On- and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice.

The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.

Boosting plant genome editing with a versatile CRISPR-Combo system.

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.

Microbial composition of Kombucha determined using amplicon sequencing and shotgun metagenomics.

Kombucha, a fermented tea generated from the co-culture of yeasts and bacteria, has gained worldwide popularity in recent years due to its potential benefits to human health. As a result, many studies have attempted to characterize both its biochemical properties and microbial composition. Here, we have applied a combination of whole metagenome sequencing (WMS) and amplicon (16S rRNA and Internal Transcribed Spacer 1 [ITS1]) sequencing to investigate the microbial communities of homemade Kombucha fermentations from day 3 to day 15. We identified the dominant bacterial genus as Komagataeibacter and dominant fungal genus as Zygosaccharomyces in all samples at all time points. Furthermore, we recovered three near complete Komagataeibacter genomes and one Zygosaccharomyces bailii genome and then predicted their functional properties. Also, we determined the broad taxonomic and functional profile of plasmids found within the Kombucha microbial communities. Overall, this study provides a detailed description of the taxonomic and functional systems of the Kombucha microbial community. Based on this, we conject that the functional complementarity enables metabolic cross talks between Komagataeibacter species and Z. bailii, which helps establish the sustained a relatively low diversity ecosystem in Kombucha.

CRISPR-Cas nucleases and base editors for plant genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.

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.

Barley Genes as Tools to Confer Abiotic Stress Tolerance in Crops.

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.

Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses.

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 (HsDHN3). HsDHN3 was found to be YSK2-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 HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 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 K1 and K2 are likely to be key factors in protection of cells.

Enhancing T-DNA Transfer Efficiency in Barley (Hordeum vulgare L.) Cells Using Extracellular Cellulose and Lectin.

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.

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 CAP3. 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).

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).

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates.

In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP)-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi. At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola, P. savastanoi pv. savastanoi and P. syringae pv. tomato. REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP- and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC) sequences failed to produce clear banding patterns in this study.

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 F(2) 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.

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 percent) contigs and 96 (10 percent) 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.

Regeneration capacity of mature embryo-derived callus in barley ( Hordeum vulgare L.).

In this study, induction of regenerable callus from mature embryos in eight Turkish barley varieties was analysed by using different plant growth regulators (PGRs). Varying concentrations (0.5-4 mg l -1 ) of 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba (3,6-dichloro-o-anisic acid) were tested for callus induction from mature embryos. Highest percent of callus induction was observed in Bornova 92 variety (98.3%) on MS medium supplemented with 4 mg l -1 dicamba. Calli were transferred to regeneration media with 0.5 mg l -1 dicamba, 0.5 mg l -1 zeatin riboside (ZR) and 2 mg l -1 thidiazuron (TDZ). Low concentrations of dicamba induced multiple shoots during callus regeneration. When the effect of precultivation with 2,4-D or dicamba on the shoot induction were evaluated, lower concentrations (< 4 mg l -1 ) of auxins have been found optimal. On the regeneration medium with 0.5 mg l -1 dicamba, shoots were able to elongate up to 20 cm and shoot numbers were between 1-23 per callus. The use of ZR led to formation of short shoot buds and somatic embryos in 2 weeks period. The effect of TDZ was different from other PGRs by inducing green solid sectors on calli surfaces (Total 51 sectors/20 callus/Akhisar variety). Five plantlets have been grown from these solid cell clumps and transferred to specific media for root formation. As a result, five varieties (Süleyman Bey, Bornova 92, Vamyk Hoca, Kaya and Akhisar) tested in our study showed the potential to produce regenerable callus by using low amounts of dicamba or TDZ. The optimization process starts from culturing embryos to plantlet formation took nearly 4 weeks.