Experiments

Below you will find information regarding the 2 Experimental Modules for this event:

Module 1: Plants fight back

Module 2: Plant's helpers

 

Module 1: Plants fight back
Plant defense mechanisms against bacterial and viral pathogens

Objectives:

  • Identify changes in the transcriptional defense response of maize caused by a bacterial virulence protein

  •  Identify Arabidopsis genes that condition resistance or enhanced susceptibility to geminiviruses

Plan Outline:

Participants will conduct RNAseq, from sample preparation to data analysis, on the maize transcriptome following infection with Pantoea stewartii, the causal agent of Stewart’s bacterial wilt and leaf blight of maize. Infections will be done with wild-type P. stewartii and a mutant strain lacking a key virulence factor - the type III secreted protein, WtsE. Preliminary data indicate that the mutant strain fails to induce 1) expression of phenylpropanoid metabolic genes and 2) a phenolic compound predicted to be produced by those genes. RNAseq will reveal a complete and quantitative measure of gene expression following P. stewartii infection with and without WtsE.

Participants will assess phenotypes following inoculation of selected Arabidopsis mutants with geminiviruses (Beet curly top virus, Cabbage leaf curl virus).  Viral genome methylation has recently been shown to be an especially potent defense against DNA viruses.  Mutants will correspond to genes known or suspected to be components of chromatin modification pathways, either as "writers" or "readers" of epigenetic marks.  Phenotypic assessment will include analysis of symptoms, viral DNA levels, and virus-derived small RNAs.  The ability of selected cellular proteins to associate with the viral genome will be assessed using chromatin immunoprecipitation (ChIP).

Specific Techniques Covered:

  • Isolation of total RNA, rRNA depletion, and RNA quantification
  • cDNA synthesis and quantitative PCR to check sample quality
  • cDNA fragmentation and barcoding
  • Acquisition and filtering of Illumina sequencing data
  • Quantification of transcript levels and identification of differentially regulated genes
  • Southern blot hybridization analysis
  • Northern blot analysis of small RNA
  • Chromatin immunoprecipitation (ChIP)

 

Module 2: Plant's Helpers
Functional genomics of plant-associated bacteria

Objectives:

  • Identify, clone, and characterize microbial genes with a defined function in plant-microbe interactions

Plan Outline:

Participants will identify DNA sequences involved in the biological control of plant pathogens. Specifically, we will engage in a multi-step process to identify functionally important genes using a combination of mutant phenotyping and genomic sequencing. Students will evaluate phenotypic data to select mutants to study, clone and sequence genes interrupted by transposon mutagenesis. They will also learn about standard annotation of bacterial genome sequences and how to use the information generated to clone and complement bacterial mutants for functional analysis. Special emphasis will be placed on the genotype-to-phenotype association.

Specific Techniques Covered:

  • PCR cloning and sequencing of amplified products
  • Plasmid isolation
  • Genome sequence annotation and operon identification
  • Local BLAST analysis
  • Primer design and targeted gene cloning
0