RNA-seq to study rice defense responses upon parasitic nematode infections. Tina Kyndt
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1 RNA-seq to study rice defense responses upon parasitic nematode infections Tina Kyndt 1
2 Rice (Oryza sativa L.) Most important staple food for at least half of the human population Monocot model plant Paddy fields: 3500 liter of fresh water needed to produce 1 kg of rice Aerobic rice 1750 liter needed to produce 1 kg of rice BUT Yield declines because of root pathogens (Pythium and nematode species)
3 3
4 Meloidogyne graminicola: the rice root knot nematode (RKN) Sedentary nematode Symptoms: hook-like galls at the root tip Very common in aerobic rice 4
5 Hirschmaniella oryzae: the rice root rot nematode (RRN) Migratory nematode Symptoms: Root necrosis, short roots Generally in flooded rice fields 5
6 Research questions Which pathways are manipulated by these nematodes to overcome host defence? mrna-seq on different tissues of infected plants Isolation of giant cells (LCM) -> mrna-seq Which defence pathways are necessary for root defence against parasitic nematodes? Infection experiments upon hormonal applications (SA/JA/ET/BR/ABA) Infection experiments on hormone mutants How can we better protect rice plants against parasitic nematodes? 6
7 Studying the rice transcriptome upon root knot and root rot nematode infection Systemic shoots - Oryza sativa cv. Nipponbare - Infection with 300 nems/plant - Material harvesting: at 3 and 7 dpi - 2/3 biological replicates - Each replicate = pool of 6 plants Systemic roots Root tips/galls Infected roots (RRN) RNA-extraction mrna-seq (Illumina) qrt-pcr 7
8 Transcriptome analysis of galls Root knot nematode infection (gall root tip) annotated rice genes novel transcriptionally active regions root knot nematode contigs Dark grey= up Light grey= down 8
9 Transcriptome analysis of RRN infected roots Root rot nematode infection (infected uninfected root) annotated rice genes novel transcriptionally active regions root rot nematode contigs Dark grey= up Light grey= down 9
10 General overview of the local transcriptional response upon infection Meloidogyne graminicola infected gall tissue (3 dpi) Hirschmanniella oryzae infected root tissue (3 dpi) Metabolism Nutrient transport Death Antioxidant activity Plant cell wall organization and biogenesis Biotic stress Hormone pathways mainly involved in plant defense: Salicylic acid pathway Jasmonate pathway and Ethylene pathway Abscissic acid pathway Hormone pathways mainly involved in plant development: Auxin pathway and Cytokinin pathway GA-pathway BR-pathway 10
11 Number of galls per plant SA, JA and ET can induce rice root defence against both nematodes a 25 b c c 5 0 Control BTH MeJA Ethephon Plants were sprayed with hormone solutions 1 day before nematode inoculation (BTH= SA-analogue; Ethephon = ET; MeJA= methyl jasmonate) 11
12 The role of the BR pathway in root susceptibility for RKN Relative expression level OsD11 OsDwarf OsBRI1 External BR application on rice plants: - qrt-pcr to detect transcriptional response of genes involved in BR-pathway - infection experiment to analyse root susceptibility BL (0.1µM) BL (1µM) BL (5µM) BL (10µM) High concentration: - negative feedback on the pathway - defence induced against RKN Number of galls/plant a b b c c Low concentration: - internal activation of the BR pathway - higher susceptibility for RKN 10 0 Control(T65) BL (0.1µM) BL (1µM) BL (5µM) BL (10µM) Internal activation of the BR-pathway is correlated with higher susceptibility for RKN 12
13 BR biosynthesis is necessary for root susceptibility to RKN 60 d 50 a a Number of galls/plant c b c b b 10 0 Control BRZ (1) d2-1 d2-1 + BL(0.1) d2-1 + BL(10) d2-2 d2-2 + BL(0.1) d2-2 + BL(10) BRZ: BR biosynthesis inhibitor d2-1 and d2-2: BR-deficient mutants 13
14 BR induction leads to suppression of the JA-pathway Relative expression level OsD11 OsDwarf OsBRI BL (0.1µM) BL (1µM) BL (5µM) BL (10µM) qrt-pcr on JA-related genes OsAOS2 OsJAmyb Internal activation of BR correlates with suppression of the (defence-inducing) JApathway Relative expression level BL (0.1µM) BL (1µM) BL (5µM) BL (10µM) BR JA 14
15 Rice thionin genes are suppressed in M.graminicola induced galls at early time points Expression in the uninfected root tips 15
16 Overexpression of OsThi7 in rice leads to reduced susceptibility to root pathogens Less colonization of the root cortex and vascular tissue 16
17 Laser Capture Microdissection of giant cells and RNA-seq Sample Total number of sequenced Reads Total number of mappings % unique mapping Uninfected vascular cells at time point 7 dai ,01 Uninfected vascular cells at time point 7 dai ,73 Giant cells at time point 7 dai ,62 Giant cells at time point 7 dai ,31 Uninfected vascular cells at time point 14 dai ,28 Uninfected vascular cells at time point 14 dai ,40 Giant cells at time point 14 dai ,32 Giant cells at time point 14 dai ,91 Total ,18 Reads (2/fragment) Coverage of the rice genome 26,86 Coverage of the rice transcriptome 97,63 17
18 Inside giant cells: Primary metabolism induced Secondary metabolism suppressed 7 dpi SA and JA-pathway strongly suppressed Chlorophyll biosynthesis induced 14 dpi 18
19 Epigenetic marker genes are induced inside giant cells Gall 7 dpi vs. Control Giant Cell 7 dpi vs. control cells Giant Cell 14 dpi vs. control cells Annotation RNAi LOC_Os01g ,02 1,98 0,73 argonaute LOC_Os01g ,58 1,37 NE AGO4-2 LOC_Os10g ,35 7,03 0,01 Dicer LOC_Os04g ,45 1,90 0,76 Dicer LOC_Os04g ,78 4,59 2,04 RNA-dependent RNA polymerase LOC_Os01g ,19 0,54-6,89 RNA-dependent RNA polymerase LOC_Os02g ,26 4,70 3,79 PINHEAD LOC_Os04g ,91 2,45 0,35 PINHEAD Histone modification LOC_Os01g ,00 3,99 NE histone-lysine N-methyltransferase SUVR3 LOC_Os01g ,16 4,25 4,25 histone-lysine N-methyltransferase, H3 lysine-9 specific SUVH1 LOC_Os07g ,09 5,87-0,15 histone-lysine N-methyltransferase LOC_Os08g ,03 7,56 4,91 histone-lysine N-methyltransferase ASHR2 LOC_Os12g ,88 4,89 2,20 histone deacetylase 6 LOC_Os05g ,88 5,53-3,11 histone deacetylase 19
20 Root knot nematode infection also leads to systemic defence suppression (next to the strong local defence suppression) At 3 dpi almost all defence-related genes are suppressed in systemic shoot tissue 20
21 Acknowledgements Anne-Sophie Petitot Diana Fernandez Ruben Verbeek Henok Zemene David De Vleesschauwer Monica Höfte 21
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