HSP70 homolog functions in cell-to-cell movement of a plant virus

Abstract

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Figure 1

(A) Diagram of the BYV genome with ORFs from 1a to 8, encoding leader proteinase (L-Pro), replication-associated proteins possessing putative methyltransferase (MET), RNA helicase (HEL), and RNA polymerase (POL) domains, 6-kDa protein (p6), HSP70 homolog (HSP70h), 64-kDa protein (p64), minor capsid protein (CPm), green fluorescent protein (GFP; ORF F, for foreign), major capsid protein (CP), 20-kDa protein (p20), and 21-kDa protein (p21). The bottom part of A is a close-up showing the reporter GFP ORF and CP ORF with adjacent promoter regions governing their expression. Selected restriction endonuclease sites are those mentioned in Materials and Methods. (B) Mutations introduced into the ORF encoding HSP70h (cross-hatched box). Point mutations and XhoI sites are shown above the box, whereas the location of the two major HSP70h domains and large deletion mutations are shown below the box.

Figure 2

Cell-to-cell movement of the GFP-expressing BYV (BYV-GFP) and its mutant variant lacking most of the HSP70h ORF (BYV-GFP-ΔXho) in mature leaves of Claytonia perfoliata. Photographs were taken with a fluorescent microscope and a GFP-specific filter. (Scale bars represent 25 μm.)

Figure 3

Size distribution analysis of the fluorescent infection foci produced by RNA transcripts generated from pBYV-GFP and its variants, possessing mutations in the HSP70h ORF at 12 d.p.i. (A) Comparison between the parental BYV variant (BYV-GFP, derived from pBYV-GFP cDNA clone) and six mutant variants (see the text for further explanation). Because all the mutants exhibited identical movement-deficient phenotypes, the corresponding data were combined. (B) Comparison between the three revertant variants in which the original mutations were changed back to the wild-type (“Yes”) variants and parental mutant variants. Because all revertants exhibited identical movement-competent phenotypes, the corresponding data were combined. n, number of analyzed infection foci.