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

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 23 hybs total

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Multi-omics analysis of small RNA, transcriptome and degradome of RSV-infected Nicotiana benthamiana - the regulatory networks of Nicothiana benthamiana genes in response to RSV infection

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