Developmentally regulated generation of a systemic signal for long-lasting defence priming in tomato

Introduction

The current food supply chain experiences major losses at the postharvest level due to both injury and infection by pathogenic fungi (Lipinski et al., 2013; Zhang et al., 2021). Tomato is a major global commodity, with 182.3 million tons of fruit produced in 2019 (FAO, 2019). However, its yield is heavily restricted due to pathogens, and 50% of yield loss occurs at the postharvest stage (FAO, 2019). Postharvest pesticide use is not permitted for tomato fruit in commercial settings (Pétriacq et al., 2018), and the main control measures at this stage are limited to cold temperature storage and strict hygiene measures (Abbey et al., 2019). However, postharvest pathogens such as Botrytis cinerea, the causal agent of grey mould, cannot be successfully controlled with these strategies. Therefore, new approaches are required. A better understanding of tomato defence mechanisms would allow researchers to design strategies to control pre- and postharvest fungal infections and reduce yield waste.

The ‘adaptive’ component of the plant immune system can be referred to as priming (Mauch-Mani et al., 2017). Unlike direct activation of defence mechanisms, which induces significant metabolic alterations, priming minimises energetic costs via targeted allocation of energy resources upon attack, thus resulting in a faster and stronger activation of defence mechanisms when required (van Hulten et al., 2006). Priming is considered to be broad spectrum and has been described in many different plant species, from Arabidopsis thaliana to Malus pumila (apple trees) (Zimmerli et al., 2000; Cohen, 2002; Reuveni et al., 2003; Cohen et al., 2010, 2016). Importantly, priming has been shown to be long-lasting (Worrall et al., 2012; Wilkinson et al., 2018; Mageroy et al., 2020; Catoni et al., 2022) and to be transmitted to following generations (Luna et al., 2011; Slaughter et al., 2011; Rasmann et al., 2012). A very well-characterised priming chemical is the nonprotein amino acid β-aminobutyric acid (BABA), first identified in the 1960s (Papavizas & Davey, 1963). BABA has subsequently been documented to be effective against both abiotic and biotic stresses in a range of species (Cohen et al., 2016). BABA-induced resistance (BABA-IR) is associated with a range of changes to the plant such as enhanced physical protection through callose deposition, PATHOGENESIS-RELATED1 (PR1) protein accumulation and increases in defence hormones such as salicylic acid (SA) and jasmonic acid (JA) (Zimmerli et al., 2000; Ton & Mauch-Mani, 2004; Hamiduzzaman et al., 2005; Ton et al., 2005; Schwarzenbacher et al., 2020). In Arabidopsis, BABA binds to an aspartyl-tRNA synthetase (Luna et al., 2014) and changes the canonical function of the enzyme into priming. In tomato and Arabidopsis, BABA can be absorbed through the roots and is then translocated to aerial tissue (Cohen & Gisi, 1994; Wilkinson et al., 2018). Although the receptor has not been identified in tomato, BABA is thought to work in a similar way in tomato to Arabidopsis (Luna et al., 2014), leading to durable enhanced resistance against B. cinerea (Luna et al., 2016). BABA treatment has been shown to lead to long-lasting protection of fruit tissue when applied at the seedling stage, thus conferring postharvest protection (Wilkinson et al., 2018; Luna et al., 2020). Therefore, long-lasting priming offers an alternative approach to fungicides towards protecting plants from postharvest pathogenic infections.

Long-lasting priming has been linked to epigenetic changes such as DNA methylation and the production of small RNAs (sRNAs), as they can contribute to changes in gene expression (Slaughter et al., 2011; Dowen et al., 2012; Rasmann et al., 2012; Catoni et al., 2022; Hannan Parker et al., 2022). For instance, analysis of Arabidopsis epigenetic recombinant inbred lines (epiRIL) demonstrated that hypomethylated loci enhanced priming of SA-dependent and SA-independent defences against virulent Hyaloperonospora arabidopsidis (Furci et al., 2019). Moreover, sRNAs produced by the plant-specific RNA-directed DNA methylation (RdDM) pathway have been associated with long-lasting and transgenerational IR in Arabidopsis (Rasmann et al., 2012). Recent work has illustrated that JA-IR is regulated by DNA-demethylation pathways, requiring an intact sRNA binding protein AGO1 to prime defence-associated genes (Wilkinson et al., 2023). BABA has also been shown to be associated with important changes in DNA methylation. In tomato, global changes to DNA methylation in the CHH cytosine context (H indicates any nucleotide other than G) have been associated with long-lasting BABA-IR in the Money-Maker cultivar. While many differentially methylated regions (DMRs) were found in promoters of differentially expressed genes (DEGs) during B. cinerea infection, the majority of primed genes were not differentially methylated (Catoni et al., 2022). Therefore, the mechanisms behind the long-lasting epigenetic nature of priming are still unclear. In addition, the long-lasting nature of BABA-IR has yet to be explored and utilised for its potential role in postharvest resistance. Interestingly, tomato plants have been shown to have different methylation profiles depending on both fruit developmental stage and tissue type: CG and CHG methylation levels are lower in fruit tissue than in 4-wk-old leaf tissue, with the reverse pattern seen in CHH context (Zhong et al., 2013). However, how changes in developmental stage-dependent DNA methylation mediate the imprinting and the maintenance of long-lasting postharvest priming is unexplored.

Here, we found that the plant's developmental stage has a major influence on the ability to establish long-lasting priming against B. cinerea. We assessed the impact of BABA treatments on a transcriptomic and epigenomic level at different developmental stages and used methylome analysis to test the hypothesis that young plants display greater epigenetic plasticity. Additionally, we found that long-lasting BABA-IR is transmissible to naive scion tissue when grafted on primed rootstock, and we investigated the association of sRNAs with resistance. Through the integration of omics analyses, we have identified markers associated with long-lasting BABA-IR in tomato for the control of B. cinerea in fruit postharvest.