Chloroplast Hsp70 isoform is required for efficient replication of Bamboo mosaic virus in old leaves of Nicotiana benthamiana
Y. HUANG (1), C. Hu (1), N. Lin (2), Y. Hsu (1) (1) Graduate Institute of Biotechnology, National Chung Hsing University, Taiwan; (2) Institute of Plant and Microbial Biology, Academia Sinica, Taiwan

Positive strand RNA virus replication occurs within modified cellular membrane-associated complexes that consist of viral replicase as well as host proteins. In this study, the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) was found in the Bamboo mosaic virus (BaMV) replicase pull-down complexes, suggesting that NbcpHsp70 was associated with BaMV replication protein either in a direct or indirect way. BaMV RNA accumulation was significantly decreased in protoplasts with NbcpHsp70-2 specifically silenced, indicating that NbcpHsp70-2 but not NbcpHsp70-1 was involved in the efficient replication of BaMV. Overexpression of NbcpHsp70-2 does not affect BaMV RNA accumulation in N. benthamiana plants; however, when the transit peptide-deletion mutant of NbcpHsp70-2 was transiently expressed, we observed a reduction in BaMV RNA accumulation, suggesting that the importation of NbcpHsp70-2 into chloroplast is important for BaMV replication. NbcpHsp70-2 is more similar to Arabidopsis AtcpHsp70-1 which has been found to preferentially import into older chloroplasts, and the age-dependent import is regulated by two consecutive positive charges of the transit peptide. We also found that BaMV infection occurs preferentially in old leaves rather than in young ones. Overexpression of the transit peptide mutant, in which Arg24 and Lys25 are both mutated to Glu (RK2425EE), of NbcpHsp70-2 down-regulated the BaMV RNA accumulation in old leaves of N. benthamiana. According to these data, we hypothesized that BaMV recruited NbcpHsp70-2 for the assembly of replicase complexes in association with chloroplast of old leaves through the direct or indirect interaction with BaMV replication protein.

Abstract Number: P7-173
Session Type: Poster