Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA...

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Virology

Background:

• Agrobacterium tumefaciens transfers single-stranded DNA (T-strand) to host nuclei for conversion to double-stranded DNA (T-DNA) intermediates.
• The precise role of T-strands versus double-stranded T-DNA intermediates in integration remains unclear.
• DNA REPLICATION FACTOR A (RFA) in yeast binds single-stranded DNA and aids in DNA repair.

Purpose of the Study:

• To investigate the integration pathway of Agrobacterium tumefaciens T-DNA.
• To determine if T-strands are direct substrates for integration or require conversion to double-stranded forms.
• To explore the potential role of DNA REPLICATION FACTOR A (RFA) in T-DNA integration.

Main Methods:

• Generated transgenic tobacco (Nicotiana benthamiana) plants overexpressing yeast RFA subunits (RFA1-RFA3).
• Assessed T-DNA expression using a β-glucuronidase intron reporter gene after Agrobacterium infection and microbombardment.
• Utilized enhanced green fluorescent protein for RFA complex localization studies.
• Evaluated the effect of RFA expression on Bean dwarf mosaic virus movement.

Main Results:

• Overexpression of RFA in tobacco plants significantly decreased T-DNA expression following Agrobacterium infection but not microbombardment.
• RFA complex localized predominantly within the plant cell nucleus.
• RFA expression inhibited the cell-to-cell movement of a single-stranded DNA virus.
• RFA expression inhibited transient T-DNA expression and reduced T-DNA integration by approximately 50%.

Conclusions:

• The yeast RFA complex functions in the plant nucleus, potentially by binding T-strands and preventing their conversion to double-stranded intermediates.
• Both single-stranded T-DNA and double-stranded T-DNA intermediates appear to play significant roles in the Agrobacterium integration process.
• The findings suggest a dual role for T-DNA forms in integration, with RFA influencing the pathway.