Molecular evolution of the AvrLm7 avirulence gene of ... - Inra

Molecular evolution of the AvrLm7 avirulence gene of
Leptosphaeria maculans under resistance gene
selection in the field is driven by its genomic location,
mating and cropping practices
M.H. Balesdent, G. Daverdin, T. Rouxel, INRA BIOGER, Grignon
L. Gout, AgroParisTech, Grignon
J.N. Aubertot INRA UMR 1248 AGIR, Toulouse
X. Pinochet, CETIOM, Grignon

Resistance genes in oilseed rape
R
r
100%
AvrLm1
avrLm1
% acreage
sown with
Rlm1 cv.
Avr
avirulent isolate
80%
60%
avr
virulent isolate
40%
20%
0%
1994 1995 1996 1997 1998 1999 2000
(Rouxel et al, 2003)
Looking for durable resistance genes
How to manage the few R genes in oilseed rape

Resistance genes in oilseed rape
• Can we demonstrate a role of agronomic practices on the generation
and/or selection of virulent isolates
• Can we explain this role with genomic data

Resistance genes in oilseed rape
• A unique opportunity to address these question in real field conditions :
• AvrLm7 / Rlm7 interaction :
> 99.9 % avirulent isolates
< 1% oilseed rape area cropped with Rlm7 in 2005
OSR area (%)
80
70
Avr alleles frequencies
60
50
40
30
20
Rlm1
Rml3
Rlm4
Rlm7
Rlm9
10
0
2001 2003 2005 2008 2010
(Balesdent et al, EJPP 2006, Stachowiack et al, EJPP 2006)
Rlm agronomic use

AvrLm7 (AvrLm4-7) is cloned
AvrLm4-7 (148 aa)
Repeats
MPLSLEIILTLLALSIPTITACREASISGEIRYPQGTCPTKTEALNDCNKVTKGLIDFSQSHQRAWGIDMTAKVQCAPCIT
TDPWDVVLCTCKITAHRYREFVPKIPYSSFSSAPGVIFGQETGLDHDPEWVVNMKARTRGCD
Will enable us to determine which molecular event(s) is (are) responsible for the gain
for virulence when Rlm7 is used
(Parlange et al., Mol. Microbiol, 2009)

The experimental design (2004-2008)
Grignon site
Versailles site
In Versailles:
standard crop sequence; ploughing
(“Cautious system”)
In Grignon:
No crop sequence; superficial
tillage
(“Risky” system)
At both sites :
one Rlm7 cv (Roxet then Exagone)
one cv without R genes (Campala)

Samplings and analyses
Differential
treatment
1 st generation
(“2006”)
2 nd generation
(“2007”)
3 rd generation
(“2008”)
Year
Growing
season
2004-2005 2005-2006 2006-2007
1967 isolates collected & purified, then:
2007-2008
- phenotyped (AvrLm7) (% of virulent isolates /location / year)
- genotyped (neutral, minisatellite markers)
- AvrLm4-7 PCR-amplified and sequenced when relevant

Results:
1-Impact of cropping practices on local populations
50
40
30
20
Frequency (%)
of virulent
avrLm7 isolates
In Grignon : , a rapid, local
increase in virulent isolates
Differential
treatment
In Versailles : , virulent
isolates remained undetectable
10
0
1998 2000 2002 2004 2006 2008 2010

Results:
2- Diversity of molecular events in virulent isolates
• 169 avirulent isolates analysed
(5 haplotypes)
• 679 virulent isolates analysed
(under-expression)
(2 haplotypes)
(2 haplotypes)
(7 haplotypes)
(98 haplotypes for 127 aligned
RIPped sequences!)

Results:
2- Diversity of molecular events in virulent isolates
• examples of protein sequence alignments :
AvrLm7
*
*
SNPs
avrLm7
*
*
*
*
*
*
RIP
*
*
1 bp del
*
*
2 bp del

Results:
3-Virulence mainly generated by sexual matings
• 679 virulent and 169 avirulent isolates analysed
(under-expression)
(2 haplotypes)
(2 haplotypes)
(7 haplotypes)
(98 haplotypes !)
• Nearly all events (97.8%) : loss or inactivation of the gene or drastic effect
on the protein structure
• 87.6 % of molecular events are generated by sexual mating and are favored
by the repeat-rich environment of the Avr gene
• Neutral markers : the same levels of gene and genetic diversity in virulent
and avirulent populations; no population differentiation

Concluding Hypothesis
• Yearly sexual matings generate each year, and locally, virulent
isolates at a high frequency, which are selected by the resistant line
• The “risky” system both increases the population size and favors
sexual matings on stem debris left on the soil
• The first molecular support of the role of cropping practices on
resistance gene durability
Daverdin et al., PLoS Pathogens 8(11): e1003020. doi:10.1371/journal.ppat.1003020

Concluding remarks
• In spite of the ability of L. maculans to generate virulent isolates and rapidly
overcome a resistance gene, the overwhelming of Rlm7 is not comparable to
that of Rlm1
80
70
60
50
40
30
20
10
0
2001 2003 2005 2008 2010
Rlm1
Rml3
Rlm4
Rlm7
Rlm9
100%
80%
60%
40%
20%
0%
100%
80%
60%
40%
20%
1994 1995 1996 1997 1998 1999 2000 2010
AvrLm1
avrLm1
Area
with Rlm1
AvrLm7
avrLm7
Area
with Rlm7
0%
2000 2001 2002 2003 2004 2005 2006
2010

Next steps
- To continue the monitoring of virulent (a7) isolates
at the national level
* new events
* success of one allele
- To understand the molecular bases of the AvrLm3 –
AvrLm7 interaction
- To model and to test in field conditions the
potential durability of the two R genes Rlm3 and
Rlm7 used in different genetic constructions or
agronomic situations

• INRA Grignon BIOGER, “Lepto team”
• Isabelle Fudal (Constructions for AvrLm4-7 complementation)
• Bénédicte Olivier (Transformations)
• Laurent Coudard (Isolate management)
• Martin Willigsecker and Bertrand Auclair (Plant management)
• Thomas Mongin (M2 2010, AvrLm4-7 Fitness)
• CETIOM
• Martine Leflon
• Julien Carpezat
• Sabrina Fouillou
(G. Daverdin Thesis)
Project ADD-Cedre
CTPS Project “EVOLEP”
Département SPE
Next projects :
CTPS2012: “Icoscop”
INRA AAP Presume: “K-Masstec”