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Plant Communications Apr 2024Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated...
Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocols and low efficiency of current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of the regenerative activity-dependent in planta injection delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues. Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues. The method was successfully used for transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (Ipomoea pes-caprae). Compared with traditional transformation methods, RAPID has a much higher transformation efficiency and shorter duration, and it does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.
Topics: Plants, Genetically Modified; Agrobacterium tumefaciens; Ipomoea batatas
PubMed: 38243598
DOI: 10.1016/j.xplc.2024.100822 -
Nature Plants Sep 2023Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes. Transfer DNA (T-DNA) originating from...
Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.
Topics: Gene Editing; Genome, Plant; Retroelements; Genes, Plant; Arabidopsis
PubMed: 37653336
DOI: 10.1038/s41477-023-01495-w -
Journal of Experimental Botany Jun 2023CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial...
CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.
Topics: Gene Editing; CRISPR-Cas Systems; Agrobacterium tumefaciens; Virulence; DNA
PubMed: 36919203
DOI: 10.1093/jxb/erad096 -
Editorial: New developments in Agrobacterium Mediated Transformation of tree fruit crops, volume II.Frontiers in Plant Science 2023
PubMed: 37502706
DOI: 10.3389/fpls.2023.1249563 -
Current Opinion in Microbiology Jun 2024The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described.... (Review)
Review
The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome. Polar incorporation of envelope components relies on homologous proteins shared by the Hyphomicrobiales, reviewed here. Ongoing and future work will reveal how unipolar growth is integrated into the alphaproteobacterial cell cycle and coordinated with other processes such as chromosome segregation and cell division.
Topics: Brucella abortus; Sinorhizobium meliloti; Bacterial Proteins; Agrobacterium tumefaciens; Cell Wall; Peptidoglycan; Cell Division
PubMed: 38569420
DOI: 10.1016/j.mib.2024.102470 -
Journal of Fungi (Basel, Switzerland) Dec 2023is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional...
is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an -mediated transformation (ATMT) system to for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase () gene or the enhanced green fluorescent protein () gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, and gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in .
PubMed: 38132759
DOI: 10.3390/jof9121158 -
BioRxiv : the Preprint Server For... Nov 2023Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which...
Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in and is found in multiple additional bacterial pathogens.
PubMed: 38014264
DOI: 10.1101/2023.11.18.567607 -
Plant Direct Jun 2024-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect...
-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect of -infiltration parameters on the transient transformation efficiency of seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre- and post-infiltration dark incubation and is less sensitive to the growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven- to eight-fold while a dark incubation pre- and post-infiltration increased transformation efficiency by five- to 13-fold. in exponential compared with stationary phase increased transformation efficiency by two-fold. Finally, we quantified the variation in our -infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6-fold in raw firefly luciferase () and raw luciferase () luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of infiltration in seedlings will facilitate the study of this important medicinal plant and will expand the application of -mediated transformation methods in other plant species.
PubMed: 38855128
DOI: 10.1002/pld3.596 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
Journal of Bacteriology Jul 2023The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii, PecS controls a range of virulence...
The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii, PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene , which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen (formerly named Agrobacterium tumefaciens), the locus is conserved. Using a strain of in which has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates fitness during its transition from the rhizosphere to the host plant. PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species.
Topics: Transcription Factors; Reactive Oxygen Species; Gene Expression Regulation, Bacterial; Agrobacterium; Agrobacterium tumefaciens; Bacterial Proteins
PubMed: 37314346
DOI: 10.1128/jb.00478-22