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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.

Shock-like water stress using hydroponics and gradual water deficit in soil are the two widely used treatments to analyze transcriptional response of many crops to drought. In this study, we investigated the effects of shock drought (ShD) (0, 0.5, 1, 4, and 8 h) and slow drought (SDD) [soil water content (SWC) of 35 and 50 %] on the expression of well-known drought-responsive genes supplemented with physiological changes in barley. Two barley cultivars with contrasting leaf relative water content (RWC) and water loss rate (WLR) values were selected as Martı (MR; 60 % RWC and 0.046 gh −1  g −1 DW) and Erginel90 (ER; 38.3 % RWC and 0.350 gh −1  g −1 DW) under 38 % of SWC condition. According to the results, 0.5 h ShD was the critical time point for stress perception in leaves defined by the increase in WLR, ion leakage and H 2 O 2 concentration. Expressions of antioxidant-related genes (Cu–Zn/ SOD , HvCAT2 , HvGST6 , HvAPX ) were rapidly induced in MR at 8 h shock, while only slightly upregulated in ER. We have also observed higher induction of expressions of HvBAS1 , HvMT - 2, HvABA7 and a photosynthesis-related gene HvLHCB during ShD compared to SDD. Contrarily, transcription factors (TFs), HvWRKY12 and HvDRF1 were expressed with lower values during shock-dehydration. Slow-drought treatments in both cultivars were characterized with high leaf RWCs and osmotic adjustment with low cell membrane damage, suggesting that barley maintains a basal tolerance to long-term water deficit. Our results confirmed that type of water stress treatment is crucial to measure gene expression, and a shock-like dehydration method should be the treatment of choice in evaluating barley plants with different physiological characteristics for water tolerance.

Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs) responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA) proteins, antioxidant enzymes, osmolytes, and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors (HvCBF4), dehydration-responsive element-binding factors (HvDREB1), and WRKYs (HvWRKY38), in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na(+)/H(+) antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures.

CRISPR-Cas9, its derived base editors and CRISPR activation systems have greatly aided genome engineering in plants. However, these systems are mostly used separately, leaving their combinational potential largely untapped. Here we develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase the powerful applications of CRISPR-Combo for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a new method to predominately enrich heritable targeted mutations. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.Pan et al. develop a versatile CRISPR-Combo platform for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants, representing a versatile genome engineering tool with promising applications in crop breeding.

Fusarium culmorum is able to cause devastating crown rot disease, particularly in barley and wheat worldwide. The aim of this study was to investigate the early physiological and molecular changes in barley roots in response to F. culmorum infection. Therefore, we have infected 3-day old barley roots with a highly pathogenic F. culmorum isolate (F16). The root length and shoot length were significantly reduced at 7 days after infection in six widely cultivated Turkish barley cultivars. Based on the disease index values, Martı (six-rowed) and Tokak 157/37 (two-rowed) were selected. Defense response was comparatively assessed with measures including H 2 O 2 production and induction of stress-induced genes at six-time points after infection (0–96 h). Fungal infection did not affect the membrane integrity of root cells while osmolality decreased and H 2 O 2 production increased. At the molecular level, antioxidant-related genes, HvCu / ZnSOD , HvGST6 , HvAPX and HvBAS1 were constitutively and strongly expressed unlikely to HvCAT2 in which transcript accumulation was slightly detected upon infection. Differential expression of HvMT2 , HvLOX1 and HvWRKY12 has been observed following the infection. Importantly, pathogenesis related (PR) genes HvPR1 , HvPR3 , HvPR4 , HvPR5 and HvPR10 were induced at different time points of infection. The transcript accumulation of HvPR4 was the highest while HvPR10 expressed in minimal levels. Our results showed unexpected cellular responses such as disruption of osmotic adjustment in barley roots and the role of PR genes in initial response under F. culmorum attack.

We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide ( Hs DHN3). Hs DHN3 was found to be YSK 2 -type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in Hs DHN3 that coincides with the K 1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K 1 segment and a novel conserved region located between the S and K 1 segments [E(D/T)DGMGGR]. We found that H. vulgare , H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K 1 and K 2 are likely to be key factors in protection of cells.

A major limitation of transforming barley tissues by Agrobacterium tumefaciens is the low frequency of T-DNA transfer due to recalcitrance of barley as a host. The effect of extracellular cellulose and lectin on Agrobacterium transformation efficiency was investigated in this study. Barley callus cultures were transformed with the AGL1 strain containing the vector pBI121 in the presence of 10 mg mL⁻¹ cellulose or 0.001, 0.05 and 0.1 mg mL⁻¹ lectin. Addition of cellulose significantly (P ≤ 0.05) increased the number of GUS spots by 50 % compared to standard conditions in the presence of only 200 μM acetosyringone (AS). Frequency of G418-resistant aggregates on the surfaces of callus cultures was 29 and 71.5 %, following AS and AS + cellulose treatments, respectively, after 4 weeks of selection. Presence of 0.05 or 0.1 mg mL⁻¹ lectin also increased the number of GUS spots and frequency of G418-resistant cells in the selection period, but the increase in blue spots was not significant. We examined the effect of lectin and cellulose on bacterial attachment to callus tissues. Both cellulose and lectin were found to have a significant positive effect on the numbers of bacteria attached to barley callus. Epifluorescence microscopy revealed that Agrobacterium cells had accumulated in the scaffolds of irregular fibrous cellulose with a mean particle size of 200 μm. Expression of nptII in transformed callus lines confirmed the stable transformation of the gene. Our study showed for the first time the binding of Agrobacterium cells to fibrous cellulose and also demonstrated how polysaccharides and glycoproteins can be used to improve T-DNA transfer in monocotyledon transformation procedures.

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs) responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA) proteins, antioxidant enzymes, osmolytes and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors (HvCBF4), dehydration-responsive element-binding factors (HvDREB1) and WRKYs (HvWRKY38), in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na+/H+ antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures.

Our study aimed to obtain clinical indication-based typical dose values and size-specific dose estimates (SSDEs) for multiphasic abdominopelvic computed tomography (CT) examinations and to review our data with published diagnostic reference levels (DRLs). In this retrospective study, multiphasic liver, kidney, pancreas, and mesenteric ischemia protocol CT scans performed at our center between January 2018 and December 2021 were analyzed. The clinical indications were hepatocellular carcinoma, renal cell carcinoma, pancreas adenocarcinoma, and mesenteric ischemia. The computed tomography dose index volume (CTDI ) and dose-length product (DLP) values were recorded, and the SSDE and effective dose (ED) values were calculated. The water-equivalent diameter (Dw) value required for the SSDE calculation was measured using the automated calculation of the Dw program. The total number of patients was 514, with 86 patients excluded from this study. The dose values were calculated for 426 patients (183 female and 243 male; 111 liver, 120 kidney, 85 pancreas, and 110 mesenteric). The median values for the CTDI , DLP, SSDE, and ED were 6.86 mGy, 683.02 mGy. cm, 8.75 mGy, and 10.45 mSv for the liver CT; 8.37 mGy, 908.37 mGy.cm, 10.37 mGy, and 13.89 mSv for the kidney CT; 7.82 mGy, 517.98 mGy.cm, 10.01 mGy, and 7.92 mSv for the pancreas CT; and 9.48 mGy, 983.68 mGy.cm, 12.78 mGy, and 13.86 mSv for the mesenteric CT, respectively. All dose values were lower than the published DRLs. The literature reveals large differences in the multiphasic abdominopelvic CT protocols, especially in the number of phases and scan length. This situation makes comparing dose values difficult. Dose studies revealing the protocol parameters in detail are needed so that institutions can compare and optimize their own protocols. Additionally, users should periodically check the dose values in their own institutions.