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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #420628
Research Project: Omics-Based Approach to Detection, Identification, and Systematics of Plant Pathogenic Phytoplasmas and Spiroplasmas

Location: Molecular Plant Pathology Laboratory

Title: CRISPR/Cas9-mediated generation of tomato RAD23 knockouts for studying phytoplasma-induced symptoms

Author
item Inaba, Junichi
item Kim, Bo Min
item ZHAO, YAN - Retired ARS Employee
item Wei, Wei

Submitted to: Phytopathogenic Mollicutes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2024
Publication Date: 3/5/2025
Citation: Inaba, J., Kim, B., Zhao, Y., Wei, W. 2025. CRISPR/Cas9-mediated generation of tomato RAD23 knockouts for studying phytoplasma-induced symptoms. Phytopathogenic Mollicutes. 15(1):9-10. https://doi.org/10.5958/2249-4677.2025.00004.7.
DOI: https://doi.org/10.5958/2249-4677.2025.00004.7

Interpretive Summary: Phytoplasmas are small plant pathogens that disrupt floral development, significantly reducing fruit production and crop yield. In Arabidopsis, the phytoplasma effector protein SAP54 interacts with RAD23 proteins to induce floral abnormalities, with rad23 mutants partially alleviating these symptoms. Building on this insight, ARS scientists in Beltsville, Maryland, explored whether a similar mechanism operates in tomatoes by using CRISPR/Cas9, a gene-editing tool, to knock out (or turn off) two RAD23 genes in tomatoes, called SlRAD23C and SlRAD23D. Special guide RNAs helped target and disrupt these genes, and tests confirmed that the genes were successfully turned off, leading to shortened proteins in the edited plants. These CRISPR-edited tomato mutants will be tested for susceptibility to floral deformation upon infection by potato purple top (PPT) phytoplasma. This research seeks to clarify the role of RAD23 proteins in phytoplasma-induced symptoms, aiming to support the development of tomato plants with improved resistance to phytoplasma infections. This study will interest crop scientists and geneticists examining pathogen-plant interactions, along with breeders and agricultural professionals working on crop resilience and sustainable disease resistance. It also offers an example of CRISPR technology for educators and students in plant pathology and agricultural biotechnology.

Technical Abstract: Phytoplasma infection often induces abnormal flower development, Phytoplasma infection often induces abnormal flower development, such as phyllody and cauliflower-like inflorescence, leading to compromised fruit development and significant crop yield reduction. In Arabidopsis, the phytoplasma effector SAP54 interacts with RAD23 proteins to induce floral abnormalities, partially alleviated in rad23 mutants. To investigate whether a similar mechanism may occur in tomatoes, in this study, CRISPR/Cas9 technology was employed to generate knockout tomato mutants of SlRAD23C and SlRAD23D. Target-specific guide RNAs (sgRNAs) were designed to disrupt the coding sequences of SlRAD23C and SlRAD23D. PCR screening and sequencing confirmed successful deletions in the targeted regions, leading to truncated protein products in mutant lines. These mutants will be further analyzed for their response to potato purple top (PPT) phytoplasma-induced flower deformation to develop tomato lines less susceptible to phytoplasma-related reproductive reduction.