2019-11_18-019 – Phloem specific responses to CLas for the identification of novel HLB resistance genes

Phloem specific responses to CLas for the identification of novel HLB resistance genes

Report Date: 12/13/2019
Project: 18-019   Year: 2019
Category: Horticultural & Management
Author: Elizabeth Rogers
Sponsor: Citrus Research and Development Foundation, Inc.

Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our 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. 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 characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. 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.
During the 4th quarter of the first year of our grant, the post-doctoral researcher, Tami Collum, continues to optimize nucleic acid extraction protocols for citrus. She traveled to the Stover lab to learn their citrus propagation and infection protocols. The Stover lab continues Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes (Carrizo, Poncirus and Hamlin sweet orange) with the His-FLAG tagged RPL18 (ribosomal protein L18) under the 35S promoter and all three phloem promoters pSUC2, pSUL and p396ss. Carrizo transgenic plants with three promoters (p35S::HF-RPL18, pSUL::HF-RPL18, and p396ss::HF-RPL18) have been shown to be expressing the transgenic RPL18 by qRT-PCR and transferred to Ft. Detrick. Carrizo with pSUC2 and Poncirus transformants are close behind with multiple lines in soil and ready to ship to Ft. Detrick in January. Hamlin transformation was intensified in last quarter and now many putative transformants are in rooting media. They will be transferred to soil early next quarter before expression testing and transfer to Ft. Detrick. For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) Dr. Dawson’s lab has moved all necessary constructs into citrus. CTV-infected plants will be shipped to Maryland in January.

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