Tomato transcriptomic response to Tuta absoluta infestation

A cherry type tomato (Solanum Lycopersicum) line putatively tolerant/partially resistant to T. absoluta, BR221 (from now on named as ‘T’) and a susceptible one, PS650 (from now on named as ‘S’), kindly provided by FARAOO S.r.l., were used in this work. The FARAOO S.r.l., company also provided the facilities to conduct experiments according to Italian legislation and ensured the formal identification of the samples.

An infestation experiment with T. absoluta adults was set up in a flying nylon tunnel (90 cm × 500 cm × 90 cm) located in a glasshouse. Environmental conditions recorded in the glasshouse in which the tunnel was located were, RH = 60–70% and T° = 25 ± 2 °C. The tunnel was divided in two sections by a plastic mesh screen. For both tomato genotypes, 20 plants, 4 weeks old were transplanted in 14 cm diameter pots filled with mixed potting soil and placed in each half-tunnel, using a randomized complete block design. The plants of one half-tunnel were exposed to 100 females and 100 males of T. absoluta, obtained from pupae reared on the tomato cultivar San Marzano nano, while plants of the second half-tunnel, not exposed to T. absoluta, served as control. Plants were inspected for egg laying and infestation level at 10–12 day interval up to 40 days after adult release, when the overall plant damage was assessed (type of leaflet damage, the number of injuries/mines/cm² of leaflets, the percentage of infested fruits), using the scoring method proposed by Resende et al. [17]. On that occasion, leaves with and without mines from each plant were singly collected and immediately frozen in liquid nitrogen for subsequent molecular analyses. The data obtained were processed using the software Statgraphics PlusR (Statgraphics Technologies Inc., The Plains, VA, USA). Data satisfying conditions of normality and homoscedasticity, or after appropriate transformation, if needed, were analyzed by a two-tailed Student’s test and the means were separated at the 0.001 level of significance.

Leaves were randomly harvested from T and S control tomato plants. Five fully expanded leaves were selected from each plant and five measurements were made on each sample. The different types of trichomes present on the two tomato genotypes, respectively belonging to classes II-VII, were identified and counted according to Channarayappa et al. [82]. Observations were made with a metallurgical microscope (Leitz Wetzlar Ortholux Microscope) equipped with an Ultropak® objective 6.5x, with a visual field area of 3.1 mm². The data obtained were analyzed by the Student’s t-test (P < 0.001).

The perturbations of tomato pathways induced by T. absoluta feeding were depicted by integrating Gene Ontology enrichment analysis and DEGs metabolic mapping. Key metabolic pathways, modulated during the herbivore-plant interaction, in the two tomato genotypes (T and S), in infested vs non-infested conditions, were mainly related to cell wall, sterol and steroid metabolism, leaf development and photosynthesis (Fig. 2c).

The activation of apoplast plant laccase, three dirigent-like proteins (DIR) (Solyc07G042300, Solyc10G055200, Solyc10G055230), two Pinoresinol-lariciresinol reductases, Solyc03g044720 (log₂FC = 0.64) and Solyc06g066160 (log₂FC = 2.17), and one cell wall peroxidase (TPX1; Solyc07g052510, log₂FC = 3,19) suggested that the lignin/lignan synthesis was promoted in T. In addition, cutin synthesis in T genotype resulted enhanced by extensive induction of glycerol-3-phosphate acyltransferases (GPAT4, GPAT6), ω-hydroxylases, such as HOTHEAD oxidase (HTH), HTH-like, or cytochrome (Fig. 3d) and cutin monomers transport by ATP-binding cassette (ABC) transporter Solyc05g018510, orthologous to Arabidopsis AtABCG32.

Divergences in the signal transduction between genotypes were also detected in the activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs) (Fig. 4c,d). In T was observed the up-regulation of SlMPK3, well known to be induced by wounding, and SlMAPK5, homologous to A. thaliana MPK4 (AT4G01370.1), a positive regulator of JA mediated signaling (Fig. 4c). Interestingly, in T also the up-regulation of SlCDPK18 (equivalent to LeCDPK1) and SlCDPK4/LeCDPK2 (Fig. 4d) was noted.

A commonly used marker for the wounding/insect response, a threonine deaminase (TD, Solyc09g008670, log₂FC = 1,73), was exclusively produced in T, as well as the polyphenol oxidase (PPO) Solyc08g074630-PPO-F (log₂FC = 5,04), while Solyc08g074680-PPO-B was expressed three times more in T (log₂FC = 8,52) than in S (log₂FC = 3,32). T genotype showed also a strong up-regulation of genes encoding defense proteins related to protease inhibitors (PIs), well known to be induced by jasmonic acid. Up to 17 PIs were activated, counting a relevant number of potato inhibitors I (PI-1) such as Solyc09g084470 (log₂FC = 2.10), late wound-response genes induced by JA, carboxipeptidase, three metallocarboxypeptidase inhibitors (Solyc07g007260, Solyc07g007250, Solyc06g061230-putative) and two defensin proteins (Solyc07g007760, Solyc07g007750). Although Solyc07g007760 was up-regulated both in S and T, another defensin Solyc07g007710 was down-regulated (log₂FC = -2.61) in S.

DEGs were identified in the pathway of glucosinolate biosynthesis in both genotypes, while a desulfoglucosinolate sulfotransferase, Solyc03g114800, was exclusively up-regulated in T. Furthermore, two 3-isopropylmalate dehydratases (Solyc03g005730 and Solyc09g090900), involved not only in leucine synthesis but also in the production of the glucosinolate methylthioalkylmalate, resulted up-regulated only in T.

Transcriptional changes in the terpenoid biosynthetic pathway of T plants were relatively higher, both in terms of DEGs number and of their log₂FCs. Interestingly, a cluster of genes located on chromosome 8, including the terpene synthases (TPS) TPS20, TPS21 and TPS18, a Dimethylallylcistransferase CPT1/NDPS1 and the cytochrome P450-oxidoreductase CYP71BN1, were specifically up-regulated in T. In addition, in T the up-regulation of TPS5 was observed, while in S it was observed for TPS46. Both genotypes up-regulated TPS7, but with a higher induction in T (Fig. 5c).

The biosynthesis of volatile benzenoid esters, mediated by two benzoyl transferases (SlAAT3, Solyc07g049660 and SlAAT5, Solyc05g015800) producing benzylbenzoate, resulted exclusively activated in T (log₂FC = 2,51 and log₂FC = 2,48). Synthesis of the toxic compound quinone 1,4-dihydroxy-2-naphthoate was promoted in T, as indicated by the up-regulation of all genes involved in the biosynthetic pathway, while only one gene was activated in S. T also showed a higher induction of genes required for the synthesis of hydroxycinnamic acid tyramine amides (Fig. 5d) and of genes involved in the production of a modified fatty acid, clustering on chromosome 12 (Fig. 5e).

Transcription factors involved in the regulation of secondary metabolites production (i.e. terpenes), trichome formation and jasmonic acid signaling, and cutin/wax metabolism were identified (Table 3). T specifically up-regulated two transcription activators, SlMYC1 and EXPRESSION OF TERPENOIDS 1 (EOT1), controlling the terpene biosynthesis in glandular trichomes and the development of type VI glandular trichome. GLABRA2 (GL2, Solyc03g120620), implicated in cell differentiation of various epidermal components, including trichomes, was also up-regulated in T and down-regulated in S. By contrast, TRIPTYCHON (SlTRY, Solyc01g095640), a negative regulator of trichome formation, was up-regulated in S while the bHLH TF (SlbHLH150, Solyc09g065100), important for marginal trichome development, was exclusively induced in T.

Jasmonic acid 1, involved in the activation of threonine deaminase (TD), was up-regulated in T (Solyc05g007180, log₂FC = 2,32). By contrast, MTB1 (bHLH113, Solyc01g096050), that is negative regulator of JA signaling, was down-regulated in T. MYB30, a key regulator of both the protective hypersensitive response (HR) and wax biosynthesis, was up-regulated in T whilst MYB41, that mediates the negative regulation of cutin biosynthesis in response to stress, was repressed in T and induced in S. Furthermore, the negative regulator of cuticle development CFL1 (Solyc01g009770, log₂FC = -1.01) resulted down-regulated in T.

The first line of defense against herbivores and pathogens is often provided by physical barriers, such as trichome density and quality, cuticle tickness, and cell wall strength [32]. A higher density of glandular trichomes, (type VI and type IV) as well as non glandular trichomes (type II and V), trait of resistance to herbivory [33, 34], was observed in T. It is conceivable that difference in frequency and quality of trichomes on the surface of S compared to T could affect the T. absoluta feeding preference. Glandular trichomes secrete flavonoids, poisonous terpenoids and alkaloids [32, 35] that act as deterrent for oviposition and feeding of arthropod pests [36], and serve as mechanoreceptors of herbivores [37]. In particular, type VI trichomes accumulate JA, an important sensor for detecting insect movement on the leaf surface [37].

In T genotype, T. absoluta attack could trigger the production of trichomes on newly emerging leaves due to the up-regulation of the gene SlMYC1 involved in type VI trichome formation and in the synthesis of mono and sesquiterpenes [38]. Indeed, SlMYC1 knocking down induced the production of smaller type VI glandular trichomes at lower densities and its knocking out led to their absence [39]. In addition, SlbHLH150 (Solyc09g065100), a transcription factor belonging to helix-loop-helix gene family [40], and GLABRA2 (GL2), encoding a homeobox leucine zipper protein, promoting leaf development and trichome formation [41–43] were also up-regulated in the T genotype. By contrast, the up-regulation in S of Solyc01g095640, a negative of regulator of trichome formation suggested that trichome morphogenesis process was reduced.

We hypothesize that the cuticle structure of T genotypes could be reinforced in response to T. absoluta attack thanks to the up-regulation of genes involved in cutin monomers formation and assembly, such as HOTHEAD (HTH), Glc-methanol-choline oxidoreductase family proteins [44] and MYB30, a crucial regulator of both wax biosynthesis and Systemic Acquired Resistance (SAR) [45]. Instead, a negative regulation of cutin biosynthesis was observed in S as indicated by the up-regulation of the genes MYB41 and CFL1 [46, 47]. T cell wall remodeling promoted reinforcement and the maintainance of cell function thanks to the induction of glycosyl transferases (GTs), glycoside hydrolases (GHs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs). Acetylation of cell wall polymers enhanced the interaction of the hemicelluloses with other wall polymers [48], to obtain a more rigid wall conformation [49]. Moreover, the activation of a higher number of fasciclin like arabinogalactan proteins, involved in cell–cell communication during cell wall remodeling, supported the idea that changes in cell wall conformation occurred. The dirigent-like (DIR) proteins activated in T may be involved both in dynamic reorganization of the cell wall and in the production of defense compounds such as lignin and lignans [50], disruptors of insect endocrine system [51]. DIR-like protein-encoding genes during the interaction of moss Physcomitrella patens with Colletotrichum gleosporioides or Pectobacterium carotovorum promoted cell wall modifications, including increased incorporation of phenolic compounds [52]. In addition, the significant overexpression of the peroxidase gene TPX1 suggested that it served as storage pool of monolignols for lignification for enhancing the rigidity and decreasing the digestibility of the cell wall [53]. The accumulation of hydroxycinnamic acid tyramine amides as well as of phenolic compounds (E-feruloyltyramine and E-p-coumaroyltyramine) in T genotype may limit the T. absoluta infestation as well as the digestibility of the cell wall [54, 55].

The extensive reshaping of gene expression in T genotype induced by T. absoluta feeding activated a signaling cascade that culminated into a complex re-arrangement of primary and secondary metabolism leading to a systemic plant defense response. The cell surface perception resulted enhanced in T by the induction of cell surface receptor-kinase ERECTA (ER, Solyc03g007050), BAK1 and SERK1, involved in plant growth, developmental and immune processes regulation [56]. Members of the ER pathway interact with BAK1 in the regulation of innate immune response, plant growth and cell wall composition [57–60]. A prompter signaling in T could be mediated by the up-regulation of the calmodulin and calcium-sensing genes, involved in the activation of calcium cascade, and of MAPKs and CDPKs, such as SlMPK3 involved in systemin-mediated defense response against insect attack [61], and SlCDPK18 that modulates the wounding signaling [62]. These signal transduction pathways lead to the activation of well-characterized proteins involved in ROS detoxification such as PPO (Solyc08g074630-Solyc08g074680), proteinase and metallocarboxypeptidase inhibitors (Solyc09g084470, Solyc07g007260, Solyc07g007250, Solyc06g061230 and defensin proteins (Solyc07g007760, Solyc07g007750) that prevent pest damage [63]. Defense signaling acted synergistically to activate a finely tuned response to increase tolerance to T. absoluta without compromise plant fitness.

Inducible defenses against herbivores include the synthesis of a wide range of species-specific toxic plant secondary metabolites (e.g., phenylpropanoids, flavonoids, anthocyanins, alkaloids, terpenoids, glucosinolates), and anti-nutritive enzymes and proteins (e.g., proteinase inhibitors (PIs), amino acid catabolizing enzymes, polyphenol oxidases, and peroxidases), mostly controlled by jasmonic acid [64–66]. T showed a much more pronounced activation of the gene arsenal involved in primary metabolism with defensive functions against insects [67], characterized by the presence of a higher number of up-regulated genes or of genes with a higher FC, such as the JA-induced defenses markers, including antinutritive and enzyme proteins TD, LAP, PPO and PI and defensins.

The divergent synthesis and composition in alkaloids, terpenes and glucosinolates between the two lines underlined the significant difference found in trichome density and type [68]. T genotype could accumulate steroidal glycoalkaloids (SGAs), as emerged from the activation of genes involved in GLYCOALKALOID METABOLISM (GAME). In particular, GAME25 gene promoted higher levels of α-tomatine and acetoxytomatine that act synergistically against pathogens to exert cytotoxic and anti-nutritional function [69]. Moreover, genes involved in cholesterol production, that serves as the precursor for the biosynthesis of SGAs as well as of two orthologs of Petunia hybrida PDR2, involved in the control of steroidal compounds accumulation in leaves and trichomes [70], were highly induced in T. This is consistent with the fact that tomato resistance to phytophagus Spodoptera litura caterpillars was weakened by reduced SGA accumulation [71].

T. absoluta feeding could increase the production of some monoterpenes and other minor products in the T genotype [72] thanks to the activation of terpene synthases including the trichome-localized TPS5/MTS1, that preferentially produces linalool [73], TPS21 that, together with CYP71BN1, leads to the formation of lycosantanolol [74], and with CPT1/NDPS1 is involved in phellandrene synthesis [75]. By contrast, the up-regulation of TPS46/GLS in S lead to the production of the diterpene geranyllinalool [76]. Finally, although both genotypes up-regulated TPS7, the stronger induction in T suggests a higher production of pinene, myrcene, sabinene and linalool [77]. A different level of glucosinolate biosynthesis in T was supposed by the up-regulation of a desulfoglucosinolate sulfotransferase (Solyc03g114800) promoting the accumulation of 3-methylthiopropyl-glucosinolate at expense of other types of glucosinolates. In addition, the formation of benzylbenzoate, a major volatile compound with ovicidal properties produced after leaf disruption and wounding [78] was exclusively promoted in T by up-regulation of two benzoyl transferases (Solyc07g049660; Solyc05g015800). T genotype also showed a higher number of up-regulated genes involved in the production of 1,4-dihydroxy-2-naphthoate (DHNA), toxic precursor toward insects [79]. A major involvement of up-regulated clustered genes, required for the production of the highly functionalized oxylipin falcarindiol [80] in T leaves, was also observed. Genes involved in jasmonic acid metabolism and signaling, in the production of terpenes, glycoalkaloids and several genes in the steroid pathway as well as Sl SSR2 (Solyc02g069490), a key enzyme in the biosynthesis of toxic SGAs derived from cholesterol were up-regulated after exposure of tomato plants to volatiles resulting in enhanced resistance to Tuta absoluta in commercial greenhouses [10].

Additional file 1:Table S1. shows information about the raw reads quality control. Figure S1. shows the sequencing data filtering. Table S2. reports a list of genes with the largest differences in fold changes between the two genotypes.

Additional file 2:Dataset S1-S2. list the up- and down-regulated genes detected in the comparison Ti vs Tni. Dataset S3-S4. list the up- and down-regulated genes detected in the comparison Si vs Sni.