Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions

Transient expression of recombinant proteins in plant tissues following Agrobacterium‐mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub‐optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10⁸, 10⁹, and 10¹⁰ CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break‐Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10⁸, 10⁹, and 10¹⁰ CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1137–1144, 2014     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile‐to‐sessile switch

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a u nip olar p olysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c‐di‐GMP) lead to surface‐contact‐independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP‐negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c‐di‐GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c‐di‐GMP phosphodiesterase also elevate UPP production and attachment, consistent with c‐di‐GMP activation of surface‐dependent adhesin deployment.     

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.     

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable ‘AgroLux’ strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.     

A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression

Agrobacterium-mediated gene transfer, or agroinfiltration, can be a highly efficient method for transforming and inducing transient transgene expression in plant tissue. The technique uses the innate DNA secretion pathway of Agrobacterium tumefaciens to vector a particular plasmid-encoded segment of DNA from the bacteria to plant cells. Vacuum is often applied to plant tissue submerged in a suspension of A. tumefaciens to improve agroinfiltration. However, the effects of vacuum application on agroinfiltration and in planta transient transgene expression have not been well quantified. Here we show that vacuum application and release act to drive A. tumefaciens suspension into the interior of leaf tissue. Moreover, the amount of suspension that enters leaves can be predicted based on the vacuum intensity and duration. Furthermore, we show that transient expression levels of an agroinfiltrated reporter gene vary in response to the amount of A. tumefaciens vacuum infiltrated into leaf tissue, suggesting that vacuum infiltration conditions can be tailored to achieve optimal transient transgene expression levels after agroinfiltration.     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

Protection Induced by Protein-Lipopolysaccharide Complexes in Tobacco Leaves: Role of Protected Tissue Free-space Solutes*

Hypersensitive confluent necrosis in tobacco leaves, caused by Pseudomonas syringae pv. aptata, is prevented by an intercellular injection of protein-lipopolysaccharide (pr-LPS) complexes 48 h earlier. An increase (48 %) in the peroxidase activity and a new low molecular weight protein (ε 17.5 kD) were found in the intercellular fluid of protected tissue (treated fluid) 48 h after pr-LPS infiltration. Up to 24 h after pr-LPS infiltration the treated fluid did not inhibit the in vitro growth of heterologous bacteria. Injected into tobacco leaves 30 min before the bacteria, the treated fluid retarded the rate of intercellular bacterial growth between 8 and 12 h after infiltration of the bacteria compared with the control fluid. Up to 4 h after injection, the attachment of live and dead bacteria to the cell walls occurred in both the protected tissue and in the control; 4 h after injection, however, the dead bacteria were only weakly attached in the protected tissue. Within 20 min after the intercellular infiltration, there was a decrease in the heterologous bacterial number in the protected tissue, 33 % less than that in the control tissue. Protected tissue free-space solutes did not have a direct antibacterial effect but activated an antibacterial response in the leaf tissue. Attachment of bacteria to the cell walls did not in itself trigger hypersensitive confluent necrosis. The protected tissue was initially more favourable for the survival of heterologous bacteria.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Exploration on the vacuum infiltration transformation of pakchoi

Agrobacterium tumefaciens mediated vacuum infiltration transformation in planta has been established in pakchoi, a kind of Chinese cabbage, but the transformation frequency in harvested seeds has varied in the range of 0.5 to 3.0 × 10⁻⁴ over several years and is much lower than the transformation frequency in Arabidopsis thaliana. To understand that, the distribution and vitality changes of A. tumefaciens in plant tissues were examined. Results revealed that there was a majority of A. tumefaciens in the flower compared with that in the stem and in the leaf at all times after infiltration. As fact of transformants in the upper part of the treated plant (T₀) stalk and fact of the survival of A. tumefaciens in the plant were proved, possibilities of optimizing the transformation conditions to increase the transformation frequency in pakchoi was discussed.     

Development of engineered biofilms on poly- L-lysine patterned surfaces

A technique has been developed to selectively attach bacteria to solid supports using poly-l-lysine. The patterned biofilms were labeled with green fluorescent protein (GFP) or a nucleic acid stain and imaged using both confocal microscopy and GFP stereomicroscopy. E. coli DH10B, E. coli MC1061, and Pseudomonas sp. GJ1 were selectively attached to regions coated with poly-l-lysine but not to uncoated regions. In contrast, E. coli DH5, W3110 and 33456 attached indiscriminately to the coated and uncoated regions of the surface. Those organisms that selectively attached to the poly-l-lysine coated regions formed biofilms twice as thick as the organisms that attached indiscriminately to the surface. This technique can be used for selectively patterning surfaces with genetically engineered microorganisms for biosynthesis of secondary metabolites and biodegradation or for developing a bacterial-based microscale medical diagnostic tool.     

A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions

Localized expression of genes in plants from T‐DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time‐consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host–environment and host–pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T‐DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus.     

Conditioned medium promotes the attachment ofAgrobacterium tumefaciens strain NT 1 to carrot cells

Summary Wild-typeAgrobacterium tumefaciens bind to carrot suspension culture cells. Avirulent strain NT 1 did not bind to carrot cells when they were incubated together in Murashige and Skoog medium. Conditioned medium was prepared by incubatingA. tumefaciens virulent strain C 58 with carrot cells and removing the bacteria and carrot cells using filter sterilization. This conditioned medium promoted the binding of NT 1 to carrot cells. Conditioned medium did not promote the nonspecific attachment ofEscherichia coli to carrot cells. These results suggest that when wild-typeA. tumefaciens are incubated with plant host cells, some substance(s) involved in bacterial attachment are released into the medium. Filter-sterilized medium from the incubation of the nonattachingchvB mutant A 1045 with carrot cells promoted the attachment of strain NT 1 even though A 1045 bacteria did not bind to the carrot cells. However, filter-sterilized medium from the incubation of the non-attachingatt mutant Att-B 123 with carrot cells was unable to promote the binding of strain NT 1. This suggests that nonattaching mutants ofA. tumefaciens can be divided into two groups on the basis of the properties of the substances released into the medium when the bacteria are incubated with carrot cells.Abbreviations MS Murashige and Skoog tissue culture medium Dedicated to the memory of Professor John G. Torrey     

2‐DE proteome maps for the leaf apoplast of Nicotiana benthamiana

We provide 2‐D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC‐MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance‐independent up‐regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant–pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.     

Agrobacterium tumefaciens mediated transient expression of plant cell wall‐degrading enzymes in detached sunflower leaves

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S‐driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA‐Based Overexpression Vector) systems. For 6‐day leaf incubations, codon‐optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two‐phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6‐fold. Production also increased up to 4.2‐fold when the leaf incubation temperature was elevated above the typical temperature, 20°C, to 30°C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co‐expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall‐degrading enzymes in sunflower. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:905–915, 2014     

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.