• Ca signalling;
  • cryptogein;
  • histone deacetylase;
  • hypersensitive response (HR);
  • plant immune signalling;
  • programmed cell death (PCD)

The sacrifice of one for the good of the many is a noble attribute in people (as well as, perhaps, altruistic ground squirrels who vocalize warning alarms at the site of a predator shortly before joining the raptor for lunch!). Perhaps, a corollary in plants is the programmed cell death (PCD) that, as an important component of the immune response to pathogens, increases organism fitness. Elucidation of some key molecular steps underlying this notion of individual (cell) sacrifice for the good of the ‘whole’ is the focus of the exciting work of Stéphane Bourque et al. (pp. 127–139) in this issue of New Phytologist.

‘Herein lies the roots of a rather adventuresome fishing expedition undertaken by Stéphane Bourque and colleagues that has landed us a very big fish indeed.’

Plant responses to pathogens include an ‘innate’ or basal immune response that occurs upon recognition of evolutionarily-conserved essential components of pathogens; pathogen (or microbe) associated molecular patterns (PAMPs or MAMPs). PAMP-triggered immunity (PTI) involves defence gene expression and generation of antimicrobial molecules. Some pathogens have (i.e. race-specific or strain-specific) effector proteins that act to impair molecular components of the plant innate immune response, leading to effector triggered susceptibility (ETS). Recognition (either directly or indirectly) of pathogen effector proteins (or the disruptions they have on basal immune system components) by plant cells evokes another layer of immune responses (effector triggered immunity (ETI)) which can include a hypersensitive response (HR) to ETS (Jones & Dangl, 2006; Thomma et al., 2011). This component of a plant’s repertoire of pathogen defence programmes involves PCD at the site of an infection. Cells neighbouring one under assault sense the presence of danger in the form of a pathogenic microbe. The danger alarm occurs through the perception of molecules associated with: the invading non-self pathogen; breakdown products derived from plant cells harmed by pathogens; and even specific signalling molecules endogenous to the plant that provide a warning alarm to reprogramme cell metabolism leading to defence responses. HR is a particularly powerful defence response because the regulated PCD occurring at a site where host cells are under assault limits proliferation of the pathogen within the plant and thereby prevents spread of an infection. The Greek philosopher Plato no doubt was considering humanity when he commented that, ‘No one knows whether death, which people fear to be the greatest evil, may not be the greatest good’ but this perspective might apply to the PCD that provides protection to the plant against proliferation of pathogens.

The delineation of specific molecular events and identification of specific proteins involved in plant immunity, that is, characterizing the signal transduction pathway that leads to PTI and ETI, is a remarkably active and vibrant area of current plant biology research (e.g. Coll et al., 2011). It is clear that a critical and early signal occurring upon pathogen perception is an elevation in cytosolic calcium ions (Ca2+), which leads to generation of the antimicrobial and signalling molecules nitric oxide and various reactive oxygen species (ROS) intermediates such as hydrogen peroxide (H2O2) (Ma et al., 2009; Krol et al., 2010; Mazars et al., 2010). This Ca2+ signal is also necessary for transcriptional reprogramming and increased expression of pathogen defence genes (Aslam et al., 2008). Further, phosphorylation events in the cytosol are also required for the pathogen defence signalling pathway leading to altered gene expression. It is likely that Ca2+-dependent kinases (CPKs) and mitogen activated kinase (MAPK) signalling networks acting downstream from CPKs, as well as independently, act to phosphorylate transcription factor proteins (WRKYs) that act as master regulators to facilitate reprogramming in the nucleus (Boudsocq et al., 2010; Mao et al., 2011). However, one point of incertitude in these newly-developed insights about specific steps in the immune signalling pathway is the question of which of these signalling events are specific to ETI. In fact, many of the intermediate events and proteins involved in these steps of signal transduction are required for both (PTI and ETI) components of plant immune response programmes.

The aforementioned notion of a multilayered plant immune response, involving PTI, ETS, and ETI which leads to HR, was conceptualized by Jones & Dangl (2006), and posits that HR occurs under specific conditions of plant–pathogen interactions. However, it is becoming increasingly clear that pathogen defence programmes leading to HR may occur under a broader range of conditions than originally conceptualized by Jones & Dangl (2006). Thus, some currently view the distinction between PTI and ETI as ‘blurred’ (Thomma et al., 2011). Examples of why the dichotomy between PTI and ETI is under re-evaluation are as follows. PAMP molecules (such as the bacterial motility organ protein, flagellin) have been found to induce HR in the absence of pathogens (Naito et al., 2008). Cryptogein is another pathogen-associated protein that can also cause HR when applied to plants in the absence of a pathogen. Cryptogein is a 10-kDa protein secreted by the plant pathogen Phytophthora cryptogea. Rather than acting to disarm components of PTI and induce ETS, cryptogein is a general plant cell toxin. But, it can clearly induce the specific Ca2+- and ROS-dependent apoptotic PCD that is a hallmark of plant cell HR to pathogens (Zhu et al., 2010). And, in this specific capacity, it is an especially powerful probe, or ‘fishing hook’ to use in order to go searching, or fishing so to speak, for specific steps in the signal transduction pathway that leads to HR. Herein lies the roots of a rather adventuresome fishing expedition undertaken by Stéphane Bourque and colleagues that has landed us a very big fish indeed. Their work provides new insights into mechanisms that lead to transcriptional reprogramming necessary for PCD occurring during HR.

As noted, previous work has already linked the early immune signals such as Ca2+ elevation to signalling cascades (e.g. involving CPKs and MAPKs) that alter gene expression associated with pathogen defence programmes. However, linking the early Ca2+ signal to events in the nucleus that alter gene expression specifically associated with HR is limited. That is the focus of the studies by Bourque et al., and so their work breaks new ground in this active area of plant biology research. Bourque, along with colleagues David Wendehenne and Alain Pugin and co-workers have been using cryptogein as a probe of signalling in plant cells responding to pathogen perception for quite some time (e.g. Lecourieux et al., 2005; Garcia-Brügger et al., 2006; Dahan et al., 2009). These prior studies led to the identification of nucleus-localized Ca2+ elevation and protein phosphorylation as key events downstream from cryptogein perception by plant cells. In the current work, they undertook a biochemical approach to develop new insights into immune signalling leading to HR. They used two-dimensional protein electrophoresis combined with H332PO4 radiolabelling to identify peptide fragments of nuclear-localized proteins phosphorylated (within minutes) specifically in response to cryptogein. Overcoming a number of technical challenges in their attempt to ‘fish’ for low abundance, nuclear-localized proteins phosphorylated in response to their cryptogein ‘hook’, they successfully identified two histone deacetylases (HDACs) as involved in this signalling pathway. They provide convincing evidence that these HDACs act as negative regulators of cryptogein-induced HR.

Bourque et al. further showed that cryptogein treatments, in addition to causing rapid HDAC phosphorylation, also repressed expression of the HDAC genes, and reduced HDAC protein levels. Using a number of complementary approaches, they linked the effects of cryptogein on HDAC phosphorylation and expression with HR. HDACs are key enzymes regulating gene expression by removing acetyl groups from the core histones. HDAC deacetylates histones, causing tighter coiling of DNA around the histone and a closed chromatin structure. The closed chromatin state makes the DNA inaccessible to transcriptional machinery, leading to repression of gene expression. Besides the role in chromatin remodelling, HDACs also interact with other non-histone proteins in the nucleus (Yu et al., 2011). The role HDACs play in the nucleus, then, is consistent with their identification by Bourque et al. as negative regulators of PCD; the insights developed in this work should lead to further characterization of PCD associated with pathogen defence through the examination of targets of HDAC deacetylase activity.

Their work also provides us with an excellent comparison between PAMP signalling that leads to basal defence programmes, and the substantially overlapping signalling cascade that, under certain conditions, can also lead to HR. That point provides the field with some new and important insights. Oligogalacturonides (OGs) are plant cell wall fragments generated during pathogen infections. OGs are examples of plant-derived breakdown products that, as already mentioned, act as danger alarm signals in a fashion similar to PAMPs and evoke PTI defence signalling cascades. These OG danger alarms evoke many of the same signalling steps as cryptogein (cytosolic Ca2+ elevation, ROS generation, MAPK cascades) except they do not lead to nuclear-localized Ca2+ elevation, which occurs in response to cryptogein. Bourque et al. show that OG application to plants does not repress HDAC expression. Thus, their work allows us to sort out the key signalling steps that lead to the aforementioned ‘noble sacrifice’, the programmed cell death/hypersensitive response that limits pathogen infection in plants.


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