Enhancing sweet orange transformation through embryogenic cell cultures using CRISPR-Cas9 mediated editing of sieve element occlusion gene

• Grant number: 23/10399-0
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: January 31, 2024
• End date: July 30, 2024
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Principal Investigator: Alessandra Alves de Souza
• Grantee: Lucas Nascimento dos Santos
• Supervisor: Manjul Dutt
• Host Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil
• Institution abroad: University of Florida, Gainesville (UF), United States
• Associated to the scholarship: 22/12483-5 - CRISPR/Cas9 system in Sieve Element Occlusion c (SEOc) gene editing, aiming at obtaining citrus plants tolerant to Huanglongbing (HLB), BP.MS

Abstract

Citrus species are crops that significantly boost Brazilian agribusiness. However, the productivity of orchards is constantly affected by biotic factors. Citrus Huanglongbing or Greening is the most important disease caused mainly by Candidatus Liberibacter asiaticus (CLas). CLas is a Gram-negative bacterium, which colonizes the phloem, causing physiological damage to citrus plants. Among the consequences of infection is the accumulation of P-proteins in the sieve elements of the phloem. Plants infected by CLas trigger a series of reprogramming of gene expression, resulting in the exaggerated production of calluses and phloem proteins (P-proteins), which act as a physical barrier trying to block the systemic spread of CLas. Based on studies approaching citrus-CLas interaction, genes associated with susceptibility to HLB were prospected and identified, such as the Sieve Element Occlusion c gene (SEOc). SEO family genes encode common phloem proteins (P-proteins) that connect to sieve plates following injury. Thus, SEOc is a suitable gene editing-target aiming to genomic silencing with commercial interest. However, it is still necessary to study efficient editing strategies for a successful methodology in citrus, and the use of embryogenic cells has proven to be a robust method of genetic transformation, since few cells with high rates of cell division will form somatic embryos, reducing the development of chimeras. Hence, our goal is to improve the transformation/editing technique through CRISPR/Cas9, using embryogenic cells from sweet orange (Citrus sinensis) varieties for editing the SEOc gene, aiming to decrease the plant damage caused by HLB.