SummaryChanging environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen-activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O 3 ) to generate ROS in the apoplast of wildtype Col-0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O 3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full-level AtMPK3 activation by O 3 . In addition, we show that AtMPK3, but not AtMPK6, responded to O 3 transcriptionally and translationally during O 3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O 3 treatment. The use of O 3 to induce apoplastic ROS formation offers a non-invasive in planta system amenable to reverse genetics that can be used for the study of stress-responsive MAPK signaling in plants.

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burstindependent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain-and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.

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Abstract:Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader's molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity.

SummaryTobacco plants (Nicotiana tabacum cv. Xanthi-nc) infiltrated with either of two pathovars of Pseudomonas syringae -an avirulent strain of P. syringae pv. tabaci (Pst) or the non-host pathogen P. syringae pv. maculicola M2 (Psm) -developed a hypersensitive response (HR). There were considerable differences in HR phenotype, timing and sequence of cell dismantling between the two pathosystems. Following Psm infiltration, the first macroscopic signs were visible at 4.5 h post-infiltration (hpi). Simultaneously, increased plasma membrane permeability was observed, suggesting that the loss of cell membrane integrity initiates the macroscopic HR evoked by Psm. In contrast, after Pst treatment there was a distinct time lapse between the first signs of tissue collapse (9 hpi) and the occurrence of plasma membrane discontinuity (12 hpi). Ultrastructural studies of cells undergoing the HR triggered by Psm and Pst revealed distinct patterns of alterations in morphology of organelles. Moreover, while different forms of nuclear degeneration were observed in leaf zones infiltrated with Pst, we failed to detect any abnormalities in the nuclei of Psm-treated tissue. In addition, application of synthetic caspase inhibitors (Ac-DEVD-CHO, Ac-YVAD-CMK) abolished HR induced by Pst, but not Psm. Our observations suggest that different cell death mechanisms are executed in response to Psm and Pst. Interestingly, pre-inoculation with Pst, but not with Psm, induced a long-distance acquired resistance (LDAR) response, even though locally a typical set of defense responses, including acquired resistance, was activated locally in response to Psm. The failure of Psm to induce LDAR may be due to the rapid degeneration of bundle sheath cells resulting from Psm infection.

During the first 24 hours of infection, Alternaria brassicicola developmental parameters such as conidial germination, germ tubes and appressoria formation on each of the five mature Brassica juncea leaves, correlated with a leaf position showing stronger development of the pathogen on older leaves than on young ones. As a consequence of fungal development, the black spot disease was observed during 96 hours of infection on a macroscopic scale, as well as via confocal microscopy. Degradation of the chloroplast thylakoids and plastoglobule appearance during infection, followed by the decrease in chlorophyll a fluorescence parameters i.e. maximum quantum yield of PSII (Fv/Fm), non‐photochemical quenching (NPQ) and chlorophyll a:b ratio, have been observed. Also, after an initial increase of carbohydrates (glucose, fructose and sucrose), content far below the respective control values was found. The content of secondary metabolites such as flavonoids and glucosinolates increased in a leaf position‐dependent manner in infected leaves, with a lower level in older leaves than in younger ones. Although, the total phenolic compounds (TPCs) content did not differ significantly in infected leaves compared to control leaves, TPCs level in both control and infected leaves was leaf position‐dependent. To the best of our knowledge, this is the first report on leaf position‐dependent effect on the B. juncea biochemical response to A. brassicicola infection.

Black spot disease, caused by Alternaria brassicicola in Brassica species, is one of the most devastating diseases all over the world, especially since there is no known fully resistant Brassica cultivar. In this study, the visualization of black spot disease development on Brassica oleracea var. capitata f. alba (white cabbage) leaves and subsequent ultrastructural, molecular and physiological investigations were conducted. Inter- and intracellular hyphae growth within leaf tissues led to the loss of host cell integrity and various levels of organelle disintegration. Severe symptoms of chloroplast damage included the degeneration of chloroplast envelope and grana, and the loss of electron denseness by stroma at the advanced stage of infection. Transcriptional profiling of infected leaves revealed that photosynthesis was the most negatively regulated biological process. However, in infected leaves, chlorophyll and carotenoid content did not decrease until 48 hpi, and several chlorophyll a fluorescence parameters, such as photosystem II quantum yield (Fv/Fm), non-photochemical quenching (NPQ), or plant vitality parameter (Rdf) decreased significantly at 24 and 48 hpi compared to control leaves. Our results indicate that the initial stages of interaction between B. oleracea and A. brassicicola are not uniform within an inoculation site and show a complexity of host responses and fungal attempts to overcome host cell defense mechanisms. The downregulation of photosynthesis at the early stage of this susceptible interaction suggests that it may be a part of a host defense strategy, or, alternatively, that chloroplasts are targets for the unknown virulence factor(s) of A. brassicicola. However, the observed decrease of photosynthetic efficiency at the later stages of infection is a result of the fungus-induced necrotic lesion expansion.