Inspection of Tuta absoluta infestation
At all sampling times the parameters scored showed highly significant differences between the T and S lines (P < 0.0001, t-test, Table 1). At 10 days after adult release (10 DAAR), the difference in number of eggs/plant laid on the two tomato lines was highly significant (t=-6.16; n=20; P < 0.001) with the average value registered on T genotype about 4 times lower than on S (Table 1). At 20 DAAR, the highly significant difference in the average number of mines/plant recorded in T and S remained in the same range (t=-5.05; n=20; P < 0.001). The plant damage assessed 20 days later (i.e. 40 DAAR) followed the same trend of significant differences between the two genotypes, both in terms of number and type of mines. In particular, an average of 0.2 mines/cm2 and 0.71 mines/cm2 of leaflets were counted on T and S, respectively (t=-9.26; n=20; P < 0.001). Moreover, while in the T genotype the large majority of mines were single and non-coalescent, for the S genotype this type of mine was recorded only on 52.8% of analyzed samples (t=-7.94; n=20; P < 0.001). The difference in the level of fruit infestation per plant was even more pronounced, with a 6.5% for T genotype and 33% for S genotype (t=-7.75; n=20; P < 0.001).
Leaf trichome analysis
The density of glandular and non-glandular trichomes on leaves of T and S lines showed significant differences (Fig. 1a,b; Table 2). In particular, T had a remarkable higher density of glandular trichomes types IV (adaxial) (t=-8.33; P < 0.001) and VI (adaxial and abaxial) (t=-6.45; P < 0.001 and t=-13.42; P < 0.001), as well as non-glandular trichomes types II (adaxial) (t=-6.96; P < 0.001) and V (adaxial and abaxial) (t=-6.60; P < 0.001 and t=-42.78; P < 0.001). On the contrary density of type VII glandular trichomes was significantly higher in S (t=3.43; P < 0.001).
Tomato transcriptional response
The changes of gene expression in tomato leaves as affected by T. absoluta feeding on T and S genotypes were determined. RNA-sequencing produced an average of 24 million reads per sample (Additional File 1: Table S1). Approximatively 19,000 and 21,500 transcripts, for T and S respectively (Additional File 1: Fig. S1a,b), were mapped to the tomato reference genome (SL.2.50) and used for further analysis. Differentially expressed genes (DEGs) were computed for each genotype (T and S) comparing T infested vs T non-infested (Ti vs Tni) and S infested vs S non-infested (Si vs Sni) conditions. The comparison Ti vs Tni showed a marked gene expression change in T genotype after T. absoluta challenge (11486 DEGs, Fig. 2a, Additional file 2: Dataset S1-S2), while for the S genotype (Si vs Sni) a total of 6,793 DEGs were obtained (Fig. 2a, Additional file 2: Dataset S3-S4). Out of the total DEGs, 2,845, and 696 genes resulted specifically up-regulated in T and S, respectively, while 3074 in T genotype and 530 in S genotype were specifically down-regulated (Fig. 2b). More than 5,000 genes (3,039 up-regulated and 2,431 down-regulated) resulted differential expressed in both genotypes.
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).
Plant development and leaf structures morphogenesis and differentiation
The T genotype showed an enrichment of up-regulated genes involved in leaf development, structural meristem formation and photosynthesis (Fig. 2c). The GO term “photosynthesis, light harvesting in photosystem I”, included many Chlorophyll a-b binding proteins. The GO terms “leaf development” and “leaf morphogenesis” enclosed an abundance of receptor like kinases (RLKs) such as Solyc08g061560, encoding a putative orthologue of ERECTA, Solyc08g014030, a gene closely related to the Arabidopsis gene encoding SHORTROOT (SHR) , which, together with a GRAS-domain TF, SCARECROW (SCR), regulates the duration of cell proliferation in leaves , the Solyc03g112750 protein tornado-TRN1 and the Solyc04g081590 CLAVATA 1, involved in meristem organization. Transcription factors (TFs) involved in meristem formation and trichome differentiation, exclusively present in T, including PHANTASTICA (Solyc09g010840) belonging to MYB family, Solyc02g092370 (SlSHRa) belonging to GRAS family, Solyc08g066500 (SlHB8) an HD-ZIP III transcription factor, and a subunit of the CAF (Solyc11g008670, FAS1) were also identified (Table 3). An enrichment of genes involved in cell wall synthesis, degradation and assembly as well in cell wall structural proteins was observed in T (Fig. 3a,b), including glycosyl transferases (GTs) families involved in cellulose (GT2), hemicelluloses (GT47), pectin (GT8) and xylan (GT43) synthesis (Fig. 3c) as well as glycoside hydrolases (GHs) belonging to GH9, GH5 and GH31 families, lyases (PL1), esterases (CE1) and expansins (Fig. 3c). Moreover, a higher number of cell wall associated genes, such as Fasciclin-like arabinogalactan (FLA) proteins (13 up-regulated in T and 5 up-regulated in S), involved in cell wall modification and assembly, and Trichome Birefringence-Like (TBL) genes (21 up-regulated in T and 9 up regulated in S) were also detected in the T genotype (Fig. 3c).
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.
Perception of damage, signal transduction and defense response
A plethora of up-regulated RLKs resulted enriched in T during T. absoluta challenge (Fig. 4a,b). Most of them belonged to families involved in defense response and growth. In particular, SERK1 Solyc04g072570 (LRR II), several NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE NIK members and LRR-XIII members (including Solyc03g007050-ERECTA-like 1 and Solyc08g061560-ERECTA) as well as specific extensin proteins were identified.
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.
Defensive secondary metabolites synthesis
The sterol metabolism resulted strongly activated in the T genotype, as indicated by the up-regulation of a sterol side chain reductase enzyme (SSR2, Solyc02g069490), catalyzing the conversion of cycloartenol (the precursor for phytosterols) to cycloartanol, the first committed step in cholesterogenesis, that, in contrast, in S was down-regulated (Fig. 5a). In addition, the core steps of sterol biosynthesis, starting from cycloartenol, resulted clearly activated in T, as shown by the specific up-regulation of Obtusifoliol 14-alpha demethylase (O14DM), a delta14-sterol reductase (D14SR) as well as of three Sterol 4-alpha-methyl-oxidases (SMO, Solyc01g091320, Solyc06g005750 and Solyc08g079570) and an Acid phosphatase-like (Solyc04g072190). Interestingly, among the SMO, the gene Solyc06g005750 was up-regulated in T and down-regulated in S (Fig. 5a). The sterol transport seemed to be activated in the T genotype, as indicated by up-regulation of two ABC(G) transporters, Solyc12g100180 and Solyc12g100190. The production of sterol glycoalkaloids (SGA) in the T genotype was promoted by up-regulation of genes involved in glycoalkaloids biosynthesis (GAME), clustering on chromosome 7 (Fig. 5b).
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).
The transcriptional regulation in response to T. absoluta attack mediated by TFs in T and S was examined in-depth, since T genotype showed a very high number of specifically differentially expressed TF, including 145 up-regulated and 192 down-regulated. Among the TF with a contrasting expression, five were up-regulated in T and down-regulated in S. On the contrary three were up-regulated in S and down-regulated in T (Fig. 6a). The classification of specific TF showed that they were distributed among different classes with many copies belonging to bHLH and MYB family (Fig. 6b).
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.