Although angiosperm plants generally react to immunity elicitors like chitin or chitosan by the cell wall callose deposition, this response in particular cell types, especially upon chitosan treatment, is not fully understood. Here we show that the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase-like 5/PMR4 is vital for chitosan-induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the molecular and cellular mechanisms underpinning the responses to two chitosan treatments, we analysed early Ca2+ and defence-related signalling, gene expression, cell wall and RH cellular endomembrane modifications. Chitosan-induced callose deposition is also present in the several other plant species, including functionally analogous and evolutionarily only distantly related RH-like structures such as rhizoids of bryophytes. Our results point to the RH callose deposition as a conserved strategy of soil-anchoring plant cells to cope with mild biotic stress. However, high chitosan concentration prominently disturbs RH intracellular dynamics, tip-localised endomembrane compartments, growth and viability, precluding callose deposition.

• Keywords
• arabidopsis, cell wall, defence, gene expression, signalling,
• MeSH
• Arabidopsis * growth & development   drug effects   metabolism   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Wall * metabolism   MeSH
• Chitosan * pharmacology   MeSH
• Glucans * metabolism   MeSH
• Glucosyltransferases metabolism   MeSH
• Plant Roots * growth & development   metabolism   drug effects   MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• callose MeSH Browser
• Chitosan * MeSH
• Glucans * MeSH
• Glucosyltransferases MeSH
• Arabidopsis Proteins * MeSH
• Calcium MeSH

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

• Keywords
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytoplasm metabolism   MeSH
• Exocytosis MeSH
• Phospholipids metabolism   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EXO70A1 protein, Arabidopsis MeSH Browser
• Phospholipids MeSH
• Arabidopsis Proteins MeSH

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

• Keywords
• HvMPK3, PR proteins, TALEN, barley, chitinases, flagellin, proteomics, root hairs,
• Publication type
• Journal Article MeSH

The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

• Keywords
• Arabidopsis thaliana, EXO70, biotic stress, exocyst, gene expression, lipid binding, redundancy, root hairs,
• Publication type
• Journal Article MeSH