Metabolic crosstalk between hydroxylated monoterpenes and salicylic acid in tomato defense response against bacteria.

Hydroxylated monoterpenes (HMTPs) are differentially emitted by tomato (Solanum lycopersicum) plants resisting bacterial infection. We have studied the defensive role of these volatiles in the tomato response to bacteria, whose main entrance is through stomatal apertures. Treatments with some HMTPs resulted in stomatal closure and pathogenesis-related protein 1 (PR1) induction. Particularly, α-terpineol induced stomatal closure in a salicylic acid (SA) and abscisic acid-independent manner and conferred resistance to bacteria. Interestingly, transgenic tomato plants overexpressing or silencing the monoterpene synthase MTS1, which displayed alterations in the emission of HMTPs, exhibited changes in the stomatal aperture but not in plant resistance. Measures of both 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) and SA levels revealed competition for MEcPP by the methylerythritol phosphate (MEP) pathway and SA biosynthesis activation, thus explaining the absence of resistance in transgenic plants. These results were confirmed by chemical inhibition of the MEP pathway, which alters MEcPP levels. Treatments with benzothiadiazole (BTH), a SA functional analog, conferred enhanced resistance to transgenic tomato plants overexpressing MTS1. Additionally, these MTS1 overexpressors induced PR1 gene expression and stomatal closure in neighboring plants. Our results confirm the role of HMTPs in both intra- and interplant immune signaling and reveal a metabolic crosstalk between the MEP and SA pathways in tomato plants.

Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure.

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Tomato geranylgeranyl diphosphate synthase isoform 1 is involved in the stress-triggered production of diterpenes in leaves and strigolactones in roots.

Carotenoids are photoprotectant pigments and precursors of hormones such as strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), three genes encode plastid-targeted GGPP synthases (SlG1 to SlG3) and three genes encode PSY isoforms (PSY1 to PSY3). Here, we investigated the function of SlG1 by generating loss-of-function lines and combining their metabolic and physiological phenotyping with gene co-expression and co-immunoprecipitation analyses. Leaves and fruits of slg1 lines showed a wild-type phenotype in terms of carotenoid accumulation, photosynthesis, and development under normal growth conditions. In response to bacterial infection, however, slg1 leaves produced lower levels of defensive GGPP-derived diterpenoids. In roots, SlG1 was co-expressed with PSY3 and other genes involved in SL production, and slg1 lines grown under phosphate starvation exuded less SLs. However, slg1 plants did not display the branched shoot phenotype observed in other SL-defective mutants. At the protein level, SlG1 physically interacted with the root-specific PSY3 isoform but not with PSY1 and PSY2. Our results confirm specific roles for SlG1 in producing GGPP for defensive diterpenoids in leaves and carotenoid-derived SLs (in combination with PSY3) in roots.

Down-regulation of tomato STEROL GLYCOSYLTRANSFERASE 1 perturbs plant development and facilitates viroid infection.

Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but instead hypersusceptible to the viroid. A combination of phenotypic, molecular, and metabolic analyses of amiRNA-expressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism, or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus demonstrating that down-regulation of SlSGT1 was responsible for the viroid-hypersusceptibility phenotype. Our results highlight the role of sterol glycosyltransferases in proper plant development and indicate that the imbalance of sterol glycosylation levels favors viroid infection, most likely by facilitating viroid movement.

Cryo-EM structure and rRNA modification sites of a plant ribosome.

Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

Revisiting the Non-Coding Nature of Pospiviroids.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure.

Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid-host interactions.

Signaling in the Tomato Immunity against Fusarium oxysporum.

New strategies of control need to be developed with the aim of economic and environmental sustainability in plant and crop protection. Metabolomics is an excellent platform for both understanding the complex plant-pathogen interactions and unraveling new chemical control strategies. GC-MS-based metabolomics, along with a phytohormone analysis of a compatible and incompatible interaction between tomato plants and Fusarium oxysporum f. sp. lycopersici, revealed the specific volatile chemical composition and the plant signals associated with them. The susceptible tomato plants were characterized by the over-emission of methyl- and ethyl-salicylate as well as some fatty acid derivatives, along with an activation of salicylic acid and abscisic acid signaling. In contrast, terpenoids, benzenoids, and 2-ethylhexanoic acid were differentially emitted by plants undergoing an incompatible interaction, together with the activation of the jasmonic acid (JA) pathway. In accordance with this response, a higher expression of several genes participating in the biosynthesis of these volatiles, such as MTS1, TomloxC,TomloxD, and AOS, as well as JAZ7, a JA marker gene, was found to be induced by the fungus in these resistant plants. The characterized metabolome of the immune tomato plants could lead to the development of new resistance inducers against Fusarium wilt treatment.

Apoplastic lipid barriers regulated by conserved homeobox transcription factors extend seed longevity in multiple plant species.

Cutin and suberin are lipid polyesters deposited in specific apoplastic compartments. Their fundamental roles in plant biology include controlling the movement of gases, water and solutes, and conferring pathogen resistance. Both cutin and suberin have been shown to be present in the Arabidopsis seed coat where they regulate seed dormancy and longevity. In this study, we use accelerated and natural ageing seed assays, glutathione redox potential measures, optical and transmission electron microscopy and gas chromatography-mass spectrometry to demonstrate that increasing the accumulation of lipid polyesters in the seed coat is the mechanism by which the AtHB25 transcription factor regulates seed permeability and longevity. Chromatin immunoprecipitation during seed maturation revealed that the lipid polyester biosynthetic gene long-chain acyl-CoA synthetase 2 (LACS2) is a direct AtHB25 binding target. Gene transfer of this transcription factor to wheat and tomato demonstrated the importance of apoplastic lipid polyesters for the maintenance of seed viability. Our work establishes AtHB25 as a trans-species regulator of seed longevity and has identified the deposition of apoplastic lipid barriers as a key parameter to improve seed longevity in multiple plant species.

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

Qualitative and Quantitative Differences in Osmolytes Accumulation and Antioxidant Activities in Response to Water Deficit in Four Mediterranean Limonium Species.

Limonium is a genus represented in the Iberian Peninsula by numerous halophytic species that are affected in nature by salinity, and often by prolonged drought episodes. Responses to water deficit have been studied in four Mediterranean Limonium species, previously investigated regarding salt tolerance mechanisms. The levels of biochemical markers, associated with specific responses-photosynthetic pigments, mono- and divalent ions, osmolytes, antioxidant compounds and enzymes-were determined in the control and water-stressed plants, and correlated with their relative degree of stress-induced growth inhibition. All the tested Limonium taxa are relatively resistant to drought on the basis of both the constitutive presence of high leaf ion levels that contribute to osmotic adjustment, and the stress-induced accumulation of osmolytes and increased activity of antioxidant enzymes, albeit with different qualitative and quantitative induction patterns. Limonium santapolense activated the strongest responses and clearly differed from Limonium virgatum, Limonium girardianum, and Limonium narbonense, as indicated by cluster and principal component analysis (PCA) analyses in agreement with its drier natural habitat, and compared to that of the other plants. Somewhat surprisingly, however, L. santapolense was the species most affected by water deficit in growth inhibition terms, which suggests the existence of additional mechanisms of defense operating in the field that cannot be mimicked in greenhouses.

Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus.

BACKGROUND: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. RESULTS: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1-silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. CONCLUSIONS: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.

Citrus exocortis viroid causes ribosomal stress in tomato plants.

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance.

RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.

Effect of Benzothiadiazole on the Metabolome of Tomato Plants Infected by Citrus Exocortis Viroid.

Benzothiadiazole (BTH) is a functional analogue of the phytohormone salycilic acid (SA) involved in the plant immune response. NahG tomato plants are unable to accumulate SA, which makes them hypersusceptible to several pathogens. Treatments with BTH increase the resistance to bacterial, fungal, viroid, or viral infections. In this study, metabolic alterations in BTH-treated Money Maker and NahG tomato plants infected by citrus exocortis viroid (CEVd) were investigated by nuclear magnetic resonance spectroscopy. Using multivariate data analysis, we have identified defence metabolites induced after viroid infection and BTH-treatment. Glycosylated phenolic compounds include gentisic and ferulic acid accumulated in CEVd-infected tomato plants, as well as phenylalanine, tyrosine, aspartate, glutamate, and asparagine. Besides, an increase of γ-aminobutyric acid (GABA), glutamine, adenosine, and trigonelline, contributed to a clear discrimination between the metabolome of BTH-treated tomato leaves and their corresponding controls. Among them, GABA was the only metabolite significantly accumulated in both genotypes after the chemical treatment. In view of these results, the addition of GABA was performed on tomato plants infected by CEVd, and a reversion of the NahG hypersusceptibility to CEVd was observed, indicating that GABA could regulate the resistance to CEVd induced by BTH.

A New Role For Green Leaf Volatile Esters in Tomato Stomatal Defense Against Pseudomonas syringe pv. tomato.

The volatile esters of (Z)-3-hexenol with acetic, propionic, isobutyric, or butyric acids are synthesized by alcohol acyltransferases (AAT) in plants. These compounds are differentially emitted when tomato plants are efficiently resisting an infection with Pseudomonas syringae pv. tomato. We have studied the defensive role of these green leaf volatile (GLV) esters in the tomato response to bacterial infection, by analyzing the induction of resistance mediated by these GLVs and the phenotype upon bacterial infection of tomato plants impaired in their biosynthesis. We observed that treatments of plants with (Z)-3-hexenyl propionate (HP) and, to a greater extent with (Z)-3-hexenyl butyrate (HB), resulted in stomatal closure, PR gene induction and enhanced resistance to the bacteria. HB-mediated stomatal closure was also effective in several plant species belonging to Nicotiana, Arabidopsis, Medicago, Zea and Citrus genus, and both stomatal closure and resistance were induced in HB-treated NahG tomato plants, which are deficient in salicylic acid (SA) accumulation. Transgenic antisense AAT1 tomato plants, which displayed a reduction of ester emissions upon bacterial infection in leaves, exhibited a lower ratio of stomatal closure and were hyper-susceptible to bacterial infection. Our results confirm the role of GLV esters in plant immunity, uncovering a SA-independent effect of HB in stomatal defense. Moreover, we identified HB as a natural stomatal closure compound with potential agricultural applications.

A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae.

Volatile organic compounds (VOCs) emitted by plants are secondary metabolites that mediate the plant interaction with pathogens and herbivores. These compounds may perform direct defensive functions, i.e., acting as antioxidant, antibacterial, or antifungal agents, or indirectly by signaling the activation of the plant's defensive responses. Using a non-targeted GC-MS metabolomics approach, we identified the profile of the VOCs associated with the differential immune response of the Rio Grande tomato leaves infected with either virulent or avirulent strains of Pseudomonas syringae DC3000 pv. tomato. The VOC profile of the tomato leaves infected with avirulent bacteria is characterized by esters of (Z)-3-hexenol with acetic, propionic, isobutyric or butyric acids, and several hydroxylated monoterpenes, e.g., linalool, α-terpineol, and 4-terpineol, which defines the profile of an immunized plant response. In contrast, the same tomato cultivar infected with the virulent bacteria strain produced a VOC profile characterized by monoterpenes and SA derivatives. Interestingly, the differential VOCs emission correlated statistically with the induction of the genes involved in their biosynthetic pathway. Our results extend plant defense system knowledge and suggest the possibility for generating plants engineered to over-produce these VOCs as a complementary strategy for resistance.

Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus.

Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.

Transgenic tomato plants overexpressing tyramine N-hydroxycinnamoyltransferase exhibit elevated hydroxycinnamic acid amide levels and enhanced resistance to Pseudomonas syringae.

Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-overexpressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection.