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Short RNAs associated to Cauliflower mosaic virus infections in Canola and Turnip

ABSTRACT: Three plants of two species in Brassica genus, i.e. B. rapa cv. "Purple To White Globe" and B. napus cv. "SariGol", highly susceptible and tolerant hosts of Cauliflower mosaic virus (CaMV), respectively, were mechanically inoculated with CaMV strain NY8153.Phosphate buffer was used as control in mock-inoculated plants. RNAs, in three biological repeats, were extracted 20 days post-inoculation (dpi) collecting ten leaves from mock-inoculated and virus-infected plants using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Five µg of total RNA were resolved in 15% SDS-PAGE gel. Total RNA pattern was visualized upon Ethidium Bromide staining and sRNAs in the size range of 20-30 nt were eluted from gel. sRNAs replicates were quantified and mixed in equal molarity. Libraries were obtained using NEXTflex Small RNA-Seq Kit v3 (Bio Scientific) according to manufacturer’s instructions. Libraries were eluted from a 6% non-denaturing PAGE. The quality of libraries was checked by BioPharma QC, and then, submitted for sequencing with a HiSeq 2500 Illumina platform.

ORGANISM(S): Brassica rapa Brassica napus

PROVIDER: GSE119398 | GEO | 2020/09/27

REPOSITORIES: GEO

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Project description:Objectives: Our work focuses on the responses of Solanaceous plants to viruses that cause economically important diseases in tree fruits. Using mock inoculated leaf tissue as a reference, we plan to compare the gene expression profiles of Nicotiana Benthamiana plants infected with one of three viruses; Plum Pox Potyvirus (PPV), Tomato Ringspot Nepovirus (ToRSV), and Prunus Nectrotic Ringspot Nepovirus (PNRSV). Our goals are as follows: (1) Identify genes that are induced/repressed in response to individual viruses. (2) Identify genes that are induced/repressed in response to all 3 viruses. (3) Compare results to existing potato array data to look for similarities in responses to other pathogens. Experimental Design: Nicotiana benthamiana plants were inoculated with one of three viruses: PPV, ToRSV, or PNRSV. 3 week old plants were inoculated by rubbing virus infected plant sap onto leaves dusted with carborundum. Control plants were mock inoculated using sap from healthy plants. All plants were maintained in a growth chamber at 22C for 18 days. 8 plants were inoculated with each virus or mock inoculated. This experiment was repeated twice. 4 biological replicates derived from 2 virus infected plants from each replica experiment (4 plants) are to be used for hybridizations. RNA from all mock inoculated plants was similarly pooled to create 4 biological replicates. Each replicate control will serve as a universal reference sample that is to be hybridized pair wise with each of the three virus infected samples. RNA extraction: After 18 days, un-inoculated leaves displaying clear symptoms were harvested and immediately frozen in liquid N2. Total RNA was purified using Trizol according to TIGRs listed protocol. RNA was subsequently treated with Turbo DNA-free RNase (Ambion cat#1907). Finally, total RNA was further purified on RNeasy columns (Qiagen) according to manufacturer’s instructions and quantified using a Nanodrop spectrophotometer. Keywords: Reference design

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Short RNAs associated to Cauliflower mosaic virus infections in Canola and Turnip

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