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Significance We communicate the rather remarkable observation that among 291 tested accessions of cultivated sweet potato, all contain one or more transfer DNA (T-DNA) sequences. These sequences, which are shown to be expressed in a cultivated sweet potato clone (“Huachano”) that was analyzed in detail, suggest that an Agrobacterium infection occurred in evolutionary times. One of the T-DNAs is apparently present in all cultivated sweet potato clones, but not in the crop’s closely related wild relatives, suggesting the T-DNA provided a trait or traits that were selected for during domestication. This finding draws attention to the importance of plant–microbe interactions, and given that this crop has been eaten for millennia, it may change the paradigm governing the “unnatural” status of transgenic crops. Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions ( Ib T-DNA1 and Ib T-DNA2) are present in the cultivated sweet potato ( Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. Ib T-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase ( iaaM ), indole-3-acetamide hydrolase ( iaaH ), C-protein ( C-prot ), and agrocinopine synthase ( Acs ) genes of Agrobacterium spp. Ib T-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. Ib T-DNA2 contained at least five ORFs with significant homology to the ORF14 , ORF17n , rooting locus ( Rol ) B/RolC , ORF13 , and ORF18/ORF17n genes of A. rhizogenes . Ib T-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

Background Recently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium -mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation. Results In the present study, we efficiently applied targeted mutagenesis in common wheat ( Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens . Seven target sites in three genes ( Pinb, waxy and DA1 ) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens -mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences. Conclusions Our results showed that our Agrobacterium -mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

Bacteria produce second messengers, such as c-di-GMP, to regulate various cellular processes, including biofilm formation, virulence, and bacterial antagonism. Diguanylate cyclases (DGCs) catalyze the biosynthesis of c-di-GMP and function to cope with changing environments through targeting specific effector proteins. In this study, we uncover that phytopathogenic agrobacteria deploy two DGC domain proteins to suppress virulence and interbacterial competition through two different regulatory pathways. One exhibits the DGC activity, enhancing global c-di-GMP concentration to elevate biofilm formation and inhibit virulence and antibacterial activity, while the other specifically suppresses virulence, independent of c-di-GMP biosynthesis. Our findings provide new insight into the distinct regulatory mechanisms of DGC domain proteins on regulating virulence and interbacterial competition, highlighting potential new strategies for controlling Agrobacterium pathogenicity.

Agrobacterium tumefaciens-mediated plant transformation is the most dominant technique for the transformation of plants. It is used to transform monocotyledonous and dicotyledonous plants. A. tumefaciens apply for stable and transient transformation, random and targeted integration of foreign genes, as well as genome editing of plants. The Advantages of this method include cheapness, uncomplicated operation, high reproducibility, a low copy number of integrated transgenes, and the possibility of transferring larger DNA fragments. Engineered endonucleases such as CRISPR/Cas9 systems, TALENs, and ZFNs can be delivered with this method. Nowadays, Agrobacterium-mediated transformation is used for the Knock in, Knock down, and Knock out of genes. The transformation effectiveness of this method is not always desirable. Researchers applied various strategies to improve the effectiveness of this method. Here, a general overview of the characteristics and mechanism of gene transfer with Agrobacterium is presented. Advantages, updated data on the factors involved in optimizing this method, and other useful materials that lead to maximum exploitation as well as overcoming obstacles of this method are discussed. Moreover, the application of this method in the generation of genetically edited plants is stated. This review can help researchers to establish a rapid and highly effective Agrobacterium-mediated transformation protocol for any plant species.

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

Seven bacterial isolates were obtained from crown gall in blueberry plants ( Vaccinium corymbosum ) growing in Serbia in 2020. Phylogenetic analysis of the novel isolates using four housekeeping genes – atpD , dnaK , glnA , and rpoB , as well as gene 16 S rRNA – revealed their distinct position, separate from other known Agrobacterium species, but closest to A. vaccinii , A. rosae and A. rubi . Genome sequencing of one of these blueberry isolates revealed less than 86% average nucleotide identity (ANI) between the blueberry isolate and type strains of Agrobacterium species, indicating that it does not belong to any previously described species. Although genome sequencing revealed absence of the tumor-inducing (Ti) plasmid, stabbing the crown region with bacterial cultures induced gall-like structures formed on blueberry plants (cultivar Duke). Phenotypically, these blueberry isolates were differentiated from closely related Agrobacterium species in their utilization of dulcitol, d-tagatose and xylitol. On the basis of these results, Serbian blueberry isolates are considered to represent a novel species of the genus Agrobacterium , for which the name Agrobacterium vacciniicorymbosi sp. nov. is proposed, with BA1120 T (NCPPB 4800 = CFBP 9290) as the type strain. This isolate and BA2520 (NCPPB 4801 = CFBP 9291) have been deposited in public collections of plant pathogenic bacteria.

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Summary Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene‐edited plants using an Agrobacterium‐mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T‐DNA in cannabis genome.

The Agrobacterium fabrum C58 is a phytopathogen able to infect numerous species of cultivated and ornamental plants. During infection, bacteria genetically transform plant cells and induce the formation of tumours at the site of invasion. Bacterial cell wall components play a crucial role in the infection process. Lipopolysaccharide is the main component of Gram-negative bacteria’s outer leaflet of outer membrane. Its lipophilic part, called lipid A, is built of di-glucosamine backbone substituted with a specific set of 3-hydroxyl fatty acids. A. fabrum incorporates a very long chain hydroxylated fatty acid (VLCFA), namely 27-hydroxyoctacosanoic acid (28:0-(27OH)), into its lipid A. A. fabrum C58 mutants deprived of this component due to mutation in the VLCFA’s genomic region, have been characterised. High-resolution mass spectrometry was used to establish acylation patterns in the mutant’s lipid A preparations. The physiological properties of mutants, as well as their motility, ability to biofilm formation and plant infectivity, were tested. The results obtained showed that the investigated mutants were more sensitive to environmental stress conditions, formed a weakened biofilm, exhibited impaired swimming motility and were less effective in infecting tomato seedlings compared to the wild strain.