Plant cells are able to respond rapidly to potential fungal or oomycete pathogens on their surface. As the pathogen infection structures develop and attempt to penetrate the epidermis, basal resistance mechanisms are mobilized and in most cases succeed in inhibiting the establishment of disease by non-adapted pathogens. Within the plant cell in contact with the pathogen, cytoplasmic streaming accelerates, cytoplasmic strands become focused on the infection site and a cytoplasmic aggregate forms under the pathogen cell [1, 13, 25]. Immunofluorescence labelling, GFP-tagging of cell components and pharmacological studies have shown that these changes in the distribution and behaviour of the cytoplasm are associated with and dependent upon reorganization of the actin cytoskeleton which becomes focused on the infection site [6, 20, 21, 26–30]. Reorganization of the actin cytoskeleton is also accompanied by rearrangement of ER and aggregation of dictyosomes and peroxisomes at the infection site [20, 31]. These changes are believed to facilitate localized secretion of cell wall material and antimicrobial compounds at the infection site [15, 16]. In addition to normal wall constituents, the thickened regions of cell wall, referred to as wall appositions, contain callose, phenolics, phytoalexins and H2O2 that together strengthen the wall to make it a more effective barrier against pathogen ingress and provide localized concentrations of toxins that inhibit and kill the invading pathogen [11, 13, 16, 32]. Basal resistance is induced in response to non-adapted, avirulent and virulent fungal and oomycete pathogens alike , suggesting the existence of a triggering factor common to all these potential pathogens that can be detected by the plant. One such trigger could be the pressure exerted by the pathogen as it attempts to penetrate the plant surface.
Plants can respond rapidly to mechanical stimulation. Within 30 minutes, touching a plant induces wide-ranging changes in gene expression, including the up-regulation of disease resistance genes [33–35]. It also leads to rapid changes in intracellular organization. Touching the cell surface with a glass or tungsten microneedle or capillary can cause chloroplast movement away from, or nuclear and cytoplasmic migration towards, the contact site [36–38]. Removal of the stimulus results in disappearance of the cytoplasmic aggregation and a return of the nucleus to its former position [37, 38]. The present study of Arabidopsis plants containing GFP-tagged cell components demonstrates that touching the plant surface also induces a reorganization of subcellular components similar to that observed during attempted infection. Within 3 to 5 minutes of touching the cotyledon surface, actin, ER and GFP-tubulin begin to form dense patches under the contact site and peroxisomes begin to cluster in the cytoplasm beneath the needle tip. The subcellular responses are highly dynamic, with the morphology of the patches of actin, ER and GFP-tubulin changing continuously, including tracking the needle tip when it moves across the surface and dispersing within minutes of removal of the needle's pressure.
Studies of actin arrays in transgenic plants expressing a number of different constructs encoding GFP-tagged actin binding domains have indicated that in some cases not all components of the actin cytoskeleton are visualized and/or that the dynamics and organization of the actin array may be disturbed [39–44]. There is, however, no evidence to indicate that these problems occur in the GFP-hTalin plants used in the present study. Indeed the rapidity with which the actin array becomes focused on the contact site, the similar rapidity with which it disperses on removal of the pressure and the fact that this response time is similar to that of the microtubule array, all suggest that actin dynamics are not hampered in this line of GFP-hTalin plants. In the experiment that generated the GFP-hTalin plants, individual lines exhibited a range of GFP fluorescence intensities . The line chosen for subsequent use was selected for having moderate levels of transgene expression, as indicated by the levels of GFP fluorescence, and no phenotypic abnormalities . In addition to the absence of any indication of disturbance of the actin arrays in these plants in our experiments, this Arabidopsis GFP-hTalin line has also been used in investigations of the role of actin in regulating the dynamics of plastid stromule morphology and behaviour . Stromule formation is a highly delicate process that is easily perturbed, however, no differences in the morphology or movement of the plastids or stromules between wild-type and the GFP-hTalin plants were found. Together, these results give evidence of the integrity and normal dynamics of the actin cytoskeleton in the transgenic plants used in the current study.
With time, the initial concentrations of actin, ER and GFP-tubulin continue to consolidate. Some existing actin cables in the region surrounding the contact site are dismantled before new, or reoriented, cables become focused on the contact site. Much accumulated evidence indicates that the radial actin array is responsible for delivery of various cell components, including peroxisomes, dictyosomes and secretory vesicles, to the site of attack [15, 17, 46–48]. In addition to achieving localized secretion of cell wall materials and toxins, proximity of peroxisomes to the site of infection is thought to facilitate their anti-microbial functions [49–51].
Continued response to touch included the development of a microtubule-depleted zone around the contact site. Similar localized regions of microtubule depolymerization have been reported during infection of parsley, soybean and barley by Phytophthora infestans, P. sojae and Erysiphe graminis f. sp.hordei, respectively [52–54]. Widespread microtubule depolymerization has also been reported to occur several hours after treatment of tobacco and Arabidopsis suspension-cultured cells with cryptogein elicitin and Verticillium toxin, respectively [55–57]. Microtubule depolymerization is likely to increase the level of tubulin monomers, dimers or oligomers in the cytoplasm, giving rise to the dense cloud of diffuse fluorescence seen below the contact site (e.g. Figs. 4H–L, 5B–E). The dynamic behaviour of the GFP-tubulin and ER clouds is likely to arise through continued actin-driven cytoplasmic motility, as indicated by peroxisome movement throughout much of the observation periods. In our study, disturbance of the ER network and microtubule cytoskeleton through mechanical stimulation also led to the appearance of diffuse strands of ER or microtubules that moved within the cortical cytoplasm. Similar strands were seen in control cells that had not been touched with the microneedle but they were much less frequent and were not confined to any particular location in the cell. The diffuse strands observed in the GFP-tubulin-expressing plants were similar to the blurry microtubules described by Cyr and colleagues in their studies of the dynamics of cortical microtubule arrays in tobacco cells expressing MDB-DsRed or YFP-TUA6 . These authors propose that the blurry images of microtubules are due to movement of microtubules that are not anchored to the plasma membrane. The diffuse microtubule strands observed in the present study were at a greater distance from the plasma membrane than the array of sharply-focused microtubules and are thus unlikely to be attached to the plasma membrane. In our studies of microtubule arrays in cells responding to mechanical stimulation, in most cases these diffuse microtubule strands subsequently disappeared. One interpretation of these data is that, having detached from the plasma membrane, the microtubules or microtubule bundles are less stable and depolymerize. This phenomenon may be part of the normal dynamics of microtubule arrays but may also be an important mechanism for remodeling microtubule arrays following mechanical stimulation by biotic or abiotic factors.
One might question whether or not a fungal hypha or other infection structure is capable of exerting a pressure or force sufficiently large to be perceived by the plant cell. In fact, there is little doubt that it can. A variety of technical approaches have now been used to calculate or measure the force exerted by fungal and oomycete hyphae and appressorial penetration pegs and the values obtained typically lie within the range of 5–100 μN [59–63]. Given the diminutive size of the structures involved, it is difficult to fully appreciate the magnitude of these forces but to put them in perspective, if we could exert the same force per unit area with a finger, we could hold a 25–500 kg weight against gravity!
The force exerted by an invading pathogen will depend on the pathogen cell's turgor pressure and the area and properties of its cell wall in contact with the underlying plant cell. Turgor pressure provides the basis of the invasive force although the actual pressure applied to the host cell will be decreased by the resistance of the pathogen wall to extension . The force (in μN) is equal to the pressure (in MPa, i.e. μN μm-2) multiplied by the contact area (in μm2). Thus, if the wall exerts minimal yield resistance, a hypha with a cross-sectional area of 300 μm2 (about 20 μm in diameter) and a turgor of 0.4 MPa would exert a force of 120 μN . In most cases, the forces that have been measured are less than those maximally possible for a given turgor pressure and it appears, at least for hyphae, that the mechanical strength of the wall results in only about 10% of the available force actually being applied by the fungal hypha . However, this may not be the case for appressorial penetration pegs, as discussed below.
Fungal and oomycete hyphae typically generate turgor pressures of 0.2–0.7 MPa . With a cross-sectional area of 80–500 μm2, these hyphae have a contact area of up to 500 times that of the penetration peg produced by appressoria of Colletotrichum graminicola or Magnaporthe grisea. Thus, in order to generate an invasive force within the range cited above, these fungal appressoria accumulate high concentrations of glycerol that generate turgor pressures of the order of 6–8 MPa, a pressure 30–40 times greater than an average car tire [64–66]. Evidence suggests that the yield threshold of the penetration peg is minimal such that essentially all the turgor pressure is applied in the generation of the invasive force by the penetration peg [62, 64]. Thus, the localized forces exerted by invading fungi and oomycetes are substantial and, like the mechanical stimulation used in the study, may be detected by the plant cell and used to trigger basal defence.
A number of studies have shown that cytoplasmic aggregation and actin reorganization can occur in the plant cell below hyphae or beneath appressoria before a penetration peg is discernible, suggesting that, at least in some cases, the plant cell can detect the presence of hyphae or appressoria on their surface before invasion begins [1, 7, 13, 21, 25]. However, given the difficulties of knowing when the pathogen has strengthened its attachment to the plant surface sufficiently to maintain contact during invasion, when an appressorium has gained full turgor or when penetration is first initiated, it is difficult to assess how quickly the plant can respond to the presence of the pathogen. In addition to demonstrating that touching the cotyledon surface with a microneedle induces similar subcellular reorganization to that observed during pathogen infection, this form of mechanical stimulation allows a more precise determination of the dynamics of the response than is possible during plant-pathogen interactions. Thus, while cytoplasmic aggregation, actin rearrangement or microtubule depolymerization have been observed to occur 15 minutes after inoculation or 20–30 minutes before a penetration peg becomes visible [1, 25], the studies reported here indicate that these subcellular responses can occur within 3–4 minutes of stimulation. Touch can induce a transient increase in cytoplasmic calcium in a similar or even shorter timeframe [67, 68]. As proposed for the mechano-response of chloroplasts , these observations are consistent with stretch-activated channels in the plant plasma membrane having a role in recognition of the pathogen's presence and induction of a signal transduction cascade involving transient calcium elevation that quickly results in the subcellular reorganization employed during the basal plant defence response.