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The role of small RNAs regulatory networks in tolerance - linking
developmental and immune signalling in potato
Kristina Gruden
Department of Biotechnology and Systems biology
National Institute of Biology, Ljubljana, Slovenia
Plant stress response operates through complex signaling network
Genotype
PhenotypeNetwork
X
or
Pathogen/
Pest
x
ENV
xMICROBIOME
Disease
Resistance
orTolerance
Potato – PVY interaction
• Differential sensitivity of different cultivars
Sensitive, tolerant, HR, ER
• Plants with perturbed immune signalling available: nahG, coi1 transgenics in different genetic backgrounds
Systems biology approachTo study underlying mechanisms
Functional gene analysis
Transcriptomicsproteomics
Data analysis, modeling
Hypothesisgeneration
The role of smallRNA network in potatoimmune signaling?
Transcriptomicsmicroarrays, qPCR,
and Proteomics
…smallRNAomics
- time series analysis
Plant small RNAs
• miRNAs ; siRNAs
• 18-24 nt long , single stranded, ncRNAs
• Targeting mRNAs , viral RNA
• plant growth, development, abiotic stressresponse and biotic reponses
• shown to be directly involved in regulation of resistance genes
• amplification of miRNA silencing -
triggering production of phasiRNAs
Degradation Translation inh
Plant inoculation
Leaf sampling 3 dpi
RNA isolation
Experimental setup
library preparation
tolerant Désirée susceptible NahG-Désirée
Identification of miRNAs / phasiRNAs/ vsiRNAs
Finding small RNA targetssmall-RNAseq
Illumina
The role of sRNAs in regulation of tolerant response to PVY?
Expression analysis
SALICYLIC
ACID
Different analyses formiRNAs/phasiRNAs/vsiRNAs identification
small RNA-seq
miRNAs can be identified by their characteristic fold-back precursors
novel miRNAs
miRCat; miRPlant
141 novel miRNAs
known miRNAs
mapping to miRNAs from miRBase
database
vsiRNAs
mapping to PVYNTN
genome
~ 58000 vsiRNAs328 known miRNAs
phasiRNA
PHASI-predictiontool
2558 phasiRNAs
Plant inoculation
Leaf sampling 3 dpi
RNA isolation
Experimental setup
library preparation
tolerant Désirée susceptible NahG-Désirée
Identification of miRNAs / phasiRNAs/ vsiRNAs
Finding small RNA targetssmall-RNAseq
Illumina
The role of sRNAs in regulation of tolerant response to PVY?
Small RNA expressionanalysis
SALICYLIC
ACID
sRNA are regulated following PVY infection
• Infection predominantly caused an increase in miRNAs levels.
• Response on the sRNA level attenuated in NahG-Désirée.
• Several sRNAs are SA-dependent
PVY regulated
SA regulated
Small RNA-seq results were confirmed by RT-qPCR
Plant inoculation
Leaf sampling 3 dpi
RNA isolation
Experimental setup
library preparation
tolerant Désirée susceptible NahG-Désirée
Identification of miRNAs / phasiRNAs/ vsiRNAs
Finding small RNA targetssmall-RNAseq
Illumina
The role of sRNAs in regulation of tolerant response to PVY?
Small RNA expression analysis
SALICYLIC
ACID
Targets of differentially expressed small RNAs
1. in silico (psRNATarget)
reverse complementary matching between small RNA and target transcript
distinguishes cleavage /translational inhibition
Experimental confirmation of degradation predictions2. PARE / degradome sequencing
parallel analysis of RNA ends
transcripts
Small RNA sequences
Integration of small RNA-target results with the expression of their targets (data available in our lab)
PVY induced changes in sRNA regulatory network at the onset of viral multiplication are controlling
multiple immune signaling components
Désirée NahG-Désirée
miRNA and phasiRNAs regulate immunereceptor genes
• Regulation of NBS-LRRs already discovered
• Novel link: regulation of LRR-RPKs
• reduced miR6022 expression level increased LRK expression only Desiree
• Relieved silencing tolerance ?
Downregulated
Upregulated
miR393-TIR/AFB network regulates diverseimmune signaling components
• miR393-mediated cleavage of transcripts encoding multiple members of TIR/AFB gene family (auxin receptor genes)
• network is targeting also other phytohormone signalling pathways
EthyleneJasmonic acidBrassinosteroidsAuxin
miRNAs and phasiRNAs negatively regulategibberellin biosynthesis
DésiréeB
IOS
YN
TH
ES
IS
DownregulatedUpregulated
GA3ox gene downregulated
vsiRNAs !!
Lower level of gibberellin in tolerant plants
Down-regulation of GA biosynthesis enzymes lower level of GA3
Désirée
BIO
SY
NT
HE
SIS
miRNAs and phasiRNAs negatively regulategibberellin biosynthesis
BIO
SY
NT
HE
SIS
DownregulatedUpregulated
Désirée NahG-Désirée
miRNAs - GA regulatory circuit identified
Désirée Analysis of miR6022 promoter region
GAMYB (StMYB33) binding sites were detected in miR6022 and miR482f promoter region
link between GA signalling and R-gene expression
SIG
NA
LL
ING
miRNA regulatory network and transcriptional regulation are tightly interconnected!
sRNA regulatory network discovered as a link between immune and developmental signaling establishment of tolerance
This regulation is under control of salicylic acid deficiency attenuates GA regulatory circuit and leads to development of disease
Additionaly similarities in response of sRNA network between this and other tolerant/ symbiotic interactions discovered
Conclusions
sRNA regulatory network
Immunesignalling
Developmentalsignalling
Disease symptoms - Tolerance
• Maja Križnik
• dr. Marko Petek dr. David Dobnik
Acknowledgments
• Funding
dr. Špela Baebler
• prof. dr. Stephan Pollmann
• dr. Jan Kreuze
Many sRNA are regulated following PVYNTN
infection
Infection predominantly caused an increase in miRNA¸and phasiRNA levels.
• Several DE miRNAs/phasiRNA found
only in Desiree—tolerance?
• Regulation of auxin, GA
pathways
Correlated with negative regulation on gene expression level, metabolite level
• Link Growth – Defence - Tolerance
miRNA
Auxinpathway
GA pathway
Immunity/
Tolerance
immunereceptors
phasiRNA
Growth
Summary
vsiRNA