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

Experimental system: Potato - PVY

potato tuber ring necrosis disease

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.

Small RNA-seq results were confirmed by RT-qPCR

6 selected miRNAs analysed

• 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