Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation.

Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance.

Identification of soybean proteins from a single cell type: the root hair.

Root hairs (RH) are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean RH cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean RH using an accurate mass and time tag approach, establishing a comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to RH formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and ras-associated binding proteins). Interestingly, some of these proteins appear to be specifically detected in RH and constitute promising candidates for further studies to elucidate unique features of this single-cell model.

Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum.

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 eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

LYK4, a lysin motif receptor-like kinase, is important for chitin signaling and plant innate immunity in Arabidopsis.

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 LysM-containing 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.

Establishment of a protein reference map for soybean root hair cells.

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.