Plant immunity from A to Z

A report of The Keystone Symposium on Plant Innate Immunity, Keystone, USA, 10-15 February 2008

CalloseMeasurer: a novel software solution to measure callose deposition and recognise spreading callose patterns

BACKGROUND: Quantification of callose deposits is a useful measure for the activities of plant immunity and pathogen growth by fluorescence imaging. For robust scoring of differences, this normally requires many technical and biological replicates and manual or automated quantification of the callose deposits. However, previously available software tools for quantifying callose deposits from bioimages were limited, making batch processing of callose image data problematic. In particular, it is challenging to perform large-scale analysis on images with high background noise and fused callose deposition signals. RESULTS: We developed CalloseMeasurer, an easy-to-use application that quantifies callose deposition, a plant immune response triggered by potentially pathogenic microbes. Additionally, by tracking identified callose deposits between multiple images, the software can recognise patterns of how a given filamentous pathogen grows in plant leaves. The software has been evaluated with typical noisy experimental images and can be automatically executed without the need for user intervention. The automated analysis is achieved by using standard image analysis functions such as image enhancement, adaptive thresholding, and object segmentation, supplemented by several novel methods which filter background noise, split fused signals, perform edge-based detection, and construct networks and skeletons for extracting pathogen growth patterns. To efficiently batch process callose images, we implemented the algorithm in C/C++ within the AcapellaTM framework. Using the tool we can robustly score significant differences between different plant genotypes when activating the immune response. We also provide examples for measuring the in planta hyphal growth of filamentous pathogens. CONCLUSIONS: CalloseMeasurer is a new software solution for batch-processing large image data sets to quantify callose deposition in plants. We demonstrate its high accuracy and usefulness for two applications: 1) the quantification of callose deposition in different genotypes as a measure for the activity of plant immunity; and 2) the quantification and detection of spreading networks of callose deposition triggered by filamentous pathogens as a measure for growing pathogen hyphae. The software is an easy-to-use protocol which is executed within the Acapella software system without requiring any additional libraries. The source code of the software is freely available at https://sourceforge.net/projects/bioimage/files/Callose

Expression patterns of FLAGELLIN SENSING 2 map to bacterial entry sites in plant shoots and roots

Expression of the flagellin receptor FLS2 is regulated in a cell/tissue-specific and stress-induced manner that correlated with sites of bacterial infection. The vasculature expresses FLS2 and responds to flagelli

Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities

Bacterial flagellin is known to stimulate host immune responses in mammals and plants. In Arabidopsis thaliana, the receptor kinase FLS2 mediates flagellin perception through physical interaction with a highly conserved epitope in the N-terminus of flagellin, represented by the peptide flg22 derived from Pseudomonas syringae. The peptide flg22 is highly active as an elicitor in many plant species. In contrast, a shortened version of the same epitope derived from Escherichia coli, flg15E coli, is highly active as an elicitor in tomato but not in A. thaliana or Nicotiana benthamiana. Here, we make use of these species-specific differences in flagellin perception abilities to identify LeFLS2 as the flagellin receptor in tomato. LeFLS2 is most closely related to AtFLS2, indicating that it may represent the flagellin receptor of tomato. Expression of the LeFLS2 gene in Arabidopsis did not result in accumulation of its corresponding gene product, as indicated by experiments with LeFLS2-GFP fusions. In contrast, expression of LeFLS2-GFP fusions in N. benthamiana, a species that, like tomato, belongs to the Solanaceae, was obviously functional. N. benthamiana plants transiently expressing a LeFLS2-GFP fusion acquired responsiveness to flg15E coli to which they are normally unresponsive. Thus, LeFLS2 encodes a functional, specific flagellin receptor, the first to be identified in a plant family other than the Brassicacea

The family of Peps and their precursors in Arabidopsis: differential expression and localization but similar induction of pattern-triggered immune responses

In Arabidopsis thaliana, the endogenous danger peptides, AtPeps, have been associated with plant defences reminiscent of those induced in pattern-triggered immunity. AtPeps are perceived by two homologous receptor kinases, PEPR1 and PEPR2, and are encoded in the C termini of the PROPEP precursors. Here, we report that, contrary to the seemingly redundant AtPeps, the PROPEPs fall at least into two distinct groups. As revealed by promoter-β-glucuronidase studies, expression patterns of PROPEP1-3, -5, and -8 partially overlapped and correlated with those of the PEPR1 and -2 receptors, whereas those of PROPEP4 and -7 did not share any similarities with the former. Moreover, bi-clustering analysis indicated an association of PROPEP1, -2, and -3 with plant defence, whereas PROPEP5 expression was related to patterns of plant reproduction. In addition, at the protein level, PROPEPs appeared to be distinct. PROPEP3::YFP (fused to yellow fluorescent protein) was present in the cytosol, but, in contrast to previous predictions, PROPEP1::YFP and PROPEP6::YFP localized to the tonoplast. Together with the expression patterns, this could point to potentially non-redundant roles among the members of the PROPEP family. By contrast, their derived AtPeps, including the newly reported AtPep8, when applied exogenously, provoked activation of defence-related responses in a similar manner, suggesting a high level of functional redundancy between the AtPeps. Taken together, our findings reveal an apparent antagonism between AtPep redundancy and PROPEP variability, and indicate new roles for PROPEPs besides plant immunit

Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis

Peer reviewe

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signalling

SummaryPlant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signalling molecules. Receptor kinases are regulated by numerous post-translational modifications. Here, using the immune receptor kinases FLS2 and EFR, we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biophysical properties and behaviour within the membrane environment. We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following perception of its ligand flg22, in a BAK1 co-receptor dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signalling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR supressed elf18 triggered signalling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents and native membrane DIBMA nanodiscs indicates that S-acylation stabilises and promotes retention of activated receptor kinase complexes at the plasma membrane to increase signalling efficiency

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex

The leucine-rich repeat receptor kinase QSK1 is a novel regulator of PRR-RBOHD complex and is employed by the bacterial effector HopF2Pto_{Pto} to modulate plant immunity

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) like EFR and FLS2, which recognize bacterial EF-Tu and flagellin, respectively. These PRRs, belonging to the leucine-rich repeat receptor kinase (LRR-RK) family, activate the production of reactive oxygen species via the NADPH oxidase RBOHD. The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogenic effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative co-immunoprecipitation analysis using EFR, FLS2, and RBOHD. We identified QSK1, an LRR-RK, as a novel component of the PRR-RBOHD complex. QSK1 functions as a negative regulator of PRR-triggered immunity (PTI) by downregulating the abundance of FLS2 and EFR. QSK1 is targeted by the bacterial effector HopF2$_{Pto}$, a mono-ADP ribosyltransferase, resulting in the reduction of FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2$_{Pto}$ reduces transcript levels of PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MIK2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide novel insights into the mechanism by which HopF2$_{Pto}$ employs QSK1 to desensitize plants to pathogen attack. One Sentence Summary: QSK1, a novel component in the plant immune receptor complex, downregulates these receptors and phytocytokines, and is exploited by bacterial effector HopF2$_{Pto}$ to desensitize plants to pathogen attack