Development of a GFP expression vector for Cucurbit chlorotic yellows virus

Area of Science:

• Plant Virology
• Molecular Biology

Background:

• Cucurbit chlorotic yellows virus (CCYV) is a bipartite crinivirus causing significant damage to cucurbit crops.
• Previous development of an infectious CCYV clone laid the groundwork for further genetic manipulation.
• Construction of crinivirus green fluorescence protein (GFP) expression vectors remains an underexplored area.

Purpose of the Study:

• To develop a functional CCYV green fluorescence protein (GFP) expression vector.
• To investigate the efficacy of different promoter strategies for GFP and coat protein (CP) expression.
• To analyze the movement and localization of CCYV within host plants using GFP expression.

Main Methods:

• Utilized an "add a gene" strategy based on the CCYV RNA2 cDNA construct.
• Generated three distinct CCYV GFP expression vector clones: pCCYVGFP_SGC, pCCYVGFP_CGC, and pCCYVGFP_CGS.
• Employed different promoters to drive GFP and CP expression in the constructed vectors.

Main Results:

• GFP fluorescence was observed in leaf veins and surrounding cells by 25 days post-inoculation (dpi).
• The pCCYVGFP_CGC clone demonstrated significant cell-to-cell spread by 25 dpi, unlike other clones.
• Phloem-limited localization of GFP expression in systemic leaves was confirmed for pCCYVGFP_CGC at later time points (30, 40, 50 dpi).

Conclusions:

• Successfully developed a CCYV GFP expression vector.
• The new vector facilitates detailed studies on CCYV movement within cucurbit plants.
• This tool is valuable for understanding crinivirus-host interactions and disease progression.