Target identification strategies and sweet orange (Citrus sinensis L. Osb.) genome editing for tolerance to Huanglongbing

Author(s)

Maiara Curtolo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2021-04-27

Examining board members

Marcos Antonio Machado; Renato Vicentini; Raquel Luciana Boscariol Camargo; Monalisa Sampaio Carneiro; Francisco de Assis Alves Mourão Filho

• Advisor: Marcos Antonio Machado

Abstract

Despite the advances in the Brazilian citrus industry, pests and diseases are still considered the main obstacles in citrus groves. Huanglongbing (HLB) or Greening is currently considered the most devastating citrus disease, since all commercial varieties are highly susceptible to this disease. Thus, the pursuit of knowledge of genetic mechanisms that trigger many biological processes involved in the response to the HLB is of utmost importance for the citrus industry. The present work aimed: a) To use different biotechnological techniques (RNA-seq and eQTL Mapping) aiming the identification of target genes related to susceptibility, tolerance and resistance to HLB. b) To develop a genome editing platform based at CRISPR / Cas9 system. Posteriorly, that platform will allow the development of citrus plants more tolerant to HLB. The transcriptomic analysis of six different genotypes led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance. The deposition of large amounts of P protein and callose on the phloem sieve plates seems to be the main alteration that determines the typical HLB symptoms. Transcriptomic analysis also indicated that calloses synthases and P proteins genes also had their expression level altered by Candidatus Liberibacter asiaticus (CLas) infection. The expression of eight callose synthase genes was evaluated in hybrids between Citrus sunki (HLB susceptible) and Poncirus trifoliata (HLB tolerant) infected with HLB. The expression data were associated with single nucleotide polymorphism (SNP) of C. sunki and P. trifoliata, allowing the identification of interesting genomic regions for citrus breeding programs. Transcriptomes indicated that Sieve Element Occlusion c (SEOc) may be related to HLB susceptibility. In addition, previous studies also demonstrated that members of the SEO family encode P-protein subunits. Thus, SEOc was the target gene chosen to be worked during the development of the genome editing platform. The CRISPR / Cas9 system was used to modify the tobacco and citrus genome. The results generated by this work indicated that optimization of the genome editing technique should be adopted in order to increase the edition rate in citrus (AU)

• FAPESP's process: 16/22133-0 - Genome editing aimed at QTLs validation associated with tolerance Candidatus Liberibacter asiaticus in citrandarins (Citrus sunki x Poncirus trifoliata)
• Grantee: Maiara Curtolo
• Support Opportunities: Scholarships in Brazil - Doctorate