Tomato (Solanum lycopersicum) is one of the most important vegetable crops worldwide. Conventional breeding methods played an important role in obtaining tomato varieties with desired traits, but selection is a time- and labor-intensive process. In recent years, CRISPR-Cas9 genome editing has emerged as a game-changing tool that offers a faster and more precise approach to improving elite tomatoes. This technique utilizes guide RNAs (gRNAs) and the Cas9 enzyme to introduce targeted modifications in the plant genome, enabling the enhancement of specific traits related to stress tolerance and fruit quality. However, CRISPR technology is not yet routinely used in the development of commercial lines. The selection of suitable targets using transcriptomic, literature, and multi-omics analyses could help to avoid adverse effects on plant growth and development. In addition, the design of efficient and specific gRNAs is crucial to escape undesired genetic alterations, which are generally referred to as off-target effects, as well as the choice of effective delivery systems for CRISPR components. In this review, the main challenges for choosing targets, designing gRNAs, and assembling constructs are illustrated. In addition, the crucial role of the appropriate delivery methods is discussed. Furthermore, screening protocols for identifying successfully edited crops and producing CRISPR-Cas9-free progeny were explored. Coupling CRISPR-Cas9 with advanced computational analyses and efficient transformation methods has enormous potential for tomato breeding in the future. However, it also faces limitations such as off-target effects, mosaicism, and delivery efficiency. Safety concerns also exist and should be considered. This constantly progressing technology is paving the way for more sustainable agricultural solutions, contributing to boosting crop productivity and quality.

Cucurbita pepo L. cultivars display high morphological traits variation. In addition, C. pepo faces numerous threats, such as viral and fungal infections, which significantly influence crop cultivation. Recent genomic advancements improved the understanding of genetic diversity and stress responses in this crop. We investigated genetic variations related to plant morphology and quality traits. Additionally, the inclusion of both powdery mildew (PM) and Zucchini yellow mosaic virus (ZYMV) susceptible and tolerant varieties facilitated the examination of genetic diversity concerning biotic stress. The sequencing of eight Cucurbita pepo varieties produced an average of 40 million raw reads with a coverage of reference genome ranging from 22 to 40X. More than 4.7 million genomic variants were identified in all genomes. Based on admixture and PCA analysis, the eight C. pepo genotypes were grouped in two clusters belonging to Cocozelle and Zucchini groups, with “Whitaker” separated from the rest of the accessions. Genes involved in pathways related to gibberellin regulation, leaf development, and pigment accumulation resulted highly affected by variation suggesting that the diversity observed among varieties in plant and fruit morphology could be related to variants identified in such genes. Each variety showed its own set of genetic differences. The genomic comparison of 381e, 968Rb and SPQ allowed the identification of variants in chromosome regions affecting response to Zucchini yellow mosaic virus (ZYMV) and powdery mildew (PM). Variants in key genes associated with resistant traits were identified, suggesting potential pathways and mechanisms involved in biotic stress response and plant immunity. Genetic variations affecting morphology and fruit quality in C. pepo emphasize their significance for breeding efforts. Furthermore, the genomic comparison of 381e, 968Rb and SPQ highlighted variants in chromosomal regions influencing zucchini’s response to PM and ZYMV. These findings could pave the way for more targeted and effective genetic improvement strategies, thereby potentially leading to increased agricultural productivity and quality.

The plant cell wall is a complex and dynamic structure that gives plants strength and flexibility, supporting growth, development, and mediating responses to environmental stimuli. It also represents an important source of renewable lignocellulosic biomass for bioenergy production. The plant cell wall has been studied for years, but the processes controlling its construction, deconstruction, and remodeling remain largely unclear. Genetic technologies have revealed new details about the network of genes regulating the production and modification of cellulose, hemicellulose, pectin, and lignin. These methods are providing us with a more complete view of the complexity of cell wall metabolism. Furthermore, innovative biotechnological tools, such as CRISPR/Cas9 and other genetic engineering techniques, have been shown to facilitate cell wall degradation, promoting the release of fermentable sugars from the biomass.In this review, we summarize recent advances in understanding the molecular composition, biosynthesis, and remodeling of cell wall polymers, highlighting how combining multi-omics data with biotechnology can improve biomass yield, composition, and digestibility. We also discuss key challenges, such as the trade-off between increasing biomass saccharification value and plant performance. Knowledge gaps regarding the link between molecular regulation and field performance under changing environmental conditions remain to be filled. Combining genetic, biochemical, and systems biology perspectives, this review outlines a framework for designing “archetypal” bioenergy crops to balance process efficiency and environmental resilience, thus promoting the sustainable conversion of lignocellulosic biomass into bioenergy and bioproducts.

Powdery mildew represents one of the most significant challenges for cucurbit crops. In recent decades, progress has been made in identifying resistance sources that improve yield and quality while reducing fungicide use. This study explored the molecular mechanisms underlying cucurbit responses to powdery mildew through comparative RNA-seq of two contrasting Cucurbita pepo cultivars: the partially resistant 968Rb and the susceptible True French. Differential expression analysis between inoculated and non-inoculated conditions identified 398 DEGs in 968Rb and 1129 in True French. In 968Rb, a stronger defense response emerged with cell wall reinforcement and upregulation of fructose-1,6-biphosphate aldolase genes, while True French showed activation of chitinase genes. Machine learning models, including Random Forest and K-means, identified expression features and gene modules linked to resistance. By combining conventional and Artificial Intelligence-based analyses, we identified a putative adaptive genetic variation, supported by a higher single nucleotide polymorphism density within expression clusters enriched for upregulated genes in the partial resistant cultivar 968Rb. The integration of Artificial Intelligence tools in our pipeline facilitated the understanding of the genetic basis of Cucurbita pepo resistance to Podosphaera xanthii, highlighting the transcriptional modules and variant patterns associated with resistance traits, and providing a scalable framework for future applications in crop improvement.

Environmental conditions greatly influence the quality of tomato fruit by affecting the expression of genes, the abundance of metabolites, and the perception of sensorial attributes. In this study, a fruit transcriptome investigation, a sensory test, and a metabolomic analysis were performed to evaluate the impact of the environment on two popular tomato cultivars grown in two Italian regions. The transcriptional profile of each cultivar, cultivated in two different areas, highlighted differential expression in genes involved in pathways related to cell wall components such as pectin, lignin, and hemicellulose and sugars as well as in amino acids, phenylpropanoids, and pigment synthesis. The cultivation area mainly affects sensory attributes related to texture and flavor and the metabolic pattern of cell wall precursors, sugars, glutamate, aspartate, and carotenoids. In the two genotypes cultivated in the same environment, some attributes and fruit-related quality processes are similarly affected, while others are differently influenced based on the specific genetic makeup of the tomato. A combination of transcriptomic, sensory, and metabolomic data obtained from the two tomato genotypes revealed that the environment has a profound effect on specific sensory traits, providing information on factors that shape the specific characteristics and genetic targets for improving tomato fruit characteristics.

Background Cucurbita pepo L. cultivars display high morphological traits variation. In addition, C . pepo faces numerous threats, such as viral and fungal infections, which significantly influence crop cultivation. Recent genomic advancements improved the understanding of genetic diversity and stress responses in this crop. We investigated genetic variations related to plant morphology and quality traits. Additionally, the inclusion of both powdery mildew (PM) and Zucchini yellow mosaic virus (ZYMV) susceptible and tolerant varieties facilitated the examination of genetic diversity concerning biotic stress. Results The sequencing of eight Cucurbita pepo varieties produced an average of 40 million raw reads with a coverage of reference genome ranging from 22 to 40X. More than 4.7 million genomic variants were identified in all genomes. Based on admixture and PCA analysis, the eight C. pepo genotypes resulted organized in two clusters belonging to Cocozelle and Zucchini groups, with “Whitaker” separated from the rest of the accessions. Genes involved in pathways related to gibberellin regulation, leaf development, and pigment accumulation resulted highly affected by variation suggesting that the diversity observed among varieties in plant and fruit morphology could be related to variants identified in such genes. Each variety showed its own set of genetic differences. The genomic comparison of 381e, 968Rb and SPQ allowed the identification of variants in chromosome regions affecting response to Zucchini yellow mosaic virus (ZYMV) and powdery mildew (PM). Variants in key genes associated with resistant traits were identified, suggesting potential pathways and mechanisms involved in biotic stress response and plant immunity. Conclusions Genetic variations affecting morphology and fruit quality in C. pepo emphasize their significance for breeding efforts. Furthermore, the genomic comparison of 381e, 968Rb and SPQ highlighted variants in chromosomal regions influencing zucchini's response to PM and ZYMV. These findings could pave the way for more targeted and effective genetic improvement strategies, thereby potentially leading to increased agricultural productivity and quality.

Dissimilar patterns of variants affecting genes involved in response to herbivory, including those leading to difference in VOC production, were identified in tomato lines with contrasting response to Tuta absoluta. Tuta absoluta is one of the most destructive insect pest affecting tomato production, causing important yield losses both in open field and greenhouse. The selection of tolerant varieties to T. absoluta is one of the sustainable approaches to control this invasive leafminer. In this study, the genomic diversity of two tomato varieties, one tolerant and the other susceptible to T. absoluta infestation was explored, allowing us to identify chromosome regions with highly dissimilar pattern. Genes affected by potential functional variants were involved in several processes, including response to herbivory and secondary metabolism. A metabolic analysis for volatile organic compounds (VOCs) was also performed, highlighting a difference in several classes of chemicals in the two genotypes. Taken together, these findings can aid tomato breeding programs aiming to develop tolerant plants to T. absoluta.

Powdery mildew (PM) is a widespread plant disease that causes significant economic losses in thousands crops of temperate climates, including Lamiaceae species. Multiple scientific studies describe a peculiar form of PM-resistance associated at the inactivation of specific members of the Mildew Locus O (MLO) gene family, referred to as mlo-resistance. The characterization of Lamiaceae MLO genes, at the genomic level, would be a first step toward their potential use in breeding programs. We carried out a genome-wide characterization of the MLO gene family in 11 Lamiaceae species, providing a manual curated catalog of 324 MLO proteins. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis and motif patter reconstruction. Our approach highlighted seven different clades diversified starting from an ancestral MLO domain pattern organized in 18 highly conserved motifs. In addition, 74 Lamiaceae putative PM susceptibility genes, clustering in clade V, were identified. Finally, we performed a codon-based evolutionary analysis, revealing a general high level of purifying selection in the eleven Lamiaceae MLO gene families, and the occurrence of few regions under diversifying selection in candidate susceptibility factors. The results of this work may help to address further biological questions concerning MLOs involved in PM susceptibility. In follow-up studies, it could be investigated whether the silencing or loss-of-function mutations in one or more of these candidate genes may lead to PM resistance.

EDITORIAL article Front. Plant Sci., 01 February 2023Sec. Plant Biotechnology Volume 14 - 2023 | https://doi.org/10.3389/fpls.2023.1106996

Until a few years ago, many studies focused on the transcriptomic response to single stresses. However, tomato cultivations are often constrained by a wide range of biotic and abiotic stress that can occur singularly or in combination, and several genes can be involved in the defensive mechanism response. Therefore, we analyzed and compared the transcriptomic responses of resistant and susceptible genotypes to seven biotic stresses (Cladosporium fulvum, Phytophthora infestans, Pseudomonas syringae, Ralstonia solanacearum, Sclerotinia sclerotiorum, Tomato spotted wilt virus (TSWV) and Tuta absoluta) and five abiotic stresses (drought, salinity, low temperatures, and oxidative stress) to identify genes involved in response to multiple stressors. With this approach, we found genes encoding for TFs, phytohormones, or participating in signaling and cell wall metabolic processes, participating in defense against various biotic and abiotic stress. Moreover, a total of 1474 DEGs were commonly found between biotic and abiotic stress. Among these, 67 DEGs were involved in response to at least four different stresses. In particular, we found RLKs, MAPKs, Fasciclin-like arabinogalactans (FLAs), glycosyltransferases, genes involved in the auxin, ET, and JA pathways, MYBs, bZIPs, WRKYs and ERFs genes. Detected genes responsive to multiple stress might be further investigated with biotechnological approaches to effectively improve plant tolerance in the field.

Background Cucurbita pepo is highly susceptible to Zucchini yellow mosaic virus (ZYMV) and the resistance found in several wild species cannot be considered as complete or broad-spectrum resistance. In this study, a source of tolerance introgressed in C. pepo (381e) from C. moschata, in True French (TF) background, was investigated 12 days post-inoculation (DPI) at transcriptomic and genomic levels. Results The comparative RNA-sequencing (RNA-Seq) of TF (susceptible to ZYMV) and 381e (tolerant to ZYMV) allowed the evaluation of about 33,000 expressed transcripts and the identification of 146 differentially expressed genes (DEGs) in 381e, mainly involved in photosynthesis, transcription, cytoskeleton organization and callose synthesis. By contrast, the susceptible cultivar TF triggered oxidative processes related to response to biotic stimulus and activated key regulators of plant virus intercellular movement. In addition, the discovery of variants located in transcripts allowed the identification of two chromosome regions rich in Single Nucleotide Polymorphisms (SNPs), putatively introgressed from C. moschata, containing genes exclusively expressed in 381e. Conclusion 381e transcriptome analysis confirmed a global improvement of plant fitness by reducing the virus titer and movement. Furthermore, genes implicated in ZYMV tolerance in C. moschata introgressed regions were detected. Our work provides new insight into the plant virus recovery process and a better understanding of the molecular basis of 381e tolerance.

A sophisticated innate immune system based on diverse pathogen receptor genes (PRGs) evolved in the history of plant life. To reconstruct the direction and magnitude of evolutionary trajectories of a given gene family, it is critical to detect the ancestral signatures. The rearrangement of functional domains made up the diversification found in PRG repertoires. Structural rearrangement of ancient domains mediated the NB-LRR evolutionary path from an initial set of modular proteins. Events such as domain acquisition, sequence modification and temporary or stable associations are prominent among rapidly evolving innate immune receptors. Over time PRGs are continuously shaped by different forces to find their optimal arrangement along the genome. The immune system is controlled by a robust regulatory system that works at different scales. It is important to understand how the PRG interaction network can be adjusted to meet specific needs. The high plasticity of the innate immune system is based on a sophisticated functional architecture and multi-level control. Due to the complexity of interacting with diverse pathogens, multiple defense lines have been organized into interconnected groups. Genomic architecture, gene expression regulation and functional arrangement of PRGs allow the deployment of an appropriate innate immunity response.

The nucleotide-binding and leucine-rich repeat (NB-LRR) genes, also known as resistance (R)-genes, play an important role in the activation of immune responses. In recent years, large-scale studies have been performed to highlight the diversification of plant NB-LRR repertories. It is well known that, to provide new functionalities, NB-LRR sequences are subject to duplication, domain fusions and acquisition and other kinds of mutations. Although some mechanisms that govern NB-LRR protein domain adaptations have been uncovered, to retrace the plant-lineage-specific evolution routes of R protein structure, a multi-genome comparative analysis was performed. This study allowed us to define groups of genes sharing homology relationships across different species. It is worth noting that the most populated groups contained well-characterized R proteins. The arsenal profile of such groups was investigated in five botanical families, including important crop species, to underline specific adaptation signatures. In addition, the dissection of 70 NB domains of well-characterized R-genes revealed the NB core motifs from which the three main R protein classes have been diversified. The structural remodeling of domain segments shaped the specific NB-LRR repertoires observed in each plant species. This analysis provided new evolutionary and functional insights on NB protein domain shuffling. Taken together, such findings improved our understanding of the molecular adaptive selection mechanisms occurring at plant R loci.

Walking through the produce section at the grocery store, you are likely to �nd tomatoes of all shapes, sizes, and colors. One variety may be large and oblong with a hint of bitter �avor while another will have small, sweet fruits. This variance is primarily the result of genetic and chemical properties of the different varieties. But it turns out, environment also plays a role. A team of Italian scientists has shown that the molecular properties of tomatoes are strongly in�uenced by environmental factors such as temperature and moisture. And changing these factors can, in turn, have pronounced effects on the physical and culinary qualities of the fruits – an important �nding considering the pace of current climate change. To tease apart the interaction between genetics, environment, and organoleptic traits, the research team grew three tomato varieties in two different locations. This exposed the plants to varying levels of moisture, soil acidity, and temperature, among other conditions. Environmental effects were gauged at three different levels: genetic, metabolic, and sensory. A panel of judges evaluated the tomatoes’ sensory traits in terms of �avor, appearance, texture, and aroma. RNA sequencing and metabolomics analyses were then used to examine environmental effects at the genetic and chemical level. The investigators found that under certain environmental conditions, the plants responded with a cascade of genetic changes. While the three varieties varied in their genetic and metabolitic responses, things like appearance and taste were consistently affected. These �ndings highlight the complex interplay between genetics and the environment and provide insights into the mechanisms that regulate fruit quality variation. The implications of this work, however, go beyond pasta sauce and tomato soup. Understanding the genomic response of tomatoes to environmental stressors can help to better understand why some varieties are more resilient than others. In the face of ongoing climate change, that knowledge could give plant breeders insights into how best to develop this important food source.

Background The South America pinworm, Tuta absoluta , is a destructive pest of tomato that causes important losses worldwide. Breeding of resistant/tolerant tomato cultivars could be an effective strategy for T. absoluta management but, despite the economic importance of tomato, very limited information is available about its response to this treat. To elucidate the defense mechanisms to herbivore feeding a comparative analysis was performed between a tolerant and susceptible cultivated tomato at both morphological and transcriptome level to highlight constitutive leaf barriers, molecular and biochemical mechanisms to counter the effect of T. absoluta attack. Results The tolerant genotype showed an enhanced constitutive barrier possibly as result of the higher density of trichomes and increased inducible reactions upon mild infestation thanks to the activation/repression of key transcription factors regulating genes involved in cuticle formation and cell wall strength as well as of antinutritive enzymes, and genes involved in the production of chemical toxins and bioactive secondary metabolites. Conclusions Overall, our findings suggest that tomato resilience to the South America pinworm is achieved by a combined strategy between constitutive and induced defense system. A well-orchestrated modulation of plant transcription regulation could ensure a trade-off between defense needs and fitness costs. Our finding can be further exploited for developing T. absoluta tolerant cultivars, acting as important component of integrated pest management strategy for more sustainable production.

Background: The leaf miner, Tuta absoluta , is a destructive pest of tomato that causes important losses worldwide. Breeding of resistant/tolerant tomato cultivars could be an effective strategy for T. absoluta management but, despite the economic importance of tomato, very limited information is available about its response to this treat. To elucidate the defense mechanisms to herbivore feeding a comparative analysis was performed between a tolerant and susceptible cultivated tomato at both morphological and transcriptome level to highlight constitutive leaf barriers, molecular and biochemical mechanisms to counter the effect of T. absoluta attack. Results: The tolerant genotype showed an enhanced constitutive barrier due to the higher density of trichomes and increased inducible reactions upon mild infestation thanks to the activation/repression of key transcription factors regulating genes involved in cuticle formation and cell wall strength as well as of antinutritive enzymes, and genes involved in the production of chemical toxins and bioactive secondary metabolites. Conclusions: Overall, our findings suggest that tomato resilience to the leaf miner is achieved by a combined strategy between constitutive and induced defense system. A well-orchestrated modulation of plant transcription regulation ensures a trade-off between defense needs and fitness costs. Our finding can be further exploited for developing T. absoluta tolerant cultivars, acting as important component of integrated pest management strategy for more sustainable production.

Tomato (Solanum lycopersicum L.) is a model system for studying the molecular basis of resistance in plants. The investigation of evolutionary dynamics of tomato resistance (R)-loci provides unique opportunities for identifying factors that promote or constrain genome evolution. Nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors belong to one of the most plastic and diversified families. The vast amount of genomic data available for Solanaceae and wild tomato relatives provides unprecedented insights into the patterns and mechanisms of evolution of NB-LRR genes. Comparative analysis remarked a reshuffling of R-islands on chromosomes and a high degree of adaptive diversification in key R-loci induced by species-specific pathogen pressure. Unveiling NB-LRR natural variation in tomato and in other Solanaceae species offers the opportunity to effectively exploit genetic diversity in genomic-driven breeding programs with the aim of identifying and introducing new resistances in tomato cultivars. Within this motivating context, we reviewed the repertoire of NB-LRR genes available for tomato improvement with a special focus on signatures of adaptive processes. This issue is still relevant and not thoroughly investigated. We believe that the discovery of mechanisms involved in the generation of a gene with new resistance functions will bring great benefits to future breeding strategies.

The Plant Resistance Genes database (PRGdb; http://prgdb.org/prgdb4/) has been greatly expanded, keeping pace with the increasing amount of available knowledge and data (sequenced proteomes, cloned genes, public analysis data, etc.). The easy-to-use style of the database website has been maintained, while an updated prediction tool, more data and a new section have been added. This new section will contain plant resistance transcriptomic experiments, providing additional easy-to-access experimental information. DRAGO3, the tool for automatic annotation and prediction of plant resistance genes behind PRGdb, has been improved in both accuracy and sensitivity, leading to more reliable predictions. PRGdb offers 199 reference resistance genes and 586.652 putative resistance genes from 182 sequenced proteomes. Compared to the previous release, PRGdb 4.0 has increased the number of reference resistance genes from 153 to 199, the number of putative resistance genes from 177K from 76 proteomes to 586K from 182 sequenced proteomes. A new section has been created that collects plant-pathogen transcriptomic data for five species of agricultural interest. Thereby, with these improvements and data expansions, PRGdb 4.0 aims to serve as a reference to the plant scientific community and breeders worldwide, helping to further study plant resistance mechanisms that contribute to fighting pathogens.

Background: Cucurbita pepo is high susceptible to Zucchini yellow mosaic virus (ZYMV) and the resistance found in several wild species does not provide complete or broad-spectrum resistance. In this study, a source of tolerance introgressed in C. pepo (381e) from C. moschata, in True French (TF) background, was investigated 12 days post inoculation (DPI) at transcriptomic and genomic levels. Results: A comparative RNA-seq experiment on TF (susceptible to ZYMV) and 381e (tolerant to ZYMV), allowed to evaluate 33,000 expressed transcripts and to identify 146 differentially expressed genes (DEGs) in 381e, mainly involved in photosynthesis, transcription, cytoskeleton organization and callose synthesis. By contrast, the susceptible line True French triggered oxidative processes related to response to biotic stimulus, and key regulators of plant virus intercellular movement. Moreover, the genome mapping of transcripts allowed the identification of two chromosome regions rich in SNPs (Single Nucleotide Polymorphisms), putatively introgressed from C. moschata, containing genes exclusively expressed in 381e. Conclusion: The transcriptome reprogramming of 381e ZYMV challenged revealed a globally restoring of cellular activities and a reduced virus movement and replication. Furthermore, genes putatively involved in ZYMV tolerance were detected in C. moschata introgressed genomic regions. Our work provides new insight into the plant virus recovery process and a better understanding of the molecular basis of 381e tolerance.