What is the relationship of aluminium-triggered signalling with basal defence?
The fact that SA signalling can be activated by aluminium and is acting in the context of basal immunity provokes a new question: Is it possible to define the event, where the actin-dependent pathway merges with the signalling deployed in the context of PTI? The operational criterion for an event located downstream of this merging point would be that inhibition of this event would block both, gene expression induced by flg22, and gene expression induced by aluminium. It seems that MAPK signalling meets this criterion, because treatment of grapevine cells with the MAPK inhibitor PD98059 can fully suppress the induction of all three tested genes involved in phytoalexin synthesis (i.e. PAL ,RS , and STS , Figure 6A ), and the same was observed for the induction of STS by both flg22 and by harpin (Chang & Nick, 2012). This inhibitor acts at the MAPKK level of the cascade (Cohen, 1997). Specifically for defence signalling, MKK1 has been suggested as target (Mészáros et al., 2006). Thus, at the second tier of MAPK signalling, flg22-triggered, harpin-triggered, and aluminium-triggered signal transduction seem to be confluent already. Since aluminium, in contrast to flg22, fails to induce extracellular alkalinisation (Suppl. Figure S3 ), the merging point must be downstream of calcium influx. Furthermore, it must be located downstream of actin, since Latrunculin B can block the induction of gene expression by aluminium (Figure 3A ).
The fact that aluminium activates a key gene of SA synthesis (isochorismate synthase) and a marker gene for SA response (PR1) does not mean that SA is part of the signalling responsible for the activation of phytoalexin synthesis genes. This is rather unlikely, due to the time course. However, SA might act in a feedback loop contributing to the persistence of defence. A straightforward mechanism would be the accumulation of intracellular ROS. The original idea that this might be caused by direct inhibition of catalase through salicylic acid (Chen et al., 1993; Klessig et al. , 2019) has meanwhile been discarded after decades of controversy, and replaced by a model, where SA is acting through a signalling network that regulates expression and activities of the enzymatic and non-enzymatic redox homeostasis (for a recent review see Klessig et al. , 2019). The link with actin might come through phospholipase D: by binding to phospholipids, the substrates of phospholipid D, aluminium inhibits the formation of phosphatidic acids, as concluded from the fact that exogenous phosphatidic acids can mitigate the effect of aluminium in Arabidopsis seedlings (Matoušková et al., 2014).
There is a seemingly paradox observation, though: In our study, several of the genes that are activated by Al3+, are also activated by Latrunculin B significantly to the same extent. While the combination of Al3+ and Latrunculin B eliminates this activation. Interestingly, the transcription factor MYB14 does not show this activation by Latrunculin B. Although it appears paradox that two compounds that act antagonistically upon actin, should exert the same effect on gene activation, this is not the first time that this has been reported for phytoalexin synthesis genes in grapevine (Qiao et al., 2010). Also for MAPK signalling, the activation of the MAP kinase SIMK by both Latrunculin B and Jaspaklinolide has revealed a similar paradox (Šamaj et al. , 2002). Activation of signalling by elimination of actin could be achieved by releasing an activator that had been tethered to actin, as it has been proposed for the MAP kinases SIMK and SAMK (Šamaj et al. , 2002; Sangwan et al. , 2002). How signalling might be stimulated by suppression of actin turnover, is more challenging to explain. One link might be Capping Protein that can either be sequestered to the plasma membrane by phosphatidic acids, the products of the phospholipase D signalling hub, or block the elongation of actin filaments by blocking the barbed end (Li et al. , 2015). Stabilisation of actin might repartition Capping Protein from the membrane towards bundling actin cables, such that phosphatidic acid would be released for other signalling functions, such as activation of the Respiratory burst oxidase Homologue (Eggenberger et al. , 2017), leading to a self-amplifying signalling loop between actin and reactive oxygen species. The fact that some genes (PAL ,RS , STS ) show both, the activation by Al3+ and by Latrunculin B, while others (MYB14 ) are not activated by Latrunculin B, indicates that the signal chains initiating from actin (one possibly through MAPK signalling, the other possibly through phosphatidic acid signalling) might be activated differentially and exert differing impact on different target genes.