Multiplex Gene Editing of Sieve Element Occlusion Genes to Obtain Huanglongbing-Tolerant Plants

• Grant number: 25/01305-7
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2025
• End date: March 31, 2029
• Field of knowledge: Agronomical Sciences - Agronomy
• Principal Investigator: Alessandra Alves de Souza
• Grantee: Lucas Nascimento dos Santos
• Host Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil
• Associated research grant: 20/07045-3 - Biotechnological and genomic strategies for quality, productivity and sustainable management of citrus, coffee and sugar cane in the State of São Paulo, AP.NPOP

Abstract

Citrus industry, like other sectors of national agriculture, holds significant economic and social importance. However, orange production has been severely threatened by Huanglongbing (HLB), a disease caused by the bacterium Candidatus Liberibacter asiaticus (CLas). From previous studies on citrus-CLas interactions, genes associated with susceptibility to HLB have been identified, such as those in the Sieve Element Occlusion (SEO) family, which encode P-proteins. These proteins are directly linked to phloem blockage, resulting in impaired translocation of sieve. In this context, the SEOc gene, along with other newly identified genes from the same family that are also induced by the disease, represent promising targets for knockout using CRISPR systems. Specifically, a multiplex editing approach allows for the generation and evaluation of plants with multiple edited genes in varying proportions. When combined with co-editing strategies and the high efficiency of embryogenic cell transformation, this multiplex approach holds great biotechnological potential for developing disease-resistant or tolerant plants. This project proposes the editing of the ALS gene as an editing marker alongside other target genes from the SEO family, utilizing the recently optimized transformation of embryogenic cells. Additionally, the project explores the application of cationic lipids for the transfection of ribonucleoprotein (RNP) complexes in protoplasts, focusing on the CsSEOc target as a promising strategy for generating transgene-free edited plants. Our validations will rely on next-generation sequencing through the Hi-TOM platform and aim to produce plants thoroughly characterized at the molecular level. These findings will also be linked to cellular characterization, aiming to describe the physiological role of SEO genes in citrus, particularly in the context of HLB. (AU)