Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.
- MeSH
- buněčná membrána metabolismus MeSH
- chromatografie kapalinová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fungální proteiny genetika metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- PAMP struktury metabolismus MeSH
- Phytophthora fyziologie MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika metabolismus mikrobiologie MeSH
- tandemová hmotnostní spektrometrie MeSH
- trypsin chemie MeSH
- Publikační typ
- časopisecké články MeSH
Few cases of spontaneously horizontally transferred bacterial genes into plant genomes have been described to date. The occurrence of horizontally transferred genes from the T-DNA of Agrobacterium rhizogenes into the plant genome has been reported in the genus Nicotiana and in the species Linaria vulgaris. Here we compare patterns of evolution in one of these genes (a gene encoding mikimopine synthase, mis) following three different events of horizontal gene transfer (HGT). As this gene plays an important role in Agrobacterium, and there are known cases showing that genes from pathogens can acquire plant protection function, we hypothesised that in at least some of the studied species we will find signs of selective pressures influencing mis sequence. The mikimopine synthase (mis) gene evolved in a different manner in the branch leading to Nicotiana tabacum and N. tomentosiformis, in the branch leading to N. glauca and in the genus Linaria. Our analyses of the genus Linaria suggest that the mis gene began to degenerate soon after the HGT. In contrast, in the case of N. glauca, the mis gene evolved under significant selective pressures. This suggests a possible role of mikimopine synthase in current N. glauca and its ancestor(s). In N. tabacum and N. tomentosiformis, the mis gene has a common frameshift mutation that disrupted its open reading frame. Interestingly, our results suggest that in spite of the frameshift, the mis gene could evolve under selective pressures. This sequence may still have some regulatory role at the RNA level as suggested by coverage of this sequence by small RNAs in N. tabacum.
- MeSH
- Agrobacterium enzymologie genetika MeSH
- bakteriální proteiny klasifikace genetika metabolismus MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- imidazoly metabolismus MeSH
- interakce hostitele a patogenu genetika MeSH
- Linaria genetika mikrobiologie MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- oxidoreduktasy působící na CH-NH vazby klasifikace genetika metabolismus MeSH
- posunová mutace MeSH
- přenos genů horizontální * MeSH
- pyridiny metabolismus MeSH
- regulace genové exprese enzymů MeSH
- sekvence nukleotidů MeSH
- selekce (genetika) MeSH
- tabák klasifikace genetika mikrobiologie MeSH
- transformace genetická MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.
- MeSH
- buněčná membrána metabolismus MeSH
- fungální proteiny metabolismus MeSH
- konfokální mikroskopie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- NADPH-oxidasy genetika metabolismus MeSH
- Phytophthora fyziologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika metabolismus mikrobiologie MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND AND AIMS: Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens. METHODS: The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange. KEY RESULTS: Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure. CONCLUSIONS: Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.
- MeSH
- alkyltransferasy a aryltransferasy genetika MeSH
- buněčná smrt MeSH
- chlorofyl metabolismus MeSH
- chloroplasty genetika metabolismus MeSH
- cytokininy genetika metabolismus MeSH
- dexamethason farmakologie MeSH
- fotosyntéza genetika MeSH
- geneticky modifikované rostliny MeSH
- interakce hostitele a patogenu * MeSH
- listy rostlin cytologie genetika fyziologie MeSH
- nekróza genetika MeSH
- oxidační stres genetika MeSH
- peroxid vodíku metabolismus MeSH
- peroxidace lipidů MeSH
- průduchy rostlin fyziologie MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- tabák genetika mikrobiologie fyziologie MeSH
- umlčování genů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plants protect themselves from pathogen invasion through the local expression of a variety of pathogenesis-related proteins. They are highly diverse in both primary structure and length, and exhibit different direct antimicrobial activity. This text reviews the knowledge of osmotin, antimicrobial protein involved in innate immunity of plants. Osmotin belongs to the fifth class of the group of pathogenesis-related (PR) proteins and has been found in different plants species, in every case osmotin is cysteine-rich protein involved in plant defense responses to several pathogens and abiotic stresses. The phylogenetic tree of amino acids compositions of osmotins from different plant species is presented and the basic similarities of clusters are discussed in this review. Osmotin gene is activated by different biotic as well as abiotic signals and has many functions. The review summarizes biochemical and structural properties, induction, functions and structural homology between osmotin and other proteins. Recent data about recombinant production in bacterial and plant cells are examined. The article indicates possible ways of osmotin application in research in the field of functional biology, medicine and agriculture.
- MeSH
- antifungální látky chemie imunologie farmakologie MeSH
- fylogeneze MeSH
- fyziologický stres MeSH
- geneticky modifikované rostliny MeSH
- houby účinky léků MeSH
- molekulární modely MeSH
- nemoci rostlin imunologie MeSH
- regulace genové exprese u rostlin * MeSH
- rekombinantní proteiny genetika imunologie farmakologie MeSH
- rostlinné proteiny genetika imunologie farmakologie MeSH
- salinita MeSH
- sekundární struktura proteinů MeSH
- tabák genetika imunologie mikrobiologie MeSH
- terciární struktura proteinů MeSH
- vodní a elektrolytová rovnováha MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Aktivní i pasivní kouření je spojeno s významně větší kolonizací dýchacího ústrojí patogenními bakteriemi a se zvýšeným rizikem akutních bakteriálních onemocnění horních i dolních cest dýchacích. Studie zaměřené na zkoumání kvality tabáku zjistily přítomnost mnoha druhů grampozitivních i gramnegativních bakterií, z nichž mnohé jsou zařazeny mezi lidské patogeny či podmíněné patogeny. Ke kontaminaci dochází při pěstební činnosti a bakterie mohou přežívat nejen v procesu výroby kuřiva, ale dokonce i expozici vysokým teplotám a plynům ze zapálené cigarety. Některé bakterie produkují bioaktivní endotoxiny, které byly zjištěny jak v cigaretovém kouři vdechovaném kuřákem, tak i v v kouři, který je emitován ze zapáleného konce do kuřákova okolí. Zjištěné nálezy jsou dalším důvodem pro striktní dodržování zákazu kouření i manipulace s kuřivem v zařízeních pro nemocné a děti, kteří jsou zvýšeně vnímaví k bakteriálním infekcím.
Both active and passive smoking are associated with significantly enhanced colonization of the respiratory tract by bacterial pathogens and with a higher risk of acute infectious diseases of upper and distal respiratory tract. Studies concerned about the measurement of tobacco quality have found many species of Gram-positive and Gram-negative bacteria, many of which are human pathogens or opportunistic pathogens. The bacterial contamination occurs during the tobacco growing process and bacterial species can survive not only the tobacco product manufacturing process, but also exposure to high temperatures and gases from a lit cigarette. Some species release bioactive endotoxins, which are present both in the mainstream and sidestream of tobacco smoke. These results are another reason for the urgent introduction of a complete ban on smoking, and on the manipulation of tobacco, in places where sick persons and children are living, as they are more vulnerable to bacterial infections.
Multi-color fluorescence emission from leaf tissues is presented as a powerful reporter on plant biochemistry and physiology that can be applied both at macro- and micro-scales. The blue-green fluorescence emission is typically excited by ultraviolet (UV) excitation. However, this approach cannot be applied in investigating intact leaf interior because the UV photons are largely absorbed in the epidermis of the leaf surface. This methodological barrier is eliminated by replacing the UV photon excitation by excitation with two infra-red photons of the same total energy. We demonstrate this approach by using two-photon excitation for microscopy of Arabidopsis thaliana leaves infected by pathogenic bacterium Pseudomonas syringae. The leaf structures are visualized by red chlorophyll fluorescence emission reconstructed in 3-D images while the bacteria are detected by the green emission of engineered fluorescence protein.
- MeSH
- Arabidopsis metabolismus mikrobiologie MeSH
- chlorofyl metabolismus MeSH
- fluorescenční spektrometrie MeSH
- listy rostlin metabolismus mikrobiologie MeSH
- mezofylové buňky cytologie metabolismus MeSH
- nemoci rostlin mikrobiologie MeSH
- Pseudomonas syringae fyziologie MeSH
- tabák metabolismus mikrobiologie MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
