Potential novel bZIP-like gene for resistance to Erysiphe necator identified in Chinese wild Vitis pseudoreticulata

In this study, a novel bZIP-like gene was isolated from Chinese wild Vitis pseudoreticulata W. T. acc. Baihe-35-1. The full-length complementary deoxyribonucleic acid (cDNA) sequence of the gene was 1583 bp including 159 bp 5’ untranslated region (UTR), 365 bp 3’ UTR and a 1083 bp ORF which encodes a polypeptide of 360 amino acids with a molecular weight of 38.662 kDa. The deduced amino acid sequence shares an overall 46 to 69.8% sequence similarity with bZIP from other plants. Therefore, we designated this gene as V. pseudoreticulata bZIP (VpbZIP-like). The expression of VpbZIP-like was induced 12 h post inoculation (hpi) by Erysiphe necator, but transiently decreased, then increased in these two genotypes and its expression was lower in highly resistant genotype Baihe-35-1 than in susceptible genotype Hunan-1 at 24, 48 and 72 hpi. We further tested whether the expression was also a response to plant signaling molecules. Results indicate that the susceptible genotype Hunan-1 showed higher expression of VpbZIP-like than the highly resistant genotype Baihe-35-1 after exogenous application of methyl jasmonate (MeJA), salicylic acid (SA) and ethephon (Eth). Moreover, tissue specific expression pattern of VpbZIP-like was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results reveal that it was in lower lever in flower than in leaf, stem, tendril and fruit. The CDS of VpbZIP-like was inserted into the prokaryotic expression construct pGEX-4T-1, and then transformed into Escherichia coli BL21-code induced by isopropyl-b-D-thiogalactopyranoside (IPTG) which resulted in the production of a Mr. 64 kDa of GST- VpbZIP-like fusion protein displayed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Key words: Chinese wild Vitis, bZIP, gene expression, signaling molecules, fusion protein expression

Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of ∼16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum

Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas

BACKGROUND: Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated. METHODOLOGY/PRINCIPAL FINDINGS: We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated. CONCLUSION/SIGNIFICANCE: IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT

Genome-Wide Identification of the ABA Receptors Genes and Their Response to Abiotic Stress in Apple

The pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) (known as PYLs for short) have been identified and characterized as the ABA receptors in some plants. However, little is known about the details regarding PYL family genes in the apple (Malusdomestica). In this study, we identified 13 apple PYLs, termed MdPYL1-13, which could be classified into four groups according to structural features of the amino acid sequence. The gene structures and conserved motifs analysis found that the majority of MdPYLs had a similar number of exons and similar conserved motif profile in the same group. In addition, 11 gene pairs were identified to exhibit synteny by synteny analysis between the apple and Arabidopsis. Furthermore, we investigated MdPYLs transcript level in various organs of the red-fleshed apple (Malussieversii f. Neidzwetzkyana (Dieck) Langenf) ‘Xinjiang No.1’. The results suggested all MdPYLs within group I were expressed at relatively higher levels in all of the organs tested. However, the genes of group IV had little or no variation. Additionally, we found various hormone and stress-related cis-elements in the promoters of MdPYLs by analyzing cis-elements. Therefore, the expression levels of all MdPYLs were further detected under ABA, PEG, salt, and cold stresses in ‘Xinjiang No.1’ seedlings. We found that all MdPYLs except for MdPYL11 were upregulated by ABA treatment, 10 genes were upregulated by PEG treatment, 12 genes were upregulated by NaCl treatment, and six genes were upregulated by cold treatment (4 °C) while seven genes were downregulated. Thus, these MdPYLs might be involved in the defense against abiotic stresses. In addition, the interaction between 13 MdPYLs and two 2C protein phosphatases in the apple (MdPP2C65 and MdPP2C72) was investigated in yeast two-hybrid assays. These results suggested that MdPYLs may bind to MdPP2C65 and MdPP2C72 in different manners and with different intensity. Our studies provide useful information for further investigating and researching the regulatory mechanisms of PYL family genes in response to abiotic stresses in the apple

Overexpression of a SBP-Box Gene (VpSBP16) from Chinese Wild Vitis Species in Arabidopsis Improves Salinity and Drought Stress Tolerance

Salinity and drought are two major abiotic stresses that limit grape productivity. Responses to stress in grape are known to be regulated by several families of transcription factors. However, little is known about the role of grape Squamosa promoter binding protein (SBP)-box transcription factor genes in response to abiotic stress. To better understand the functions of the grape SBP-box genes in abiotic stress tolerance, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP16 was amplified from Chinese wild grapevine Vitis pseudoreticulata clone “Baihe-35-1”. We observed that the VpSBP16 protein fused to the green fluorescent protein (GFP) reporter accumulated in the nucleus when transiently expressed in onion epidermal cells. Moreover, VpSBP16 was shown to have transcriptional activation activity using a yeast trans-activation assay. We performed a VpSBP16 functional analysis through the characterization of transgenic Arabidopsis thaliana plants constitutively over-expressing VpSBP16. The transgenic lines had longer roots and the seeds had a higher germination rate than the wild type (WT) under osmotic stress. In addition, the accumulation of reactive oxygen species (ROS) of transgenic seedlings was significantly lower than WT in the transgenic lines, as was electrolyte leakage. VpSBP16 overexpression also elevated expression levels of stress-response genes involved in the salt overly sensitive (SOS) pathway. These results indicate that overexpression VpSBP16 in A. thaliana enhances tolerance of salt and drought stress during seed germination, as well in seedlings and mature plants, by regulating SOS and ROS signaling cascades

A Modified Impact-Increment-Based State Enumeration Method and its Application in Power Distribution Systems

Reliable planning and operation of power distribution systems are of great significance. In this paper, the impact-increment based state enumeration (IIBSE) method is modified to adapt to the features of distribution systems. With the proposed method, the expectation, probabilistic, and duration reliability indices can be accurately obtained with a lower enumerated order of contingency states. In addition, the time-consuming optimal power flow (OPF) calculation can be replaced by a simple matrix operation for both independent and radial series failure states. Therefore, the accuracy and efficiency of the assessment process are improved comprehensively. The case of RBTS bus 6 system and IEEE 123 node test feeder system are utilized to test the performance of the modified IIBSE. The results show the superiority of the proposed method over Monte Carlo (MC) sampling and state enumeration (SE) methods in distribution systems