Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.

BACKGROUND: Rhizobium radiobacter is a Gram-negative pathogen present in soil and plants. Cases of R radiobacter infection in immunocompromised hosts have been sporadically reported. However, septic shock caused by R radiobacter is rarely seen.
METHODS: Here, we describe an elderly patient with a rapid progression of watery diarrhea, anorexia, fever, weakness, oliguria, and shock. Blood results showed increased total white blood cell count and C-reactive protein. Arterial blood gas results showed hypoxia and elevated lactate level. The Sequential Organ Failure Assessment score was 11. Blood culture at admission showed Gram-negative bacteria, which were later confirmed as R radiobacter.
METHODS: Septic shock caused by R Radiobacter.
METHODS: The patient was treated with intravenous cefoperazone/sulbactam and sequential oral levofloxacin.
RESULTS: The patient recovered completely.
CONCLUSIONS: R radiobacter may be considered as a potential opportunistic pathogen that may cause severe sepsis in elderly patients, especially those with multiple underlying diseases.

暂无摘要。

Rhizobium Radiobacter (RR) has rarely been associated with human infection, mainly sepsis or bacteremia, and an unique case of prosthetic aortic endocarditis has been reported. We present a case of native mitral valve endocarditis to RR, to our knowledge the first clinical report of such infection.

Rhizobium radiobacter is a Gram-negative, nitrogen-fixing bacterium, which is found mainly on the ground. It rarely causes infections in humans. It has been associated with bacteremia, secondary to colonization of intravascular catheters, in immunocompromised patients. The aim of this paper was to report the case of an infective endocarditis caused by R. radiobacter, in a 47-year-old male, diagnosed with chronic kidney disease stage 5, on replacement therapy with hemodialysis and who attended the medical center with fever of two weeks duration. The patient was hospitalized and samples of peripheral blood were taken for culture. Empirical antibiotic therapy was started with cefotaxime plus vancomycin. The transthoracic echocardiogram revealed fusiform vegetation on the tricuspid valve, with grade III-IV/IV regurgitation. On the seventh day after the start of antibiotic therapy, the patient had a clinical and paraclinical improvement. The bacterium identified by blood culture was Rhizobium radiobacter, ceftriaxone-resistant and sensitive to imipenem, amikacin, ampicillin and ampicillin/sulbactam. Because of the clinical improvement, it was decided to continue treatment with vancomycin and additionally, with imipenem. At 14 days after the start of antibiotic therapy, the patient was discharged with outpatient treatment with imipenem up to six weeks of treatment. The control echocardiogram showed the absence of vegetation on the tricuspid valve. This case suggests that R. radiobacter can cause endocarditis in patients with intravascular catheters.

Rhizobium radiobacter is an uncommon opportunistic pathogen present in soil. It has been particularly associated with indwelling intravascular devices in immunocompromised patients. Reported herein is a case of R. radiobacter bloodstream infection associated with an implantable vascular access port, which was easily controlled with antimicrobial treatment and did not require removal of the intravascular device, in a child diagnosed with acute lymphoblastic leukemia. Also included is a review of the pertinent literature regarding the clinical presentation and management of R. radiobacter infections in the childhood period.

暂无摘要。

BACKGROUND: Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules.
RESULTS: Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration.
CONCLUSIONS: The fast and efficient strategies described here should have broad applications, in particular for the study of \"difficult\" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents.

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first-rate production of plant-based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high-value plant-derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate-limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by-product formation can be accomplished either through single-step engineering or through the multi-step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low-volume, but high-value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary-metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from \'-omic\' platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide-ranging views of DNA sequence-to-metabolite passageway networking and their control to obtain desired results.