dc.description.abstract
The present study investigated the effect of a preceding, short cold treatment (4 °C, 24 h) on the resistance of Arabidopsis thaliana against hemibiotrophic Pseudomonas syringae pv. tomato (Pst) and necrotrophic Botrytis cinerea. For this aim, two different experimental cold setups were compared. In the first setup (cold pre-treatment; CT), the pathogen infection occurred directly after the cold treatment. The second experimental setup (cold priming; CP) included a memory phase between the cold treatment and the infection, whereby the impact of a priming memory on the resistance against pathogens was investigated. Arabidopsis benefited from the cold treatment and exhibited significantly increased resistance against Pst and B. cinerea. For plant defense against Pst, cold priming and cold pre-treatment led to increased resistance, while against B. cinerea only cold pretreatment resulted in transient resistance increase. To identify the immune signaling pathways responsible for the increased resistance, transcript analyses and pathogen growth experiments were conducted with Arabidopsis (Accession: Col-0) and selected mutant lines. The experiments demonstrated that cold priming-mediated resistance in Arabidopsis against virulent Pst is independent of the plant immune regulator Enhanced Disease Susceptibility 1 (EDS1) and does not alter transcript levels of pathogen-triggered Pathogenesis-related 1 (PR1), Isochorismate Synthase 1 (ICS1), FLG22-induced receptor-like Kinase 1 (FRK1), and NDR1/HIN1-like 10 (NHL10). These results, but also the observation that cold priming does not confer resistance against avirulent Pst avrRPS4 and Pst avrRPM1, indicated that cold-mediated resistance against Pst is mainly independent from effector-triggered immunity (ETI), hypersensitive response (HR), PAMPs-triggered immunity (PTI) and salicylic acid (SA) signaling. Cold pretreatment-mediated resistance against B. cinerea was also without cold signatures on the transcript levels of Pathogenesis-related 1 (PR1), Pathogenesis-related 4 (PR4), Plant Defensin 1.2a (PDF1.2a) and Phytoalexin Deficient 3 (PAD3). This suggested that cold exposure-mediated resistance against B. cinerea is independent of SA signaling, jasmonic acid (JA) signaling, and camalexin biosynthesis. Increased accumulation of reactive oxygen species (ROS) was observed in Arabidopsis leaves after the cold pre-treatment and subsequent B. cinerea infection indicating a central function of ROS in the crosstalk between the prior cold and pathogen infection. Besides other functions, ROS contribute to lignin and callose formation. Enhanced callose formation and lignification could be detected immediately after the cold exposure. In contrast, B. cinerea did not alter plant lignin amounts and pathogen-triggered callose deposition was not further affected by prior cold treatment. We extended our analyses by investigating the impact of cold-pre-treatments on the establishment of systemic aquired resistance (SAR). The induction of SAR in cold-pretreated plants was successful, but the prior cold treatments did not provide additional benefits for SAR against Pst. Interestingly, a syringe infiltration with the mock solution instead of the SAR-triggering primary Pst inoculation enhanced the susceptibility of Arabidopsis after a cold pre-treatment against Pst. The most pivotal finding of this work demonstrated that the chloroplast-localized ROS-scavenging enzymes stromal ascorbate peroxidase (sAPX) and thylakoid ascorbate peroxidase (tAPX) are essential for the wildtype-like enhanced resistance against Pst and B. cinerea after prior cold treatments. CP-mediated resistance was also confirmed in the immunocompromised null mutant enhanced disease susceptibility 1 (eds1-2) crossed with stroma ascorbate peroxidase knockout (eds1 sapx). This work, furthermore, showed that sAPX is essential for cold-induced callose formation and dispensable for pathogen-induced callose. Finally, sapx, but not thylakoid ascorbate peroxidase knockout (tapx), showed a different ROS generation pattern, with significantly more ROS being generated than in cold-treated sapx or compared to wildtype plants without cold pre-treatment. These results highlight the relevance of the chloroplast antioxidant system in cold sensing and its impact on the plant immune system.
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