Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.
The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.
Raghupathy
Mohan B
MB
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford St., London, Ontario, Canada N5V 4T3.
Griffiths
Jonathan S
JS
Stobbs
Lorne W
LW
Brown
Daniel C W
DC
Brandle
James E
JE
Wang
Aiming
A
D017360:Arabidopsis / Q000235:genetics* / Q000821:virology; D002480:Cellulase / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D018613:Microscopy, Confocal; D010935:Plant Diseases / Q000235:genetics / Q000821:virology*; D018515:Plant Leaves; D017801:Plum Pox Virus / Q000235:genetics*; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection
D004279:DNA, Viral; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D002480:Cellulase; C018506:diacetylfluorescein