Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

Establishing Physalis as a Solanaceae model system enables genetic reevaluation of the inflated calyx syndrome.

The highly diverse Solanaceae family contains several widely studied models and crop species. Fully exploring, appreciating, and exploiting this diversity requires additional model systems. Particularly promising are orphan fruit crops in the genus Physalis, which occupy a key evolutionary position in the Solanaceae and capture understudied variation in traits such as inflorescence complexity, fruit ripening and metabolites, disease and insect resistance, self-compatibility, and most notable, the striking inflated calyx syndrome (ICS), an evolutionary novelty found across angiosperms where sepals grow exceptionally large to encapsulate fruits in a protective husk. We recently developed transformation and genome editing in Physalis grisea (groundcherry). However, to systematically explore and unlock the potential of this and related Physalis as genetic systems, high-quality genome assemblies are needed. Here, we present chromosome-scale references for P. grisea and its close relative Physalis pruinosa and use these resources to study natural and engineered variations in floral traits. We first rapidly identified a natural structural variant in a bHLH gene that causes petal color variation. Further, and against expectations, we found that CRISPR-Cas9-targeted mutagenesis of 11 MADS-box genes, including purported essential regulators of ICS, had no effect on inflation. In a forward genetics screen, we identified huskless, which lacks ICS due to mutation of an AP2-like gene that causes sepals and petals to merge into a single whorl of mixed identity. These resources and findings elevate Physalis to a new Solanaceae model system and establish a paradigm in the search for factors driving ICS.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

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.

An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Pathogen elicitor peptide (pep), systemin, and their receptors in tomato: sequence analysis sheds light on standing disagreements about biotic stress signaling components.

Peps are endogenous damage-associated polypeptides that evoke defense responses in plants. Like other damage-associated molecular patterns, Pep signals are transduced by receptors. PEPRs are the receptors that transduce Pep danger signals. This paper identifies new putative Peps in the Solanaceae (including Solanum spp., Nicotiana spp., and Petunia spp.) and Coffea and explores their properties. Using these newly identified Peps we derive sequence logos that present a refinement of the current understanding of the importance of specific residues in the Pep signaling molecules in Solanaceae, including several arginines, prolines that restrict peptide's conformations, and C-terminal asparagine. We examine the degree of disorder in Pep, which is likely important to the mechanism of Pep perception. This work also calls into question some of the evolutionary relationships between Peps in Solanaceae and specific Arabidopsis Peps published in previous literature, culminating in a conclusion that SlPep should not be named SlPep6 due to the lack of conservation of protein sequences in AtPROPEP6 and SlPROPEP, and that SlPep probably does not have two receptors in tomato, based on phylogenetic analysis. Our analyses advance understanding of the Pep signaling system in Solanaceae.

Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

Complex evolution of novel red floral color in Petunia.

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

Fields of flowers with few strikes: how oligolectic bees manage their foraging behavior on Calibrachoa elegans (Solanaceae).

In specialized plant-pollinator associations, partners may exhibit adaptive traits, which favor the maintenance of the interaction. The association between Calibrachoa elegans (Solanaceae) and its oligolectic bee pollinator, Hexantheda missionica (Colletidae), is mutualistic and forms a narrowly specialized pollination system. Flowers of C. elegans are pollinated exclusively by this bee species, and the bees restrict their pollen resources to this plant species. The pollen presentation schedules of C. elegans were evaluated at the population level to test the hypothesis that H. missionica females adjust their foraging behavior to the resource offering regime of C. elegans plants. For this, the number of new flowers and anthers opened per hour (as a proxy for pollen offering) was determined, and pollen advertisement was correlated with the frequency of flower visits during the day. Preferences of female bees for flowers of different stages were also investigated, and their efficiency as pollinators was evaluated. Pollen offering by C. elegans was found to be partitioned throughout the day through scattered flower openings. Females of H. missionica indeed adjusted their foraging activity to the most profitable periods of pollen availability. The females preferred new, pollen-rich flowers over old ones and gathered pollen and nectar selectively according to flower age. Such behaviors must optimize female bee foraging efficiency on flowers. Female bees set 93% of fruit after a single visit. These findings guarantee their importance as pollinators and the persistence of the specialized plant-pollinator association.

Withanolide Profile and Acetylcholinesterase Inhibitory Activity of Two Argentinean Jaborosa Species.

Acetylcholinesterase (AChE) inhibitors are still an important option for managing symptoms of mild to moderate Alzheimer's disease. In this study, we aimed to evaluate the potential in vitro AChE inhibitory activity of two Argentinian endemic Solanaceae species, Jaborosa bergii and J. runcinata. UHPLC-DAD-HRMS metabolite profiling revealed the presence of withanolides in the active CH2Cl2 subextracts. Their fractionation led to the isolation and identification of two known spiranoid withanolides from J. runcinata and three new withanolides with a skeleton similar to that of trechonolide-type withanolides from J. bergii. The known compounds showed moderate AChE inhibitory activity, while the new ones were inactive.

A Review on the Chemical Properties, Plant Sources, Anti-shock Effects, Pharmacokinetics, Toxicity, and Clinical Applications of Anisodamine.

Alkaloids are natural products that occur widely in many herbal plants. Anisodamine, widely present in the Solanaceae family, is an alkaloid extracted from the roots of the Anisodus tanguticus Maxim. It is an antagonist to M-choline receptors and exhibits diverse pharmacological effects, such as cholinolytic effect, calcium antagonist effect, anti-oxygenation effect. Anisodamine, a prominent constituent of the tropine alkaloid family, exhibits a range of pharmacological effects akin to those of atropine and scopolamine. owing to its low toxicity and moderate efficacy in clinical to wide applications, especially for varieties of shock treatment. However, there remains a dearth of research regarding the in vivo pharmacokinetics, mechanism of action, and toxicity of anisodamine. Consequently, this paper provides a comprehensive review of the anti-shock effects, toxicity, and pharmacokinetic characteristics of anisodamine to increase the understanding of its medicinal value, and provide reference and inspiration for the clinical application and further in-depth research of anisodamine.

Molecular mechanisms of self-incompatibility in Brassicaceae and Solanaceae.

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. SI is controlled by a single S-locus with multiple haplotypes (S-haplotypes). When the pistil and pollen share the same S-haplotype, the pollen is recognized as self and rejected by the pistil. This review introduces our research on Brassicaceae and Solanaceae SI systems to identify the S-determinants encoded at the S-locus and uncover the mechanisms of self/nonself-discrimination and pollen rejection. The recognition mechanisms of SI systems differ between these families. A self-recognition system is adopted by Brassicaceae, whereas a collaborative nonself-recognition system is used by Solanaceae. Work by our group and subsequent studies indicate that plants have evolved diverse SI systems.

Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress.

Calmodulin-binding transcription activator (CAMTA) is an important calmodulin-binding protein with a conserved structure in eukaryotes which is widely involved in plant stress response, growth and development, hormone signal transduction, and other biological processes. Although CAMTA genes have been identified and characterized in many plant species, a systematic and comprehensive analysis of CAMTA genes in the Solanaceae genome is performed for the first time in this study. A total of 28 CAMTA genes were identified using bioinformatics tools, and the biochemical/physicochemical properties of these proteins were investigated. CAMTA genes were categorized into three major groups according to phylogenetic analysis. Tissue-expression profiles indicated divergent spatiotemporal expression patterns of SmCAMTAs. Furthermore, transcriptome analysis of SmCAMTA genes showed that exposure to cold induced differential expression of many eggplant CAMTA genes. Yeast two-hybrid and bimolecular fluorescent complementary assays suggested an interaction between SmCAMTA2 and SmERF1, promoting the transcription of the cold key factor SmCBF2, which may be an important mechanism for plant cold resistance. In summary, our results provide essential information for further functional research on Solanaceae family genes, and possibly other plant families, in the determination of the development of plants.

Chromosome-level pepino genome provides insights into genome evolution and anthocyanin biosynthesis in Solanaceae.

Pepino (Solanum muricatum, 2n = 2x = 24), a member of the Solanaceae family, is an important globally grown fruit. Herein, we report high-quality, chromosome-level pepino genomes. The 91.67% genome sequence is anchored to 12 chromosomes, with a total length of 1.20 Gb and scaffold N50 of 87.03 Mb. More than half the genome comprises repetitive sequences. In addition to the shared ancient whole-genome triplication (WGT) event in eudicots, an additional new WGT event was present in the pepino. Our findings suggest that pepinos experienced chromosome rearrangements, fusions, and gene loss after a WGT event. The large number of gene removals indicated the instability of Solanaceae genomes, providing opportunities for species divergence and natural selection. The paucity of disease-resistance genes (NBS) in pepino and eggplant has been explained by extensive loss and limited generation of genes after WGT events in Solanaceae. The outbreak of NBS genes was not synchronized in Solanaceae species, which occurred before the Solanaceae WGT event in pepino, tomato, and tobacco, whereas it was almost synchronized with WGT events in the other four Solanaceae species. Transcriptome and comparative genomic analyses revealed several key genes involved in anthocyanin biosynthesis. Although an extra WGT event occurred in Solanaceae, CHS genes related to anthocyanin biosynthesis in grapes were still significantly expanded compared with those in Solanaceae species. Proximal and tandem duplications contributed to the expansion of CHS genes. In conclusion, the pepino genome and annotation facilitate further research into important gene functions and comparative genomic analysis in Solanaceae.

A lineage-specific arginine in POS1 is required for fruit size control in Physaleae (Solanaceae) via gene co-option.

Solanaceae have important economic value mainly due to their edible fruits. Physalis organ size 1/cytokinin response factor 3 (POS1/CRF3), a unique gene in Solanaceae, is involved in fruit size variation in Physalis but not in Solanum. However, the underlying mechanisms remain elusive. Here, we found that POS1/CRF3 was likely created via the fusion of CRF7 and CRF8 duplicates. Multiple genetic manipulations revealed that only POS1 and Capsicum POS1 (CaPOS1) functioned in fruit size control via the positive regulation of cell expansion. Comparative studies in a phylogenetic framework showed the directional enhancement of POS1-like expression in the flowers and fruits of Physaleae and the specific gain of certain interacting proteins associated with cell expansion by POS1 and CaPOS1. A lineage-specific single nucleotide polymorphism (SNP) caused the 68th amino acid histidine in the POS1 orthologs of non-Physaleae (Nicotiana and Solanum) to change to arginine in Physaleae (Physalis and Capsicum). Substituting the arginine in Physaleae POS1-like by histidine completely abolished their function in the fruits and the protein-protein interaction (PPI) with calreticulin-3. Transcriptomic comparison revealed the potential downstream pathways of POS1, including the brassinosteroid biosynthesis pathway. However, POS1-like may have functioned ancestrally in abiotic stress within Solanaceae. Our work demonstrated that heterometric expression and a SNP caused a single amino acid change to establish new PPIs, which contributed to the co-option of POS1 in multiple regulatory pathways to regulate cell expansion and thus fruit size in Physaleae. These results provide new insights into fruit morphological evolution and fruit yield control.

The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids.

Cocaine and hyoscyamine are two tropane alkaloids (TA) from Erythroxylaceae and Solanaceae, respectively. These famous compounds possess anticholinergic properties that can be used to treat neuromuscular disorders. While the hyoscyamine biosynthetic pathway has been fully elucidated allowing its de novo synthesis in yeast, the cocaine pathway remained only partially elucidated. Recently, the Huang research group has completed the cocaine biosynthetic route by characterizing its two missing enzymes. This allowed the whole pathway to be transferring into Nicotiana benthamiana to achieve cocaine production. Here, besides highlighting the impact of this discovery, we discuss how TA biosynthesis evolved via the recruitment of two distinct and convergent pathways in Erythroxylaceae and Solanaceae. Finally, while enriching our knowledge on TA biosynthesis, this diversification of the molecular actors involved in cocaine and hyoscyamine biosynthesis opens perspectives in metabolic engineering by exploring enzyme biochemical plasticity that can ease and shorten TA pathway reconstitution in heterologous organisms.

The evolutionary history of small RNAs in Solanaceae.

The Solanaceae or "nightshade" family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family's small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.

Anatomical and biophysical basis for graft incompatibility within the Solanaceae.

Interspecies grafting is an economically relevant technique that allows beneficial shoot and root combinations from separate species to be combined. One hypothesis for the basis of graft compatibility revolves around taxonomic relatedness. To test how phylogenetic distance affects interspecific graft compatibility within the economically important Solanaceae subfamily, Solanoideae, we characterized the anatomical and biophysical integrity of graft junctions between four species: tomato (Solanum lycopersicum), eggplant (Solanum melongena), pepper (Capsicum annuum), and groundcherry (Physalis pubescens). We analyzed the survival, growth, integrity, and cellular composition of the graft junctions. Utilizing various techniques, we were able to quantitatively assess compatibility among the interspecific grafts. Even though most of our graft combinations could survive, we show that only intrageneric combinations between tomato and eggplant are compatible. Unlike incompatible grafts, the formation of substantial vascular reconnections between tomato and eggplant in the intrageneric heterografts likely contributed to biophysically stable grafts. Furthermore, we identified 10 graft combinations that show delayed incompatibility, providing a useful system to pursue deeper work into graft compatibility. This work provides new evidence that graft compatibility may be limited to intrageneric combinations within the Solanoideae subfamily. Further research amongst additional Solanaceous species can be used to test the extent to which our hypothesis applies to this family.