Membrane microdomains play vital roles in the process of bacterial infection. The membrane microdomain-associated protein Flot1 acts in an endocytic pathway and is required for seedling development, however, whether Flot1 is a part of host defense mechanisms remains unknown. During an analysis of callose deposition, we found that Flot1 amiRNAi mutants exhibited defects in response to flg22. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), structured illumination microscopy (SIM) and fluorescence cross spectroscopy (FCS), we determined that the dynamic behavior of GFP-Flot1 in Arabidopsis thaliana cotyledon epidermal cells changed significantly in plants treated with the elicitor flg22. Moreover, we found that Flot1 was constitutively recycled via an endocytic pathway and that flg22 could promote endocytosis. Importantly, targeting of Flot1 to the late endosome/vacuole for degradation increased in response to flg22 treatment; immunoblot analysis showed that when triggered by flg22, GFP-Flot1 was gradually degraded in a time-dependent manner. Taken together, these findings support the hypothesis that the changing of dynamics and oligomeric states can promote the endocytosis and degradation of Flot1 under flg22 treatment in plant cells.

• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• endocytóza účinky léků   genetika   fyziologie   MeSH
• flagelin farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• kotyledon genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Recognition of pathogen-associated molecular patterns (PAMPs) is crucial for plant defence against pathogen attack. The best characterized PAMP is flg22, a 22 amino acid conserved peptide from flagellin protein. In Arabidopsis thaliana, flg22 is recognized by the flagellin sensing 2 (FLS2) receptor. In this study, we focused on biotic stress responses triggered by flg22 after exposure to temporary heat stress (HS). It is important to study the reactions of plants to multiple stress conditions because plants are often exposed simultaneously to a combination of both abiotic and biotic stresses. Transient early production of reactive oxygen species (ROS) is a well-characterized response to PAMP recognition. We demonstrate the strong reduction of flg22-induced ROS production in A. thaliana after HS treatment. In addition, a decrease in FLS2 transcription and a decrease of the FLS2 presence at the plasma membrane are shown after HS. In summary, our data show the strong inhibitory effect of HS on flg22-triggered events in A. thaliana. Subsequently, temporary HS strongly decreases the resistance of A. thaliana to Pseudomonas syringae. We propose that short exposure to high temperature is a crucial abiotic stress factor that suppresses PAMP-triggered immunity, which subsequently leads to the higher susceptibility of plants to pathogens.

• MeSH
• alarminy metabolismus   MeSH
• Arabidopsis účinky léků   genetika   imunologie   mikrobiologie   MeSH
• flagelin farmakologie   MeSH
• genetická transkripce účinky léků   MeSH
• imunita rostlin * účinky léků   MeSH
• nemoci rostlin imunologie   mikrobiologie   MeSH
• odolnost vůči nemocem imunologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Pseudomonas syringae účinky léků   fyziologie   MeSH
• reakce na tepelný šok * účinky léků   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• respirační vzplanutí účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

• MeSH
• Arabidopsis enzymologie   imunologie   mikrobiologie   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfolipasy typu C fyziologie   MeSH
• Golgiho aparát enzymologie   MeSH
• imunita rostlin fyziologie   MeSH
• klonování DNA MeSH
• konfokální mikroskopie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• nemoci rostlin imunologie   mikrobiologie   MeSH
• proteiny huseníčku fyziologie   MeSH
• protoplasty enzymologie   MeSH
• Pseudomonas syringae * MeSH
• reaktivní formy kyslíku MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Background and Aims: Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated. Methods: Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed. Key Results: Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter. Conclusions: Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root hair growth response is triggered in the range of hours after bacterial contact with roots and can be modulated by FLS2 signalling. Bacterial stimulation of root hair growth requires functional ethylene signalling and an efficient exocyst-dependent secretory machinery.

• MeSH
• Arabidopsis genetika   růst a vývoj   mikrobiologie   MeSH
• genový knockout MeSH
• interakce hostitele a patogenu * MeSH
• kořeny rostlin růst a vývoj   mikrobiologie   MeSH
• mutace MeSH
• proteinkinasy genetika   MeSH
• proteiny huseníčku genetika   MeSH
• Pseudomonas syringae * MeSH
• receptory buněčného povrchu genetika   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

• MeSH
• Arabidopsis chemie   metabolismus   MeSH
• arabinonukleosidy chemie   farmakologie   MeSH
• cytokininy chemie   farmakologie   MeSH
• imunita rostlin účinky léků   MeSH
• listy rostlin účinky léků   MeSH
• MAP kinasový signální systém účinky léků   MeSH
• mitogenem aktivované proteinkinasy genetika   metabolismus   MeSH
• molekulární struktura MeSH
• PAMP struktury farmakologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH