Methods S1: Vector constructions and transformation of yeast and fungal isolates
pBIN, a vector suitable for transient expression experiments, was used to express
AvrLm4-7 in tobacco leaves, alone or fused to eGFP. eGFP coding sequence was amplified
from plasmid peGFP (Clontech, Mountain View, CA, USA) using primers pBINeGFPXbaUp (which introduces a XbaI restriction site) and pBINeGFP-SacLo (which introduces a
SacI restriction site). eGFP PCR product was digested by XbaI and SacI and ligated into
XbaI-SacI digested pAG1 (Bourreau et al., 2006). AvrLm4-7 fragment without signal peptide
was amplified from cDNA of v23.1.3 mycelium using respectively primers pBINAvr47noPSXbaUp (which introduces a XbaI restriction site) and pBINAvr47noStop-XbaLo or
pBINAvr47Stop-XbaLo (which introduces also a XbaI restriction site). The PCR products
were digested by XbaI and ligated into XbaI digested pBIN-eGFP or pBIN. The constructs,
termed pBIN-A47noPS-eGFP and pBIN-A47noPS were cloned in E. coli, reextracted and
sequenced. Finally, the constructs were introduced into A. tumefaciens strain C58 by
electroporation at 1.5 kV, 200 ohms and 25 µF. A. tumefaciens strain C58 containing carrying
pBIN-eGFP construct was used as control of efficient transient expression in tobacco leaves.
The vector pPIC9 (Invitrogen, Cergy Pontoise, France) was used to express AvrLm47, without signal peptide and with an histidine-tag in the C terminal part of the protein, in P.
pastoris. AvrLm4-7 fragment was amplified from cDNA of v23.1.3 or v23.1.2 mycelium
using primers PIC9-A47-Up (which introduces a SnaBI restriction site) and PIC9-A47-Lo
(which introduces a NotI restriction site and a sequence encoding for a hexa-histidine tag).
AvrLm4-7 PCR product was digested by SnaBI and NotI. AvrLm4-7 fragment was ligated
into SnaBI-NotI digested pPIC9. The construct, termed pPIC9-A47, was cloned in E. coli,
reextracted and sequenced. Preparation of yeast electrocompetent cells and transformation of
P. pastoris GS115 were performed as described by the supplier (Invitrogen, K1710-01).
pPIC9-A4-7 plasmid was extracted from E. coli and digested with SalI, and the linear plasmid
DNA (5 μg) was transformed into 80 μl of competent GS115 cells by pulsed electroporation
using a BTX electro cell manipulator (1,500 V, 25 µF, 200 ohms). Transformed cells were
incubated on minimal dextrose (MD) plates at 30 °C for 4–6 days until colonies appeared.
Phenotyping of transformants was performed by patching the colonies in replica-plating on
minimal dextrose vs. minimal methanol plates. PCR was used to confirm the positive yeast
recombinants containing AvrLm4-7.
AvrLm4-7 and its mutated versions M1 (RAWG motif mutated to qAWG), M2
(HRYRE motif mutated to HqYqE) and M3 (RAWG and HRYRE motif mutated to qAWG
and HqYqE respectively) were synthesized by GenScript (Piscataway, NJ, USA) with codon
optimization for expression in E. coli and introduced in vector pUC57. Constructs were then
transferred into pDONR (Invitrogen, Cergy Pontoise, France) and finally transferred into
other vectors as needed. sAvrLm4-7 constructs were secreted via a soybean PR1a leader
(Anderson et al., 2012) while mAvrLm4-7 constructs lacked a secreted leader sequence. For
flow cytometry experiments, native AvrLm4-7 and the three mutant genes were ligated into
pGAPz-Alpha, in frame with the S. cerevisiae mating factor signal sequence followed by
C-terminal myc epitope and hexa-histidine tags. Linearized pGAPz-Alpha containing the
respective gene of interest was transformed into P. pastoris strain GS115 and selected under
100 ug/mL Zeocin.
The binary vector pPZPNat1, which carries the nourseothricin acetyltransferase nat1
gene (conferring resistance to the antibiotic nourseothricin) under control of the A. nidulans
trpC promoter as a selectable marker, was used for A. tumefaciens-mediated transformation of
L. maculans. Vector pPZPNat1-A4A7 which carries v23.1.3 allele of AvrLm4-7 and vector
pPZPNat1-a4A7 which carries v23.1.2 allele of AvrLm4-7 were as described by Parlange et
al. (2009). PCR-mediated site-directed mutagenesis was performed on these two constructs.
Using the pPZPnat1-a4A7 construct as template, the primers MD1-Up (with a C299 instead of
G299), MD2-Up (with a C305 instead of T305) or MD3-Up (with a A336 instead of C336) were
used in combination with primer AVR47-JN2-Lo (Table S1) in a PCR reaction using the high
fidelity DNA polymerase Taq Phusion (Finnzymes) to generate respectively 487-bp fragment
MP1, 484-bp fragment MP2 or 452-bp fragment MP3. MP1, MP2 or MP3 was then used as a
megaprimer in combination with AVR47-JN2-Up in a Taq Phusion PCR reaction to generate
1843-bp products termed v23.1.3-MD1, v23.1.3-MD2, v23.1.3-MD3, v23.1.2-MD1, v23.1.2MD2 and v23.1.2-MD3. Following restriction with SnaBI and BstXI and gel purification, the
fragments were used to replace the excised corresponding sequence of pPZPNat1-a4A7. The
constructs, termed pPZPNat1-a4A7-MD1, pPZPNat1-a4A7-MD2 and pPZPNat1-a4A7-MD3
were cloned in E. coli, reextracted and accuracy of the point mutations checked by
sequencing. The same strategy was adopted to generate mutated versions of KGLI, RAWG
and RYRE motifs by using primers RxLRMD1-Up (with a C157 instead of A157), RxLRMD2Up (with a G157 instead of A157), RxLRMD3-Up (with a AA157-158 instead of GT157-158),
RxLRMD4-Up (with a AAA157-156 instead of CGT156-158) and RYRE-MD1-Up (with a CA292293
and A299 instead of AG292-293 and G299) and pPZPNat1-A4A7 construct as template.
Finally, the constructs were introduced into A. tumefaciens and used for transformation of
isolate G07E106 (virulent towards Rlm4 and Rlm7). The constructions were then introduced
into A. tumefaciens strain C58 by electroporation at 1.5 kV, 200 ohms and 25 µF.
Transformation of L. maculans was performed as described by de Groot et al. (1998) with
minor modifications (Gout et al., 2006). Fungal transformants were selected on 50 µg/ml
nourseothricin (WERNER BioAgents, Jena, Germany).
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