Molecular cloning,expression pattern and phylogenetic analysis of Guanine nucleotide exchange factor Vav2 in lamprey,Lampetra japonica

The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.

Genetic analysis of GEFs and GDIs in rice reveals the roles of OsGEF5,OsGDI1,and OsGEF3 in the regulation of grain size and plant height

Guanine nucleotide exchange factors(GEFs)and guanine nucleotide-dissociation inhibitors(GDIs)regulate small GTPase proteins,which function as molecular switches in various signaling pathways,but their identification and functions in plants are not well understood.Using in-silico analysis and transgenic approaches,respectively,we dissected the evolutionary relationships and functions of all GEF and GDI genes in rice.Intron-exon distribution and phylogenetic analyses identified 30 GEF and 10 GDI genes in rice that shared close evolutionary relationships with other eukaryotes.Tissue-specific expression and co-expression analyses revealed that phylogenetically related genes had similar expression patterns.GEF and GDI genes were highly expressed in panicles,hulls,and stamens.Co-expression network analysis identified panicle and stamen-specific modules of co-expressed genes in both families.Mapping of these genes in known protein interactomes further identified two and one small G-protein sub-networks.A mutant library of GEF and GDI families was constructed by CRISPR knockout of each gene,and their genotypes and phenotypes were confirmed.Phenotype changes occurred with the mutation of only three genes(OsGEF5,OsGDI1,and OsGEF3).OsGEF5 and OsGDI1 single mutants exhibited significantly reduced height and longer and thinner grains,whereas OsGEF3 mutants had reduced grain length compared to the wild type.Haplotype and eGWAS analyses showed that natural variations in the three genes affected gene expression in reproductive tissues that were significantly associated with the phenotypic variation.BiFC assays demonstrated that GDI1 and GEF3 interacted with grain-size protein GS3,pointing to a role of these genes in the regulation of grain size and plant architecture connected to heterotrimeric G-proteins in rice.

Structure and Expression of Several Putative Cdc42-Interacting Proteins in Magnaporthe grisea

MgCdc42 （Cdc42 in Magnaporthe grisea）, with high homology to ScCdc42 （Cdc42 in Saccharomyces cerevisiae）, has been demonstrated to involve in the morphogenesis and infection process. To further understand the signaling network, the putative MgCdc42-interacting proteins were analyzed. ScCdc42-interacting protein sequences were first used to BLAST against the M. grisea genome database to retrieve their corresponding analogs. Subsequently, conserved domains of these proteins were compared and expression patterns of their encoding genes in different MgCdc42 mutation states were analyzed by semiquantitative RT-PCR. All retrieved analogs of ScCdc42-interacting proteins from the M. grisea database have conserved domains as those in S. cerevisiae. Expression of their encoding genes increased in MgCdc42CA mutant and decreased in MgCdc42KO mutant. However, MgBeml, Chin1, and MgGicl in MgCdc42DN mutant had the same expression level as that in the wild type, although MgBem4, MgBoi2, MgCdc24, MgGic2, MgRgal, and Mst20 had decreased expression level, as expected. Overall, it is concluded that there may exist a similar Cdc42 signal pathway in M. grisea as in S. cerevisiae and MgCdc42 plays a key role in the pathway.

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex.During infection,rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia.However,the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive.Here,we found that small GTPases(MtRac1/MtROP9 and its homologs)are required for root hair development and rhizobial infection in Medicago truncatula.Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs.In turn,phosphorylated MtRopGEF2 can activate MtRac1.Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP.Taken together,our results support the hypothesis that MtRac1,LYK3,and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.

Crystal structure of the C-terminal domain of the ε subunit of human translation initiation factor eIF2B

Eukaryotic translation initiation factor eIF2B,the guanine nucleotide exchange factor(GEF)for eIF2,catalyzes conversion of eIF2·GDP to eIF2·GTP.The eIF2B is composed of five subunits,α,β,γ,δandε,within which theεsubunit is responsible for catalyzing the guanine exchange reaction.Here we present the crystal structure of the C-terminal domain of human eIF2Bε(eIF2Bε-CTD)at 2.0-Åresolution.The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified.When compared to yeast eIF2Bε-CTD,one area involves highly conserved AA boxes while the other two are only partially conserved.In addition,the previously reported mutations in human eIF2Bε-CTD,which are related to the loss of the GEF activity and human VWM disease,have been discussed.Based on the structure,most of such mutations tend to destabilize the HEAT motif.