C. Jacyn Baker

An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue

Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cells temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.

A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defence response to infection.

Pseudomonas syringae strains translocate effector proteins into host cells via the hrp‐encoded type III protein secretion system (TTSS) to facilitate pathogenesis in susceptible plants. However, the mechanisms by which pathogenesis is favoured by these effectors are not well understood. Individual strains express multiple effectors with apparently distinct activities that are co‐ordinately regulated by the alternative sigma factor HrpL. Genes for several effectors were identified in the P. syringae pv. tomato DC3000 genome using a promoter trap assay to identify HrpL‐dependent promoters. In addition to orthologues of avrPphE and hrpW, an unusual allele of avrPphD was detected that carried an IS52 insertion. Using this avrPphD::IS52 allele as a probe, a wild‐type allele of avrPphD, hopPtoD1, and a chimeric homologue were identified in the DC3000 genome. This chimeric homologue, identified as HopPtoD2 in the annotated DC3000 genome, consisted of an amino terminal secretion domain similar to that of AvrPphD fused to a potential protein tyrosine phosphatase domain. Culture filtrates of strains expressing HopPtoD2 were able to dephosphorylate pNPP and two phosphotyrosine peptides. HopPtoD2 was shown to be translocated into Arabidopsis thaliana cells via the hrp‐encoded TTSS. A ΔhopPtoD2 mutant of DC3000 exhibited strongly reduced virulence in Arabidopsis thaliana. Ectopic expression of hopPtoD2 in P. syringae Psy61 that lacks a native hopPtoD2 orthologue delayed the development of several defence‐associated responses including programmed cell death, active oxygen production and transcription of the pathogenesis‐related gene PR1. The results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defence responses.

Early physiological responses associated with race-specific recognition in soybean leaf tissue and cell suspensions treated with Pseudomonas syringae pv. glycinea

Abstract Pseudomonas syringae pv. glycinea race 4 (Psgr4) induces a compatible reaction on soybean cv. Mandarin leaves while race 6 (Psgr6) induces an incompatible hypersensitive response (HR). A single Psgr6 avirulence gene, avrA, when transformed into Psgr4, causes the resultant Psgr4(pAVRA) to induce a HR on Mandarin leaves. Mandarin leaf discs and suspension cells were inoculated and monitored for the K + H + and active oxygen responses, two early physiological events believed to be associated with recognition of HR-causing bacteria. Mandarin leaf discs exhibited a strong K + H + response following infiltration with either Psgr6 or Psgr4(pAVRA) but not Psgr4. Suspension cells derived from Mandarin hypocotyls reacted similarly when tested for the K + H + response to the Psg isolates. Luminol-dependent chemiluminescence (LDC) assays with suspension cells revealed active oxygen production concomitant with the K + H + response for both Psgr6- and Psgr4(pAVRA)-treated cells, but no such response was observed for Psgr4-treated cells. The data indicate that the K + H + and active oxygen responses are tightly correlated with recognition of the Psgr6 avrA gene or its products by Mandarin soybean leaves and suspension cells.

An improved method for monitoring active oxygen in bacteria-treated suspension cells using luminol-dependent chemiluminescence

Abstract Modification of luminol-dependent chemiluminescence (LDC) assays resulted in a reliable and highly reproducible method for the measurement of active oxygen levels in bacteria-treated soybean suspension cells. Optimization of the LDC assay for use at pH 7·0 required the addition of horseradish peroxidase (HRP) and the continuous monitoring of chemiluminescence to detect accurate maximum peak heights. With this improved technique, we were able to detect changes in active oxygen in soybean cell suspension cultures inoculated with Pseudomonas syringae pv. glycinea races 4 and 6. During the first hour after inoculation, increased active oxygen was observed in cells inoculated with either race. A second peak occurred after 3–4 h in suspension cell cultures inoculated with race 6, which causes a hypersensitive reaction in soybean leaves.

Redox-cycling and H2O2 generation by fabricated catecholic films in the absence of enzymes.

Phenolic matrices are ubiquitous in nature (e.g., lignin, melanin, and humics) but remain largely intractable to characterize. We examined an abiotic phenol-polysaccharide matrix fabricated by the anodic grafting of catechol to chitosan films. Previous studies have shown that catechol-modified chitosan films are redox-active and can be repeatedly interconverted between oxidized and reduced states. Here we developed quantitative electrochemical methods to characterize biorelevant redox properties of the catechol-modified chitosan films. Our analysis demonstrates that these films can (i) accept electrons from biological reductants (e.g., ascorbate and nicotinamide adenine dinucleotide phosphate, NADPH) and (ii) donate electrons in a model biological oxidation process. Furthermore, these films can donate electrons to O(2) to generate H(2)O(2). The demonstration that abiotic catechol-chitosan films possess catalytic activities in the absence of enzymes suggests the possibility that phenolic matrices may play an important role in redox cycling and reactive oxygen species (ROS) signaling in biology and the environment.

Inhibition of the hypersensitive response in tobacco by pectate lyase digests of cell wall and of polygalacturonic acid

Abstract A close association exists between induction of the bacterial hypersensitive response (HR) and stimulation of a prolonged net H+ uptake/K+ efflux in tobacco cells. The key role played by K+ in cellular metabolism suggests that this specific loss of K+ may play a critical role in HR. We previously reported that pectate lyase:(1) prevents the HR in tobacco leaves; and (2) stimulates a transient H+ decrease (pH increase) and K+ increase in supernatants of suspension cells. In addition we found that subsequent exposure of suspension cells to pectate lyase did not elicit additional transient K + H + responses. The latter result suggested that pectate lyase pretreatment may prevent HR by preventing cells from undergoing the prolonged K + H + response. The present study demonstrates that treatment of tobacco cell walls with this enzyme results in a heat stable digest which closely mimics the enzyme itself in ability to alter these plant responses. A similarly acting digest can be generated from polygalacturonic acid (PGA) by the same enzyme; digests with as little as 0·5 μg carbohydrate ml−1 will stimulate a transient pH response and 15 μg ml−1 will inhibit the HR. The magnitude of the transient pH increase is directly proportional to the concentration of digests of either walls or PGA; however, the increase in K+ did not increase proportionally, suggesting that the mechanism of this response was different from the K + H + response associated with bacteria-induced HR. Once tobacco suspension culture cells have undergone a transient pH response, their response to further additions of the digest is greatly diminished. The effects of the PGA digest on the transient pH response of suspension cells and the HR of leaf tissue were compared to effects of heat-killed bacteria which classically have been demonstrated to inhibit the HR. Experiments with leaf discs pretreated with either PGA digest or heat-killed bacteria and subsequently challenged with an HR-causing bacteria, demonstrate that although HR is inhibited, the associated prolonged K + H + response is not. This suggests that the K + H + response in itself does not lead to cell death. Understanding the simplified model systems described here should provide new insight into the complex reactions involved in the hypersensitive response and plant/bacteria recognition.

MONITORING THE RATE OF OXYGEN CONSUMPTION IN PLANT CELL SUSPENSIONS

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered.

Plasma membrane alteration during bacteria-induced hypersensitive reaction in tobacco suspension cells as monitored by intracellular accumulation of fluorescein

Alteration of plant cell plasma membrane lipid phase during bacteria-induced hypersensitive reaction has been reported. To improve the detection of membrane lipid phase alteration during bacteria-induced hypersensitive reaction we monitored the accumulation of the fluorescent molecule fluorescein, from added fluorescein diacetate. Cells exposed to fluorescein diacetate accumulate fluorescein by rapid diffusion of fluorescein diacetate through the lipid phase of the plasma membrane and intracellular release of the acetate moieties by enzymes having esterase activity. Therefore, alterations of the lipid phase which affect fluorescein accumulation by changing the diffusion rate of fluorescein diacetate through the membrane can be monitored. The hypersensitive reaction was induced in tobacco suspension cultured cells by addition of the pathogenic bacterium, Pseudomonas syringae pv. syringae . Dramatic reduction of fluorescein accumulation was observed after 2 h. Tobacco cells exposed to a Tn5 insertion mutant of the pathogen, which does not induce the hypersensitive reaction, or the compatible pathogen Pseudomonas syringae pv. tabaci , did not show a reduction in fluorescein accumulation. Evaluation of possible mechanisms indicated that decreased fluorescein accumulation resulted from decreased membrane permeability to fluorescein diacetate. Since fluorescein diacetate is a lipid-soluble, non-polar molecule, the lowered plasma membrane permeability to fluorescein diacetate implies that alteration of plasma membrane lipid phase occurs during the hypersensitive reaction of tobacco to P. syringae pv. syringae .

A novel open-system technique to monitor real-time oxygen consumption during early phases of seed germination

A novel technique allows long-term monitoring of real-time oxygen consumption during seed germination in an open system. Most current techniques used to detect oxygen consumption by seeds measure the decrease in oxygen concentration in a closed chamber. This is not ideal for long-term experiments because the chamber must be replenished with air periodically, subjecting the seeds to abrupt changes in oxygen concentration. The current technique employs an open system, in which seeds are submerged in a continuously aerated aqueous environment. Oxygen electrodes are used to measure the steady-state concentration of oxygen in the solution, which is a function of both the rate of oxygen consumption by the seed and the rate of aeration from the atmosphere. The rate of aeration is directly dependent on the oxygen concentration of the bathing solution; therefore, previous calibration of the system allows the direct conversion of steady-state oxygen concentrations into oxygen consumption rates. Because oxygen is not limiting, the experimental design described here can monitor the same sample non-intrusively every minute for more than 24 h, allowing for greater precision than hourly readings often reported with current techniques. Multiple treatments and/or replicates can be run simultaneously, allowing sensitive comparison of various seed treatments or seed types. To illustrate its potential application, the technique was used to follow the rehydration and pre-emergence phases of germination of cucumber ( Cucumis sativum ), pea ( Pisum sativum ) and mustard ( Brassica juncea ) seeds, detect the inhibitory effects of surface sterilization techniques on seed respiration of cucumber, and follow the interaction of a bacterial biocontrol agent with germinating cucumber and pea seeds.

Application of a modified EDTA-mediated exudation technique and guttation fluid analysis for Potato spindle tuber viroid RNA detection in tomato plants (Solanum lycopersicum).

Potato spindle tuber viroid (PSTVd) is a small plant pathogenic circular RNA that does not encode proteins, replicates autonomously, and traffics systemically in infected plants. Long-distance transport occurs by way of the phloem; however, one report in the literature describes the presence of viroid RNA in the xylem ring of potato tubers. In this study, a modified method based on an EDTA-mediated phloem exudation technique was applied for detection of PSTVd in the phloem of infected tomato plants. RT-PCR, nucleic acid sequencing, and Southern blot analyses of RT-PCR products verified the presence of viroid RNA in phloem exudates. In addition, the guttation fluid collected from the leaves of PSTVd-infected tomato plants was analyzed revealing the absence of viroid RNA in the xylem sap. To our knowledge, this is the first report of PSTVd RNA detection in phloem exudates obtained by the EDTA-mediated exudation technique.