Investigations into the mechanisms of activation of the MrRUN1 and - PowerPoint PPT Presentation

Investigations into the mechanisms of activation of the MrRUN1 and MrRPV1 resistance proteins and the signal transduction pathways leading to resistance to grapevine powdery and downy mildew Ian Dry, Ling Yin, Laura Davies, Junjie Qu, XinLong Li, Hayden Burdett, Simon Williams, Jiang Lu & Bostjan Kobe CSIRO Agriculture & Food

Portan + MrRUN1 Portan Tempranillo Tempranillo + MrRPV1

MrRUN1 & MrRPV1 encode TIR-NB-LRR proteins TIR NB LRR Signal ATP hydrolysis Effector recognition transduction TIR = Toll/Interleukin receptor domain Current model of TIR-NB-LRR activation ( based on L6 rust resistance protein ) NB = Nucleotide-binding domain LRR = Leucine-rich repeat domain TIR Signaling partner(s) Resting Activation self-association recruitment effector Immune Immune responses responses ADP bound ATP bound “OFF” “ON”

MrRUN1 & MrRPV1 encode TIR-NB-LRR proteins TIR NB LRR Signal ATP hydrolysis Effector recognition transduction Current model of TIR-NB-LRR activation ( based on L6 rust resistance protein ) Resting Activation effector ADP bound ATP bound “OFF” “ON”

MrRUN1 & MrRPV1 encode TIR-NB-LRR proteins TIR NB LRR Signal ATP hydrolysis Effector recognition transduction Current model of TIR-NB-LRR activation ( based on L6 rust resistance protein ) TIR Signaling partner(s) Resting Activation self-association recruitment effector Immune Immune responses responses ADP bound ATP bound “OFF” “ON”

LRR domain is involved in pathogen effector recognition Transgenics recovered  MrRUN1:MrRPV1-LRR TIR NB LRR  MrRPV1:MrRUN1-LRR TIR NB LRR Downy mildew inoculation Powdery mildew inoculation 70 % PCD in penetrated cells 100 60 % Sporulation 80 50 40 60 30 40 20 20 10 0 0

MrRUN1 & MrRPV1 encode TIR-NB-LRR proteins TIR NB LRR  Signal Effector recognition ? transduction Current model of TIR-NB-LRR activation ( based on L6 rust resistance protein ) TIR Signaling partner(s) Resting Activation self-association recruitment effector Immune Immune responses responses ADP bound ATP bound “OFF” “ON”

The TIR domain induces cell death in tobacco LRR TIR NB MrRGA2-TIR:YFP MrRPV1-Full Length TIR TIR YFP MrRPV1-TIR Agro only MrRPV1-TIR:YFP N. tabacum cv. White Burley – 5 dpi

Crystal structure of MrRUN1 and MrRPV1 TIR domains reveals a conserved dimeric interface α AE Interface αE αA αA αE

Dimer formation is required for TIR signal transduction MrRUN1 MrRPV1 MrRPV1-TIR R49A MrRPV1-TIR H42A D41 D44 S171 S177 αE αE αA αA MrRPV1-TIR E170 E176 D41A H42 H45 H42 H45 MrRPV1-TIR αA αA MrRPV1-TIR E170 E176 S171 S177 αE αE Agro alone D41 D44 D41-S171 & H42-E170 D44-S177 & H45-E176 Williams, Yin et al . (2016) Front. Plant Sci. 7:1850

MrRUN1 & MrRPV1 encode TIR-NB-LRR proteins TIR NB LRR   Signal Effector recognition transduction Current model of TIR-NB-LRR activation ( based on L6 rust resistance protein ) TIR Signaling partner(s) Resting Activation self-association recruitment effector Immune Immune responses responses ADP bound ATP bound “OFF” “ON”

Many organisms have proteins with TIR domains Ve et al . (2015) Apoptosis 20:250-61

SARM1 = sterile alpha and Toll/interleukin-1 receptor motif-containing 1 Gerdts et al . (2016) Neuron 89: 449-460 Essuman et al . (2017) Neuron 93, 1334-1343 OH SARM1-TIR NADase NAD + ADP-ribose Nicotinamide

A conserved glutamate residue is required for NAD cleavage activity in SARM1-TIR  B  C  C MrRUN1-TIR MrRUN1-TIR E100A L6-TIR L6-TIR E135A MrRPV1-TIR MrRPV1-TIR E100A

Proposed mode of action of MrRUN1 & MrRPV1 TIR Signalling Resting Activation oligomerization effector NADase Immune activity? responses ADP bound ATP bound “OFF” “ON” Tempranillo Tempranillo + MrRPV1 Portan Portan + MrRUN1

Effector-triggered MrRPV1-dependent gene expression in transgenic downy mildew resistant Shiraz • 85 genes found to be significantly upregulated in MrRPV1- Shiraz vs control Shiraz at 18hpi with downy mildew Stilbene synthases PR proteins Receptor-like kinases Transcription factors Heat-shock 70s Calcium-binding proteins Phenylalanine ammonia lyases Cytochrome P450s Ascorbate oxidases Other

Is effector-triggered MrRPV1 resistance mediated by stilbenes? Stilbene Pv growth biosynthesis inhibited Activated RESISTANCE MrRPV1 Reduced Programmed availability cell death of nutrition