Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-22000-022-018-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 1, 2019
End Date: Mar 31, 2024
Objective:
The overall goal of this project is to characterize the molecular interactions among potato, potato psyllid, and Liberibacter. Specific objectives include: 1) identify salivary gland transcripts from Liberibacter-infected and uninfected psyllids, 2) identify genes that encode for secreted proteins, and 3) test which of these proteins can manipulate plant defenses.
Approach:
Salivary glands of psyllids will be dissected and pooled in RNA later to be sequenced and contiguous sequences (contigs) and unassembled reads (singletons) will be assembled. The annotation of unigenes will be run by NCBI blast against a pooled database of non-redundant (nr) sequences. Annotation of contigs and singletons will be performed using Blast2GO software that categorizes putative biological functions to genes. By comparing transcriptomes from psyllids, we expect to observe genes that are differentially express among psyllid haplotypes, and between Liberibacter infected and uninfected psyllids. Differential expression of up to 10 genes will be confirmed using reverse transcriptase qPCR.
Sequences will be analyzed using the Tuxedo Suite in the computational infrastructure Cyverse. Highly expressed genes will be mined to identify genes encoding putative secreted proteins based on sequence similarity to known secreted proteins identified in other arthropods, as well as by identifying sequences encoding signal peptides typically found in secreted proteins. The expression profile of the best candidates (high expression level and presence of a predicted signal peptide) will be validated by qRT-PCR by Krey and Cooper. Specifically, expression in the salivary glands will be compared to expression in other organs such as bacteriocytes or gut, to select salivary gland-specific genes. Expression will be compared between Lso-infected and Lso-free psyllids to evaluate if psyllid infection with Lso changes the expression profile of the candidate genes. Gene expression verification will be performed. To evaluate if the candidate genes can manipulate the plant defenses, the full length of each candidate will be amplified by PCR from psyllid cDNA and cloned into the vector pEarlyGate 101, under the control of the Cauliflower mosaic virus 35S promoter. Each plasmid will be sequenced to validate the constructs. Each construct will be independently transferred to Agrobacterium tumefaciens strain LBA4404 for transient expression analyses. A minimum of ten candidates will be tested. The constructs will be transferred separately to six-week old Nicotiana benthamiana plants by leaf infiltration using syringe. As controls, leaves will be also infiltrated with a BAX construct that induces hypersensitive response (HR) and with a GFP construct that does not induce HR. The development of HR for each construct will be visually evaluated at the site of infiltration by comparing to the controls: specifically, development of extended cell death in the infiltrated region. This will identify psyllid proteins that can induce plant defenses. Then, individually, each construct will be co-infiltrated with the BAX construct. Candidates that produce reduced cell death when co-infected with BAX when compared to the cell death produced by BAX alone are candidates able to disrupt plant defenses. The best candidates identified, i.e. induce strong responses and can disrupt HR in conjunction with BAX, will be tested on potato.
