Identification of MAMP-triggered immunity (MTI)-suppressing RXLR effectors from Phytophthora infestans and functional characterization of the calmodulin-binding effector SFI5

An important branch of the plant immune system is based on the sensing of potential pathogens by the recognition of highly conserved microbe-associated molecular patterns (MAMPs), such as the peptide epitope flg22 from bacterial flagellin, and the activation of complex defense signaling events yielding a generic anti-microbial response, which is called MAMP-triggered immunity (MTI). The successful establishment of infection relies on the pathogen’s capability to deliver effectors that subvert plant immunity. Although some effectors from eukaryotic filamentous pathogens have been identified as MTI-compromising factors, our general understanding of the effector-target biology and the molecular mechanisms underlying the mode of action of these effectors is still in its infancy. A large repertoire of candidate effector genes, including hundreds of putative host-targeting RXLR effectors, is present in the genome of Phytophthora infestans, the causal agent of potato and tomato late blight. In this thesis, we used protoplast-based high-throughput assays to identify and characterize RXLR effectors interfering with the early stages of MAMP-induced immune signaling responses e.g. calcium and oxidative burst, post-translational MAP kinase activation and transcriptional up-regulation of MAMP-inducible genes. Among 33 RXLR effectors tested, eight were identified as Suppressor of early Flg22-induced Immune responses (SFI effectors) in tomato protoplasts. Epistatic analysis showed that three RXLR effectors (SFI5-SFI7) disturb flg22-mediated signaling at- or upstream of the MAP kinase cascade, concomitant with their localization at the host plasma membrane. The remaining five RXLR effectors (SFI1-4 and SFI8) act downstream of the MAP kinase cascade, four of them are localized in the host nucleus. Furthermore, we provide evidence that all but one SFI effectors enhance host susceptibility to P. infestans infection. We have identified the calcium sensor calmodulin (CaM) as an interacting plant protein of SFI5 using bioinformatics, proteomics and biochemical approaches. Structure-function analyses with SFI deletion and point mutants showed that the CaM-binding motif in the C-terminal part of SFI5 is crucial for the plasma membrane (PM) localization, MTI-suppressing activity and virulence function of SFI5. In addition, a predicted ATP/GTP-binding site motif (P-loop) at the N-terminus of SFI5 was demonstrated to be necessary for the effector activity but has no influence on CaM binding and PM localization. Our current model predicts a two-step activation mechanism of SFI5 with CaM serving as a co-factor and regulating SFI5 to target potential MTI components at the PM. Altogether, we have shown that P. infestans contains functionally redundant effectors to inhibit MAMP-dependent early signal transduction during host infection. Our results present a conceptual advance in the understanding of the biology of effectors originated from eukaryotic plant pathogens and show parallels with the strategies developed by prokaryotic pathogens

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

The Role of the S40 Gene Family in Leaf Senescence

Senescence affect different traits of plants, such as the ripening of fruit, number, quality and timing of seed maturation. While senescence is induced by age, growth hormones and different environmental stresses, a highly organized genetic mechanism related to substantial changes in gene expression regulates the process. Only a few genes associated to senescence have been identified in crop plants despite the vital significance of senescence for crop yield. The S40 gene family has been shown to play a role in leaf senescence. The barley HvS40 gene is one of the senescence marker genes which shows expression during age-dependent as well as dark-induced senescence. Like barley HvS40, the Arabidopsis AtS40-3 gene is also induced during natural senescence as well as in response to treatment with abscisic acid, salicylic acid, darkness and pathogen attack. It is speculated that rice OsS40 has a similar function in the leaf senescence of rice

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

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

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

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