Inhibition of MAMP-inducible reporter gene activation by PiRXLR effectors.

<p>Luciferase reporter gene activity in flg22-challenged <i>S. lycopersicum</i> and <i>A. thaliana</i> protoplasts expressing PiRXLR effector genes. Mesophyll protoplasts were co-transfected with a <i>p35S-effector</i> construct (or a <i>p35S-GFP</i> control vector) and the two reporter gene constructs <i>pFRK1-Luc</i> and <i>pUBQ10-GUS</i>. Reporter gene activity was assessed 3 or 6 h later for <i>S. lycopersicum</i> and <i>A. thaliana</i>, respectively. For each data set, flg22-induced luciferase activity was calculated relative to the untreated sample and was normalized by the corresponding GUS activities in flg22 and untreated sample (<i>pFRK1-Luc</i> activity (+flg22/−flg22)). AvrPto was used as a positive control for <i>pFRK1-Luc</i> activity suppression. Four independent biological experiments were carried out per effector. Within each experiment three technical replicates were performed. Pooled data are presented as mean ± SEM. Differences in luciferase/GUS activity between control and effector gene-expressing protoplasts were determined using one-way ANOVA followed by Dunnett's multiple comparison test. An asterisk marks data sets with a p-value<0.05.</p

Summary of PiRXLR effectors with suppressing activity on MTI.

<p>S: Suppression No: No suppression E: Enhanced n.d.: not determined.</p

MAP kinase activation upon flg22 treatment in protoplasts expressing SFI effector genes.

<p>(<b>A, C</b>) Immunoblotting of phosphorylated MAP kinase in <i>p35S-effector</i>-transfected <i>S. lycopersicum</i> (<b>A</b>) and <i>A. thaliana</i> (<b>C</b>) protoplast samples collected 0, 15 and 30 min after flg22 treatment. Antibody raised against activated MAP kinase p44/p42 was used for detection. The experiments are representative of at least two repeats. Ponceau S staining served as a loading control. (<b>B</b>) MAP kinase <i>in vitro</i> kinase assay in <i>S. lycopersicum</i> protoplasts. GFP, AvrPto, SFI5, SFI6 or SFI7 were co-expressed with hemagglutinin (HA)-tagged tomato MAP kinase SlMPK1 or SlMPK3. HA-tagged MAP kinase were immunoprecipitated with anti-HA antibody for an <i>in vitro</i> kinase assay with [γ-³²P] ATP and myelin basic protein (MBP) as phosphorylation substrate. The lower panel presents an immunoblot with anti-HA antibody showing the expression of HA-tagged proteins. The upper panel shows an autoradiography visualizing MBP phosphorylation (MBP ³²P) in the presence of immunoprecipitated MAP kinase. The experiment was repeated twice with similar results.</p

Transcriptional profiling of MAMP-inducible genes in <i>A. thaliana</i> protoplasts transfected with SFI effector constructs.

<p>(<b>A–C</b>) Relative gene expression for the flg22-inducible genes <i>FRK1</i> and <i>WRKY17</i> (<b>A, B</b>) and the housekeeping gene <i>EF1α</i> (<b>C</b>) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) 0, 1 and 3 h after protoplasts were exposed to flg22. Transcript levels of the analyzed genes were normalized to the levels of the <i>Actin</i> transcript. GFP was used as a negative and AvrPto as a positive control for suppression of gene expression. One representative independent experiment out of four is plotted. Data is presented as mean ± SEM from three technical replicates.</p

Effect of GFP-SFI5, GFP-SFI6 and GFP-SFI7 on PCD triggered by INF1 or by co-expression of Cf-4 with <i>Cladosporium fulvum</i> Avr4.

<p>(<b>A</b>) Percentage of inoculation sites showing confluent cell death at 7 days post-infiltration of <i>Agrobacterium</i> strains expressing each GFP-effector fusion protein with a strain expressing Cf-4 and Avr4. (<b>B</b>) Percentage of inoculation sites showing confluent cell death at 7 days post-infiltration of <i>Agrobacterium</i> strains expressing each GFP-effector fusion protein with a strain expressing INF1. Results in (<b>A</b>) and (<b>B</b>) represent five biological replicates, each involving 18 inoculation sites. Error bars represent SEM. * represents statistical significance (p<0.01) using one-way ANOVA.</p

Effect of transient expression of SFI effectors enhances <i>P. infestans</i> colonization of <i>N. benthamiana</i>.

<p>Mean lesion diameter (<b>A</b>) and typical disease development symptoms (<b>B</b>) are shown for <i>P. infestans</i> 7 days post-inoculation over sites on leaves where an effector construct or empty vector was agro-infiltrated 1 day earlier. Each effector was expressed as an N-terminal GFP fusion protein as indicated, except for SFI8. Error bars represent SEM, and significant difference (* = p<0.001) in lesion size compared to empty vector control was determined by one-way ANOVA. Three biological replicates were performed, each using 24 inoculation sites per construct.</p

Epistatic analysis of MAP kinase activation upon flg22 treatment in <i>S. lycopersicum</i> protoplasts expressing SFI effector genes.

<p>Immunoblotting of phosphorylated MAP kinase in <i>p35S-effector</i>- and <i>p35S-SlMEK2-DD-GFP-</i> (<b>A</b>) and in <i>p35S-effector</i>- and <i>p35S-SlMAP3Kα-KD-GFP-</i> (<b>B</b>) co-transfected <i>S. lycopersicum</i> protoplast samples collected 0, 15 and 30 min after flg22 treatment. Antibody raised against activated MAP kinase p44/p42 was used for detection. The experiments are representative of at least two repeats. Ponceau S staining served as a loading control.</p

NtPOB1 is a negative regulator of avr9 –Cf9 mediated HR-PCD.

<p><b>A.</b> RT-PCR analysis of the <i>NtPOB1</i> transcript levels in silenced and non-silenced tobacco plants 3 days after Agroinfiltrations with stated hairpin constructs. <i>ACTIN</i> transcript levels were used as loading controls. <b>B.</b> Leaf segments expressing vector only, HG: 00, or <i>NtPOB1</i>-silencing HG:NtPOB1 construct were infiltrated with Avr9 peptide 3 days after agroinfiltrations. Pictures were taken 36 hours after Avr9 treatment. At this time point <i>NtPOB1</i>-silenced leaf segments showed much earlier onset of HR-PCD than leaf segments expressing vector only. HR-PCD assays were repeated in at least three different leaves in three different <i>Cf-9</i> tobacco plants. Representative leaf image is shown of HR-PCD that was observed in 80% (8/10) of the leaves tested.</p

NtPUB17 directly interacts with NtPOB1.

<p><b>A.</b> Yeast-2-Hybrid assays indicating that full length and ARM domain of NtACRE276 (NtPUB17) interact with NtPOB1. The various indicated proteins were expressed as AD- and BD- fusions in AH109 yeast cells. Transformed AH109 cells were grown on selective media (Leu^-, Trp^-, His^- and X-α-Gal) to test for interacting partners and on nonselective media (Leu^-, Trp^-) to test for the transformation efficiency. BD-SV40 and AD-p53 fusion proteins were used as positive controls for interaction in yeast. <b>B.</b> Immunoprecipitation analysis indicates that GFP-NtPUB17 and HA-NtPOB1 interact <i>in planta</i>. HA- and GFP fusions of NtPOB1 and PUB17 respectively were expressed in tobacco leaves using Agrobacterium-mediated transient assays. Total protein was extracted after 3 days and incubated with anti-GFP agarose beads to immunoprecipitate GFP-PUB17. The protein extracts (input) as well as the immunoprecipitates (IP) were analyzed by western blot using anti-HA and anti-GFP antibodies.</p

POB1 dimerisation is critical for cell death suppression and PUB17 turnover.

<p><b>A.</b> POB1 D146A mutant protein cannot dimerize in yeast. Wildtype POB1 and POB1^D146A mutant proteins were expressed as AD- and BD- fusions in AH109 yeast cells. Transformed AH109 cells were grown on selective media (Leu-, Trp-, His- and X-α-Gal) to test for interacting partners and on nonselective media (Leu-, Trp-) to test for the transformation efficiency. BD-SV40 and AD-p53 fusion proteins were used as positive controls. <b>B</b> Immunoprecipitation analysis of the POB1^D146A mutation on POB1 dimerization. HA- and GFP fusions of the wild-type POB1 and mutant POB1 ^D146A were expressed in <i>N</i>. <i>benthamiana</i> leaves. The protein extracts and immunoprecipitates (IP) were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. As indicated barely any HA-POB1^D146A co-immunoprecipitates with the GFP-POB1^D146A. <b>C.</b> Transient over-expression of GFP-POB1 and GFP-POB1^D146A in Cf-9 tobacco. Agrobacteria containing the indicated constructs were infiltrated into Cf-9 tobacco leaf segments. Avr9 peptide was infiltrated into the leaf segments 3 days after agroinfiltrations. HR-PCD assays were repeated in at least three different leaves in three different <i>Cf-9</i> tobacco plants. Representative leaf image is shown where HR developed in leaves (9/20) expressing detectable AtPOB1 ^D146A. Pictures were taken 2 days after Avr9 treatment. <b>D.</b> Graph of HR-PCD of Cf4/Avr4 co-expressed with GFP-POB1 or GFP-AtPOB1^D146A. Statistical analysis was carried out using ANOVA with pairwise comparisons performed with a Holm-Sidak test; and letters denote whether results are statistically significantly different, p≤0.001 (n = 99); error bars show standard error. The graph represents the combined data from 4 biological reps. <b>E.</b> Immunoprecipitation analysis of the POB1^D146A mutation on POB1 interaction with PUB17. GFP fusions of the wild-type POB1 and mutant POB1 ^D146A were expressed in <i>N</i>. <i>benthamiana</i> leaves along with HA-PUB17 and treated with proteasome inhibitor MG132. The protein extracts (input) and immunoprecipitates (IP) were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. As indicated barely any GFP-POB1^D146A immunoprecipitatied with PUB17-HA.</p