Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-22000-022-005-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 1, 2020
End Date: Dec 31, 2021
Objective:
Objective 1: Identify salivary gland transcripts from Liberibacter-infected and uninfected potato psyllids of the Northwestern, Western, and Central haplotypes. I will test the hypotheses that gene expression in the salivary glands of potato psyllids differs between infected and uninfected psyllids, and among psyllid haplotypes with dissimilar host preferences. Objective 2: Use CRISPR gene silencing to determine the function of salivary transcripts in psyllid feeding and pathogen transmission. I will test the hypotheses that silencing certain salivary transcripts in potato psyllid prevents psyllids from feeding and limits the transmission and establishment of the zebra chip pathogen. Objective 3: Mentor an undergraduate student on a complimentary research project to identify salivary proteins of potato psyllid.
Approach:
Objective 1: Salivary glands of psyllids reared in a lab colony will be dissected in RNAse-free phosphate-buffered saline and 75 pairs of salivary glands per combination of psyllid haplotype and Liberibacter-infection state will be pooled in RNAlater to preserve RNA. Four replications will be included for each of the 6 treatments (36 samples). The glands will be RNA extracted using a Qiagen RNeasy mini kit. Samples will be shipped to Novogene (www.novogene.com) for RNA sequencing using 250 bp insert cDNA library and HiSeq platform, 150 bp paired-end following rigorous quality control measures. Objective 2: Late instar females (50 per treatment) will be injected with CRISPR components (100 ng/ µL sgRNA’s, with 200 ng/µL of Cas9 protein, and 0.1 ng/ µL BAPC) targeting Thirodoxin to produce knock-out G0 and G1 mutants. Insects will be injected ventrally, off center of midline in the abdomen using an Eppendorf FemtoJet 4i. Post injections psyllids will be transferred to a potato plant to oviposit. Control insects will be injected with carrier solution with CRISPR components. After optimizing CRISPR by targeting Thirodoxin, CRISPR products will be designed based on mRNA sequences obtained from Obj. 1, and products will be delivered as determined in Obj. 2a. Psyllid feeding will be assessed using standard bioassays, stylet pathways analysis, electronic penetration graphs, or fluorescence in situ hybridization.
