Virus Isolation and Rice Protoplast Infection.

Area of Science:

• Plant Pathology
• Molecular Virology
• Agricultural Science

Background:

• Rice (Oryza sativa) is crucial for global food security but faces threats from diseases like rice stripe disease, caused by rice stripe virus (RSV).
• Traditional methods for studying plant antiviral defenses, such as natural or artificial infections and transgenic approaches, are time-consuming, often requiring months for plant transformation.

Purpose of the Study:

• To develop a rapid, transgene-independent protocol for analyzing rice stripe virus-host interactions.
• To significantly accelerate the screening of antiviral mechanisms and resistance genes in rice.

Main Methods:

• The protocol involves polyethylene glycol (PEG)-based precipitation of RSV virions from infected tissues.
• Purification is achieved through differential ultracentrifugation with optimized glycerol cushions.
• High-efficiency transfection of purified RSV virions into rice protoplasts is performed using PEG-mediated delivery, followed by RT-qPCR and immunoblotting for viral replication assessment.

Main Results:

• The streamlined protocol enables the analysis of RSV-host interactions within three days, a substantial reduction from conventional methods.
• The method allows for the simultaneous introduction of exogenous plasmids for functional studies without relying on stable transgenic lines.
• Purified RSV virions demonstrated strong infection capacity in rice protoplasts when co-transfected with exogenous plasmids.

Conclusions:

• This transgene-independent protocol significantly accelerates antiviral determinant profiling from months to just three days.
• The rapid analysis facilitates quicker exploration of antiviral mechanisms and efficient screening for rice resistance genes.
• This method offers a valuable tool for advancing research on rice disease resistance and crop improvement.