Christopher, D. | bioRxiv Science

bioRxiv2025

Genome editing of papaya using both Cas9 and Cas12a

Papaya (Carica papaya L.) is an economically important tropical crop that produces papain and highly nutritious fruit, which are used in the grocery, cosmetic, pharmaceutical, and food processing industries. However, various destructive pathogens severely threaten its production. Furthermore, limited natural genetic variation restricts breeding efforts for crop improvement. Therefore, we turned to gene editing as a tool to address these problems. We utilized two CRISPR systems (Cas9 and Cas12a) and two papaya genes, CpPDS (phytoene desaturase) and CpMLO6 (Mildew Locus O 6), to establish efficient genome editing systems of papaya. The systems were delivered by an optimized protocol of Agrobacterium-mediated transformation (AMT) of embryogenic callus suspension cultures derived from hypocotyls. Accordingly, we transformed papaya with five plasmid constructs, each of which expressed one or two guide RNAs (gRNAs) for gene editing using either Cas9 or Cas12a. All except two T0 transgenic plants tested produced mutations with the majority containing indels of over 90%. Furthermore, successful mutation of the CpPDS gene using both Cas9 and Cas12a produced albino phenotypes as expected for disrupting a gene for carotenoid biosynthesis. Successful mutagenesis was achieved with seven out of eight gRNAs. Homozygous and/or biallelic mutants were generated from transformation using all five constructs, suggesting the feasibility of obtaining transgene-free homozygous segregating mutants by selfing in the second generation. Taken together, a robust and reliable papaya genome editing system was established, which enables genetic modification in various genomic environments to meet the diverse needs of basic scientific research and tropical crop improvement.

plant biology↗

bioRxiv2023

An adaptive biomolecular condensation response is conserved across environmentally divergent species

Cells must sense and respond to sudden maladaptive environmental changes--stresses--to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide an unprecedented view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein--a core marker of stress granules--condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry.

cell biology↗

bioRxiv2022

Collection of triatomines from sylvatic habitats by a Chagas-positive, trained scent-detection canine in Texas, USA

BackgroundTriatomine insects, vectors of the etiologic agent of Chagas disease (Trypanosoma cruzi), are challenging to locate in sylvatic habitats. Collection techniques used in the United States often rely on methods to intercept seasonally dispersing adults or on community scientists encounters. Neither method is suited for detecting nidicolous habitats, which is important for vector control. Furthermore, manual inspection of suspected harborages is unlikely to reveal novel locations and host associations. Similar to a team that used a trained dog to detect sylvatic triatomines in Paraguay, we worked with a trained scent detection dog to detect sylvatic locations of triatomines across Texas. Principle methodology/FindingsZiza, a 3-year-old German Shorthaired Pointer previously naturally infected with T. cruzi, was trained to detect triatomines. Over the course of 6 weeks in the fall of 2017, the dog and her handler visited 18 sites across Texas. The dog detected 60 triatomines at 7 locations; an additional 50 triatomines were contemporaneously collected at these sites without the assistance of the dog. Approximately 0.98 triatomines per hour were found when only humans were conducting searches; when working with the dog, approximately 1.71 triatomines per hour were found. In total, 3 adults and 107 nymphs of four species (Triatoma gerstaeckeri, Triatoma protracta, Triatoma sanguisuga, and Triatoma indictiva) were collected. PCR testing of a subset revealed T. cruzi infection, including DTUs TcI and TcIV, in 28% of nymphs (n=100) and 66% of adults (n=3). Blood meal analysis of a subset of triatomines (n=5) revealed feeding on Virginia opossum (Didelphis virginiana), Southern plains woodrat (Neotoma micropus) and eastern cottontail (Sylvilagus floridanus). Conclusion/SignificanceA trained scent detection dog enhanced triatomine detections in sylvatic habitats. This approach is especially effective at detecting triatomine nymphs and nidicolous locations. With new knowledge of specific sylvatic habitats and key hosts fed upon by triatomines, there are opportunities for continued exploration of novel vector control methods to block the transmission of T. cruzi to humans and domestic animals. Author summaryTriatomine insects, also known as kissing bugs, are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease in humans, dogs, and other mammals. Triatomines are found throughout the Americas, but little is known about where these blood-sucking insects spend their early life stages. Knowing more is important to vector control initiatives aimed at interrupting Chagas disease transmission. Scent detection dogs have been trained to detect many pests, including one study of a triatomine detection dog in South America. In this study, we used a dog to detect triatomines in their natural environments throughout Texas. Over 6 weeks, the dog identified 60 triatomines at 7 different locations; an additional 50 triatomines were collected without the dogs assistance. Nearly twice as many triatomines were collected per hour when the dog was searching as compared to only humans searching. Of all the triatomine nymphs collected, 28% were positive for T. cruzi, and the blood meal analysis revealed kissing bugs had fed on Southern plains woodrat, opossum, and eastern cottontail. This study outlines a strategy that can be replicated in the United States to enhance the detection and control of habitats where triatomines spend their early life stages.

ecology↗