Identification of resistance genes against Plasmopara viticola in Vitis

vinifera cultivars for breeding program

Dr. De Lorenzis Gabriella

Dr. Toffolatti Silvia Laura

Maryam Sargolzaei

2nd YEAR - XXXIII CYCLE – A.A. 2018/2019

Prof. Dr. Failla Osvaldo

Biotic and abiotic stresses threat its productivity

are

the causal agent of downy mildew.

infects all the green parts

Introduction: Grapevine Main Diseases Grapevine downy mildew, caused by the obligate biotrophicoomycetePlasmoparaviticola

Introduction: Grapevine Main Diseases

Introduction: Plasmopora viticola

Seeds

Buds and Crowns

1. pre-formed constructive barriers (structural barriers)

✓ Strong cell wall

✓ A prominent wax layer

✓ Dense hairiness

✓ Antimicrobial toxins

2. Specifically induced defense pathway

✓ Induced defense pathway

✓ Triggered immunity (PTI)

✓ R-(resistance)-genes

Code receptors to recognize and bind to effectors

Effectors of P. viticola have recently been described on RNA and genomic sequence level but their function remains to be shown

Introduction: Plant strategies to cope with pathoges:

Agronomic effortsand

fungicides spraying

Introduction

Approachto reduce

the need for fungicides

Resistance sources

identification

Enhance the effectiveness of crop breeding

Mgaloblishvili (Vitis vinifera),

Introduction:

(signal transduction)

Limitation of P. viticolagrowth and sporulation

Resistance response

The key genes in each level of plant fungi interaction:

Cytochrome P45087A3(terpene biosynthesis)MLP-like protein 34 (pathogen related protein biosynthesis)Endo-1,3;1,4-beta-D-glucanase (antifungal recognition)Rust resistance kinase Lr10 (fungal recognition)Ethylene-responsive transcription factor 1B (signal transduction)

Introduction: Putative Resistance Representation Scheme in Mgloblishvili

Introduction: Morphological/ Microscpical Study of P. viticola colonization in leaf

SV = substomatal vesicle; M = mycelium; Ha = haustorium; S = sporangiophore; CA = callose deposition..

1. To characterize 2 functional genes via genome editing to identify genes function

2. To biochemical study of leaf tissue response to pathogen

3. To identify loci related to resistance by QTL analysis

Characterization of two functional genes via genome editing

to identify genes function

Plant Propagation:

Methodology: In vitro culture

Somatic embryogenic culture:

Callus induction media

➢ Bud initiation medium

➢ Nb2 medium

Greenhouse propagated plants (Shoot samples) MS Medium

Bud Open budMicroscope

1 2 3 4 5

Callosity 0% 25% 50% 75% 100%

1 2 3

Color white Bej Black

Consistancy Friable Optimal Aqueous

Methodology: Reference genes: Rust Resistance Kinase & Ethylen responce genes

https://www.ncbi.nlm.nih.gov/gene/?term=XM_002263215.4

>XM_002263215.4 PREDICTED: Vitis vinifera rust resistance kinase Lr10 (LOC100244615), mRNA

>XM_002267534.4 PREDICTED: Vitis vinifera LEAF RUST 10 DISEASE-RESISTANCE LOCUS RECEPTOR-LIKE PROTEIN KINASE-like 2.2 (LOC100262212), mRNA

Ethylene response gene

Rust Resistance kinase

https://www.ncbi.nlm.nih.gov/gene/?term=XM_002263215.4

Single plasmidAll-in-one vector

Cas protein Target-specific gRNA

Bacterial chromosome

plasmid

Co-cultivation

Knock out the genes

Ultimate Outcome

Methodology : Perspective for Future Work

CRISPR-P 2.0. online servicehttp://cbi.hzau.edu.cn/cgi-bin/CRISPR

CR

ISP

R(C

luste

red

Re

gu

larly

Inte

rsp

ace

dS

ho

rtP

alin

dro

mic

Re

pe

ats

)

CLONING1. Vector linearization2. Blunting reaction3. Adding T to the carrier4. Ligation5. Transformation of E. coli for heat shock6. Plasmid DNA extraction7. Sequencing

http://cbi.hzau.edu.cn/cgi-bin/CRISPR

Leaf Gro

wth

An

alysis by D

un

can test

Leaf

Nu

mb

er A

nal

ysis

by

On

e w

ay A

NO

VA

Result : MS Media selection for each variety

Mgloblishvili

Pinot Noir

Bianca

Icy

Result : Acclimatization

Hydroponic chamber

MS mediaWell rooted plantlet

Greenhouse

L22

Mgloblishvili Bianca Pinot Noir

Result : Embryogenic cells propagation

Data collection

R Studio v 1.1.463 analysis

In Progress

Characterization of Terpen accumulation in leaves

inoculated with Plasmopora viticola

Methodology :SPME-GC-MS analysis, Expression study

Bianca

Mgaloblishvili

GC-MS analysisVolatiles quantitative data

Statistical analysisSampels quantification

SPME conditions (fibber: DVB/CAR/PDMS 50/30 µm)• Incubation: 30 min at 80°C• Extraction: 60 min at 80°C• Desorption: 20 min

✓ 3 genotypes✓ 3 biological repeat✓ 4 time points:

• T0: 0• T1: 1 dpi• T2: 2 dpi• T3: 3 dpi

Real time quantitative PCR

Gene nameGenBank

Accession

Calculated

primer

efficiency

Forward primer sequence Reverse primer sequence

VvGwECar2 A HM807374.1 1.813 TGCCTCAGCTGTTGAATGCT TGAGGACGGTCATCGGAACA

VvGwaBer HM807376.2 1.560 CCTAGCATTTGGGGCAATAC CCGTTGAACTGCATCGATAA

VvCSaFar HM807379.1 1.824 GGGTGCACGTTGCTTCTAGT TGGCATCAGCACTGGTGTAG

VvCSbOciM B HM807387 1.913 GGAACATCACTGGATGAGTTGA ATCTCCATGCTGATACATGCAC

VvTer AY572987.1 1.837 AGAGTCTCCATTCCCTGAAACA GGGCTCAACGAGTAATGACAA

VvPNGer HM807399.1 1.986 ATCTTCCTTTGTCGCTCCTT CCGCATGTGGAGATAGAGTT

, 11-Feb-2019 + 18:56:47Grape leaf 100 mg - terpenes

3.01 8.01 13.01 18.01 23.01 28.01 33.01 38.01 43.01Time0

100

%

110219_4 Scan EI+ TIC

1.16e911.30

3.24

6.85

25.16

19.20 24.25

27.21

29.04

, 13-Feb-2019 + 23:07:08Grape leaf 100 mg - terpenes non inoculato

3.01 8.01 13.01 18.01 23.01 28.01 33.01 38.01 43.01Time0

100

%

130219_5 Scan EI+ TIC

1.25e911.22

6.87

3.26

24.86

16.66

21.8619.66

27.17

25.13

28.97

27.25 29.04

Violet: Mgloblishvili, resistant variety

not-inoculated inoculated

Results :

Total 502 identified volatile compounds (VOCs)

32 selected VOCs

Typical substance classes

• Alkanes• Alkenes• Aldehydes• Ketones• Aromatic compounds • Terpenes

Scatter Plot

In Progress

Identifying

the loci related to resistance by

QTL analysis

Plant material collection

• 95 individual of green house propagated self-pollinated ‘’Mgaloblishvili’’

• Wooden cutting and propagation in climate chamber for at least 4 weeks

Molecular analysis

• DNA extraction

• QTL external service

➢ to identify genomic regions associated with resistance responce

➢ Chip array 18K SNP for grape are available

phenotypical study

• leaf disc assay

• Microscopically study 7 dpi

Methodology : QTL identification

In Progress

PhD Secondement

Institute for Grapevine Breeding Geilweilerhof(Coordinate: 49.218368, 8.047236)

Prof. Dr. Eva Zyprian

Identification and characterization of the Plasmopora viticola secretome by de novo transcriptome analysis

Maryam Sargolzaei*, Sarah Fröbel and Eva Zyprian#

Julius Kühn-Institut – Federal Research Centre for Cultivated Plants, Institute for Grapevine Breeding

Geilweilerhof, 76833 Siebeldingen, Germany

*Current address: Department of Agriculture and Environmental Science, University of Milan, Milan, Italy

Introduction: Plasmopora Viticola

https://www.ncbi.nlm.nih.gov/genome/genomes/11018

https://www.ncbi.nlm.nih.gov/genome/genomes/11018

2008-059-121 (Rpv10/Rpv3)(GF.GA-52-42x ‘Solaris’)

2008-059-020 (Rpv-/Rpv-) 2011-003-013 (Rpv10/Rpv10)(self-pollination of ‘Solaris’)

Methodology: Plant samples

1. Plant Sample

2. Plant-Pathogen Inoculation

3. RNA sequencing NGC Illumina technology_HiSeq 2500 technology

• Library preparation• Deep sequencing• Data analysis

Methodology: Gene Prediction

EffectorP

ApoplastP

SignalP

AGUSTUS

NGC Sequencing

Bioinformatic Analysis

Secretome Prediction

Methodology: Gene Prediction

Map Reads to Vitis vinifera Reference genomePN40024 (12Xv2Vv_Genome_edit)

Molecular Biology Tools

NGS Core Tools

Trim Sequences

Not mapped Reads

Map Reads to Plasmopara viticola genome (MTPI01.1_AGUSTUS-v331)

Selection (RPKM>2)

Mapped Reads

Map Reads to Plasmopara_viticola_INRA-PV221.assembly.PBv1

Annotated

Un-Annotated

3rd Package

2nd Package1st Package

Pv_augustus-v331_model_organism

Methodology: Gene Identification & DEG & Pathogen development in host

Solaris (Rpv10/Rpv3)Gutedel (Rpv-/Rpv-) Rondo (Rpv10/Rpv-)

Zarya Severa × St. Laurent

1. Plant samples

2. Leaf disc inoculation

3. RNA extraction

4. RT-qPCR

5. Microscopically study

1. S

amp

ling

2. S

po

re c

olle

ctio

n

3. L

eaf

dis

c in

ocu

lati

on

Sp.

6 hpi

24 hpi

7 dpi

Fluorescent microscopy

RNA extraction

Methodology: Spore Collection and Leaf Disc Assay (P. viticola development screening)

Natural diseased leaves

HealthyPropagated in Greenhouse_JKI

Spores in all cells /Cells no. x Chamber depth (0.1 mm) x Dillution (1) = spores. µ-3

Results: Map to P. viticola genome (MTPI01.1-v331)

✓ Row reads trimming: 99.8%✓ Map to Vitis vinifera reference genome: 88-90% of reads✓ Map to Plasmopara viticola genome: 8-14% of reads

C

A

B

A: Apoplastic effectors B: Signal C: Effector Other Total

0 hpi 6 hpi 0 hpi 6 hpi 0 hpi 6 hpi 0 hpi 6 hpi 0 hpi 6 hpi

2008-059-02_ Rpv-_Rpv- 8 156 5 224 3 349 25 2148 41 2877

2008-059-121-Rpv10_Rpv3 6 197 10 279 4 436 5 3230 19 4142

2011-003-013_Rpv10_Rpv10 5 201 4 246 2 362 27 2362 38 3171

A

BC

6 hpi0 hpi

Results: Genes Identification and putatie characterization using BLAST algorithm

Results: Common Regions between P. viticola genome, annotated by RNA-Seq reads, ROI selected by LGRM Algorithm

Results: GOI analysis

ORF finding

Motif analysis

GOI identification

Submission no. genes activity QC Accession Proteins2264165 Avirulent 63% XM_009537882 Effectors2265656 Hypothethical avirulent protein 32% XM_008901824.1 Effectors2265660 Dehydrogenase, plasmopora trasmembrane protein 51% XM_024721234.1 Signal2265662 Cellulose synthase 92% GQ258973.1 Apoplast Effector2265665 Ras GTPase-activating-like protein and signalling protein 57% XM_002905745.1 Effector2265671 Regulator G protein signaling 61% XM_002898161.1 SignalDEG analysis

Genotype Gene

ID

6 hpi 24 hpi

Expression change

2008-059-020 sus 2264165 11,571 up 16,151 up2265656 4,457 up 11,614 up2265660 14,512 up 5,681 down2265662 30,071 up 240,599 up2265665 0,682 inconsiderable 0,375 inconsiderable2265671 0,537 inconsiderable 0,027 inconsiderable

2011-003-013

Rpv10/Rpv10

2264165 6,034 up 1,940 down2265656 2,007 up 0,810 down2265660 3,474 up 2,330 down2265662 72,491 up 35,777 down2265665 0,098 inconsiderable 0,092 inconsiderable2265671 0,164 inconsiderable 0,060 inconsiderable

2008-059-121 Rpv10/Rpv3 2264165 13,397 up 1,856 down2265656 2,853 up 1,112 down2265660 4,769 up 0,685 inconsiderable2265662 498,056 up 10,125 down2265665 0,407 up 0,094 inconsiderable2265671 0,140 inconsiderable 0,014 inconsiderable

DEG analysis

Results: GOI analysis

✓ WS21-Pv and UBC-Pv reference✓ Calculated as 2-ΔΔCT

0,000

5,000

10,000

15,000

20,000

25,000

30,000

a b c d e a b c d e a b c d e a b c d e a b c d e a b c d e

Gutedel (-/-) Rondo (Rpv10/-) Solaris(Rpv10/Rpv3)

2008-059-020sus

2011-003-013Rpv10/Rpv10

SolarisRpv10/Rpv3

6 hpi 24 hpi

Exp

ress

ion

FC

-200

-100

0

100

200

300

400

500

600

700

Gutedel (-/-) Rondo (Rpv10/-) Solaris(Rpv10/Rpv3)

Gutedel (-/-) Rondo (Rpv10/-) Solaris(Rpv10/Rpv3)

6 24

Results: P. viticola development in leaf tissue

A: Gutedel (Rpv–/Rpv–) B: Rondo (Rpv10/-) C: Solaris (Rpv10/Rpv3)

Mycellium (M) Sporangiophores (S)

Fluorescent microscopy

H

H

H

H

H

H

M M

M

Conclusion

The results can lead to

• a better understanding of the pathogenicity mechanism of P. viticola

• provide insight into improvement of the resistance strategies against pathogen

Further investigation is needed to study the effectors, apoplast and signaling proteins in pathogen, to characterize them in detail

Emas

cula

tio

nP

olli

nat

ion

Cross breeding

Dr. Oliver Trapp

See

ds

pla

nta

tio

n

Mo

lecu

lar

anal

ysis

Cross breeding

Supplementary Data

Introduction: Plasmopora Viticola

https://www.ncbi.nlm.nih.gov/genome/genomes/11018

https://www.ncbi.nlm.nih.gov/genome/genomes/11018

Introduction: Plant-Pathogen Interaction

SA

JA ETAuxin BR

plant plasma membrane

Pathogen Effectors:

Apoplastic Area:

First compartment when Pathogen-host

interaction occur

ET signaling

Pathogen effectors

Effectores recognition

Ultimately defence

mechanism activation

ETI

Rust resistance Nucleus

Ethylen-responsive transcription

ACS

ACC

ET

specifically recognizing effectors of the fungus

Resulting weakening of fungal cell walls

Introduction: