EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen...

Area of Science:

• Plant Pathology
• Molecular Biology
• Genomics

Background:

• Plant pathogenic microbes secrete effector proteins to manipulate host plants, influencing infection and defense.
• Identifying these effectors is crucial for understanding plant-microbe interactions and developing disease resistance strategies.

Purpose of the Study:

• To develop and apply a general functional genomics approach for identifying extracellular effector proteins from plant pathogens.
• To discover novel effector proteins from Phytophthora infestans using a combination of data mining and functional expression assays.

Main Methods:

• Development of PexFinder, an algorithm for automated identification of extracellular proteins from expressed sequence tags (ESTs).
• Application of PexFinder to EST data from Phytophthora infestans, followed by proteomic validation.
• High-throughput functional expression assays using a potato virus X (PVX)-based system in plants to test identified cDNAs for effector function.

Main Results:

• PexFinder identified 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs from 2147 ESTs, with 55% being novel.
• Functional assays led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2.
• crn1 and crn2 encode extracellular proteins expressed during host colonization, with crn2 inducing defense responses in tomato.

Conclusions:

• Combining data mining with PexFinder and PVX-based functional assays is an effective strategy for discovering novel pathogen effector proteins.
• This approach can be broadly applied to various eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.
• The identified crn genes represent a significant discovery in understanding Phytophthora effector mechanisms and plant defense.