Michèle C. Heath

Hypersensitive response-related death.

The hypersensitive response (HR) of plants resistant to microbial pathogens involves a complex form of programmed cell death (PCD) that differs from developmental PCD in its consistent association with the induction of local and systemic defence responses. Hypersensitive cell death is commonly controlled by direct or indirect interactions between pathogen avirulence gene products and those of plant resistance genes and it can be the result of multiple signalling pathways. Ion fluxes and the generation of reactive oxygen species commonly precede cell death, but a direct involvement of the latter seems to vary with the plant-pathogen combination. Protein synthesis, an intact actin cytoskeleton and salicylic acid also seem necessary for cell death induction. Cytological studies suggest that the actual mode and sequence of dismantling the cell contents varies among plant-parasite systems although there may be a universal involvement of cysteine proteases. It seems likely that cell death within the HR acts more as a signal to the rest of the plant rather than as a direct defence mechanism.

Nonhost resistance and nonspecific plant defenses

In the past year, most of the advances in our understanding of nonhost resistance to plant pathogens have been incremental. Highlights include the discovery of a general bacterial elicitor of plant defenses, the description of more similarities between the hypersensitive response and animal programmed cell death, and a growing appreciation of the cell wall as the site of initiation and expression of nonhost resistance towards fungi.

Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments.

It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants.

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea–cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.

Apoptosis, programmed cell death and the hypersensitive response

Apoptosis is typically a morphologically identifiable form of programmed cell death in mammals that is regulated by genes with homologues in other animal Phyla. Although both plants and fungal plant pathogens exhibit forms of developmental programmed cell death, demonstrated morphological or genetic homologies with mammalian apoptosis are still generally lacking. Because of its ubiquity and the involvement of signal transduction pathways in its induction, a strong case is developing that the hypersensitive response is a specific form of plant programmed cell death evolved as a defense against microbial parasites. Data suggest that separate signalling pathways may lead to the cell death and the defense gene activation that characterize this response and that parasite-specific resistance genes represent only one of many types of genes involved in response regulation. However, despite some biochemical similarities between the hypersensitive response, forms of developmental programmed cell death in plants, and animal apoptosis, no unique and consistent markers for the hypersensitive response (or plant programmed cell death in general) have yet been found. Whether any of these forms of plant cell death should be called apoptosis depends on how the term is defined. Assuming the hypersensitive response is a form of programmed cell death and is the ‘default state’ upon pathogen entry into a cell, it seems likely that intracellular biotrophic plant pathogens resemble some animal viruses in being able to suppress this response in susceptible hosts.

Plasma Membrane–Cell Wall Adhesion Is Required for Expression of Plant Defense Responses during Fungal Penetration

Fungal pathogens almost invariably trigger cell wall–associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane–cell wall adhesion as shown by the loss of the thin plasma membrane–cell wall connections known as Hechtian strands, reduced the expression of cell wall–associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall–associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane–cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall–associated defenses, there was no change, or an increase, in plasma membrane–cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall–associated defenses and a localized decrease in plasma membrane–cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane–cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall–associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane–cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.

Light and electron microscope studies of the interactions of host and non-host plants with cowpea rust— Uromyces phaseoli var. vignae

Abstract A comparison of infection of host plants and eight non-host angiosperm species by cowpea rust revealed that uredospores usually germinated equally well on hosts and non-hosts. On most of the latter, however, fewer germ tubes contacted stomata, apparently because of the reduced efficiency of directional growth. Compared with both resistant and susceptible host cultivars, at least twice as many hyphae in non-host leaves stopped growing during the very early stages of intercellular development. Nevertheless, the majority of infection hyphae continued to grow to the stage at which the first haustorial mother cell would be expected to form. However, further intercellular growth was rare and few, if any, infection hyphae formed haustoria. Ultrastructural investigation of four non-hosts suggested that haustorium formation could be inhibited by at least three mechanisms: deposition of osmiophilic material on adjacent non-host walls ( Phaseolus spp.), loss of contact between haustorial mother cell and non-host cell ( Vicia faba ), or fungal death prior to haustorium initiation ( Pisum sativum ). If haustoria were formed, the death of both haustorium and invaded cell rapidly followed although the initial response to the one haustorium observed ultrastructurally resembled that described previously for susceptible interactions. The diversity of cellular responses induced in resistant hosts and non-hosts by cowpea rust, described in this and earlier work, suggests that there are a number of stages during infection at which an interaction between this fungus and the higher plant can take place. It is proposed that each stage represents a “switching point” and that responses at each determine the subsequent progress of infection in both compatible and incompatible associations.

An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility.

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.

Changes in the cytoskeleton accompanying infection‐induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi

During the infection of cowpea (Vigna unguiculata) by the cowpea rust fungus (Uromyces vignae, race 1 ) the plant nucleus moves towards and away from the invading hypha and eventually moves close to the fungus in the susceptible cultivar while it remains away in two cultivars which subsequently respond by resistance gene-dependent plant cell death (the hypersensitive response, HR). The role of plant cytoskeleton in these responses was investigated by fluorescent microscopy and treatments with anticytoskeletal drugs. Observations of microtubule organization prior to cell death revealed that the sequence of events leading to protoplast collapse differed between the two resistant cultivars, suggesting a possibility of multiple pathways for cellular degradation during the HR. Different fixations produced two different microfilament patterns: a filament network and cables. Microfilament network remained visible even at later stages of cell death. Oryzalin and taxol reduced the incidence of autofluorescence that develops late in the death process, indicating a role of microtubules in the deposition of phenolics by adjacent living cells. Cell death and nuclear movements were not affected by oryzalin and taxol but were inhibited by cytochalasin E, suggesting that the microfilaments are required for the HR.

A comparative study of non-host interactions with rust fungi

Abstract The interactions of non-host plants with sunflower, corn and cowpea rusts were compared with those described previously for other cowpea rust-non-host combinations. Compared with the situation on the respective host plant, all three rusts made fewer attempts at penetration into the leaf on half or more of the non-hosts examined. Nevertheless, in no case could such surface behavior completely account for non-host resistance since at least a few attempts at penetration were observed for all the non-host-rust combinations. In only one non-host, cabbage, was linear growth inside the leaf significantly less than that in the respective susceptible plant during the first 24 h after inoculation. Since, for cowpea and sunflower rusts at least, growth in this non-host was also less than that achieved on artificial membranes, it was concluded that non-host resistance of cabbage leaves involved an inhibition of fungal growth. In contrast, a detailed study using cowpea rust showed that the extent and pattern of growth in most of the other non-hosts was similar, apart from a greater frequency of haustorial mother cells, to that on the membranes. Thus it seemed that these non-hosts had little influence on the development of the fungus except in the induction of the haustorial mother cell septum. Susceptible plants, however, appeared to stimulate the formation of secondary hyphae, and it is postulated that the resistance of many non-hosts to this rust, and others, may be due to the absence of any similar stimulation by the plant. The importance of the haustorium in this stimulation could not be clearly determined. The possibility that the non-host plant may actively inhibit haustorium formation, however, was suggested by the fact that their absence in many non-host-rust combinations could be correlated with the darkening of the plant wall adjacent to the haustorial mother cell. The general lack of specificity of non-host responses to rust fungi was indicated by the observation that where a given non-host-rust combination was characterized by a particular feature, such as the presence of wall darkening or the relatively high frequency of haustoria, then a similar feature was often seen following infection of the same non-host with the other two rusts. The observation of a few instances where this was not the case, however, suggested that the nature of the fungus may have a modifying effect on the plant response.