Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via Agrobacterium tumefaciens. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T1 progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis (Arabidopsis thaliana) NADPH oxidase respiratory burst oxidase homolog D (RBOHD) is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase Pbl13-interacting RING domain E3 ligase (PIRE). Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in tracheophytes and expanded in angiosperms. We investigated the role of tomato (Solanum lycopersicum) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using genome editing, we generated mutants in 2 S. lycopersicum PIRE (SlPIRE) homologs. SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens P. syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soil-borne pathogens. These results indicate that PIRE regulates RBOHs in multiple plant species and is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated pathogenic interactions.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Bacterial spot, caused by Xanthomonas species, is a devastating disease of tomato (Solanum lycopersicum) and pepper (Capsicum annuum) (Schwartz et al., 2015). The recessively inherited resistance, bacterial spot 5 (bs5), in pepper (hereafter referred to as Cabs5) can confer resistance against different Xanthomonas strains (Jones et al., 2002). The Cabs5 resistance is characterized by the absence of disease symptoms, faint chlorosis at the site of infection, and reduced bacterial growth. Remarkably, commercial pepper varieties containing the bs5 allele show durable resistance, effectively impeding hypervirulent strain emergence in agricultural fields (Vallejos et al., 2010). The CaBs5 gene, together with its paralog CaBs5-like (CaBs5L), has recently been cloned (Sharma et al., 2023; Szabó et al., 2023). CaBs5 encodes a 92 amino acid long protein possessing a cysteine-rich transmembrane (CYSTM) domain, which is implicated in various biotic and abiotic responses. Typically, the CYSTM domain contains conserved residues composed of four consecutive cysteines, followed by two hydrophobic amino acids. A recent study suggested that Cabs5 mediating the resistance against bacterial spot lacks these two conserved leucine residues within the CYSTM domain (Szabó et al., 2023). Tomatoes and peppers are close relatives in the Solanaceae family and commonly susceptible to Xanthomonas infection. Based on the current findings in pepper, we hypothesized that modifying the ortholog of CaBs5 in tomato could confer resistance against Xanthomonas. Consequently, putative Bs5 (SlBs5) and Bs5L (SlBs5L) were identified in tomato based on homology to CaBs5. Both SlBs5 and SlBs5L were located on chromosome 9 with the same head-to-head orientation as their pepper homologues on chromosome 3 (Figure 1a). Despite short and highly similar amino acid sequences of SlBs5 and SlBs5L (Figure 1b), the conserved synteny and gene order in pepper and tomato genomes allowed the assignment of orthology for Bs5 and Bs5L. The mechanism by which the double leucine deletion in Cabs5 leads to resistance against Xanthomonas remains elusive (Figure 1b). Yet, this deletion in the conserved CYSTM domain could potentially impair CaBs5's native functionality (Abell and Mullen, 2011). Following this assumption, we postulated that knocking out SlBs5 would produce similar outcomes to Cabs5. We aimed to disrupt both SlBs5 and SlBs5L to prevent possible functional complementation by SlBs5L, given their greater amino acid sequence similarity compared to CaBs5 and CaBs5L (Figure 1b). We constructed a binary vector for Cas9 and a single-guide RNA (sgRNA) targeting conserved sequences present in both SlBs5 and SlBs5L (Figure 1c). Tomato variety Fla. 8000 was transformed with Agrobacterium. From the progeny of successful transformants, we selected two homozygous lines, Slbs5-1 and Slbs5-2, containing frameshift mutations in both genes (Figure 1c). These mutant lines were self-pollinated or backcrossed to the wild-type parent variety to segregate the T-DNA containing the Cas9-sgRNA cassette. The resistance of the two selected mutant lines was qualitatively evaluated against Xanthomonas perforans GE485 with dip inoculation assays (Figure 1d). At 21 days post-inoculation, the wild-type leaves were covered by black spots indicative of Xanthomonas infection, while both Slbs5-1 and Slbs5-2 retained green leaves with fewer visible symptoms. These phenotypes remained consistent in inoculations of X. perforans 4B and Xanthomonas gardneri 153 (Figure S1). Quantitative evaluation of bacterial growth further supported these findings. At 5 days post-infiltration with a low-density bacterial suspension, Slbs5-1 showed significant decreases in Xanthomonas populations compared to wild-type plants (Figure 1e). Such reductions were consistently observed for Slbs5-2 (Figure S2). However, Slbs5-1 could not significantly hinder Pseudomonas population growth. We additionally examined the growth penalty associated with Slbs5-1 and Slbs5-2 in controlled conditions (Figure 1f). The height of plants was measured at two different time points, but no significant differences were observed between the wild type and the two mutant lines (Figure 1f; Figure S3). This suggested that the resistance to Xanthomonas species comes at no developmental cost in the vegetative stage in the laboratory setting. Although Cabs5-mediated immunity is subtle, it has shown practical value in commercial pepper cultivation. To examine the commercial potential of Slbs5, field trials were conducted with both Slbs5-1 and Slbs5-2 lines at the Gulf Coast Research and Education Center in Florida, a major state for tomato production. Along with naturally occurring Xanthomonas populations, a two-isolate cocktail of X. perforans race T4 was inoculated in the field to heighten disease pressure. Plants were grown with recommended fertilizers and pest management programs, excluding the use of any bactericides or activators of systemic acquired resistance. Despite seasonal variations, Slbs5 mutant lines consistently maintained reduced disease symptoms (Figure 1g). Additionally, no developmental defects, such as stunting, were observed in these mutants (Figure 1h). Quantification of disease severity, based on visible symptoms caused by Xanthomonas infection on plant leaf surfaces, revealed higher percentages of Slbs5-2 leaves with reduced disease symptoms than wild-type leaves in all tested seasons (Figure 1i; Figure S4). Notably, the Slbs5-2 mutants demonstrated effective resistance during three periods of elevated disease pressure, Spring 2018, Fall 2019, and Fall 2023. The marketable yield of fruits is a critical consideration in tomato cultivation. We quantified total marketable yield across five seasonal trials, except for two seasons impacted by a hurricane (Fall 2022) and extremely dry weather (Spring 2023). Throughout all seasons, there was no statistically significant difference in marketable fruit yields between Slbs5-2 and the wild-type plants (Figure 1j; Figure S5). However, during the three periods of increased disease prevalence in Spring 2018, Fall 2019, and Fall 2023 (Figure 1i), the mutants consistently showed a tendency to produce a greater quantity of marketable tomatoes (Figure 1j). This possibly suggests a correlation between Xanthomonas resistance of the mutant lines and improved fruit yields. Overall, this study shows that a knockout of SlbBs5 and SlBs5L in tomatoes represents a promising strategy to achieve broad-spectrum resistance to bacterial spot disease. Compared to stronger sources of resistance, the resistance mediated by Slbs5 and Slbs5L may be considered subtle. However, our mutant lines consistently led to a reduced population of Xanthomonas in laboratory and field conditions. This decrease in pathogen populations could lessen the likelihood of hypervirulent strain emergence. Furthermore, when these mutants are combined with other sources of downstream resistance genes, they may serve as a prior layer of defence. This initial protection has the potential to diminish the probability of pathogen effectors directly interacting with and overcoming the resistance genes, possibly extending the efficacy of durable resistance in the agricultural field. This research was funded by the Innovative Genomics Institute Founders Fund and the 2Blades Foundation. A.O. and B.J.S. conceptualized the project. B.J.S. supervised the project. A.O., D.D. and B.J.S. designed the experiments and helped analyze the data. K.S. and E.S performed bioinformatics analyses. K.S. led statistical analyses and designed the figures. A.S. helped plan the project, designed and tested the guide RNAs and did preliminary genotyping and bacterial disease assays. A.O. did further genotyping, guide RNA testing and conducted disease and phenotype assays of progeny. D.P.T.T., J.V.W., J.B.J, G.M, E.S.O and D.D. performed supplemental bacterial growth assays. E.S. and S.H. conducted field trials. M.J.C. supervised the generation of tomato mutant lines. E.Z. and J.P. conducted tomato transformations. A.O., K.S. and D.P.T.T. analyzed the data and wrote the manuscript. The data that support the findings of this study are available on request from the corresponding author. Appendix S1 Materials and Methods. Figure S1 Qualitative evaluation of disease symptoms with a dip inoculation assay. Figure S2 Quantitative evaluation of bacterial growth after inoculation. Figure S3 Height comparison between wild type and mutant plants. Figure S4 The disease symptoms on wild type and Slbs5-1 plant leaves in the field trials. Figure S5 Fruit yields of wild type and Slbs5-1 plants in the Fall 2023 field trial. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao . Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T 0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

2020 Meeting of the Society for In Vitro Biology

PLANT POSTER ABSTRACTS BIOTECHNOLOGY P-3000 Characterization of the ERECTA Gene as QTL for Tolerance to Environmental Stresses Y. V. Berchembrock, University of Arkansas, V. Srivastava, F. B. S. Botelho, and S. Zhao P-3001 Expression of the Mutant ERECTA and Phenotypic Correlation in Soybean Yasmin Berchembrock, University of Arkansas, Vibha Srivastava, and Flavia Botelho P-3002 Adjuvant Properties of Plant Virus Particles with Different Shape Ekaterina Evtushenko, Lomonosov Moscow State University, Ekaterina Ryabchevskaya, Joseph Atabekov, Nikolai Nikitin, and Olga Karpova P-3003 Micrografted Tomato Plants Improve the Response to Fusarium oxysporum f. sp. lycopersici Dora J. García-Jaramillo, Universidad de Caldas Colombia, Silvia P. López-Zapata, Santiago BustamanteGranada, María Camila Zuluaga-Naranjo, Manuela Robledo-Gómez, Oswaldo Murillo-Ramírez, Jairo CastañoZapara, Nelson Ceballos-Aguirre, Walter R. López, and Lucía Atehortúa-Garcés P-3004 Genome Edited BdVDAC Gene Enable to Inhibit GA Pathway in Germination Stage Sang Yong Park, Kongju National University, Woo Joo Jung, Jin Seok Yoon, and Jae Yoon Kim P-3005 A Novel Plant Virus Platform for Biotechnology TatyanaManukhova, Lomonosov Moscow State University, Ekaterina Evtushenko, Nikolai Nikitin, and Olga Karpova P-3006 Production and Molecular Characterization of New Lemon Hybrids Ahmad A. Omar, University of Florida, Elahe Ahadi Dolatsara, Azza H. Mohamed, and Jude W. Grosser P-3013 PVX-based Vector as a Candidate for In Vitro Transient Gene Expression and for Large-scale Production of Recombinant Proteins in Plants Yana Sindarovska, Institute of Cell Biology and Genetic Engineering

Plants that lack chlorophyll are rare and typically restricted to holoparasites that obtain their carbon, water and mineral resources from a host plant. Although not parasites in the traditional sense, albino foliage, such as the sprouts that sometimes develop from redwood tree trunks, are comparable in function. They occur sporadically, and can reach the size of shrubs and in rare cases, trees. Albino redwoods are interesting because in addition to their reduced carbon resources, the absence of chloroplasts may impede proper stomatal function, and both aspects may have upstream consequences on water transport and xylem quality. We examined the water relations, water transport and xylem anatomical attributes of albino redwoods and show that similar to achlorophyllous and parasitic plants, albino redwoods have notably higher stomatal conductance than green sprouts. Given that stem xylem tracheid size as well as water transport efficiency are nearly equivalent in both albino and green individuals, we attribute the increased leaf water loss in albino sprouts to lower leaf to xylem area ratios, which favour improved hydration relative to green sprouts. The stems of albino redwoods were more vulnerable to drought-induced embolism than green stems, and this was consistent with the albino's weaker tracheids, as characterized by wall thickness to lumen diameter measures. Our results are both complementary and consistent with previous research on achlorophyllous plants, and suggest that the loss of stomatal control and photosynthetic capacity results in substantial vascular and anatomical adjustments.