Functionally Redundant RXLR Effectors from <em>Phytophthora infestans</em> Act at Different Steps to Suppress Early flg22-Triggered Immunity

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans

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

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

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

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

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

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