In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and...

Area of Science:

• Plant pathology
• Molecular biology
• Biotechnology

Background:

• Powdery mildew infection triggers specific gene expression in barley.
• Understanding these host responses requires isolating infection-specific messenger RNAs (mRNAs).
• Existing methods may lack the precision to target these specific cellular events.

Purpose of the Study:

• To develop and validate a technique for cloning cDNAs of mRNAs specifically expressed at plant pathogen infection sites.
• To identify barley genes involved in the response to powdery mildew infection.
• To adapt this technique for analyzing pathogen-derived mRNAs.

Main Methods:

• Application of polymerase chain reaction (PCR) combined with pricking microinjection.
• In situ synthesis of first-strand cDNAs within infected barley epidermal cells.
• Subcloning amplified cDNAs into a plasmid vector to create a cDNA library.
• In vitro transcription of antisense RNAs for labeling and microinjection.
• Identification of target cDNAs using in situ hybridization.

Main Results:

• Successful isolation of cDNAs corresponding to mRNAs expressed at powdery mildew infection sites in barley.
• Demonstration of the technique's efficacy in identifying specific host-pathogen interactions.
• Validation of the method for amplifying and identifying cDNAs from targeted pathogen infection structures.

Conclusions:

• The combined PCR and microinjection technique is effective for isolating infection-specific cDNAs from host plant tissues.
• This method provides a powerful tool for studying gene expression during plant-pathogen interactions.
• The technique is adaptable for analyzing both host and pathogen gene expression at infection sites.