Transcriptome analysis reveals that SlNPR1 mediates tomato fruit resistance against Botrytis cinerea by modulating phenylpropanoid metabolism and balancing ROS homeostasis

Highlights

• Knockout of SlNPR1 conferred disease resistance against B. cinerea to tomato fruit.

• Knockout of SlNPR1 enhanced activities of defense enzymes including PAL, GLU, and CHI.

• ROS homeostasis in slnpr1 was maintained by increased POD, SOD, GST and decreased CAT.

• RNA-seq data showed that phenylpropanoid biosynthesis was induced in slnpr1 fruit.

Abstract

Tomato is the fourth most popular fresh-market fruit, whereas most commercial tomato cultivars are particularly susceptible to B. cinerea. Nonexpressor of pathogenesis-related gene 1 (NPR1) is a critical regulator in plant resistance against various pathogens. However, the underlying mechanism of how SlNPR1 influences the defense against B. cinerea in tomato fruit remains unclear. In this study, two independent lines carrying homozygous mutation in SlNPR1 were used for studying its role in the interaction between tomato fruit and B. cinerea. Our results showed that knockout of SlNPR1 decreased the disease development of B. cinerea in tomato fruit. slnpr1 fruit exhibited smaller lesion sizes, higher activities of defense enzymes, and upregulated expressions of defense genes compared to wild type (WT). In addition, reactive oxygen species (ROS) homeostasis in slnpr1 fruit was balanced by increased activities of peroxidase (POD), superoxide dismutase (SOD) and glutathione S-transferase (GST), as well as decreased activity of catalase (CAT). Furthermore, SlNPR1-mediated differential expression genes (DEGs) were significantly enriched in the secondary metabolic pathways, represented by phenylpropanoid biosynthesis. Taken together, these findings revealed that knockout of SlNPR1 resulted in increased activities of defense enzymes, changes in ROS homeostasis and activation of phenylpropanoid biosynthesis and some other signaling pathways, which contributes to resistance against B. cinerea in tomato fruit.

Introduction

Botrytis cinerea, the causal agent of gray mold, is the second most important phytopathogen responsible for pre- and post- harvest decay and fruit quality deterioration in greenhouse, open field and even during cold storage (0−10 °C) (Dean et al., 2012). Tomato (Solanum lycopersicum L.) is the fourth most popular fresh-market fruit as an excellent source of vitamin C, lycopene, and other antioxidants (Wang et al., 2019), whereas most commercial tomato cultivars are particularly susceptible to B. cinerea (Smith et al., 2014).

Plants are endowed with sophisticated defense responses after recognition of invading pathogens, such as cell wall modifications, production of reactive oxygen species (ROS), and accumulation of various secondary metabolites and phytohormones, including salicylic acid (SA), jasmonate (JA), and ethylene (ET) (Mulema and Denby, 2012). Generally, SA signaling pathway is crucial in defense against biotrophic and hemi-biotrophic pathogens (Shah, 2003), whereas JA/ET signaling pathway is essential for immune responses against herbivores, insects, and necrotrophic pathogens, including B. cinerea (Backer et al., 2019). Growing evidences have documented that phytohormones SA, JA, ET, and auxin, either alone or in different combinations, serve as the critical regulators in activating plant defense against pathogens (Robert-Seilaniantz et al., 2011; Wang et al., 2013). Moreover, several previous studies have investigated the interactions between B. cinerea and Arabidopsis thaliana (Mulema and Denby, 2012), lettuce (De Cremer et al., 2013), cucumber (Kong et al., 2015) or tomato (Rezzonico et al., 2017; Smith et al., 2014; Vega et al., 2015). Many defense genes induced by B. cinerea are involved in production of pathogenesis-related proteins (PRs), ROS homeostasis, hormone biosynthesis and signal transduction, and induction of phenylpropanoid metabolism (Kong et al., 2015; Naoumkina et al., 2010); the latter activates the biosynthesis of secondary metabolites with antimicrobial qualities (De Cremer et al., 2013).

Nonexpressor of pathogenesis-related gene 1 (NPR1) serves as a SA receptor and master component of plant defense mechanisms (Dong, 2004; Li et al., 2019a; Zhang and Li, 2019). NPR1 plays a positive role in SA-mediated defense against biotrophs, and it also affects JA- and ET- mediated defense against necrotrophs (Backer et al., 2019; Spoel et al., 2003). Overexpression of AtNPR1 in crops such as wheat (Gao et al., 2013), soybean (Matthews et al., 2014), and cotton (Parkhi et al., 2010) improves certain disease resistance and even crop yield without any negative phenotypic effects. However, El Oirdi et al. (2011) have reported that overexpression of AtNPR1 decreased the resistance against B. cinerea in tomato plants, which draws extensive attention on the role of SlNPR1 in B. cinerea resistance of tomato fruit. Furthermore, previous studies have reported that B. cinerea fails to develop in unripe tomato fruit. However, it induces fruit rapid breakdown once fruit start the ripening program and become ripe (Cantu et al., 2009; Petrasch et al., 2019b). Ripening involves a series of significant shifts in the physiological and biochemical properties of the fruit, such as modification of cell wall structure and composition, conversion of starch into sugars, and inefficiency of antimicrobial metabolites (Carrari and Fernie, 2006; Li et al., 2019b). All these changes lead to the unique status of energy metabolism, which plays a critical role in disease resistance of postharvest horticultural produce (Zhang et al., 2017). Additionally, tomato has emerged as a model organism to study the molecular basis of postharvest fruit defense against necrotrophic fungi, in particular to B. cinerea (Cantu et al., 2008; Flors et al., 2007). However, our understanding of the interactions between tomato fruit and B. cinerea and how SlNPR1 influences the defense mechanisms against B. cinerea are still limited.

In this study, two homozygous slnpr1 mutants were used to investigate the role of SlNPR1 in the interaction between B. cinerea and tomato fruit. Disease resistance of slnpr1 and wild type (WT) fruit were evaluated by disease symptoms, activities of defense enzymes, ROS accumulation, and expression of defense genes after inoculation. Based on the transcriptome changes, many defense genes responding to B. cinerea infection were identified. Moreover, multiple metabolic pathways including biosynthesis of secondary metabolites, represented by phenylpropanoid biosynthesis, were significantly induced in slnpr1 fruit compared to WT. Additionally, a cluster of differential expression genes (DEGs) between slnpr1 and WT fruit inoculated with B. cinerea were found to be involved in several other disease signaling pathways. These results greatly enrich our understanding of the regulatory mechanisms underlying tomato fruit defense against B. cinerea, especially the role of SlNPR1 in this process.