Virology 260, 173–181 (1999) Article ID viro.1999.9807, available online at http://www.idealibrary.com on Subcellular Localization of the HSP70-Homolog Encoded by Beet Yellows Closterovirus Vicente Medina,* Valery V. Peremyslov,† Yuka Hagiwara,† and Valerian V. Dolja†,‡,1 *Department de Producio Vegetal I Ciencia Forestal, Universitat de Lleida, Avenida Alcalde Rovira Roure 177, 25198 Lleida, Spain; and †Department of Botany and Plant Pathology and ‡Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331 Received March 12, 1999; returned to author for revision April 11, 1999; accepted May 12, 1999 Closteroviridae is the only viral family coding for a homolog of HSP70 (HSP70h). Polyclonal antiserum to recombinant beet yellows closterovirus (BYV) HSP70h was generated and used for immunogold labeling of the leaf samples derived from the infected Nicotiana benthamiana plants. Ultrastructural analysis revealed the preferential accumulation of BYV in phloem, although occasional infection of the leaf mesophyll cells was also observed. The strongest HSP70h-specific labeling was associated with virion aggregates and vesicles harboring scattered virions. HSP70h was also observed in close proximity of plasmodesmata and inside the plasmodesmatal channels. The possible role of the BYV HSP70h in RNA encapsidation was tested in tobacco protoplasts. A BYV mutant possessing an inactivated HSP70h gene exhibited no detectable encapsidation defects. Collectively, the obtained results suggested that closteroviral HSP70h escorts the virions to their destinations inside the infected cells and possibly participates in the intercellular translocation of BYV. © 1999 Academic Press INTRODUCTION lov et al., 1998). Other possibilities included involve- ment of the HSP70h in viral translocation from cell to The proteins from the HSP70 family of molecular cell (Karasev et al., 1992; Agranovsky et al., 1998), chaperones are conserved among unicellular and mul- systemic transport, assembly of the filamentous viri- ticellular organisms (Tatusov et al., 1997; Chervitz et ons, or transmission of virus by insects (Tian et al., al., 1998; Guy and Li, 1998). Many DNA and RNA 1999). viruses of bacteria, plants, and animals recruit cell HSP70s at various stages of the life cycle (e.g., Mace- Knowledge of intracellular localization provides impor- jak and Sarnow, 1992; Jindal and Young, 1992; Cripe et tant clues as to the functions of the viral proteins and al., 1995) or regulate expression of the nuclear HSP70 mechanisms of their interaction with cell components genes (Phillips et al., 1991; Wainberg et al., 1997). In (for recent examples, see Rodriguez-Cerezo et al., 1997; particular, a plant potyvirus specifically induces tran- Schaad et al., 1997; Ward et al., 1997; Medina et al., 1998; scription of one of the host HSP70 genes in cells that Heinlein et al., 1998). The ultrastructure of the BYV-in- support active viral replication (Aranda et al., 1996). fected cells and tissues of Beta vulgaris and Tetragonia However, viral-encoded HSP70 homologs (HSP70h) expansa has been examined extensively to reveal pref- are found only among the plant closteroviruses. The erential association of virus with the phloem and its presence of the HSP70h gene in all members of the ability to exit into mesophyll cells late in infection (Esau, diverse family Closteroviridae (Agranovsky et al., 1991; 1960a, 1960b; Esau and Hoefert, 1971a, 1971b, 1971c). Pappu et al., 1994; Klaassen et al., 1995; Karasev et al., These and following studies described the cytopathology 1996; Tian et al., 1996; Jelkmann et al., 1997; Ling et al., of closterovirus infection, with the formation of cytoplas- 1998; Zhu et al., 1998; D’Ann Rochon, personal com- mic vesicles containing virions as its most striking fea- munication) suggests that this gene was acquired by ture (Lesemann, 1988). This early work, however, did not the common ancestor of closteroviruses via recombi- address the distribution of closterovirus proteins in in- nation with the cell mRNA (Dolja et al., 1994b). It was fected cells. demonstrated recently that although HSP70h of beet Here we report the subcellular localization of the yellows closterovirus (BYV) is expressed early in in- BYV HSP70h in Nicotiana benthamiana. It was found fection (unpublished results), it is not required for that HSP70h is associated with the virions present in amplification of the viral positive-strand RNA genome cytoplasm, in the nucleus, and in the vicinity of plas- or for transcription of the subgenomic RNAs (Peremys- modesmata. In addition, HSP70h was observed inside the plasmodesmata. Based on these results, we sug- gest that HSP70h accompanies virions and probably 1 To whom reprint requests should be addressed. Fax: (541) 737- targets them to plasmodesmata for translocation into 3573. E-mail: [email protected]. adjacent cells. 0042-6822/99 $30.00 173 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 174 MEDINA ET AL. FIG. 1. Cytopathology of a BYV-infected phloem companion cell. (A) Transverse section of a minor vein from young N. benthamiana leaf. The cell types are designated as S (sieve elements), CC (companion cells), PP (phloem parenchyma), BS (bundle sheath), and T (tracheary elements). The rectangle marks the area shown in B. (B) A region in companion cell containing abundant BYV-induced vesicles (Ve) and virion aggregates (V). Ch, chloroplast. Bar in A, 7.3 mm; in B, 0.91 mm. RESULTS strictly limited to, the phloem, a pattern previously de- scribed for sugar beet (Esau, 1960a, 1960b) and T. ex- Histological distribution of BYV in the leaves of pansa (Esau and Hoefert, 1971a, 1971b). Nicotiana benthamiana Although N. benthamiana is known to be one of the Colocalization of the HSP70h with BYV virions most permissive hosts for plant viruses, it has not been previously reported as a BYV host. We found that N. The IGL analysis of HSP70h distribution in BYV-in- benthamiana was readily infected by BYV after aphid- fected cells revealed that the most conspicuous associ- assisted transmission or mechanical inoculation, exhib- ation of gold particles was with masses of virions (Figs. iting vein clearing, leaf deformation, and eventually sys- 3A and 3B). To test this apparent colocalization of the temic necrosis and plant death (data not shown). Elec- virions and HSP70h, serial sections of the same infected tron microscopic (EM) examination of the vascular cell were subjected to IGL using anti-HSP70h or anti-CP bundles in infected leaves revealed formation of charac- serum. The highest density of gold particles was ob- teristic virus-induced vesicles (Fig. 1). The cytosol of a served in similar locations on both images (cf. Figs. 3C companion cell shown in Fig. 1B is packed by the vesi- and 3D). The relatively low overall density of HSP70h- cles intermingled with scattered filamentous virions and specific IGL can be attributed to the down-regulation of virion aggregates. Immunogold labeling (IGL) using an- the HSP70h production resulting in ;100-fold difference tiserum to BYV capsid protein (CP) resulted in specific in expression levels of the CP and HSP70h (unpublished association of gold particles with virions but not with the observation). thicker filaments presumably formed by the phloem pro- Further support for the association between HSP70h tein (Pp in Fig. 2A). In addition to their most consistent and virions was obtained using statistical analysis of the presence in the phloem companion and parenchyma distribution of gold particles in various areas of the cells, vesicles and virions were also observed in the infected and noninfected cells (Table 1). As expected, mesophyll cells (Fig. 2B). These results demonstrated application of IGL with anti-CP serum revealed the high- that in N. benthamiana BYV was associated with, but not est specific labeling of virion masses and virions scat- SUBCELLULAR LOCALIZATION OF VIRAL HSP70 175 FIG. 2. Immunogold labeling of BYV-infected cells using anticapsid protein serum. (A) Companion cell (bottom left) showing strong labeling of virion masses (V) and virions scattered in cytoplasm. Note that the fibrous material in the center presumably formed by the phloem protein (Pp) is not labeled. CW, cell wall between companion and bundle sheath (top right) cells. N, nucleus; M, mitochondrion; Ch, chloroplast. (B) Mesophyll cell showing labeled virion masses and vesicles (Ve). The size of gold microspheres is 30 nm. Bar in A, 1.5 mm; in B, 2.3 mm. tered among vesicles in the cytoplasm. Some CP label- vicinity of the plasmodesmata (Fig. 4C). Examination of ing was observed in the nuclei and chloroplasts but not infected cells using the CP-specific IGL revealed only in mitochondria or cell walls (Table 1). Application of the marginal levels of labeling of plasmodesmatal channels HSP70h-specific IGL revealed a very similar pattern of (1.06 6 1.66 and 0.8 6 1.23 for 10 and 30 nm gold, distribution that was reproduced using two sizes of gold respectively). Given the much higher overall abundance particles: 30 and 10 nm (Table 1). Similar analysis of of the CP compared with that of HSP70h, these results noninfected cells revealed very low background, demon- suggest that HSP70h is a resident plasmodesmatal pro- strating the specificity of the labeling. Taken together, tein, whereas association of the CP and/or virions with these results clearly indicate that the virions and HSP70h intercellular channels is only weak or transient. colocalize in BYV-infected cells of N. benthamiana. RNA encapsidation analysis using the HSP70h- Relation of the HSP70h and virions to plasmodesmata deficient BYV mutant Inspection of the HSP70h-specific IGL images re- Colocalization of HSP70h and virions could be inter- vealed consistent labeling of the intercellular plasmod- preted as indication of an HSP70h role in virion assem- esmatal channels (Figs. 4A and 4B). Analysis of 30 plas- bly. To test this possibility, we used a mutant in which the modesmata found in infected cells yielded a mean of start codon in the ORF encoding HSP70h has been 2.6 6 2.3 gold particles per plasmodesma.