Unknown,Transcriptomics,Genomics,Proteomics

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Application of Functional Genomics to the Study of Solanacea's Defense Mechanisms against Xanthomonas sp. Pathogenicity

ABSTRACT: Xanthomonas campestris pv. campestris (Xcc) is a major bacterial pathogen of cruciferous plants worldwide. The pathogen produces polysaccharides including extracellular cyclic glucan, xanthan, and extracellular enzymes that are key virulence factors. Different Xcc mutants (8397-defective in xanthan and 8523- defective in extracellular glucans) have been obtained and characterized in previously, wich shown to be less infective than the wild type strain when inoculated in N.benthamiana, wich has shown to be an excellent model for the study of Xcc-plant interaction. The objective of this work is evaluate this compounds functions in the plant-pathogen interaction, in particular in the plant transcriptoma modulation to confer susceptibility or resistance to the infection. Plant gene expression profiles would be obtained from independently inoculated leaves of N.benthamiana with the following strains of xanthomonas: Xcc. 8004 (wild type), Xcc. 8397 (xanthan minus), Xcc. 8523 (glucan minus), ant water (control). Leaves discs will be collected at 24 hs post infection and immediately submerged in liquid N2. Total RNA will be extracted with plant RNA specific kits (RNA easy QIAGen), treated with DNase, purified and quantified. Keywords: Reference design 11 hybs total

ORGANISM(S): Nicotiana benthamiana

SUBMITTER: Jia Liu

PROVIDER: E-GEOD-8276 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 23 hybs total

Project description:Treatment of the seeds. The Solanum lycopersicum var. Money Maker wild type (MM) and fri1-1 mutant seeds were incubated on distilled-water saturated cotton wool in clear plastic boxes wrapped in black plastic sheets in darkness during 3 hours at 25°C. After that, the seeds were treated as follows: a) 24 hr of continuous irradiation with far-red light, FRc; b) 24 hr of FRc follow by a 10 min red light pulse (Rp), FRc+Rp; and c) complete darkness. After the light treatments, the seeds were incubated in darkness (25°C) until sampling. Red light (35umol/m2sec) was provided by 40W fluorescent lamps (Phillips 40/15) in combination with a red translucid acrylic. FRc light (100 umol/m2sec) was provided by a set of 150W incandescent internal reflector lamps (Phillips) in combination with a red acetate filter, six 2mm thick blue acrylic filters Paolini 2031 and 10cm water filter. Total RNA isolation. The seeds were grinded in liquid nitrogen and extracted with two volumes of extraction buffer (1% SDS, 6% p-aminosalycilic acid, 1% NaCl, 6% water-saturated phenol and 2% 2-mercaptoethanol) and two volumes of acid phenol:chloroform solution. The aqueous phase was sequentially extracted with one volume of acid phenol:chloroform solution and one volume of chloroform. The nucleic acids in the aqueous phase were precipitated with 0.15 volumes of 3M NaCl and 2.5 volumes of 100% ethanol at –80°C for 20 minutes. After centrifugation, the pellet was washed with 70% ethanol, resuspended in RNase-free water and precipitated with one volume of 4M LiCl over night at 4°C. The LiCl was removed after centrifugation and the pellet was rinsed with 2M LiCl and resuspended in RNase-free water. The total RNA was treated with RNase-free DNase (Promega) and cleaned up with mini spin columns (RNeasy Plant Mini Kit, Qiagen). The mRNA was amplificated with Message Amp II aRNA Amplification Kit (Ambion) and concentrated by vacumm centrifugation. Keywords: Direct comparison 21 hybs total

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Application of Functional Genomics to the Study of Solanacea's Defense Mechanisms against Xanthomonas sp. Pathogenicity

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