Chitin is commonly found in fungal cell walls and is one of the well-studied microbe/pathogen-associated molecular patterns. Previous studies showed that lysin motif (LysM)-containing proteins are essential for plant recognition of chitin, leading to the activation of plant innate immunity. In Arabidopsis (Arabidopsis thaliana), the LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1) was shown to be essential for chitin recognition, whereas in rice (Oryza sativa), the LysMcontaining protein, CEBiP (for chitin elicitor-binding protein), was shown to be involved in chitin recognition. Unlike LYK1/ CERK1, CEBiP lacks an intracellular kinase domain. Arabidopsis possesses three CEBiP-like genes. Our data show that mutations in these genes, either singly or in combination, did not compromise the response to chitin treatment. Arabidopsis also contains five LYK genes. Analysis of mutations in LYK2, -3, -4, or -5 showed that LYK4 is also involved in chitin signaling. The lyk4 mutants showed reduced induction of chitin-responsive genes and diminished chitin-induced cytosolic calcium elevation as well as enhanced susceptibility to both the bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the fungal pathogen Alternaria brassicicola, although these phenotypes were not as dramatic as that seen in the lyk1/cerk1 mutants. Similar to LYK1/CERK1, the LYK4 protein was also localized to the plasma membrane. Therefore, LYK4 may play a role in the chitin recognition receptor complex to assist chitin signal transduction and plant innate immunity.

Root hairs are single tubular cells formed from the differentiation of epidermal cells on roots. They are involved in water and nutrient uptake and represent the infection site on leguminous roots by rhizobia, soil bacteria that establish a nitrogen-fixing symbiosis. Root hairs develop by polar cell expansion or tip growth, a unique mode of plant growth shared only with pollen tubes. A more complete characterization of root hair cell biology will lead to a better understanding of tip growth, the rhizobial infection process, and also lead to improvements in plant water and nutrient uptake. We analyzed the proteome of isolated soybean (Glycine max) root hair cells using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and shotgun proteomics (1D-PAGE-liquid chromatography and multidimensional protein identification technology) approaches. Soybean was selected for this study due to its agronomic importance and its root size. The resulting soybean root hair proteome reference map identified 1,492 different proteins. 2D-PAGE followed by mass spectrometry identified 527 proteins from total cell contents. A complementary shotgun analysis identified 1,134 total proteins, including 443 proteins that were specific to the microsomal fraction. Only 169 proteins were identified by the 2D-PAGE and shotgun methods, which highlights the advantage of using both methods. The proteins identified are involved not only in basic cell metabolism but also in functions more specific to the single root hair cell, including water and nutrient uptake, vesicle trafficking, and hormone and secondary metabolism. The data presented provide useful insight into the metabolic activities of a single, differentiated plant cell type.

Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eightplex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS 1 analysis using HCD and decision tree guided CID/ETD strategy. Root hairs are known to play an important role in increasing the root surface area for water and nutrient uptake from the soil (1). Found on the surface of the maturation zone of primary and secondary roots, root hairs develop from specialized epidermal cells (trichoblast). New root hair cells continuously develop in the elongation zone, elongating and maturing as the root grows (2). In addition to the critical role in nutrient uptake, the root hair is the primary infection site for symbiotic bacteria (rhizobia) in legume plants. During the first stages of the legume-rhizobium interaction, (iso)flavonoids secreted by the legume induce the rhizobia to synthesize the Nod factor, a specific lipo-chito-oligosaccharide. This bacterial signal molecule elicits a variety of very rapid (within minutes) responses in the root hair cell, including depolarization of the membrane potential and induction of calcium oscillations (3). The root hair then curls to form a shepherd's crook structure, where the rhizobia become entrapped within the root hair cell wall (4), leading subsequently to an invagination of the root hair plasma membrane and the formation of the tubular infection thread structure by which the bacteria ultimately gain access to the root cortex (5).Root hair physiology under both controlled and biotic/abiotic stress conditions has been studied intensively using a variety of approaches. Numerous root hair mutants have been identified and subsequently linked to the function of various 1 The abbreviations used are: nanoRPLC-MS/MS, nano reversedphase liquid chromatography-tandem mass spectrometry; HCD-CID/ ETD, high energy/collision-induced dissociation/electron transfer dissociation; Ni-NTA, Nickel-nitriloacetic acid; iTRAQ, Isobaric tag for relative and absolu...