Plants employ a sophisticated multilayered immune system to defend themselves from pathogenic microbes (Jones and Dangl, 2006; Dodds and Rathjen, 2010). Early defenses are activated upon recognition of conserved molecular patterns of potential pathogens. Recognition of microbe-associated molecular patterns (MAMPs) activates MAMP triggered immunity (MTI) which entails strengthening of the cell wall, transcriptional activation of pathogenesis related (PR) proteins and secretion of low molecular weight antimicrobial substances (Boller and Felix, 2009). MTI is effective against pathogens from several kingdoms and is often sufficient to halt the intruder from colonizing the plant. Microbial co-evolution with plants has provided selective pressure for overcoming MTI and thus increases the possibility to proliferate on or in the plant tissue and cause disease. Adapted pathogens have developed means to suppress MTI. This often comprises the secretion of so called effector proteins, which can interfere with plant defense signaling (Dodds and Rathjen, 2010). In turn, plants have evolved resistance (R) proteins to detect the presence or activity of pathogenic effectors. Recognition of effectors results in a strong and robust defense known as effector triggered immunity (ETI), which often includes the so called hypersensitive response (HR). ETI provides faster, stronger and more specific response to a pathogenic threat than MTI. Though more efficient at stopping adapted pathogens, ETI responses highly overlap those of MTI, the most prominent differential feature being HR leading to localized cell death at the site of infection (Tsuda and Katagiri, 2010). ETI also induces systemic transcriptional reprogramming and defense enhancement through the activation of systemic acquired resistance (SAR; Spoel and Dong, 2012) and long term immunity by epigenetic mechanisms (Molinier et al., 2006; Alvarez et al., 2010).

Phospholipase D (PLD) is a family of enzymes with prominent lipolytic activity in plant tissues that has been recognized for a long time (Hanahan and Chaikoff, 1947; Li et al., 2009). PLD cleaves phospholipids to produce phosphatidic acid (PA) and a free alcohol from the phospholipid head group. The former is known to be a potent second messenger in plants and other organisms (Wang, 2004; Li et al., 2009). PLD and PA dependent signals are implicated in responses to a wide range of abiotic and biotic stresses in higher plants (Laxalt and Munnik, 2002; Li et al., 2009). PA can also be produced by phosphorylation of diacylglycerol (DAG) by DAG kinase (DAGK). DAG in its turn can be produced by phospholipase C (PLC) mediated degradation of phosphoinositides (Wang, 2004). Both pathways are implicated in PA production in response to abiotic as well as biotic stress. PLD dependent PA production can be “inhibited” by primary alcohols, whereas secondary alcohols are inefficient. The effect of primary alcohols is attributable to the preferential use of a primary alcohol for transphosphatidylation by PLD giving rise to an artificial phospholipid rather than PA.

The Arabidopsis thaliana (hereafter Arabidopsis) genome contains 12 genes encoding PLDs (Li et al., 2009). The PLDs are grouped according to their co-factor requirements and substrate preferences in α, β, γ, δ, ε, and ζ families. The Arabidopsis PLDs have roles in responses to various biotic and abiotic stresses. Several of the Arabidopsis PLDs have been implicated in responses to abiotic stress (Bargmann and Munnik, 2006): PLDα in drought-, salt- and PLDα, and PLDδ in cold stress (Sang et al., 2001b; Li et al., 2004, 2008; Hong et al., 2008). PLDα has also been implicated in senescence (Fan et al., 1997).

It is well known that PA accumulates in plant cells in response to both MTI and ETI (Bargmann and Munnik, 2006). The relative contribution of PLD and PLC-DAGK to the PA formation during MTI and ETI seems to differ between plant pathogen systems. PLD was previously directly linked to the induction of the HR after recognition of Pseudomonas syringae pv. tomato (Pst) and Xanthomonas campestris effectors (Andersson et al., 2006; Kirik and Mudgett, 2009). PA production is also associated with MTI (van der Luit et al., 2000) and inhibition of PLD was shown to increase the success of a non-adapted powdery mildew in cell wall penetration in Arabidopsis (Pinosa et al., 2013).

The individual contribution of different PLD isoforms to plant defense responses is poorly understood. Transcripts of PLDα are strongly induced by both virulent and avirulent strains of Pst and isoforms of PLDβ are transiently induced by the same strains, whereas PLDγ isoforms are induced only after recognition of the avirulent strain (Zabela et al., 2002). Treatment with the fungal elicitor xylanase as well as both avirulent and virulent strains of Pst induce transcriptional activation of PLDβ1 (Laxalt et al., 2001; Zhao et al., 2013). Recently we described how PLDδ is involved in the penetration resistance of Arabidopsis against the non-adapted fungal pathogen Blumeria graminis Sp Hordei (Bgh), the causal agent of powdery mildew on barley (Pinosa et al., 2013). The reduced penetration resistance also extended to the more adapted pathogen Erysiphe pisi (Ep), responsible for the powdery mildew disease of the garden pea. In contrast to the previously described instances where PA generated by PLD seems to act as a positive regulator of plant defense induced by both MTI and ETI, a recent study suggested that PLDβ1 acts like a negative regulator of resistance responses to biotrophic pathogens, HR and salicylic acid dependent defenses in Arabidopsis (Zhao et al., 2013).

We herein show that several different Arabidopsis PLD isoforms contribute to HR induced by recognition of the Pst effector AvrRpm1. On the other hand, cell wall based MTI triggered by the pea powdery mildew Ep, which is a non-host pathogen for Arabidopsis, is exclusively regulated by a single PLD isoform, PLDδ. To the best of our knowledge, this is the first complete reverse genetics screen of knock outs of all Arabidopsis PLD genes for involvement in defense against virulent and avirulent phytopathogenic bacteria.

As PLDs are clearly involved in both PTI and ETI, we decided to test the involvement of individual Arabidopsis PLD genes on defense responses triggered by recognition of a bacterial effector. The tomato pathovar of Pseudomonas syringae DC3000 is normally highly virulent on wild type Arabidopsis. However, if the pathogen carries the AvrRpm1 effector gene, the AvrRpm1 protein is recognized by the Arabidopsis R-protein RPM1 (Grant et al., 1995). This recognition triggers induction of HR and programmed cell death in the plant. The latter can be measured as loss of electrolytes from leaf tissue into an aqueous solution (Hibberd, 1987; Mackey et al., 2002). To verify the involvement of PLD in HR triggered by AvrRpm1 recognition in Arabidopsis, leaf tissue infiltrated with Pst DC3000:AvrRpm1 was incubated in solution with different concentrations of n- or tert-butanol and the rate of cell death determined by measuring the electric conductance of the bathing solution (Figure 1). The primary alcohol n-butanol is known to inhibit PLD dependent formation of PA, as the alcohol is preferred over water to form an artificial phosphatidylalcohol by transphosphatidylation (Ella et al., 1997). Tert-butanol, on the other hand, is unable to do this. A concentration of 0.6% (v/v) n-butanol caused a decrease in the HR induced by AvrRpm1 recognition by about 40%, whereas 0.8% (v/v) of n-butanol caused an almost complete loss in cell death. Tert-butanol had only a slight effect on the HR as measured by electrolyte leakage, the effect of tert-butanol was apparent only at the two highest concentrations used.

To test whether the alcohol by itself had an effect on the bacteria, exponentially growing Pst DC3000:AvrRpm1 were incubated with n- or tert-butanol for 6 h and thereafter serially diluted, cultivated on solid medium and the number of colonies determined after 2 days (Figure S1). The alcohol treatment caused a significant growth inhibition over 6 h compared to mock treatment. However, there was no significant difference between treatments with tert- or n-butanol. Thus, the effect of n-butanol on the HR is unlikely to be caused by the growth inhibition of the bacteria as the latter did not differ from tert-butanol treatment which did not affect the HR related cell death. To further test that the n-butanol treatment really caused significant formation of PBut during the HR triggered by recognition of AvrRpm1, lipids were extracted from plant tissue treated with tert- or n-butanol 4 h following infiltration with Pst DC3000:AvrRpm1 and the amount of PBut formed analyzed by LC-MS/MS (Figure 2). There was a clear induction of PBut formation following recognition of AvrRpm1 in the presence of n-butanol at the same (0.6%) concentration that caused a significant decrease in HR related cell death. This thus confirms activation of PLD during the HR and formation of PBut in the presence on n-butanol. These results taken together support previously published data (Andersson et al., 2006; Kirik and Mudgett, 2009) that the HR induced by recognition of bacterial effectors in Arabidopsis is strongly dependent on formation of PA by PLD.

The HR was described a century ago, only recently has the molecular details of the process from the recognition of pathogenic effectors to the “auto destruction” of the host cell begun to be elucidated (Mur et al., 2008). PLDs have been shown to play an important role in the induction of HR following recognition of pathogenic effectors. This was previously demonstrated using a system where the Pst effector AvrRpm1 was expressed in planta after the selective inhibition of PLD dependent production of PA by primary alcohols (Andersson et al., 2006). We herein show that the same holds true for effector delivered by live Pst bacteria infiltrated into the tissue. Alcohol treatment caused a reduction in growth of Pst cultured in vitro. However, as the growth inhibition effect did not differ between tert- and n-butanol, and since n-butanol had a strong effect on the HR, we deem it most likely that this non-specific effect of the alcohol is not the primary cause of the inhibition of the HR related cell death. Furthermore, as formation of PBut could be observed in tissue after inoculation with Pst DC3000:AvrRpm1 in the presence of n-butanol, it is likely that the effect of n-butanol was primarily exerted on PA production by PLD.

Phosphatidic acid has been shown to directly cause oxidative damage and cell death when infiltrated into plant tissue (Sang et al., 2001a; Park et al., 2004; Andersson et al., 2006). However, the contribution of PLD derived signals needed for the induction of HR and programmed cell death seems to vary between different effectors. HR induced by recognition of AvrRpm1 is highly PLD dependent, but is also inhibited by inhibition of PLC (Andersson et al., 2006). It was thus proposed that PLD activation is dependent of PLC activity in the case of AvrRpm1 triggered HR. On the other hand HR induced by AvrRpt2 is only inhibited if both PLD and PLC activity are affected at the same time and AvrBsT is of intermediate sensitivity to PLD inhibition (Kirik and Mudgett, 2009). This highlights that different effector recognition events trigger slightly different intracellular signaling pathways. Even though many components may be shared between the different effector response pathways, the extent to which specific signal transducers are involved in the responses appears to vary.

Of the 12 different PLDs encoded by the Arabidopsis genome no single gene knockout led to a decrease in HR induced after recognition of AvrRpm1. Since chemical inhibition of PLD dependent PA formation by primary alcohols strongly affects the HR, this point to a high degree of redundancy among the PLD genes in induction of HR following effector recognition. MTI, on the other hand, as tested here and previously (Pinosa et al., 2013) was found to be affected by the loss of a single gene, PLDδ. This difference is fully compatible with the notion that ETI is characterized by robust and redundant activation of intracellular signaling, whereas signaling leading to MTI is associated with a lower degree of redundancy (Sato et al., 2010). The lack of discernable HR phenotype of the tested PLD knockouts was also reflected in that none of the tested mutants displayed any difference in ability to restrict growth of Pst expressing AvrRpm1.

When combined with partial inhibition of the HR by n-butanol induced transphosphatidylation, several of the single knockout mutants revealed a decreased HR after recognition of AvrRpm1. Specifically, pldα1, pldα2, pldβ1, pldβ2, pldδ, pldζ1, pldζ2, and pldε all displayed a conditional HR phenotype in the presence of n-butanol. We interpret this as that multiple PLDs are activated and contribute to PA production which stimulates the HR induced by AvrRpm1 recognition in Arabidopsis. However, alternative explanations exist such as that certain PLDs become more active in the absence of other isoforms. This regulation could be both at a transcriptional and/or at a post translational level. A small decrease in HR following AvrRpm1 recognition was previously reported for the double mutant pldα1 pld δ. This finding was reported in a doctoral thesis, but never formally published in a journal (http://dare.uva.nl/record/281626). Although we found no phenotype of the double mutant, it can easily be envisioned that a small phenotype might sometimes be present as these two PLDs represent the most abundant PLD transcripts in Arabidopsis. Taken together, our data points to that the small contributions of many PLDs together provide enough PA to form an active signal. A likely activation mechanism for the multiple PLDs is the very strong and sustained increase in cytosolic calcium observed to follow recognition of bacterial effectors (Grant et al., 2000).

We found no evidence of that the pldβ1 would contribute to increased HR in response to AvrRpm1 recognition as reported for recognition of AvrRpt2 (Zhao et al., 2013). This could be due to differences between the signaling pathways induced by different effector types. The pen3 mutant for example demonstrates different phenotypes depending on whether it is treated with Pst expressing AvrRpm1 or AvrRpt2 (Kobae et al., 2006; Johansson et al., 2014). The previously reported strongly decreased proliferation in leaf tissue of virulent Pst DC3000 in the pldβ1 mutant (Zhao et al., 2013) was not apparent in our hands. There are differences in the experimental setup such as light intensity and density of the bacterial inoculum which could influence the outcome. It should however be noted that the pldβ1-1 line used herein is identical to the T-DNA line used in the study by Zhao et al. (2013). Further studies are needed to clarify this point and further investigate among other factors the effect of different bacterial titers on the defense reaction and the phenotype of the mutant.

To conclude, we herein report that at least eight different PLD isoforms in Arabidopsis contribute to signaling in HR triggered by AvrRpm1 recognition. In contrast, loss of just one of the major PLD isoforms is sufficient to significantly affect MTI dependent defense responses.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.