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.

中文翻译：

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发育调节的产生系统信号，用于番茄的持久防御启动

 介绍

由于病原真菌的伤害和感染，当前的食品供应链在收获后层面遭受了重大损失（Lipinski 等 人，2013 年;Zhang et al.， 2021）。西红柿是一种主要的全球商品，2019 年生产了 1.823 亿吨水果（粮农组织，2019 年）。然而，由于病原体，其产量受到严重限制，50% 的产量损失发生在收获后阶段（粮农组织，2019 年）。在商业环境中不允许对番茄果实使用采后杀虫剂（Pétriacq et al.， 2018），现阶段的主要控制措施仅限于低温储存和严格的卫生措施（Abbey et al.， 2019）。然而，采后病原体，如灰霉病的病原体，无法通过这些策略成功控制。因此，需要新的方法。更好地了解番茄防御机制将使研究人员能够设计策略来控制采前和采后真菌感染并减少产量浪费。

植物免疫系统的“适应性”成分可以称为启动（Mauch-Mani et al.， 2017）。与直接激活防御机制不同，防御机制会引起显着的代谢改变，而启动通过在攻击时有针对性地分配能量资源来最大限度地减少能量消耗，从而在需要时更快、更强地激活防御机制（van Hulten 等 人，2006 年）。引发被认为是广谱的，并且已在许多不同的植物物种中进行了描述，从拟南芥到 Malus pumila（苹果树）（Zimmerli 等 人，2000 年;Cohen， 2002;Reuveni et al.， 2003;Cohen et al.， 2010， 2016）。重要的是，启动已被证明是持久的（Worrall 等 人，2012 年;Wilkinson等 人，2018 年;Mageroy et al.， 2020;Catoni et al.， 2022）并传递给后代（Luna et al.， 2011;Slaughter 等 人，2011 年;Rasmann et al.， 2012）。一种非常明确的引发化学物质是非蛋白质氨基酸β-氨基丁酸（BABA），首次在1960年代被发现（Papavizas & Davey，1963）。随后被证明 BABA 对一系列物种的非生物和生物胁迫都有效（Cohen 等 人，2016 年）。 BABA 诱导的抗性 （BABA-IR） 与植物的一系列变化有关，例如通过胼胝质沉积增强物理保护、PATHOGENESIS-RELATED1 （PR1） 蛋白积累以及水杨酸 （SA） 和茉莉酸 （JA） 等防御激素的增加（Zimmerli 等 人，2000 年;Ton & Mauch-Mani， 2004;Hamiduzzaman 等 人，2005 年;Ton 等 人，2005 年;Schwarzenbacher等 人，2020 年）。在拟南芥中，BABA 与天冬氨酰-tRNA 合成酶结合 （Luna et al.， 2014） 并将酶的经典功能转变为启动。在番茄和拟南芥中，BABA可以通过根吸收，然后转移到气生组织中（Cohen & Gisi， 1994;Wilkinson et al.， 2018）。尽管尚未在番茄中鉴定出受体，但人们认为 BABA 在番茄中的作用方式与拟南芥相似（Luna et al.， 2014），导致对灰葡萄孢杆菌的持久增强耐药性（Luna et al.， 2016）。BABA 处理已被证明在幼苗阶段应用时可对果组织产生持久的保护，从而赋予采后保护（Wilkinson等 人，2018 年;Luna等 人，2020 年）。因此，长效引发提供了一种替代杀菌剂的方法，以保护植物免受采后病原感染。

持久的启动与表观遗传变化有关，例如 DNA 甲基化和小 RNA （sRNA） 的产生，因为它们可以促进基因表达的变化（Slaughter 等 人，2011 年;Dowen et al.， 2012;Rasmann et al.， 2012;Catoni et al.， 2022;Hannan Parker et al.， 2022）。例如，对拟南芥表观遗传重组自交系 （epiRIL） 的分析表明，低甲基化基因座增强了对有毒的拟南芥透明质吻孢菌的 SA 依赖性和 SA 非依赖性防御的启动（Furci等 人，2019 年）。此外，由植物特异性 RNA 导向的 DNA 甲基化 （RdDM） 途径产生的 sRNA 与拟南芥中的持久和跨代 IR 有关（Rasmann等 人，2012 年）。最近的工作表明，JA-IR 受 DNA 去甲基化途径的调节，需要完整的 sRNA 结合蛋白 AGO1 与引物防御相关基因（Wilkinson等 人，2023 年）。BABA 也被证明与 DNA 甲基化的重要变化有关。在番茄中，CHH 胞嘧啶背景下 DNA 甲基化的全局变化 （H 表示除 G 以外的任何核苷酸） 与 Money-Maker 品种中持久的 BABA-IR 有关。虽然在灰葡萄孢杆菌感染期间，在差异表达基因 （DEG） 的启动子中发现了许多差异甲基化区域 （DMR），但大多数引发基因没有差异甲基化（Catoni等 人，2022 年）。因此，引发的长期表观遗传性质背后的机制仍不清楚。 此外，BABA-IR 的长期性质尚未被探索和利用，因为它在采后抗性方面的潜在作用。有趣的是，番茄植株已被证明具有不同的甲基化谱，具体取决于果实发育阶段和组织类型：果组织中的 CG 和 CHG 甲基化水平低于 4 周龄的叶组织，在 CHH 环境中可以看到相反的模式（Zhong et al.， 2013）。然而，发育阶段依赖性 DNA 甲基化的变化如何介导印记和维持持久的收获后启动尚未探索。

在这里，我们发现植物的发育阶段对建立针对灰霉菌的持久启动能力有重大影响。我们评估了 BABA 处理在不同发育阶段对转录组和表观基因组水平的影响，并使用甲基化组分析来检验年轻植物表现出更大的表观遗传可塑性的假设。此外，我们发现当嫁接到引物砧木上时，长效 BABA-IR 可传播到幼稚的接穗组织，并且我们研究了 sRNA 与抗性的关系。通过组学分析的整合，我们确定了番茄中与长效 BABA-IR 相关的标志物，用于控制采后果实中的 B. cinerea。