Introduction
Although static with respect to their location, plants live in a dynamic environment, which means that stress adaptation is crucial for their survival (Mittler, 2006). A central aspect of stress resilience is the immunity against pathogens. While plants lack the adaptable immunity, found in mammals, they have evolved two tiers of innate immunity (Jones & Dangl, 2006). For the first tier, plants sense and respond to microbes including non-pathogens via conserved Pathogen, Microbe and Damage-Associated Molecular Patterns (PAMPs, MAMPs, or DAMPs, respectively). Binding of the eliciting molecule to specific pattern recognition receptors (PRRs triggers a broadband basal immunity (Bigeard et al. , 2015) termed as PAMP triggered immunity (PTI). Since PAMPs are essential for the survival of the pathogen, selective pressure by the host towards the loss of these treacherous molecules is buffered by the selective pressure to maintain these molecules, because they are essential. This balance of antagonistic selective pressures favoured the evolution of effectors that can inhibit PTI and re-install pathogenicity. In response, plants have acquired specific receptors recognising these effectors leading to the second tier of immune system called effector-triggered immunity (ETI), which is often specific for a certain pathogen, or even to a given strain of a pathogen (Tsuda & Katagiri, 2010).
The recognition of PAMPs by PRRs is known to trigger calcium influx, followed by activation of mitogen-activated protein kinase (MAPK) cascades, in parallel with apoplastic oxidative burst. In consequence, transcription factors are activated that will stimulate the expression of defence genes (Withers & Dong, 2017). Some of these events are shared with ETI (Tsuda & Katagiri, 2010), but the relative timing seems to differ. For instance, while calcium influx precedes oxidative burst during flagellin-triggered PTI of grapevine cells, the temporal order of these two stress inputs is reversed after elicitation with harpin, a bacterial trigger of an ETI-like response culminating in programmed cell death (Chang & Nick, 2012).
The two levels of immunity differ also with respect to regulation by phyto-hormones. While salicylic acid (SA) seems to activate ETI as effective strategy against biotrophic pathogens, jasmonic acid (JA) is involved in the basal defence against necrotrophic pathogens. The antagonistic role of the two phyto-hormones is reflected as antagonistic relationship of PTI versus ETI (Ramirez-Prado et al., 2018). In suspension cells from the North American grape speciesVitis rupestris , JA accumulated in response to PTI triggered by flg22, but not in response to harpin, a trigger of ETI-like defence (Chang et al., 2017). It should be noted, however, that during ETI, this antagonism of JA and SA signalling can sometimes be replaced by a synergistic interaction. Activation of the SA receptors NPR3 and NPR4 inArabidopsis can also activate JA response and synthesis genes through degradation of JAZ proteins (Liu et al., 2016), a mechanism proposed to prevent the spread of necrotrophic pathogens in organs, where biotrophic pathogens have been warded off by hypersensitive cell death.
As described above, the early signalling, and also the regulation of PTI and ETI by phyto-hormones can overlap to a certain extent, leading to the question, whether there are early cellular events that are associated with the dichotomy of the two immunity layers. Comparative studies, where the responses to flg22 (triggering PTI), and harpin (a bacterial elicitor triggering an ETI-like type of defence) were compared side by side in suspension cells from either grapevine (Chang & Nick, 2012), or tobacco BY-2 (Guan et al., 2013), suggest that rapid reorganisation of actin filaments seems to qualify as early marker for cell-death related immunity. During harpin-triggered ETI-like response, the NADPH-dependent oxidase Respiratory burst oxidase Homologue (RboH) is activated and generates superoxide. How this primary signal is transduced into cell death and/or gene activation, has remained elusive. A few hints exist, however: A role of superoxide, and phospholipase D (PLD) for actin remodelling in response to harpin has been inferred from inhibitor studies (Chang et al. , 2015). Furthermore, pharmacological modulation of actin filaments is accompanied by elevated expression of defence genes (Qiao et al. , 2010). Modulations of actin dynamics have been shown to alter SA synthesis and signalling (Matoušková et al., 2014). Although these findings indicate that actin filaments participate in defence signalling, the functional context is far from clear. Is actin remodelling necessary and sufficient for the activation of phytoalexin genes? Is actin bundling inevitably linked with cell death, or is it possible to separate these events?
A strategy to address these questions would be to trigger actin remodeling in the absence of a pathogen-related signal and to test, whether this would activate defence responses. Actin filaments respond to numerous intracellular or extracellular signals (reviewed in Wasteneys & Yang 2004). In particular, abiotic stress factors that induce oxidative burst often cause actin bundling, and actin is also involved in tolerance to these factors. For instance, aluminium as abundant metal, and therefore of agricultural impact, is able to cause oxidative burst (reviewed in Panda et al. , 2009), and can induce actin bundling in tobacco seedlings (Ahad & Nick, 2007). Moreover, tobacco mutants generated by activation tagging that are tolerant to aluminium showed constitutive bundling of actin, even in absence of aluminium as stressor (Ahad & Nick, 2007). Therefore, we used in the current study the rationale to trigger actin bundling by Al3+. To follow actin responses, we used a grapevine cell line expressing the fluorescent actin marker GFP-AtFABD2(Akaberi et al., 2018), and we investigated the resulting defence responses in cells and plants of grapevine. We show that Al3+ causes actin reorganisation dependent on RboH, but does not induce programmed cell death. This actin reorganisation in response to Al3+ activates genes for the synthesis of SA and phytoalexins supporting a model, where actin participates in a pathway involved in basal immunity by connecting the input from oxidative burst with the activation of defence genes.