The leaves of the wild tomato Solanum galapagense harbor type-IV glandular trichomes (GT) that produce high levels of acylsugars (AS), conferring insect resistance. Conversely, domesticated tomatoes (S. lycopersicum) lack type-IV trichomes on the leaves of mature plants, preventing high AS production, thus rendering the plants more vulnerable to insect predation. We hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of adult plants could be insect-resistant. We introgressed the genetic determinants controlling type-IV trichome development from S. galapagense into cv. Micro-Tom (MT) and created a line named “Galapagos-enhanced trichomes” (MT-Get). Mapping-by-sequencing revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further genetic mapping showed that S. galapagense alleles in chromosomes 1, 2, and 3 were sufficient for the presence of type-IV trichomes on adult organs but at lower densities. Metabolic and gene expression analyses demonstrated that type-IV trichome density was not accompanied by the AS production and exudation in MT-Get. Although the plants produce a significant amount of acylsugars, those are still not enough to make them resistant to whiteflies. We demonstrate that type-IV glandular trichome development is insufficient for high AS accumulation. The results from our study provided additional insights into the steps necessary for breeding an insect-resistant tomato.

Main conclusion Cultivated tomatoes harboring the plastid-derived sesquiterpenes from S. habrochaites have altered type-VI trichome morphology and unveil additional genetic components necessary for piercing-sucking pest resistance. Abstract Arthropod resistance in the tomato wild relative Solanum habrochaites LA1777 is linked to specific sesquiterpene biosynthesis. The Sesquiterpene synthase 2 (SsT2) gene cluster on LA1777 chromosome 8 controls plastid-derived sesquiterpene synthesis. The main genes at SsT2 are Z-prenyltransferase (zFPS) and Santalene and Bergamotene Synthase (SBS), which produce α-santalene, β-bergamotene, and α-bergamotene in LA1777 round-shaped type-VI glandular trichomes. Cultivated tomatoes have mushroom-shaped type-VI trichomes with much smaller glands that contain low levels of monoterpenes and cytosolic-derived sesquiterpenes, not presenting the same pest resistance as in LA1777. We successfully transferred zFPS and SBS from LA1777 to cultivated tomato (cv. Micro-Tom, MT) by a backcrossing approach. The trichomes of the MT-Sst2 introgressed line produced high levels of the plastid-derived sesquiterpenes. The type-VI trichome internal storage-cavity size increased in MT-Sst2, probably as an effect of the increased amount of sesquiterpenes, although it was not enough to mimic the round-shaped LA1777 trichomes. The presence of high amounts of plastid-derived sesquiterpenes was also not sufficient to confer resistance to various tomato piercing-sucking pests, indicating that the effect of the sesquiterpenes found in the wild S. habrochaites can be insect specific. Our results provide for a better understanding of the morphology of S. habrochaites type-VI trichomes and paves the way to obtain insect-resistant tomatoes.

Glandular trichomes are involved in the production of food- and medicine-relevant chemicals in plants, besides being associated with pest resistance. In some wild Solanum species closely related to the cultivated tomato (S. lycopersicum), the presence of type-IV glandular trichomes leads to the production of high levels of insecticide acylsugars (AS). Conversely, low AS production observed in the cultivated tomato is attributed to its incapacity to develop type-IV trichomes in adult organs. Therefore, we hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of mature plants can be pest resistant. We introgressed into the tomato cultivar Micro-Tom (MT) the capability of S. galapagense to maintain the development of type-IV trichomes throughout all plant stages, thus creating a line named "Galapagos enhanced trichomes" (MT-Get). Mapping-by-sequencing of MT-Get revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further mapping reveled that S. galapagense alleles on chromosomes 1, 2 and 3 are sufficient for the presence of type-IV trichomes, but in lower densities. GC-MS, LC-MS, and gene expression analyses demonstrated that the increased density of type-IV trichomes was accompanied by high AS production and exudation in MT-Get. Moreover, MT-Get did not differ from MT in its susceptibility to whitefly (Bemisia tabaci). Our findings demonstrates that type-IV glandular trichome development along with AS production and exudation are partially uncoupled at the genetic level. The MT-Get genotype represents a valuable resource for further studies involving the biochemical manipulation of type-IV trichome content through either genetic introgression or transgenic approaches.

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Type-VI glandular trichomes of wild tomato Solanum habrochaites PI127826 produce high levels of the sesquiterpene 7-epizingiberene and its derivatives, making the plant repellent and toxic to several pest insects and pathogens. How wild tomato trichomes achieve such high terpene production is still largely unknown. Here we show that a cross (F1) with a cultivated tomato produced only minute levels of 7-epizingiberene. In the F2-progeny, selected for the presence of the 7-epizingiberene biosynthesis genes, only three percent produced comparable amounts the wild parent, indicating this trait is recessive and multigenic. Moreover, trichome density alone did not explain the total levels of terpene levels found on the leaves. We selected F2 plants with the “high-production active-trichome phenotype” of PI127826, having trichomes producing about 150 times higher levels of terpenes than F2 individuals that displayed a “low-production lazy-trichome phenotype”. Terpene quantities in trichomes of these F2 plants correlated with the volume of the storage cavity and shape of the gland. We found that trichome morphology is not a predetermined characteristic, but cavity volume rather depended on gland-cell metabolic activity. Inhibitor assays showed that the plastidial-precursor pathway (MEP) is fundamental for high-level production of both cytosolic as well as plastid-derived terpenes in tomato trichomes. Additionally, gene expression profiles of isolated secretory cells showed that key enzymes in the MEP pathway were higher expressed in active trichomes. We conclude that the MEP pathway is the primary precursor-supply route in wild tomato type-VI trichomes and that the high-production phenotype of the wild tomato trichome is indeed a multigenic trait.

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To deal with arthropod pests the tomato wild relatives produce a variety of defense compounds in their glandular trichomes. In Solanum habrochaites LA1777, a functional cluster of genes on chromosome 8 controls plastid-derived sesquiterpene synthesis not found in cultivated tomatoes. The main genes at the cluster are Z-prenyltransferase (zFPS) that produces Z-Z-farnesyl diphosphate (Z,Z-FPP), and Santalene and Bergamotene Synthase (SBS) that uses Z,Z-FPP to produce α-santalene, β-bergamotene, and α-bergamotene in type-VI glandular trichomes. Both LA1777 and cultivated tomatoes have type-VI trichomes, but the gland in cultivated tomato is much smaller containing low levels of monoterpenes and cytosolic-derived sesquiterpenes, which do not provide tomato with the same pest resistance as in LA1777. We successfully transferred the plastid-derived sesquiterpene pathway from LA1777 to type-VI trichomes of a cultivated tomato (cv. Micro-Tom, MT) by a back-crossing approach. The trichomes of the introgressed line named MT-Sesquiterpene synthase 2 (MT-Sst2) produced even higher levels of α-santalene, β-bergamotene, and α-bergamotene than the type-VI glandular trichomes of LA1777. We also noticed that the type-VI trichome internal storage-cavity size increases in MT-Sst2, probably as an “inflated balloon” effect of the increased amount of sesquiterpenes. Surprisingly, the presence of high amounts of plastid-derived sesquiterpenes was not sufficient to confer resistance to various tomato pests in MT-Sst2. Since MT-Sst2 made the same sesquiterpenes as LA1777, this points to additional factors, outside the genomic region thought to be the metabolic cluster, necessary to obtain arthropod-resistant tomatoes. Our results also provide a better understanding of the morphology of S. habrochaites type-VI trichomes.One-sentence summaryCultivated tomatoes harboring the plastid-derived sesquiterpenes from S. habrochaites need additional genetic components necessary to convert them into effective insecticides.