Characterisation of HvVIP1 and expression profile analysis of stress response regulators in barley under Agrobacterium and Fusarium infections

Arabidopsis thaliana’s VirE2-Interacting Protein 1 (VIP1) interacts with Agrobacterium tumefaciens VirE2 protein and regulates stress responses and plant immunity signaling occurring downstream of the Mitogen-Activated Protein Kinase (MPK3) signal transduction pathway. In this study, a full-length cDNA of 972bp encoding HvVIP1 was obtained from barley (Hordeum vulgare L.) leaves. A corresponding 323 amino acid poly-peptide was shown to carry the conserved bZIP (Basic Leucine Zipper) domain within its 157th and 223rd amino acid residue. 13 non-synonymous SNPs were spotted within the HvVIP1 bZIP domain sequence when compared with AtVIP1. Moreover, minor differences in the bZIP domain locations and lengths were noted when comparing Arabidopsis thaliana and Hordeum vulgare VIP1 proteins through the 3D models, structural domain predictions and disorder prediction profiling. The expression of HvVIP1 was stable in barley tissues infected by pathogen (whether Agrobacterium tumefaciens or Fusarium culmorum), but was induced at specific time points. We found a strong correlation between the transcript accumulation of HvVIP1 and barley PR- genes HvPR1, HvPR4 and HvPR10, but not with HvPR3 and HvPR5, probably due to low induction of those particular genes. In addition, a gene encoding for a member of the barley MAPK family, HvMPK1, showed significantly higher expression after pathogenic infection of barley cells. Collectively, our results might suggest that early expression of PR genes upon infection in barley cells play a pivotal role in the Agrobacterium-resistance of this plant

Exploiting a wheat EST database to assess genetic diversity

Expressed sequence tag (EST) markers have been used to assess variety and genetic diversity in wheat (Triticum aestivum). In this study, 1549 ESTs from wheat infested with yellow rust were used to examine the genetic diversity of six susceptible and resistant wheat cultivars. The aim of using these cultivars was to improve the competitiveness of public wheat breeding programs through the intensive use of modern, particularly marker-assisted, selection technologies. The F2 individuals derived from cultivar crosses were screened for resistance to yellow rust at the seedling stage in greenhouses and adult stage in the field to identify DNA markers genetically linked to resistance. Five hundred and sixty ESTs were assembled into 136 contigs and 989 singletons. BlastX search results showed that 39 (29%) contigs and 96 (10%) singletons were homologous to wheat genes. The database-matched contigs and singletons were assigned to eight functional groups related to protein synthesis, photosynthesis, metabolism and energy, stress proteins, transporter proteins, protein breakdown and recycling, cell growth and division and reactive oxygen scavengers. PCR analyses with primers based on the contigs and singletons showed that the most polymorphic functional categories were photosynthesis (contigs) and metabolism and energy (singletons). EST analysis revealed considerable genetic variability among the Turkish wheat cultivars resistant and susceptible to yellow rust disease and allowed calculation of the mean genetic distance between cultivars, with the greatest similarity (0.725) being between Harmankaya99 and Sönmez2001, and the lowest (0.622) between Aytin98 and Izgi01

Differential physiological and molecular responses of three-leaf stage barley (Hordeum vulgare L.) under salt stress within hours

Salt stress is first perceived by the plant roots and inhibits plant growth in the short-term by inducing osmotic stress caused by decreased water availability. In this study, 160 mM NaCl was applied to three-leaf-stage barley plants (Hordeum vulgare L. cv. Marti) for a short period (0, 2, and 26 h) Osmolyte accumulation and ion leakage was significantly increased after salt stress treatment compared with control conditons in both leaf and root tissues within 2 h. We have also found that expressions of transcription factors HvDRF2 and HvWRKY12, associated with abiotic stress including salinity and drought stress, were quite low in root and shoots in control conditions; however, salt stress resulted into upregulation of HvDRF2 expression as 28.8- and 26.6-fold in roots and leaves, respectively, within 26 h. While salt stress-induced significantly upregulation of HvPR1A (26.4-fold) HvNHX1 (sevenfold) in 2 h at P < 0.05 level, significant upregulation of HvMT2 (8.2-fold) and HvDHN3 (4.7-fold) was observed at 26 h after salt treatment in roots. In leaves, HvMT2 (12.7-fold), HvNHX1 (12.1-fold) and HvBAS1 (3.4-fold) were significantly upregulated under salt stress. Only HvLHCB mRNA level was significantly decreased as 2- and 5.6-fold in leaf tissues with salinityin 2 and 26 h, respectively

Genetic diversity and mating types of Fusarium culmorum and Fusarium graminearum originating from different agro-ecological regions in Turkey

Fusarium culmorum and F. graminearum are the major pathogens for dryland root/foot-rot and head-blight diseases in economically important grain crops. This study was aimed at the molecular characterization of Fusarium spp. isolates, which have been collected from cereal fields in three agro-ecological regions in Turkey. Genetic diversity has been analyzed by generating RFLP markers from the intergenic spacer (IGS) region of ribosomal RNA. The selection of restriction enzymes for IGS-RFLP studies has been found critical to maximize polymorphic markers. Only 3 of 14 restriction endonucleases were useful in differentiating Fusarium spp. isolates. PstI was the most efficient enzyme to produce a maximum of nine DNA markers in one individual and total 22 polymorphic representative banding patterns. Polymorphism based on IGS-RFLP was high and average 88% in both species. There was no association between IGS diversity and geographic locations from which the samples were taken. Both MAT-1 and MAT-2 sequences were amplified in F. graminearum similarly to previous reports. Most of the F. culmorum isolates carried either MAT-1 or MAT-2 sequences, and differently two isolates carried both sequences. Mating type determination was helpful to distinguish F. pseudograminearum from F. graminearum, which cannot be discriminated by SCAR markers or morphological assessment. High genetic diversity by IGS-RFLP markers in F. culmorum was discussed in relation to its fitness as the most common pathogen in dryland root rot complex (DLRRC)

Analysis of expressed sequence tags from cDNA library of Fusarium culmorum infected barley (Hordeum vulgare L.) roots

Fusarium culmorum is one of the most common and globally important causal agent of root and crown rot diseases of cereals. These diseases cause grain yield loss and reduced grain quality in barley. In this study, we have analyzed an expressed sequence tag (EST) database derived from F. culmorum infected barley root tissues available at the National Center for Biotechnology Information (NCBI). The 2294 sequences were assembled into 1619 non-redundant sequences consisting of 359 contigs and 1260 singletons using the program CAPS. BLASTX analysis for these sequences was conducted in order to find similar sequences in all databases. Gene Ontology search, enzyme search, KEGG mapping and InterProScan search were done using Blast2GO 3.0.7 tool. By BLASTX analysis, 41.7%, 7.7%, 3.2% and 47.4% of ESTs were categorized as annotated, unannotated, not mapping and without blast hits, respectively. BLASTX analysis revealed that the majority of top hits were barley proteins (43.5%). Based on Gene Ontology classification, 38.3%, 31.3%, and 16% of ESTs were assigned to molecular function, biological process, and cellular component GO terms, respectively. Most abundant GO terms were as follows: 157 sequences were related to response to stress (biological process), 207 sequences were related to ion binding (molecular function), and 160 sequences were related to plastid (cellular component). Furthermore, based on KEGG mapping, 369 sequences could be assigned to 264 enzymes and 83 different KEGG pathways. According to Enzyme Commission (EC) distribution; 94 sequences were transferases (EC2) while 70 sequences were hydrolases (EC3)

Discovery of Dhn3 variants in wild barley (Hordeum spontaneum) by high-resolution melting (HRM) technology

The analysis of allelic variation in model plant species and their wild relatives, such as Hordeum vulgare and Hordeum spontaneum, is useful for relating genetic determinants and important phenotypic traits such as stress tolerance. High resolution melting (HRM) analysis is a cost-effective, rapid, and high-throughput assay for mutation screening and genotyping without sequencing. The present study describes an HRM analysis of natural sequence variation within Dhn3 alleles from different H. spontaneum accessions. Small PCR-derived amplicon assays were developed for exon 1 and exon 2 of Dhn3. The efficiency of the HRM procedure was affected by various factors, including specificity and efficiency of PCR, amplicon length and position, and DNA template quality. In addition to these factors, the use of PCR product rather than genomic DNA in HRM increased the quality of melting curves, thus affecting the accuracy and sensitivity of the assay. HRM classified 5-6 groups of variants carrying deletion mutants and single and multiple SNPs consistent with the sequencing data. 18-bp deletion variants were distinguished from the reference sample according to HRM analysis of 207-bp fragment of exon 1. A/T conversions were difficult to discriminate variants, whereas A/G or T/C transitions were easily grouped because they required high T-m differences. The conditions and parameters for an optimized HRM assay were outlined, and its potential application in allelic variation research of stress-related genes was discussed

The Single-tooth Implant Treatment of Congenitally Missing Maxillary Lateral Incisors Using Angled Abutments: A Clinical Report

The maxillary lateral incisor is the second most common congenitally absent tooth. There are several treatment options for replacing the missing maxillary lateral incisor, including canine substitution, tooth-supported restoration, or single-tooth implant. Dental implants are an appropriate treatment option for replacing missing maxillary lateral incisor teeth in adolescents when their dental and skeletal development is complete. This case report presents the treatment of a patient with congenitally missing maxillary lateral incisors using dental implants with angled abutments