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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.

Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone–effector complex (Tap-1–Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1–PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1–Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1–Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

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.

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.

Agrobacterium tumefaciens (A. tumefaciens), the causal agent of crown gall disease on several plant species, is responsible for substantial yield losses worldwide. The limitations of conventional pesticides in controlling this disease highlight the need for alternative antibacterial solutions. Phage biocontrol can be an option, effectively managing bacterial plant diseases, by reducing pathogen loads while driving evolutionary trade-offs, often enhancing synergy with other antibacterial strategies. In this study, we aimed to explore and develop a sustainable strategy to control A. tumefaciens, by combining Agrobacterium phage PAT1 with the natural antimicrobial peptide “Ascaphin 8” and leveraging the fitness trade-offs resulting from phage resistance. In vitro and in planta investigations showed that PAT1 in combination with Ascaphin 8 at the sublethal concentration of 3 μM could effectively eradicate A. tumefaciens in YPG broth and reduce tumor formation by 46.33% on tomato plants, unlike their individual applications, indicating that the combination was synergistic against A. tumefaciens. This synergy was attributed to the fitness trade-offs in A. tumefaciens induced by phage resistance, which led to increased sensitivity to antimicrobial peptides, slower growth rate, and an 89.96% attenuation of virulence in the PAT1-resistant mutant (AT-M1). Transmission electron microscopy analyses showed that treatment with 1 µM of Ascaphin 8 induced cytoplasmic condensation in 80% of AT-M1 cells, whereas only 16% of the wild-type CFBP 5770 cells exhibited similar alterations under identical conditions. Furthermore, proteomic analyses performed on AT-M1 and CFBP 5770 revealed that the mutant AT-M1 exhibited a loss of DNA-binding protein HupB and downregulation of SDR family oxidoreductase and superoxide dismutase. These molecular alterations are potentially associated with the reduced virulence and heightened AT-M1 sensitivity. This study investigated the fitness costs associated with phage resistance in A. tumefaciens and laid the first foundation for potential biocontrol of plant bacterial diseases, particularly A. tumefaciens infections, using phage–peptide combination.

The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales . To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens , we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens , we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales - and Rhodobacterales -specific l , d -transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d -amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. IMPORTANCE Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu , grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases ( l , d -transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l , d -transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales , Actinomycetales , and other polarly growing bacterial pathogens. Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacterium , grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases ( l , d -transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l , d -transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales , Actinomycetales , and other polarly growing bacterial pathogens.

Bacterial adhesion onto mineral surfaces and subsequent biofilm formation play key roles in aggregate stability, mineral weathering and the fate of contaminants in soils. However, the mechanisms of bacteria-mineral interactions are not fully understood. Atomic force microscopy (AFM) was used to determine the adhesion forces between bacteria and goethite in water and to gain insight into the nanoscale surface morphology of the bacteria-mineral aggregates and biofilms formed on clay-sized minerals. This study yields direct evidence of a range of different association mechanisms between bacteria and minerals. All strains studied adhered predominantly to the edge surfaces of kaolinite rather than to the basal surfaces. Bacteria rarely formed aggregates with montmorillonite, but were more tightly adsorbed onto goethite surfaces. This study reports the first measured interaction force between bacteria and a clay surface and the approach curves exhibited jump-in events with attractive forces of 97 ± 34 pN between E. coli and goethite. Bond strengthening between them occurred within 4 s to the maximum adhesion forces and energies of −3.0 ± 0.4 nN and −330 ± 43 aJ (10 −18  J), respectively. Under the conditions studied, bacteria tended to form more extensive biofilms on minerals under low rather than high nutrient conditions.

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.

The Arabidopsis (Arabidopsis thaliana) transcription factor WRKY33 is essential for defense toward the necrotrophic fungus Botrytis cinerea. Here, we aimed at identifying early transcriptional responses mediated by WRKY33. Global expression profiling on susceptible wrky33 and resistant wild-type plants uncovered massive differential transcriptional reprogramming upon B. cinerea infection. Subsequent detailed kinetic analyses revealed that loss of WRKY33 function results in inappropriate activation of the salicylic acid (SA)-related host response and elevated SA levels post infection and in the down-regulation of jasmonic acid (JA)-associated responses at later stages. This down-regulation appears to involve direct activation of several jasmonate ZIM-domain genes, encoding repressors of the JA-response pathway, by loss of WRKY33 function and by additional SA-dependent WRKY factors. Moreover, genes involved in redox homeostasis, SA signaling, ethylene-JA-mediated cross-communication, and camalexin biosynthesis were identified as direct targets of WRKY33. Genetic studies indicate that although SA-mediated repression of the JA pathway may contribute to the susceptibility of wrky33 plants to B. cinerea, it is insufficient for WRKY33-mediated resistance. Thus, WRKY33 apparently directly targets other still unidentified components that are also critical for establishing full resistance toward this necrotroph.