Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Unraveling the diversity and cultural heritage of fruit crops through paleogenomics.

Abundant and plentiful fruit crops are threatened by the loss of diverse legacy cultivars which are being replaced by a limited set of high-yielding ones. This article delves into the potential of paleogenomics that utilizes ancient DNA analysis to revive lost diversity. By focusing on grapevines, date palms, and tomatoes, recent studies showcase the effectiveness of paleogenomic techniques in identifying and understanding genetic traits crucial for crop resilience, disease resistance, and nutritional value. The approach not only tracks landrace dispersal and introgression but also sheds light on domestication events. In the face of major future environmental challenges, integrating paleogenomics with modern breeding strategies emerges as a promising avenue to significantly bolster fruit crop sustainability.

A gradient of the HD-Zip regulator Woolly regulates multicellular trichome morphogenesis in tomato.

Homeodomain (HD) proteins regulate embryogenesis in animals such as the fruit fly (Drosophila melanogaster), often in a concentration-dependent manner. HD-leucine zipper (Zip) IV family genes are unique to plants and often function in the L1 epidermal cell layer. However, our understanding of the roles of HD-Zip IV family genes in plant morphogenesis is limited. In this study, we investigated the morphogenesis of tomato (Solanum lycopersicum) multicellular trichomes, a type of micro-organ in plants. We found that a gradient of the HD-Zip IV regulator Woolly (Wo) coordinates spatially polarized cell division and cell expansion in multicellular trichomes. Moreover, we identified a TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) transcription factor-encoding gene, SlBRANCHED2a (SlBRC2a), as a key downstream target of Wo that regulates the transition from cell division to cell expansion. High levels of Wo promote cell division in apical trichome cells, whereas in basal trichome cells, Wo mediates a negative feedback loop with SlBRC2a that forces basal cells to enter endoreduplication. The restricted high and low activities of Wo pattern the morphogenesis of tomato multicellular trichomes. These findings provide insights into the functions of HD-Zip IV genes during plant morphogenesis.

Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process.

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

An atlas of the tomato epigenome reveals that KRYPTONITE shapes TAD-like boundaries through the control of H3K9ac distribution.

In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.

Insights into stereoselective ring formation in canonical strigolactone: Identification of a dirigent domain-containing enzyme catalyzing orobanchol synthesis.

Strigolactones (SLs) are plant apocarotenoids with diverse roles and structures. Canonical SLs, widespread and characterized by structural variations in their tricyclic lactone (ABC-ring), are classified into two types based on C-ring configurations. The steric C-ring configuration emerges during the BC-ring closure, downstream of the biosynthetic intermediate, carlactonoic acid (CLA). Most plants produce either type of canonical SLs stereoselectively, e.g., tomato (Solanum lycopersicum) yields orobanchol with an α-oriented C-ring. The mechanisms driving SL structural diversification are partially understood, with limited insight into functional implications. Furthermore, the exact molecular mechanism for the stereoselective BC-ring closure reaction is yet to be known. We identified an enzyme, the stereoselective BC-ring-forming factor (SRF), from the dirigent protein (DIR) family, specifically the DIR-f subfamily, whose biochemical function had not been characterized, making it a key enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We first confirm the precise catalytic function of the tomato cytochrome P450 SlCYP722C, previously shown to be involved in orobanchol biosynthesis [T. Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. We then show that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid, forming orobanchol. Our methodology combines experimental and computational techniques, including SRF structure prediction and conducting molecular dynamics simulations, suggesting a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for the stereoselective BC-ring formation in orobanchol. This study sheds light on the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato.

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range.

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing tomato pericarp cells.

Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.

SlMYC2 promotes SlLBD40-mediated cell expansion in tomato fruit development.

As a plant-specific transcription factor, lateral organ boundaries domain (LBD) protein was reported to regulate plant growth and stress response, but the functional research of subfamily II genes is limited. SlMYC2, a master regulator of Jasmonic acid response, has been found to exhibit high expression levels in fruit and has been implicated in the regulation of fruit ripening and resistance to Botrytis. However, its role in fruit expansion remains unknown. In this study, we present evidence that a subfamily II member of LBD, namely SlLBD40, collaborates with SlMYC2 in the regulation of fruit expansion. Overexpression of SlLBD40 significantly promoted fruit growth by promoting mesocarp cell expansion, while knockout of SlLBD40 showed the opposite result. Similarly, SlMYC2 knockout resulted in a significant decrease in cell expansion within the fruit. Genetic analysis indicated that SlLBD40-mediated cell expansion depends on the expression of SlMYC2. SlLBD40 bound to the promoter of SlEXPA5, an expansin gene, but did not activate its expression directly. While, the co-expression of SlMYC2 and SlLBD40 significantly stimulated the activation of SlEXPA5, leading to an increase in fruit size. SlLBD40 interacted with SlMYC2 and enhanced the stability and abundance of SlMYC2. Furthermore, SlMYC2 directly targeted and activated the expression of SlLBD40, which is essential for SlLBD40-mediated fruit expansion. In summary, our research elucidates the role of the interaction between SlLBD40 and SlMYC2 in promoting cell expansion in tomato fruits, thus providing novel insights into the molecular genetics underlying fruit growth.

Interplay among photoreceptors determines the strategy of coping with excess light in tomato.

This study investigates photoreceptor's role in the adaption of photosynthetic apparatus to high light (HL) intensity by examining the response of tomato wild type (WT) (Solanum lycopersicum L. cv. Moneymaker) and tomato mutants (phyA, phyB1, phyB2, cry1) plants to HL. Our results showed a photoreceptor-dependent effect of HL on the maximum quantum yield of photosystem II (Fv/Fm) with phyB1 exhibiting a decrease, while phyB2 exhibiting an increase in Fv/Fm. HL resulted in an increase in the efficient quantum yield of photosystem II (ΦPSII) and a decrease in the non-photochemical quantum yields (ΦNPQ and ΦN0) solely in phyA. Under HL, phyA showed a significant decrease in the energy-dependent quenching component of NPQ (qE), while phyB2 mutants showed an increase in the state transition (qT) component. Furthermore, ΔΔFv/Fm revealed that PHYB1 compensates for the deficit of PHYA in phyA mutants. PHYA signaling likely emerges as the dominant effector of PHYB1 and PHYB2 signaling within the HL-induced signaling network. In addition, PHYB1 compensates for the role of CRY1 in regulating Fv/Fm in cry1 mutants. Overall, the results of this research provide valuable insights into the unique role of each photoreceptor and their interplay in balancing photon energy and photoprotection under HL condition.

A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

Solanum pennellii (LA5240) backcross inbred lines (BILs) for high resolution mapping in tomato.

Wild species are an invaluable source of new traits for crop improvement. Over the years, the tomato community bred cultivated lines that carry introgressions from different species of the tomato tribe to facilitate trait discovery and mapping. The next phase in such projects is to find the genes that drive the identified phenotypes. This can be achieved by genotyping a few thousand individuals resulting in fine mapping that can potentially identify the causative gene. To couple trait discovery and fine mapping, we are presenting large, recombination-rich, Backcross Inbred Line (BIL) populations involving an unexplored accession of the wild, green-fruited species Solanum pennellii (LA5240; the 'Lost' Accession) with two modern tomato inbreds: LEA, determinate, and TOP, indeterminate. The LEA and TOP BILs are in BC2F6-8 generation and include 1400 and 500 lines, respectively. The BILs were genotyped with 5000 SPET markers, showing that in the euchromatic regions there was one recombinant every 17-18 Kb while in the heterochromatin a recombinant every 600-700 Kb (TOP and LEA respectively). To gain perspective on the topography of recombination we compared five independent members of the Self-pruning gene family with their respective neighboring genes; based on PCR markers, in all cases we found recombinants. Further mapping analysis of two known morphological mutations that segregated in the BILs (self-pruning and hairless) showed that the maximal delimited intervals were 73 Kb and 210 Kb, respectively, and included the known causative genes. The 'Lost'_BILs provide a solid framework to study traits derived from a drought-tolerant wild tomato.