intergeneric transfer and functionality of a major apple ... transfer and functionality of a major...

Intergeneric transfer and functionality of a major apple scab

resistance gene

NOM DE L’AUTEUR JOUR / MOIS / ANNEE

Perchepied L, Vergne E, Gaillard S., Pelletier S., Ravon

E., Berthelot P., Cournol R., Chevreau E

Institut of Research on

Horticulture and Seeds

8th International Rosaceae Genomics Conference, 21-24 June 2016, Angers, France

Scab : the most important fungal disease of apple and pear

symptoms of Venturia inaequalis

on apple

symptoms of Venturia pirina

on European pear

Non-host resistance : V. inaequalis on pear and V. pirina on apple (co-adaptation)

8th International Rosaceae Genomics Conference, 21-24 June 2016, Angers, France1

Context and objectives : apple and pear scab

M. domestica / V. inaequalis : a well-known

pathosystem

many major genes and QTLs

P. communis / V. pirina : few genetic

information

strong interactions isolates/ varieties

Objectives of the project:

1) determine the degree of functional transferability of

Rvi6 (= HcrVf2) from apple to pear, towards V. pirina

2) compare the mode of action of Rvi6 in transgenic

apple and pear plants in interaction with V. inaequalis

and V. pirina at different levels

Hypothesis:It is possible to extend the natural range of a plant R gene if:

- homologous effectors are recognized

- a downstream signalization pathway is retained, leading to efficient defenses

Context and objectives: functional transfer of an apple R gene

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State of the art (Wulf et al. 2011 Curr Opinion plant Biol, 14:468-476)

- transfer of R genes between sexually incompatible species: mostly among Solanaceae

- in general, transfer of resistance to a pathogen of the same species


Belfanti et al. 2004, PNAS 101:886-890

- pear variety Conférence (scab susceptible)

- Agrobacterium-mediated transformation with pMF1 (from PRI)

cds Rvi6pMdRbc tMdRbc

Results: production of pear transgenic clones

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78 transgenic clones (12% transfo)

- PCR analysis + cytometry control: 13 independent clones selected

- rooting and greenhouse acclimatization

Very efficient transformation


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Results: expression of the transgene Rvi6

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Q-RT-PCR on 11 clones: high expression

Ratios similar to those observed on transgenic Gala + prom MdRbc-Rvi6 (Joshi et al 2011)

spring 2013 autumn 2013


- scab inoculation on own-rooted plants in greenhouse

- 3 strains of V. pirina 98, 102, 137, all aggressive on Conférence

- inoculum concentration: 200 000 spores/ml

- final observation at 42 days after inoculation

Results: scab resistance evaluation

8th International Rosaceae Genomics Conference, 21-24 June 2016, Angers, France25

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VP102

All transgenic clones are resistant to strain VP102 (test in spring 2013)

Apple qualitative scale (Chevalier et al 1991) Apple quantitative scale (Croxall et al 1952)

Results similar to those on transgenic Gala + prom MdRbc-Rvi6 inoculated with several

strains of V. inaequalis (Joshi et al 2011)

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)Days after inoculation

control CF

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VP102

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VP137

VP98

Results: scab resistance evaluation


At least one strain of V. pirina

overcome Rvi6 resistance in

transgenic pears (test in autumn 2013)

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Transgenic pear resistance takes various forms

control CF + VP102, at d+21

strong sporulation on adaxial side

strong sporulation on abaxial side

transgenic clone + VP102, at d+21

pin-points on adaxial side

chlorosis and crinkling

on adaxial side

slight sporulation

on abaxial side

Results: macroscopic observations


Transgenic pear resistance blocks conidiophore development

numerous conidiophores bearing conidia

aborted conidiophores

Results: microscopic observations


- samples fixed in 4% glutaraldéhyde and observed with scanning-electronic microscope

- samples stained with solophenyl flavine and observed with wide-field epifluorescence microscope

control CF + VP102, at d+14 transgenic clone + VP102, at d+14

RNAseq on pear samples

Design of the first expression microarray

covering all the pear transcripts: “pear V1”

(43 906 probes)

Results: transcriptomic analysis


Existing apple microarray “AryANE V2”

(74 214 probes)

(Gaillard and Celton et al. 2014 New Phytol 203: 287-299)

samples collected at T0, 8h, 24h, 72h and 7 days after inoculation

Genotype Scab strain Class of

interaction

Genotype Scab strain

Class of

interaction

Conférence V. pirina 102 susceptible Conférence V. inaequalis EUB05 non host

60AU V. pirina 102 resistant 60AU V. inaequalis EUB05 non host

Gala V. pirina 102 non host Gala V. inaequalis EUB05 susceptible

Gala-Vf V. pirina 102 non host Gala-Vf V. inaequalis EUB05 resistant

- hybridization on « pear V1 » and « AryANE V2 »

- identification of significant differential expression

1st step: construction of a complete pear microarray

3rd step: microarray analyses (underway)

2nd step: extraction of RNA from a complete range of interactions

PMdRbc HcrVf2-11

from PRI

Main findings so far:

Next steps:

- are the defense pathways activated by Rvi6 in apple and in pear similar ?

- are the non-host mechanisms (interactions apple/V. pirina and pear/V. inaequalis)

similar to the host resistance mediated by Rvi6 ?

- what is the conserved effector between V.pirina and V.inaequalis which is

recognized by Rvi6 ?

- what is the evolutionary signification of these findings ?

Conclusion and perspectives


- first example of a successful intergeneric transfer of R gene among Rosaceae, with a

resistance gene functioning towards another species of pathogen

- Rvi6 resistance to V. pirina in pear is morphologically similar to Rvi6 resistance to V.

inaequalis in apple

- at least one strain of V. pirina possesses virulence towards Rvi6

Team IRHS-ResPom

Patricia Berthelot

Raphaël Cournol

Laure Perchepied

Elisa Ravon

Emilie Vergne

Greenhouse team IRHS

(INEM)

Microscopy platform (IMAC)

Mayeul Millien

Fabienne Simoneau

Team IRHS-BioInfo

Sylvain Gaillard

Sandra Pelletier

Team IRHS-EcoFun

Marie-Noëlle Bellanger

Pascale Expert

Bruno Le Cam

Thanks to:Thanks to:

PRI (Wageningen)

Henk Shouten

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CF 60AT 60AU Gala GalVf2

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Non-host interactions : qualitative note at d+42

V. pirina 102 V. inaequalis EU B05

non-host interaction non-host interaction

Non-host interactions : macro and microscopic observations on pear


pin points on Rvi6 pear

clone + V. inaequalis EUD42

frequently symptomless

D+ 21 D+ 14

conidia

appressorium + local HR

branching

germination

chlorosis on control Conference +

V. inaequalis EUD42

Non-host interactions : macro and microscopic observations on apple


D+ 21 D+ 14

conidia

appressorium + local HR

branching germination

and pin point

Macroscopically : symptomless

on Gala + V. pirina 102 or

Gala Vf + V. pirina 102

intergeneric transfer and functionality of a major apple ... transfer and functionality of a major...

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