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The replicase associated Cucumber necrosis virus p33 targets to peroxisomes and is associated with the induction of necrosis in agro-infiltrated plants Singh, Bhavana

Abstract

Replication of Cucumber necrosis virus (CNV) RNA requires two viral-encoded proteins, p33 and its read-through product p92, both of which are believed to be components of CNV replicase enzyme. In this study we have investigated the subcellular location of CNV p33 in order to gain further insight into the CNV replication process. A p33/GFP fusion protein was cloned in an Agrobacterium tumefaciens binary vector and used to agroinfiltrate leaves of Nicotiana benthamiana. Co-infiltration experiments using a YFP-labeled peroxisomal marker (pYFP-SKL) along with confocal microscopy showed that the p33/GFP fusion protein targets to the peroxisomal membrane. In addition, peroxisomes of p33/GFP inoculated cells often showed a high level of aggregation. We also report that patches of necrosis-like symptoms develop on p33/GFP infiltrated leaves suggesting that p33 contributes to the necrotic symptoms typically observed in CNV-infected plants. Similar agroinfiltration experiments were conducted with CNV p20 which is the viral suppressor of gene silencing and a protein previously suggested to modulate symptom induction. The results indicate that p20 is not capable of inducing necrotic-like symptoms on its own in agroinfiltrated plants. Phenol red assays to assess'hydrogen peroxide production in leaf samples failed to show a clear correlation between necrosis and levels of peroxide accumulation in control and p33/GFP infiltrated plants. However, the DAB staining method indicated that peroxide accumulation occurred in p33/GFP infiltrated and CNV infected plants but not control plants. The association of CNV p33 with peroxisomes and its ability to induce necrosis-like symptoms in infiltrated plants suggests that p33 may contribute to the necrotic-like reaction via disruption of peroxisomal function. However, the involvement of peroxide accumulation in necrosis will require further experimentation.

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