Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.

The regulation of glutathione s-transferases by gibberellic acid application in salt treated maize leaves.

Plant hormones and antioxidant system changes occur during plants' exposure to stress conditions. Although the interactions of some plant hormones (abscisic acid, salicylic acid, jasmonic acid, nitric oxide, and ethylene) with the glutathione s-transferase (GST) enzyme, which is one of the antioxidant enzymes, have already been reported, the influence of gibberellic acid (GA3) on this enzyme under saline conditions has not yet been reported. Plant material for the experiments was obtained from M14G144 cultivar of maize (Zea mays L.) plants grown as a soil culture in growth chambers at 22 °C, 65-70% moisture, 16-h light/8-h dark conditions, and with full strength Hoagland solution for 8 days under controlled growth conditions. Then, the plants were exposed to salt stress (350 mM NaCl and 100, 300, and 500 ppm GA3) simultaneously. In maize leaves, GA3 treatment alleviated the physiological parameters under salt stress. Specifically, the treatments with 100 and 500 ppm of GA3 were able to trigger GST enzyme and isoenzyme activities as well as hydrogen sulfide accumulation and anthocyanin content, although the lowest malondialdehyde, hydrogen peroxide, and superoxide radical content were under the treatment of 300 ppm of GA3. Besides this, GST gene expression levels were found to be upregulated between 1.5 and fourfold higher in all the plants treated with GA3 at different concentrations in proportion to salt stress. These results first indicated that the reason for the changes in GA3-treated plants was the stimulating role of this hormone to maintain GST regulation in maize plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01269-2.

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris).

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks < 1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume.

Genome-Wide Identification of the Aconitase Gene Family in Tomato (Solanum lycopersicum) and CRISPR-Based Functional Characterization of SlACO2 on Male-Sterility.

Tomato (Solanum lycopersicum) is one of the most cultivated vegetables in the world due to its consumption in a large variety of raw, cooked, or processed foods. Tomato breeding and productivity highly depend on the use of hybrid seeds and their higher yield, environmental adaption, and disease tolerance. However, the emasculation procedure during hybridization raises tomato seed production costs and labor expenses. Using male sterility is an effective way to reduce the cost of hybrid seeds and ensure cultivar purity. Recent developments in CRISPR genome editing technology enabled tomato breeders to investigate the male sterility genes and to develop male-sterile tomato lines. In the current study, the tomato Acotinase (SlACO) gene family was investigated via in silico tools and functionally characterized with CRISPR/Cas9-mediated gene disruption. Genome-wide blast and HMM search represented two SlACO genes located on different tomato chromosomes. Both genes were estimated to have a segmental duplication in the tomato genome due to their identical motif and domain structure. One of these genes, SlACO2, showed a high expression profile in all generative cells of tomato. Therefore, the SlACO2 gene was targeted with two different gRNA/Cas9 constructs to identify their functional role in tomatoes. The gene was mutated in a total of six genome-edited tomato lines, two of which were homozygous. Surprisingly, pollen viability was found to be extremely low in mutant plants compared to their wild-type (WT) counterparts. Likewise, the number of seeds per fruit also sharply decreased more than fivefold in mutant lines (10-12 seeds) compared to that in WT (67 seeds). The pollen shape, anther structures, and flower colors/shapes were not significantly varied between the mutant and WT tomatoes. The mutated lines were also subjected to salt and mannitol-mediated drought stress to test the effect of SlACO2 on abiotic stress tolerance. The results of the study indicated that mutant tomatoes have higher tolerance with significantly lower MDA content under stress conditions. This is the first CRISPR-mediated characterization of ACO genes on pollen viability, seed formation, and abiotic stress tolerance in tomatoes.

Genome-based identification of beet curly top Iran virus infecting sugar beet in Turkey and investigation of its pathogenicity by agroinfection.

Beet curly top disease (BCTD) is a yield-limiting viral infection of sugar beet (Beta vulgaris) throughout the arid and semi-arid regions of the world. Two virus species, belonging to two different genera of the family Geminiviridae (Curtovirus and Becurtovirus) had been described as the disease's causative agents on sugar beet. Despite the detection of the BCTD in some sugar beet fields of Turkey sixty years ago, the genome based characterization of BCTD-associated viruses have not been studied previously. In this study, 628 sugar beet plants exhibiting BCTD symptoms were collected from fourteen cities in central Anatolia, the major sugar beet production areas in Turkey. PCR assays of these samples using the respective Curtovirus and Becurtovirus genus-specific primers indicated that the Turkish sugar beet samples' viral sequences belong only to the genus Becurtovirus. The results of sequencing and phylogenetic analysis of the partial genome of the virus obtained from fourteen cities confirmed that BCTD-associated virus in Turkish sugar beet fields is beet curly top Iran virus (BCTIV-Becurtovirus) species. The whole genome of the collected viruses from fourteen cities were amplified by the rolling circle amplification (RCA) and the five most phylogenetically diverse viruses obtained from Afyon, Ankara, Adapazari, Yozgat and Aksaray were sequenced. The results of whole genome sequence analysis indicated >98 % sequence identities with that of a BCTIV variants reported from Urmia province (bordering Turkey) of Iran. A virus genome from Yozgat city had a genomic sequence identity of >97 % with those of BCTIV isolated from cowpea, tomato, pepper and sugar beet in the northern part of Iran. These results suggested that the spread of BCTIV through the region could create a significant threat to the production of sugar beet as well as other agricultural crops. A tandem dimer of a BCTIV-Turkish variant isolated from Ankara city was cloned into Agrobacterium plasmid to be used for agro-infection studies. Agroinoculation of this construct on sugar beet leaves generated severe BCTD symptoms (84 %) which were also confirmed by RCA and qPCR analysis. These results constituted the first genome based characterization of BCTIV Turkish variants and the first report of BCTIV spreading out of Iran.

Genome-wide identification of the BURP domain-containing genes in Phaseolus vulgaris.

Plant-specific BURP domain-containing proteins have an essential role in the plant's development and stress responses. Although BURP domain-containing proteins have been identified in several plant species, genome-wide analysis of the BURP gene family has not been investigated in the common bean. In the present study, we identified 11 BURP family members in the common bean (Phaseolus vulgaris) genome with a comprehensive in silico analysis. Pairwise alignment and phylogenetic analyses grouped PvBURP members into four subfamilies [RD-22 like (3), PG1ß-like (4), BNM2-like (3), and USP-like (1)] according to their amino acid motifs, protein domains and intron-exon structure. The physical and biochemical characteristics of amino acids, motif and intron-exon structure, and cis-regulatory elements of BURPs members were determined. Promoter regions of BURP members included stress, light, and hormone response-related cis-elements. Therefore, expression profiles of PvBURP genes were identified with in silico tools and qRT-PCR analyses under stress (salt and drought) and hormone treatment (ABA, IAA) in the current study. While significant activity changes were not observed in BURP genes in RNA-seq data sets related to salt stress, it was determined that some BURP genes were expressed differently in those with drought stress. We identified 12 different miRNA, including miRNA395, miRNA156, miRNA169, miRNA171, miRNA319, and miRNA390, targeting the nine PvBURP genes using two different in silico tools based on perfect or near-perfect complementarity to their targets. Here we present the first study to identify and characterize the BURP genes in common bean using whole-genome analysis, and the findings may serve as a reference for future functional research in common bean. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01052-9.

Overexpression of Ks-type dehydrins gene OeSRC1 from Olea europaea increases salt and drought tolerance in tobacco plants.

Agricultural production is greatly affected by environmental stresses, such as cold, drought and high-salinity. It is possible to produce tolerant genotypes by transferring genes encoding protective proteins or enzymes from other organisms. In this regard, the current study was aimed to clone a novel OeSRC1 gene identified during the transcriptome profiling of olives (Olea europaea L.) and to investigate the function of this gene in tobacco plants. Functional evaluation of OeSRC1 gene in putative transgenic tobacco plants were carried out under drought, cold and salt stress conditions by using molecular and biochemical tools. It was observed that the transgenic tobacco plants exhibited higher seed germination and survival rates, better root and shoot growth under cold, salt and drought stress treatments compared to wild type plants. Our results also demonstrated that, under stress conditions, transgenic plants accumulated more free proline while no significant changes were observed regarding electrolyte leakage. Ascorbate peroxidase activity of OeSRC1-overexpressing plants was higher than those of the WT plants under different stress conditions. The overall results demonstrate the explicit role of OeSRC1 gene in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional role of olive OeSRC1 gene looks good to enhance environmental stress tolerance in diverse plants.