The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical processes associated with defense and susceptibility in tomato (Solanum lycopersicum) fruit. Using unripe and ripe fruit inoculated with three fungal pathogens, we identified common pathogen responses reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. We established that the magnitude and diversity of defense responses do not significantly impact the interaction outcome, as susceptible ripe fruit mounted a strong immune response to pathogen infection. Then, to distinguish features of ripening that may be responsible for susceptibility, we utilized non-ripening tomato mutants that displayed different susceptibility patterns to fungal infection. Based on transcriptional and hormone profiling, susceptible tomato genotypes had losses in the maintenance of cellular redox homeostasis, while jasmonic acid accumulation and signaling coincided with defense activation in resistant fruit. We identified and validated a susceptibility factor, pectate lyase (PL). CRISPR-based knockouts of PL, but not polygalacturonase (PG2a), reduced susceptibility of ripe fruit by >50%. This study suggests that targeting specific genes that promote susceptibility is a viable strategy to improve the resistance of tomato fruit against fungal disease.

Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.

We review the latest information related to the control of fruit softening in tomato and where relevant compare the events with texture changes in other fleshy fruits. Development of an acceptable texture is essential for consumer acceptance, but also determines the postharvest life of fruits. The complex modern supply chain demands effective control of shelf life in tomato without compromising colour and flavour.The control of softening and ripening in tomato (Solanum lycopersicum) are discussed with respect to hormonal cues, epigenetic regulation and transcriptional modulation of cell wall structure-related genes. In the last section we focus on the biochemical changes closely linked with softening in tomato including key aspects of cell wall disassembly. Some important elements of the softening process have been identified, but our understanding of the mechanistic basis of the process in tomato and other fruits remains incomplete, especially the precise relationship between changes in cell wall structure and alterations in fruit texture.

Social Impact Statement: Tomato fruit is an important and popular commodity producing $95.62 billion worldwide. Tomato fruit losses in the supply chain vary between 25% and 42% depending on the production area and the availability of postharvest technologies. For many decades, conventional tomato breeding programs have focused on extending the shelf‐life of fresh‐market varieties. However, in many instances, consumer‐based quality traits were not considered a priority. Consumers are now demanding safe, nutrient‐rich, high‐flavor, and convenient fruit. Here, we demonstrate the use of gene editing to improve fruit shelf‐life and positively impact quality, which can help significantly reduce tomato fruit losses and meet consumer expectations.Summary: Finding alternative ways to extend tomato fruit shelf‐life without reducing the quality is critical to ensure the accessibility and likeability of this commodity worldwide. Improving fruit firmness in tomato fresh‐market varieties directly impacts their shelf‐life potential. We simultaneously knocked out two pectin‐degrading enzymes, polygalacturonase (SlPG2a) and pectate lyase (SlPL), key for tomato fruit softening. We expected this gene‐editing approach to result in longer‐lasting fruit without negatively impacting consumer‐based quality attributes. By generating the double clustered regularly interspaced short palindromic repeats (CRISPR) knockout PGPL, we evaluated the combined functions of SlPG2a and SlPL on fruit quality, including shelf‐life attributes like firmness and water loss, fruit marketability, and disease incidence. We also assessed additional attributes impacting consumer acceptance, such as taste and aroma. We revealed that the enzymes SlPG2a and SlPL act additively, significantly impacting fruit firmness and shelf‐life, with the double CRISPR knockout PGPL outperforming the wild‐type fruit. Additionally, fruit quality traits, such as sugar: acid ratio, aroma volatiles, and skin color, were improved or not affected in the double CRISPR knockout PGPL compared to the wild‐type. The discoveries of this research provide new insights into the influence of pectin backbone degradation on fruit physiology and postharvest quality, which can be used in crop improvement programs to make fruit more resilient in the supply chain without compromising quality.