A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant...

Area of Science:

• Plant Pathology
• Molecular Biology
• Genetics

Background:

• Identifying pathogen effectors is crucial for understanding plant-pathogen interactions.
• Functional cloning methods are essential for discovering genes encoding virulence factors.
• The hypersensitive response (HR) is a plant defense mechanism often triggered by pathogen elicitors.

Purpose of the Study:

• To develop and validate a novel, high-throughput functional cloning strategy.
• To isolate cDNAs from plant pathogens that elicit a hypersensitive response (HR) in host plants.
• To identify specific elicitors and other HR-inducing proteins from Cladosporium fulvum.

Main Methods:

• Construction of a cDNA library from Cladosporium fulvum RNA using a potato virus X (PVX)-based expression vector.
• Transformation of the library into Agrobacterium tumefaciens for functional screening.
• High-throughput inoculation of 9600 individual clones onto tomato and Nicotiana plants to identify HR-inducing cDNAs.

Main Results:

• Four cDNAs were identified in tomato, including AVR4, a race-specific elicitor targeting the Cf-4 resistance gene.
• Three other cDNAs encoded proteins homologous to bZIP transcription factors, inducing non-genotype-specific HR.
• Inoculation on Nicotiana revealed four HR-inducing cDNAs encoding the extracellular protein ECP2.

Conclusions:

• The developed functional screening method is a versatile and effective strategy for identifying pathogen cDNAs encoding elicitors.
• This approach successfully identified both race-specific and non-specific HR-inducing proteins from plant pathogens.
• The method demonstrates broad applicability across different plant-pathogen systems.