Norton M. Mock

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.

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.

Gene Expression Profiling in Viable but Nonculturable (VBNC) Cells of Pseudomonas syringae pv. syringae

Pseudomonas syringae infects diverse crop plants and comprises at least 50 different pathovar strains with different host ranges. More information on the physiological and molecular effects of the host inhibitory environment on the pathogen is needed to develop resistant cultivars. Recently, we reported an in vitro model system that mimics the redox pulse associated with the oxidative burst in plant cells inoculated with Pseudomonas syringae pv. syringae. Using this system, we demonstrated that oxidation of acetosyringone, a major extracellular phenolic compound induced in some plants in response to bacteria, rendered Pseudomonas syringae pv. syringae to a “viable but nonculturable” (VBNC) state. Here we performed a large scale transcriptome profiling of P. s. pv. syringae in the VBNC state induced by acetosyringone treatment and identified bacterial genes and pathways presumably associated with this condition. The findings offer insight into what events occur when bacterial pathogens are first encountered and host defense responses are triggered. The acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance. We believe that this is the first work on global gene expression profiling of VBNC cells in plant pathogenic bacteria.

The dynamics of apoplast phenolics in tobacco leaves following inoculation with bacteria.

This study demonstrates that the accumulation of apoplastic phenolics is stimulated in planta in response to bacterial inoculation. Past studies have shown that levels of extracellular phenolics are elicited in plant cell suspensions in response to bacteria, and that tomato plants infected with viroids showed changes in apoplastic phenolics. The method described here monitored changes in apoplastic phenolics in tobacco leaves following bacterial inoculation of the same tissue. Inoculation with a saprophyte, Pseudomonas fluorescens, which does not cause visible symptoms or physical damage, was used to elicit phenolics and examine the effects of variable parameters on phenolic composition. Location of the inoculation on the leaf, position, or developmental age of the leaf on the plant, and inoculum concentration were standardized for further experiments. The patterns of phenolic change in the apoplast were compared for tobacco inoculated with P. syringae pathovars, pv. syringae, which causes a resistant HR reaction within 15 h, and pv. tabaci, which causes a susceptible reaction with delayed visible symptoms. Both pathogens elicited lower increased levels of acetosyringone compared to the saprophyte, P. fluorescens but had greatly increased levels of the chlorogenic acid derivatives. The latter metabolites appear to have come from the intracellular stores, which could indicate a weakening of the apoplast/symplast barrier. This unexpected aspect will require further study of intracellular phenolics.

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.