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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #432646

Research Project: Exotic Pathogens of Citrus: Curation, Diagnostics, and Interactions

Location: Molecular Plant Pathology Laboratory

Project Number: 8042-22000-301-00-D
Project Type: In-House Appropriated

Start Date: Apr 9, 2017
End Date: Apr 8, 2022

Objective:
Objective 1: Evaluate patterns of gene expression in mixed infections between CTV and HLB in citrus, with the goal of discovering both cross-protecting strains and potential synergistic interactions. Objective 2: Develop new diagnostic reagents for emerging citrus pathogens and evaluate them as research reagents for the citrus community.

Approach:
Host gene expression with two phloem limited pathogens - We intend to define citrus genes expressed in response to infection by phloem limited pathogens Citrus tristeza virus (CTV) and 'Ca. Liberibacter asiaticus' (CaLas). RNA Seq: Asymptomatic, young leaf tissue will be ground to a powder in liquid nitrogen and RNA will be isolated using a Trizol protocol. RNA will be used for RNA sequencing (Illumina 2500). Paired-end reads will be mapped to Citrus sinensis ‘Valencia’ reference genome. Differentially expressed transcripts will be identified. P < 0.01 and log2 fold change (log2FC) =¦1¦will be set as cut-off values. Curation of Exotic Pathogens of Citrus Collection: Each CTV isolate is composed of different genotypes, and we do not have detailed information on the CTV strain composition within each isolate. We will obtain this information by extracting RNA, preparing cDNA and performing multiplex PCR to identify mixed genotypes to characterize the CTV isolates. We will then extract dsRNA and use it as template for single read RNASeq and CTV genome assembly of CTV isolates of interest. ‘Ca. Liberibacter asiaticus’: We will determine the genome sequence of the 14 strains of ‘CaLas’ present in the EPPC. The parasitic plant dodder, Cuscuta indecora, is itself parasitized by ‘Ca. Liberibacter asiaticus’. The original genomic sequence data for ‘Ca. Liberibacter asiaticus’ was obtained from a single super-infected psyllid that provided a high ratio of CaLas to psyllid DNA required for shotgun sequencing. We can’t use psyllids under the conditions of our permit from USDA APHIS. We have shown that 2 cm segments of dodder stem infected with CaLas vary greatly in the concentration of CaLas, reaching concentrations of 109/g. We will infest citrus inoculated with CaLas from our collection with dodder and allow it to establish. We will harvest the dodder, cut them in 2 cm segments, and extract DNA. The extracts will be tested for the CaLas using our standard assay. We expect to find segments which have very high concentrations of CaLas (Cq<16). These will be sent to Jianchi Chen at Parlier for sequencing following amplification using his established protocol. We have recombinant antibodies that recognize surface antigens of CaLas. We have MTRAs with USDA APHIS CPHST and PathSensors, Inc (Baltimore, MD) to use our antibodies to develop a ‘CANARY’ assay for ‘Ca. Liberibacter asiaticus’. CANARY enables serologically based detection of pathogens in three minutes starting from an environmental sample. Direct tissue blot immunoassay (DTBIA) – The DTBIA is a well established technique for localizing proteins in plant tissue. We have used a rabbit polyclonal antibody for DTBIA of CaLas. We therefore expressed and purified the same antigens used to generate scFv to immunize New Zealand white rabbits to produce conventional polyclonal antisera. The DTBIA format preserves the localized concentration of CaLas observed in phloem cells and works in leaf midribs as well as in fruit petioles and peduncles, seed, stem and root tissues. These new antibodies will be used in DTBIA and the results compared with those obtained with anti-OmpA antibodies.