Bozkurt Et Al. REM1.3Perihaustorial Targeting and Disease Susceptibility

Bozkurt et al. REM1.3perihaustorial targeting and disease susceptibility

SUPPLEMENTAL DATA FILES

Figure S1. Phylogeny of N. benthamiana remorins and design of a REM1.3 VIGS silencing construct.

(A) A parsimony tree of remorin proteins from A. thaliana (red), tomato (blue), potato (yellow) and N. benthamiana (green) built from a 101 amino-acids alignment of a conserved region in the Remorin_C domain. Bootstrap values for 100 replicates are indicated along the main branches. The sequence identifiers correspond to identifiers from Raffaele et al. 2007 for A. thaliana remorins and Solgenomics sequence identifiers otherwise. StREM1.3 orthologs targeted by the Virus-Induced gene silencing (VIGS) construct used in this work is indicated. (B) Nucleotide alignment of StREM1.3 and its N. benthamiana orthologs. The StREM1.3 sequence used as a silencing construct is indicated with green line. Sites predicted to be targeted by 21nt siRNA are shown in red.

Figure S2.Characterization of N. benthamiana plants silenced for REM1.3.

(A) Test for the functionality of the VIGS construct in plants constitutively expressing YFP:REM1.3. Plants were infiltrated with the viral constructs including TRV RNA1 (pBINTRA) and either the remorin silencing vector (pTV00 REM) or an empty vector (pTV00 e.v.) and observed on the day of infiltration and 18 dpi. Silencing of YFP:REM1.3 was assessed by the loss of fluorescence, measured with identical settings. Leaves infiltrated with pTV00 REM and located 6 leaves above on infiltrated plants did not show any fluorescence at 18 dpi. (B) General aspect of plants infiltrated by pTV00 NbPDS (N. benthamiana phytoene desaturase), pTV00 Rem and pTV00 e.v. at 30 dpi. The pTV00 Rem construct did not cause any visible defect beyond what is seen on pTV00 e.v. infiltrated plants. Dpi, days post inoculation.

Figure S3. Details of the molecular and phenotypic characterization of tomato transgenic lines misexpressing tomato REM1.3 ortholog (SlREM1.2).

(A) Western blot analysis of a representative set of plants for the verification of REM1.3 level. The top panel shows anti-REM1.3 Western blot used for the quantification of REM1.3 levels, middle panel shows Ponceau Red staining used for the quantification of total proteins, bottom histogram shows relative REM1.3 level normalized based on total proteins in extracts and expressed as a % or REM1.3 level in WT plant#3. Sense plants with REM1.3 level <150% and antisense plants with REM1.3 level >80% were not included in the next steps of the analysis. (B) Dot plot showing the relationship between the relative REM1.3 level as estimated by Western blot and the relative size of lesions at 4 days post inoculation by P. infestans 88069 for one complete (out of three) biological replicate. Only Sense plants with REM1.3 level <150% and antisense plants with REM1.3 level >80% were included at this stage. Error bars show standard deviation for 3 or 4 lesions measured on each plant. AS, antisense; SD, standard deviation; SE, sense; WB, Western blot; WT, wild type.

Supplementary file 1. Multiple sequence alignment used for the generation of the parsimony tree of figure S1.

Supplementary movie 1. 3D imaging ofYFP:REM1.3 with RFP:AVRblb2 co-localization at the EHM using super-resolution microscopy. Images are obtained at 3 dpi.

Supplementary movie 2. 3D imaging of discrete EHM domains marked by RFP:REM1.3 with GFP:SYT1 at the EHM using super-resolution microscopy. Images are obtained at 3 dpi.

Supplementary movie 3. 3D imaging of discrete EHM domains marked by RFP:AVRblb2 with GFP:SYT1 at the EHM using super-resolution microscopy. Images are obtained at 3 dpi.