrytis cinerea. Sci
Rep 8(1):7320
11. Zhang DD, Wang XY, Chen JY, Kong ZQ, Gui
YJ, Li N, Bao YM, Dai XF (2016) Identification and characterization of a pathogenicityrelated gene VdCYP1 from Verticillium dahliae. Sci Rep 6:27979
12. Niehl A, Soininen M, Poranen MM, Heinlein
M (2018) Synthetic biology approach for plant
protection using dsRNA. Plant Biotechnol J 16
(9):1679–1687
13. Kettles GJ, Hofinger BJ, Hu P, Bayon C, Rudd
JJ, Balmer D, Courbot M, Hammond-Kosack
KE, Scalliet G, Kanyuka K (2019) sRNA
profiling combined with gene function analysis
reveals a lack of evidence for cross-kingdom
RNAi in the wheat – Zymoseptoria tritici Pathosystem. Front Plant Sci 10:892
14. Koch A, Ho ¨fle L, Timo Werner B, Imani J,
Schmidt A, Jelonek L, Kogel KH (2019)
SIGS vs HIGS: a study on the efficacy of two
dsRNA delivery strategies to silence genes in
infected host and non-host plants. Molecular
Plant Pathology 20 (12):1636–1644
238
Matteo Galli et al.