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Microbiology JoVE (Open Access) Citable · DOI

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

DOI: 10.3791/55150-v
What you'll learn
  • ✓ Collect infected plant tissue samples from field-grown hosts
  • ✓ Perform yeast two-hybrid screening to identify effector protein interactions
  • ✓ Analyze and validate positive clones from selective growth plates
  • ✓ Interpret results to determine bacterial effector binding partners
Protocol

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Difficulty
advanced
Total time
~5–7 days (sample collection ~2 hrs; yeast screening ~3–5 days; clone analysis ~1–2 days)
Model organism
Apple tree (Malus domestica) infected with non-cultivable bacterial pathogen
Biosafety
BSL-1

Steps

1
Collect root and leaf samples from infected apple trees

Harvest diseased root and leaf tissue from naturally infected apple trees in the field. Samples are used as source material for pathogen detection and downstream molecular analysis.

▶ 00:48
2
Perform yeast two-hybrid screen for effector binding partners

Conduct a yeast two-hybrid assay using bacterial effector protein as bait to screen a plant cDNA library and identify interacting host proteins. Grow yeast on selective media to isolate positive clones.

▶ 02:22
3
Analyze clones from selective growth plates

Characterize and verify positive yeast clones using colony PCR, sequencing, or re-transformation assays to confirm genuine effector–host protein interactions.

▶ 07:47
4
Interpret results to identify bacterial effector interactions

Evaluate yeast two-hybrid data to determine which host plant proteins bind the effector, linking molecular interactions to pathogenicity mechanisms in the non-cultivable bacterial pathogen.

▶ 08:50
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