This project addresses standard program area 4: "environmental effects of GE relative to non-GE organisms in the context of production systems". Our focus is on program area 4d as this project is a "comparative assessment of environmental impacts of agricultural production systems using organic and/or conventional methods with those involving plant, animal, or microbial biotechnology". Here GE-derived tomato plants are compared with CRISPR mutants of tomato that express the same phenotype, increased plant defense. Our goal is to identify environmental hazards associated with the GE lines. Tomato lines will be cultured under agricultural conditions with four plantings over two years. In objective 1, plant and soil microbiome changes will be determined in field-grown GE lines in the field compared to the other lines. Microbiome analysis will include full-length rRNA operon and shotgun metagenomic sequencing. In objective 2, the effects of GE lines on disease pressure, symptom ratings, yield, horticultural characteristics, soil nutrients, and beneficial microbial populations will be measured. In objective 3, all the data from objectives 1 and 2 will be integrated using multiple hazards and XGBoost-based models. Hazard ratios of genotype by environment interactions will be systematically compared to provide federal regulators with decision-making tools. Our hypothesis is that the environmental effects of the GE lines will be insignificant compared to the CRISPR mutant lines. Both the GE and CRISPR lines are expected to show similar decreased and increased levels of pathogens and beneficial microbes, respectively, in bulk soil, rhizosphere, and apoplast compared to the unmodified wild-type lines.Specific Objectives:Objective 1. Define changes to the microbiome of bulk soil, rhizosphere, phyllosphere, fruit, and apoplast in tomato with samples collected from four field plantings over 2 years when planted with the CRISPR mutant lines, GE lines versus their parent line. These analyses will identify changes in bacterial and fungal pathogen populations both in relative and absolute abundance.Objective 2. Assess impact of the GE and non-GE plant defense lines on disease pressure, disease symptom ratings, mycorrhiza populations, yield, and soil nutrient levels.Objective 3. An integrated analysis of data generated from the many variables measured in objectives 1 and 2 will be performed including Cox proportional hazards modeling to identify hazards associated with using the GE-derived tomato genotypes compared to the wild-type parent and CRISPR mutants.Approach: The transgenic tomato lines to be used will express the Arabidopsis genes NPR1, LecRK-I.8, LecRK-VI.2, or ELP4. These genes are all driven by the 35S CMV promoter. The CRISPR-generated lines have a single base mutation in NPR3 or NPR4 or both. These mutations result in increased NPR1 expression. The corresponding wild-type line of each GE or non-GE line will also be used. Changes to soil nutrient levels and to microbial communities on the phyllosphere, apoplast (endophytes), rhizosphere, and bulk soil will be assessed through metagenomics at three time points during the growing season. These analyses will identify changes in bacterial and fungal pathogen populations both in relative and absolute abundance. Yield will be measured at the end of the growing season. Over time, tomato pathogens may develop resistance to these GE-derived or CRISPR mutant lines with increased plant defense. Although this is considered unlikely since these genome alterations take place in genes deep within a signaling pathway, we plan to assess the frequency of spontaneous resistance of pathogens to these altered lines.