Project Number: 6034-21000-018-23-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Dec 1, 2018
End Date: Nov 30, 2021
This proposal aims to identify genes expressed in citrus phloem that show differential regulation in huanglongbing HLB susceptible and resistant citrus cultivars. Candidatus Liberibacter asiaticus (CLas) is a phloem limited pathogen that manipulates phloem gene regulation to facilitate its life cycle. Phloem tissues are buried in the plant and are extremely difficult to physically separate and study in isolation. The inability to accurately characterize phloem responses to CLas infection remains a significant impediment to understanding CLas-host interactions and to identifying gene targets for engineering resistance.
Recently, our team developed a new methodology for woody crops that allows gene expression profiling of phloem infected tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating messenger Ribonucleic acid (mRNAs) from phloem specific tissues. The approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Huanglongbing-susceptible sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of existing phloem-specific promoters. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.