Effects of Atmospheric Plasma Corona Discharge on Agrobacterium tumefaciens Survival

Soil-borne pathogenic microorganisms are known to cause extensive crop losses. Agrobacterium tumefaciens, a member of the Proteobacteria, causes the neoplastic crown gall disease in plants. Plant protection is mainly based on toxic chemicals that are harmful to the environment. The use of cold atmospheric-pressure plasma is an attractive method for microbial eradication. Its antimicrobial mechanism includes the formation of large quantities of reactive oxygen species (ROS). The advantages of eradicating bacteria using cold plasma are not needed for chemicals, short treatment, and environmental temperatures. This study examined the impact of plasma corona discharge exposure on A. tumefaciens viability, membrane permeability, relative cell size, and ROS formation. The results showed that 90 s of plasma exposure led to a reduction by four orders of magnitude when the initial concentration was 1 × 107 CFU/mL and in a dry environment. When the initial concentration was 1 × 106 CFU/mL, 45 s of exposure resulted in total bacterial eradication. In a liquid environment, in an initial concentration of 2.02 × 106 CFU/mL, there was no complete bacterial eradication even at the most prolonged examined exposure (90 s). The influence of plasma treatment on the membrane permeability of A. tumefaciens, and their possible recovery, were analyzed using flow cytometer analysis using propidium iodide (PI). When the plasma-treated bacteria were suspended in Luria–Bertani (LB) (rich medium), the PI-positive count of the plasma-treated bacteria after two hours was 12 ± 3.9%. At the 24th hour, this percentage was only 1.74 ± 0.6%, as the control (0.7 ± 0.1%). These results may indicate the repair of the plasma-treated bacteria that were suspended in LB. At the 24th hour, the relative cell size of the treated bacteria shifted to the right, to ~3 × 104 forward side scatter (FSC), about 0.5-fold higher than the untreated cells. Measurement of the ROS showed that the intracellular fluorescence of the 90-s plasma-treated cells led to significant fluorescence formation of 32 relative fluorescence units (RFU)/cell (9 × 104 fold, compared to the nontreated cells). This study showed that cold plasma is a useful method for A. tumefaciens eradication. The eradication mechanism involves ROS generation, membrane permeability, and changes in cell size.

LaCl3 Treatment Improves Agrobacterium-mediated Immature Embryo Genetic Transformation Frequency of Maize

Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive calluses, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40% to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.