HaRxL44 interacts with and destabilizes MED19a in a Proteasome-dependant manner.

<p>(A) Schematic representation of the relevant interactions obtained by Y2H between HaRxL44 and <i>Arabidopsis</i> cDNA library. Data extracted from Mukhtar et al. (2011) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001732#pbio.1001732-Mukhtar1" target="_blank">[17]</a>. (B) Subcellular localisation of GFP-BOI and GFP-MBR1–like determined by transient expression in <i>N. benthamiana.</i> (C) Immunoblotting of protein extracted from <i>N. benthamiana</i> leaves after transient assay, in presence or not of MG132 for 4 h. (D) Immunoblotting of protein extracted from <i>N. benthamiana</i> leaves after transient assay. Note the co-immunoprecipitation (Co-IP) of HA-HaRxL44 with GFP-MED19a in the presence of proteasome inhibitor.</p

MED19a is a positive regulator of nuclear immunity against <i>Hpa</i>.

<p>(A) Schematic diagram of T-DNA insertions in <i>MED19a</i>. (B) <i>MED19a</i> expression in <i>med19a-1</i> and <i>med19a-2</i> mutants. (C) Representative images of the phenotype observed in 4-wk-old floral stem of Col-0, <i>med19a-1</i>, <i>med19a-2</i>, and <i>med19a</i> mutant complemented line C1. (D) Developmental phenotype of <i>Arabidopsis</i> transgenic lines OE-MED19a compared to Col-0. (E) Immunoblot of the Co-immunoprecipitation analysis between GFP-MED19a and MED6 and MED7. Arrows point out the interaction detected between GFP-MED19a and MED6 and MED7. (F) Subcellular localisation of GFP-MED19a in <i>Arabidopsis</i> plant. Scale bar, 5 µm. (G) Immunoblot of proteins extracted from two independent lines expressing GFP-MED19a. Stars indicate the expected size for GFP-MED19a. Notice the upper bands in the blot that might suggest posttranscriptional modifications. (H) Monitoring of <i>Hpa</i> sporulation at 5 DAI in control lines (Col-0 and GFP), <i>med19a</i> mutant complemented lines (C1 and C2), Mediator mutants, and MED19a OE lines. Error bars represent the standard error of the mean. Asterisks represent the significance of individual unpaired <i>t</i> tests comparing the given column with the control (<i>p</i> value<0.01).</p

Interaction between MED19a and HaRxL44 is important for HaRxL44–induced MED19a degradation via proteasome.

<p>(A) Immunoblotting of proteins, extracted from <i>N. benthamiana</i> leaves after transient assay. Note the absence of Co-IP of HA-HaRxL44^M with GFP-MED19a. (B) Co-localisation analysis between GFP-MED19a and RFP-HaRxL44 or HaRxL44^M determined by transient assay in <i>N. benthamiana</i>. Note the lack of GFP-MED19a in the presence of RFP-HaRxL44 (arrow) but not HaRxL44^M. (C) Quantification of the number of fluorescent nucleoplasm observed in nucleus transformed with GFP-MED19a in the presence or not of RFP-HaRxL44 or RFP-HaRxL44^M. All the confocal pictures were taken with PMT 1 (494–541 nm) at Gain: 864 and PMT 2 (591–649 nm) at Gain: 844. Note the decrease in GFP-MED19a transformed cells in the presence of RFP-HaRxL44 in comparison with RFP alone or RFP-HaRxL44^M.</p

HaRxL44 destabilizes MED19a <i>in planta.</i>

<p>(A) Subcellular localisation of GFP-HaRxL44 (in green), RFP-MED19a (in red), and YFPc-HaRxL44 + YFPn-MED19a (BiFC, yellow) obtained by transient expression in <i>N. benthamiana</i>. n, nucleus. (B) Western blot analysis of protein extracted after transient expression of DEX::HaRxL44-GFP with RFP or RFP-MED19a in the presence or not of dexamethazone (DEX). Note the decrease in the level of MED19a observed in the presence of HaRxL44. (D) Co-localisation analysis between GFP-MED19a and nuclear-HaRxLs determined by transient assay in <i>N. benthamiana</i>. Note the lack of GFP-MED19a in the presence of RFP-HaRxL44 (arrow).</p

HaRxL44-expressing lines, <i>med19a</i> mutants show elevated JA/ET signalling, which is also observed after <i>Hpa</i> infection.

<p>(A and B) qRT-PCR results on <i>PDF1.2</i> marker gene. Data are presented as average fold induction compared with control of three biological replicates ± SD. (C) Expression pattern of <i>PDF1.2</i> during a time course of <i>Hpa</i> Waco9 infections in <i>Arabidopsis</i> Col-0 extracted from expression profiling experiment. (D) Expression pattern of <i>HaRxL44</i> during a time course of <i>Hpa</i> Waco9 infection in <i>Arabidopsis</i> Col-0 analysed by qRT-PCR. Data are presented as average fold induction compared with control of three biological replicates ± SD. (E) Monitoring of <i>Pst</i> growth in <i>Arabidopsis</i> Col-0. DC_Emp, <i>Pst</i> DC3000 strain carrying EDV vector; COR-_Emp, <i>Pst^COR</i>⁻ strain carrying EDV vector, COR-_44, <i>Pst^COR</i>⁻ strain carrying EDV-HaRxL44. Error bars represent the standard error of the mean (Tukey–Kramer test, <i>p</i> value<0.01). (F) Monitoring of <i>Botrytis cinerea</i> growth 5 DAI in transgenic lines expressing HaRxL44 under the control of DEX inducible promoter (D44 lines) in the presence or not of dexamethazone and under the control of 35S promoter (44 lines). Col-0, HUB1 OE, as well as <i>hub1</i> KO mutants were used as controls. Error bars represent the standard error of the mean. Asterisks represent the significance of individual unpaired <i>t</i> tests comparing the given column with the control (<i>p</i> value<0.01).</p

HaRxL44 is a nuclear-HaRxL that enhances plant susceptibility to <i>Hpa.</i>

<p>(A) <i>In silico</i> prediction of HaRxL44 protein organization. SP, signal peptide; RFL, RxLR motif. (B) Monitoring of <i>Hpa</i> Waco9 sporulation at 5 d after inoculation in transgenic lines expressing HaRxL44 under the control of 35S promoter (44 lines), under the control of an “haustoriated-cell specific” promoter (dP2–44 lines), under the control of DEX inducible promoter (D44 lines). For D44 lines, plants were treated with DEX 2d after <i>Hpa</i> infection, in order to induce HaRxL44 expression. Expression of HaRxL44 in all the lines was monitored by Western blot (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001732#pbio.1001732.s003" target="_blank">Figure S3</a>). Error bars represent the standard error of the mean. Asterisks represent the significance of individual unpaired <i>t</i> tests comparing the given column with the control. (C) Subcellular localisation of GFP-HaRxL44 4 DAI with <i>Hpa</i>. The green colour corresponds to the GFP signal, and the red colour corresponds to chloroplast autofluorescence. Asterisks indicate the position of the haustorium. n, nucleus.</p

HaRxL44 expression, MED19a mutation suppresses <i>PR1</i> induction.

<p>(A–C) qRT-PCR on SA marker genes from 5-wk-old <i>Arabidopsis</i> plants. (D–F) qRT-PCR on <i>PR1</i> marker gene 8 h after SA treatment (200 µM) from 5-wk-old <i>Arabidopsis</i> plants. Data are presented as average fold induction compared with control of three biological replicates ± SD.</p

<i>Hpa</i> suppresses PR1 induction in infected cells.

<p>(A) qRT-PCR on <i>PR1</i> gene during a time course of infection of <i>Hpa</i> Waco9 in <i>Arabidopsis</i> Col-0. Data are presented as average fold induction compared with control of three biological replicates ± SD. (B) GUS staining of pro(<i>PR1</i>)::GUS in <i>Arabidopsis</i> leaves 6 DAI <i>Hpa</i> Waco9. Red arrows indicate <i>Hpa</i> hyphae's print surrounded by GUS stained cells. Note that no GUS was detected in <i>Hpa</i>-haustoriated mesophyll cell (black stars), while GUS staining was restricted to nonhaustoriated mesophyll cells (red stars). (C) qRT-PCR on <i>PR1</i> gene 6 DAI <i>Hpa</i> Waco9 in <i>Arabidopsis</i> Col-0, <i>med19a-1</i>, and <i>med19a-2</i> KO mutants. (D) Western blot on proteins extracted from <i>med19a</i> mutant complemented with GFP-MED19a after <i>Hpa</i> infection in comparison with mock treatment, using GFP antibody.</p

A Golden Gate Modular Cloning Toolbox for Plants

Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant <i>Nicotiana benthamiana</i> is discussed

Results of <i>Arabidopsis thaliana</i> Sanger sequencing.

<p>From the ranked list of all SNPs predicted by Bubbleparse in contigs of over 200 nt, the top 48, as well as 16 from 25%, 50% and 75% down the list were tested with Sanger sequencing. This confirmed all but 5 as being real SNPs between Col-0 and Ler-1. The remaining five all had sequencing problems – such as the sequence ending before the SNP was reached – so are not confirmed as false postives.</p