Nejvíce citovaný článek - PubMed ID 14586469
Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.
- MeSH
- Arabidopsis * metabolismus MeSH
- buněčná stěna metabolismus MeSH
- nemoci rostlin mikrobiologie MeSH
- odolnost vůči nemocem genetika MeSH
- protein - isoformy genetika metabolismus MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- rostlinné proteiny metabolismus MeSH
- trichomy genetika metabolismus MeSH
- vezikulární transportní proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- EXO70H4 protein, Arabidopsis MeSH Prohlížeč
- protein - isoformy MeSH
- proteiny huseníčku * MeSH
- rostlinné proteiny MeSH
- vezikulární transportní proteiny MeSH
Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis (Arabidopsis thaliana). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121ΔC, which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding.
- MeSH
- Arabidopsis cytologie genetika růst a vývoj metabolismus MeSH
- buněčná membrána metabolismus MeSH
- exocytóza fyziologie MeSH
- geneticky modifikované rostliny MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteiny Qa-SNARE genetika metabolismus MeSH
- proteiny R-SNARE genetika metabolismus MeSH
- proteiny SNARE genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- EXO70A1 protein, Arabidopsis MeSH Prohlížeč
- PEN1 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- proteiny Qa-SNARE MeSH
- proteiny R-SNARE MeSH
- proteiny SNARE MeSH
- VAMP721 protein, Arabidopsis MeSH Prohlížeč
BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. METHODS: To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. KEY RESULTS: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. CONCLUSIONS: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.
- Klíčová slova
- Arabidopsis thaliana, biotic stress, callose, isochorismate synthase 1, pathogens, phytohormones, pi4kβ1β2/PI4Ks, salicylic acid,
- MeSH
- 1-fosfatidylinositol-4-kinasa * MeSH
- Arabidopsis * MeSH
- kyselina salicylová MeSH
- mutace MeSH
- nemoci rostlin MeSH
- proteiny huseníčku genetika MeSH
- Pseudomonas syringae MeSH
- regulace genové exprese u rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-fosfatidylinositol-4-kinasa * MeSH
- AT5G09350 protein, Arabidopsis MeSH Prohlížeč
- AT5G64070 protein, Arabidopsis MeSH Prohlížeč
- kyselina salicylová MeSH
- proteiny huseníčku MeSH
Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.
- MeSH
- Arabidopsis genetika imunologie metabolismus mikrobiologie MeSH
- Ascomycota patogenita MeSH
- brefeldin A imunologie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- chitin imunologie metabolismus MeSH
- cyklopentany metabolismus MeSH
- exocytóza účinky léků imunologie MeSH
- fosfolipasa D genetika metabolismus MeSH
- kyseliny fosfatidové metabolismus MeSH
- nemoci rostlin imunologie mikrobiologie MeSH
- oxylipiny metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- přirozená imunita * účinky léků MeSH
- proteiny huseníčku metabolismus MeSH
- proteiny Qa-SNARE metabolismus MeSH
- proteiny R-SNARE metabolismus MeSH
- proteiny SNARE genetika metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce imunologie fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- brefeldin A MeSH
- chitin MeSH
- cyklopentany MeSH
- fosfolipasa D MeSH
- jasmonic acid MeSH Prohlížeč
- kyseliny fosfatidové MeSH
- oxylipiny MeSH
- PEN1 protein, Arabidopsis MeSH Prohlížeč
- peroxid vodíku MeSH
- phospholipase D delta MeSH Prohlížeč
- proteiny huseníčku MeSH
- proteiny Qa-SNARE MeSH
- proteiny R-SNARE MeSH
- proteiny SNARE MeSH
- reaktivní formy kyslíku MeSH
- REM1 protein, Arabidopsis MeSH Prohlížeč
- VAMP721 protein, Arabidopsis MeSH Prohlížeč
- VAMP722 protein, Arabidopsis MeSH Prohlížeč
Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence.
- MeSH
- Arabidopsis imunologie mikrobiologie MeSH
- DNA bakterií MeSH
- interakce hostitele a patogenu * MeSH
- inzerční mutageneze MeSH
- nemoci rostlin imunologie MeSH
- proteiny huseníčku fyziologie MeSH
- Pseudomonas syringae fyziologie MeSH
- techniky dvojhybridového systému MeSH
- upregulace MeSH
- vezikulární transportní proteiny fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- Exo70B2 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- T-DNA MeSH Prohlížeč
- vezikulární transportní proteiny MeSH
Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C approximately 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat-barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H-7H. Thus, the barley genome can be dissected into fractions representing only about 6-12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.
