Molecular architecture determines tomato plant resistance to its main pest

An international study investigating why wild tomatoes are more resistant to the tomato leafminer (Phthorimaea absoluta) than cultivated varieties revealed that the secret lies in the spatial structure of a plant defense molecule. The researchers mapped the biochemical origin of this difference and demonstrated that altering four amino acids in a single enzyme is sufficient to alter the structure of the substance and increase its toxicity against the pest. This finding paves the way for creating resistant tomatoes through gene editing.

The research, supported by FAPESP and published in the journal Nature Communications, was coordinated by Jiancai Li, a professor at the Shanghai Institute of Plant Physiology and Ecology and at the University of the Chinese Academy of Sciences in Beijing. José Maurício Simões Bento, a professor at the Luiz de Queiroz College of Agriculture at the University of São Paulo (ESALQ-USP), also participated in the research.

Li’s team works on genetics, biochemistry, and the biosynthesis of specialized plant metabolites, while Bento’s group focuses on chemical ecology and plant-insect interactions. “Integrating these fields was crucial. Once the genetic and biochemical mechanisms responsible for the different molecular forms were identified, it became crucial to grasp the biological ramifications of those differences and evaluate whether a slight alteration in the three-dimensional structure of a molecule could impact a plant’s capacity to defend itself against a pest,” says Bento.

The collaboration between the Chinese and Brazilian groups originated from a call for proposals issued in 2024 by FAPESP and the National Natural Science Foundation of China (NSFC), following FAPESP Week China.

The study analyzed steroidal glycoalkaloids (SGAs), natural substances produced by certain plants, such as tomatoes, potatoes, and eggplants, of the genus Solanum. These substances play a role in the plants’ chemical defense against herbivorous insects and pathogens.

One of the main SGAs present in the leaves of cultivated tomatoes (Solanum lycopersicum) is α-tomatine. However, in the leaves of wild tomatoes (Solanum pennellii), filotomatine predominates. Both substances have the same sugar content, but the three-dimensional structure of their molecular cores is different.

This is where chirality comes into play. A molecule is called chiral when its structure cannot be perfectly superimposed onto its mirror image. The classic example is the right and left hands: they have the same parts arranged in a mirror-image fashion, yet no translation or rotation allows one to fit perfectly over the other. Similarly, a chiral molecule can exist in two forms, such as “right” and “left” versions of the same structure. These two forms constitute a pair of enantiomers, or chiral molecules in mirror-image forms.

“Life itself is a highly chiral environment. Amino acids and sugars, for example, predominantly exist in one of their enantiomeric forms. Therefore, when faced with a specific biological target, only one configuration can properly fit into the cavity or groove of a protein while the other can’t,” Li explains.

This general principle was demonstrated in experiments with insects. Filotomatine, predominant in wild tomato leaves, provided greater protection against the tomato leafminer than α-tomatine, predominant in cultivated varieties. This result was observed in both plant experiments and bioassays using an artificial diet. In the latter, the insects were fed a laboratory-prepared diet to which the compounds to be tested were added separately. This setup allowed for a direct comparison of the effects of the compounds on the insects without interference from other plant characteristics.

“One of the most interesting aspects of the study is that it shows that in plant defense, it’s not only the presence or quantity of a substance that matters but also how its molecules are organized in space,” says Bento. “α-tomatine and filotomatine are extremely similar molecules, but they differ in their three-dimensional configuration. The study showed that that difference is biologically relevant.”

According to the ESALQ researcher, the results demonstrate just how sophisticated plants’ chemical defenses can be. “The shape of the molecule can facilitate or hinder its interaction with specific structures within the insect’s body.”

The exact molecular target of these glycoalkaloids in insects is not yet known. However, Li believes that stereospecific recognition – the ability of a biological target to distinguish between different spatial configurations – is likely behind the observed difference.

“This phenomenon has received considerable attention in the development of drugs derived from natural plant products. However, the ecological forces that shape the chirality of metabolites in nature are still poorly understood. With this study, we hope to draw more attention to the ecological functions of chirality in plant-derived natural products and their potential applications in plant protection,” the Chinese researcher emphasizes.

Four amino acids make the difference

To discover the source of the difference between the two types of tomatoes, the researchers investigated the GAME8 gene, which encodes an enzyme involved in the biosynthesis of these glycoalkaloids.

Wild tomatoes have two very similar GAME8 genes, GAME8a and GAME8b. The enzyme produced by GAME8a predominantly directs the formation of the configuration associated with filotomatine. In contrast, the enzyme produced by GAME8b favors the configuration associated with α-tomatine. In cultivated tomatoes, the two GAME8 genes have identical coding sequences, and their enzymes predominantly favor the configuration associated with α-tomatine.

It is at this point that the genetic difference becomes a chemical difference. These enzymes act on a glycoalkaloid precursor molecule and determine which spatial configuration will be formed at a decisive stage in the reaction chain. In wild tomatoes, the enzyme produced by GAME8a favors the R configuration, leading to filotomatine, while the enzyme produced by GAME8b favors the S configuration, leading to α-tomatine. In cultivated tomatoes, the enzymes produced by the two identical GAME8 genes favor the S configuration and consequently α-tomatine.

The letters R and S correspond to the Latin words rectus (“right”) and sinister (“left”), the terms used to designate the two possible spatial configurations around a chiral center.

The researchers then took it a step further, seeking to discover what within the structure of these enzymes caused one to favor the R configuration and the other to favor the S configuration. Although GAME8a and GAME8b are very similar, the researchers identified four amino acids that play a decisive role in this distinction. “Those four amino acids are located around the enzyme’s catalytic cavity and determine the orientation of the precursor molecule inside it. That orientation defines which hydrogen atom will be modified and consequently the final configuration of the product,” Li explains.

The mechanism could then be explained at the molecular level. The precursor molecule fits into a cavity within the enzyme, and the four amino acids help determine its position. Depending on its orientation within this space, one or the other hydrogen atom of the precursor molecule ends up in the proper position to be modified by the enzyme. This small difference determines whether the product will have an R or S configuration. Therefore, the chirality of tomato glycoalkaloids can be traced through a fairly straightforward sequence: differences in the gene, differences in the enzyme, different positioning of the precursor molecule, R or S configuration, and filotomatine or α-tomatine.

With the necessary caveats, one can imagine the enzyme as a structure that receives a molecule and holds it in a specific position while the chemical reaction takes place. Minimal alterations to the walls of this structure change the orientation of the substrate and consequently the spatial outcome of the reaction. “The Chinese team managed to reveal the molecular mechanism with great precision,” Bento comments.

For Li, this discovery paves the way for the deliberate modification of the chirality of natural plant products. In principle, enzymes could be designed to favor producing the molecular configuration with the highest activity and lowest toxicity, which is also of interest to synthetic biology.

Tailored defense

One of the most interesting findings of the study emerged when the researchers tested the two molecules against another insect. Filotomatine and α-tomatine exhibited similar defensive effects against Spodoptera litura, a generalist herbivore capable of feeding on numerous plant species and commonly known as the tobacco cutworm or cotton leafworm. Therefore, the superiority of filotomatine is not absolute. It depends on which insect is attempting to feed on the plant.

According to Li, this finding supports the hypothesis that the activity of each molecular configuration depends on its interaction with specific targets within the insect’s body. “Our results suggest that filotomatine isn’t simply more toxic in a general sense. If that were the case, we’d expect all wild tomato species to accumulate filotomatine,” he argues.

This is not the case. Different wild species accumulate different configurations of these compounds, suggesting that each chemical profile may offer specific advantages against the herbivores found in a given environment. “That suggests that plants’ chemical defenses may evolve in response to very specific interactions with certain herbivores rather than simply to make plants more toxic in general,” Bento comments.

The results raise an interesting evolutionary question. S. pennellii possesses the genetic ability to produce both isomers; however, it predominantly accumulates filotomatine in its leaves and α-tomatine in other tissues. Since leaves are one of the main targets of herbivores, this distribution may not be coincidental.

“Currently, our evidence is correlational. We observed that filotomatine provides greater resistance to P. absoluta, which naturally interacts with S. pennellii. Since leaves are the organs most exposed to herbivorous pests, we interpret that as a likely result of long-term coevolution between the plant and its specialized herbivores,” Li notes. However, he points out that obtaining definitive proof of such an evolutionary scenario is extremely difficult.

More resistant tomatoes

Identifying the four amino acids that control the GAME8 enzyme’s preference moves the research from fundamental biology to potential agricultural applications. In principle, it would be possible to edit the cultivated tomato’s gene to produce filotomatine and increase its resistance to P. absoluta.

“From a technical standpoint, modifying GAME8 so that the tomato plant accumulates filotomatine is perfectly feasible. We can do that by editing the four key amino acids using current biotechnologies without introducing exogenous genes,” says Li.

This possibility is particularly relevant because P. absoluta is a pest that specifically targets tomato plants. However, turning this discovery into a new commercial variety requires caution. The experiment showed that a configuration more effective against one insect species is not necessarily superior against others. It will be necessary to test its effects on other major tomato pests, such as whiteflies.

There is also another problem. Glycoalkaloids are of interest not only for their defensive function. Altering their metabolism can affect agronomic and nutritional characteristics. Fruit quality, flavor, and antinutritional properties must be evaluated before modifications of this nature can be used in cultivars intended for consumption.

Therefore, the outlook still depends on a long series of tests. However, the study establishes a broader principle: improving a plant’s chemical defense does not necessarily require increasing the amount of a toxic substance. In certain cases, modifying the spatial orientation of a molecule the plant already produces may be sufficient.

About São Paulo Research Foundation (FAPESP)

The São Paulo Research Foundation (FAPESP) is a public institution with the mission of supporting scientific research in all fields of knowledge by awarding scholarships, fellowships and grants to investigators linked with higher education and research institutions in the State of São Paulo, Brazil. FAPESP is aware that the very best research can only be done by working with the best researchers internationally. Therefore, it has established partnerships with funding agencies, higher education, private companies, and research organizations in other countries known for the quality of their research and has been encouraging scientists funded by its grants to further develop their international collaboration. You can learn more about FAPESP at www.fapesp.br/en and visit FAPESP news agency at www.agencia.fapesp.br/en to keep updated with the latest scientific breakthroughs FAPESP helps achieve through its many programs, awards and research centers. You may also subscribe to FAPESP news agency at http://agencia.fapesp.br/subscribe.

 

 

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