AHL induced systemic resistance against Xanthomonas translucens

In modern agriculture, concepts of biological pest control and enhancement of plants resistance are implemented (Berg, 2009). Chemical (e.g.: β-Aminobutyric acid, probenazole, phosphite) and biological (e.g. mycorrhizal fungi, PGPRs, algal extracts) activators, for which a growing market exist, were found to confer resistance of crop plants towards various pathogens (Berg, 2009; Walters et al., 2013). The colonization of the plant root by PGPRs led to resistance in distal parts of various plants to different bacterial and fungal pathogens, whereby the term rhizobacteria-induced systemic resistance (ISR) arose (van Loon, 1998;

De Vleesschauwer and Höfte, 2009; Balmer et al., 2012). This preparation of the plant to efficiently combat any further biotic or abiotic attack is termed priming and is characterized by an augmented sensitization and activation of cellular defense mechanism, which may lead to enhanced resistance (Conrath et al., 2002; Conrath, 2011). Several PGPR-mediated ISRs are based on this priming state and provide plants with an enhanced cellular defense response against upcoming pathogens (Pieterse et al., 2014). Diverse microbial-derived molecules have been determined as elicitors of the rhizobacteria-induced systemic resistance, among them lipopolysaccharides, siderophores, exopolysaccharides, and also AHLs (De Vleesschauwer and Höfte, 2009; Balmer et al., 2012). In the present study, the

DISCUSSION

93 application of 10 µM short- and long-chain AHL to the root system of barley caused a systemic reduction of the biotrophic pathogen Xtc in barley leaves compared to controls, thus inducing systemic resistance. The mean value of the 4 biological replicates shows credible Xtc titer reduction after previous 24 and 96 h of AHL treatment. If all single experiments were analyzed separately, also the 72 h incubation with AHL led to credible reduction of Xtc, but this effect was lost when creating the mean value over all conducted experiments.

Furthermore, it was observable that the bacterial titer of controls and AHL treatments decreased between the 24 h and 96 h time point (fig. 3.20 B). As AHLs did not interfere with the Xtc growth and also the control treatment showed this total titer decrease, the findings of Dey et al. (2014) that Xtc is mobile in barley, could be causal.

The involvement of AHLs in PGPR-mediated ISR was already discovered in the interaction of tomato with the AHL-producing rhizobacteria Serratia liquefaciens MG1, which enhanced the systemic resistance against the necrotrophic fungus Alternaria alternata in tomato. The resistance was induced after 72 h of the inoculation with the PGPR strain.

Serratia liquefaciens MG44, a bacterial mutant impaired in AHL production, did not induce resistance in distal leaf parts (Schuhegger et al., 2006). Accordingly, Serratia liquefaciens MG1 led to a reduction of the spreading of Pseudomonas syringae pv. tomato DC3000 in A.

thaliana leaves, but this effect was not significant (von Rad et al., 2008). Further investigations with Serratia plymuthica HRO-C48 achieved ISR to the necrotrophic Botrytis cinerea, which causes the grey mold disease in bean and tomato plants, whereas AHL-deficient mutants of this bacterial strain showed weaker infection containment (Pang et al., 2009). Also here, an incubation of 3 days with the PGPR strain conferred the systemic resistance. Serratia plymuthica HRO-C48 also rescued cucumber plants against damping-off disease caused by the oomycete Pythium aphanidermatum. These data reveal that bacterial signaling compounds are required to increase systemic resistance to pathogens. Serratia liquefaciens MG1 produces the short-chain AHLs C4- and C6-HSL, while Serratia plymuthica HRO-C48 produces the same AHLs and additionally oxo-C6-HSL, showing that short-chain AHLs are able to confer resistance induction. In this context, the oxo-C8-HSL producing bacterial strain Rhizobium etli 11541 was not effective in resistance induction against the hemibiotrophic Pseudomonas syringae pv. tomato DC3000 in A. thaliana (Zarkani et al., 2013), whereas in the present study, the purified short-chain C8-HSL is sufficient to enhance systemic resistance against Xtc in Hordeum vulgare. Moreover, the authors investigated that Sinorhizobium meliloti Rm2011, producing the long-chain oxo-C14-HSL, stopped the spreading of the tomato bacterial speck caused by Pseudomonas syringae pv. tomato DC3000 (Zarkani et al., 2013). These data give reason to the assume that rhizobacteria, which produce short- or long-chain AHLs are able to induce resistance, but that this effect is dependent on the corresponding host plant and pathogen lifestyle. Moreover, recent

DISCUSSION

94 investigations displayed that the application of commercially produced AHLs is enough to increase the resistance against various pathogens, which reinforce the results of this present study. A 3-day oxo-C14-HSL-treatment of A. thaliana and barley enhanced the defense against the obligate biotrophic powdery mildews Golovinomyces orontii and Blumeria graminis, respectively (Schikora et al., 2011). Resistance reinforcement against the hemibiotrophic Pseudomonas syringae pv. tomato DC3000 in A. thaliana was also induced after 3 days of oxo-C12-, hydroxy-C14- and oxo-C14-HSL, whereas the strongest effect was achieved by oxo-C14-HSL. But, no resistance effect turned out after C6-HSL application (von Rad et al., 2008; Schenk et al., 2012; Schenk et al., 2014), revealing an AHL chain length and substitution dependent effect in A. thaliana. To sum up, application of commercially produced long-chain AHLs (oxo- C12-/ C14-HSL and hydroxyl-C14-HSL) reduced disease symptoms of hemi- and biotrophic pathogens, but not of necrotrophic ones, while rhizobacteria, producing short-chain (C4-, C6- and oxo-C6-HSL) and long-chain AHLs (oxo-C14) did so against necrotrophic, biotrophic, and hemibiotrophic pathogens. Commercially available C6-HSL was not able to induce the plant defense against Pseudomonas syringae pv. tomato DC3000 in A. thaliana (von Rad et al., 2008). In the present in vitro system, commercial C8- and C12-HSL reduced the titer of the biotrophic leaf pathogen Xtc, thus no chain length dependent effect could be displayed. Furthermore, the endophyte Gluconacetobacter diazotrophicus caused the protection to Xanthomonas albilineans in its beneficial interaction with sugarcane (Arencibia et al., 2006). The authors discuss that the endophyte could possess and/or produce elicitors, which induce the sugarcane defense response. Recently, Nieto-Peñalver et al. (2012) demonstrated that Gluconacetobacter diazotrophicus produces AHLs, among them also C8- and C12-HSL, which gives reason to speculate that these microbial signaling molecules could be involved in the abovementioned induced resistance and resemble the same AHL molecules that are applied in the present study.

Interestingly, in all above mentioned PGPR / AHL-inoculation experiments an exposure of 3 days was necessary to show ISR. The application of 10 µM C8- and C12-HSL credibly reduced the Xtc titer in barley leaves after 24 h, lost in strength at an exposure time of 48 h, and reached the ISR effect after 96 h again, while in the single biological experiments the bacterial titer reduction was already achieved after 72 h of AHL exposure.

Partly, the present results are in accordance with the 3-day achieved resistance, but PGPR application even conferred resistance in radish after 1 day (Leeman et al., 1995b).

Schikora et al. (2011) proved that root-applied oxo-C14-HSL was not detectable in the leaf tissue, which is in accordance to the findings of Götz et al. (2007) and Sieper et al.

(2014), that only short-chain AHLs are transported but long-chain AHLs are not. If the AHL-transport is the crucial factor for resistance induction, why and/or how could the long-chain

DISCUSSION

95 AHL induce resistance barley leaves? Since C8- and C12-HSL induced ISR in barley, a root-to-shoot signal acting as second messenger should exist. Thus, the NO accumulation that occurred in barley roots after AHL treatment (chapter 3.2.1) could be a possible mediator for ISR induction. NO is a diffusible gas and is known to act as a systemic signaling compound.

A root derived stimulus of NO-donor solution led to a rapid activation of kinases in leaves (Capone et al., 2004), while reportedly mitogen-activated protein kinases (MAPK) are involved in systemic resistance (Viterbo et al., 2005). Also, Schikora et al. (2011) demonstrated that MAPKs are necessary for an AHL-induced resistance in A. thaliana.

Furthermore, NO increases SA levels which is the key regulator in SAR and was reported to be involved in induced systemic resistance upon rhizobacteria inoculation (Durner et al., 1998; Wendehenne et al., 2001; Schuhegger et al., 2006). Elevated SA concentrations were detected after 4 h of 10 µM C8- and C12-HSL treatment (chapter 3.3.2). Moreover, as already discussed in chapter 4.1.4, short chain AHLs elevated intracellular Ca²⁺

concentrations in A. thaliana roots and, reportedly, NO production follows Ca²⁺ bursts (Ali et al., 2007; Song et al., 2011). But interestingly, long-chain AHL signaling seems to be calmodulin independent in the root tissue (Zhao et al., 2015) All these facts lead to the assumption that short-chain AHL induced resistance occurs at first by a rapid induction of a Ca²⁺ burst, triggering the production of NO, and the accumulation of SA in leaves, which then activates further signaling cascades (e.g. defense gene regulation), leading to the establishment of a systemic resistance against biotrophic pathogens like Xtc. For long-chain AHLs it seems that they trigger an NO accumulation in the root tissue, induce MAPKs in leaves and activates further signaling cascades (e.g. defense gene regulation), via an accumulation of SA and ABA in leaves, which then leads to the establishment of a systemic resistance against biotrophic pathogens like Xtc.

DISCUSSION

96 Figure 4.5 Model summarizing AHL induced reactions in barley. AHLs are recognized in the root tissue via a yet unknown mechanism and induce a membrane hyperpolarization in root epidermal cells, which is likely to activate root K⁺ uptake. Higher nutrient uptake and a NO-dependent lateral root formation are possibly responsible for root and shoot biomass gain. Additionally, NO accumulates in roots after AHL application, probably in consequence to a Ca²⁺ burst that was investigated by Song et al. (2011). NO probably acts as a root-to-shoot signal, leading to a systemic priming effect. Also, short-chain AHL transport (Götz et al., 2007; Sieper et al., 2014) and MAPKs (Schikora et al., 2011) could be involved. As a consequence, SA and ABA are accumulated and result in PAL and defense gene activation. It is suggested that priming and a SA-dependent ISR are the basis for this AHL-induced resistance.