Induction of HSP70 and polyubiquitin expression associated with plant virus replication

Abstract

By examining the front of virus invasion in immature pea embryos infected with pea seed-borne mosaic virus (PSbMV), the selective control of different host genes has been observed. From our observations, the early responses to PSbMV replication can be grouped into three classes, inhibited host gene expression, induced host gene expression, and no effect on a normal host function. The expression of two heat-inducible genes encoding HSP70 and polyubiquitin was induced coordinately with the onset of virus replication and the down-regulation of two other genes encoding lipoxygenase and heat shock cognate protein. The down-regulation was part of a general suppression of host gene expression that may be achieved through the degradation of host transcripts. We discuss the possibilities of whether the induction of HSP70 and polyubiquitin genes represents a requirement for the respective protein products by the virus or is merely a consequence of the depletion of other host transcripts. The former is feasible, as the induction of both genes does result in increased HSP70 and ubiquitin accumulation. This also indicates that, in contrast to some animal virus infections, there is not a general inhibition of translation of host mRNAs following PSbMV infection. This selective control of host gene expression was observed in all cell types of the embryo and identifies mechanisms of cellular disruption that could act as triggers for symptom expression.

Figure 1

Selective regulation of host gene expression following PSbMV infection of pea cotyledonary tissues. The orientation of the pea cotyledon sections is shown in (A). B–E, F and G, H–K, and L and M were from four separate embryos. Sections B–G and H–M are from healthy and PSbMV-infected cotyledons, respectively. The sections were subjected to in situ hybridization with probes for PSbMV −ve RNA (B and H), PSbMV +ve RNA (C, F, I, and L), PsHSP71.2 RNA (D and J), polyubiquitin RNA (E and K), and PsHSC71.0 RNA (G and M). The zone of HSP70 and polyubiquitin induction correspond with the front of virus invasion and the onset of virus replication (H–K, arrows). In contrast, HSC70 expression is reduced at the front of virus invasion (L and M, white arrows).

Figure 2

Spatial correlation between the zones of transcript depletion and HSP70 induction. B–D were cut from the same PSbMV-infected cotyledon in the area shown in A; B and C are adjacent sections. Sections were subjected to in situ hybridization with probes for lox1:Ps:2 RNA (B), PsHSP71.2 RNA (C) and both probes together (D). The transcript depletion seen for lox1:Ps:2 appears to involve a rapid loss of transcript from the cytoplasm and the nucleus (B, Inset: compare the signal in the cytoplasm, c, and the nucleus, n, on the right and left of the panel, i.e., outside and inside the infection front, respectively). The zone of PsHSP71.2 induction corresponds with the first few cells of the zone of lox1:Ps:2 RNA depletion (B and C, arrows). When both probes are combined (D) the transcript-depleted zone gets correspondingly narrower. These data are summarized graphically (E).

Figure 3

Accumulation of viral and host proteins at the front of virus invasion. B and C and D–F, respectively, were cut from two different infected embryos. The orientation of the sections is illustrated in A. Sections were subjected to in situ hybridization with probes for PSbMV +ve RNA (B) or 18S rRNA (C), or immunohistochemistry with antibodies for PSbMV coat protein (D), HSP70 (E), or ubiquitin (F). rRNA was uniformly distributed throughout the infected cotyledon. Cells at the front of virus invasion (arrows) accumulated viral and induced host proteins.

Figure 4

Selective control of host gene expression in the infected embryonic radicle. B–E and F–I were sectioned from single healthy and infected embryos, respectively, as illustrated in A. Sections were subjected to in situ hybridization with probes for PSbMV +ve RNA (B and F), polyubiquitin RNA (C and G), lox1:Ps:2 RNA (D and H) or PsHSP71.2 RNA (E and I). As observed in cotyledons, coordinate induction of polyubiquitin and HSP70 and down-regulation of lipoxygenase expression were observed. Note, to visualize the tissue structure in the absence of hybridization signal, these sections were photographed under joint-transmitted and epifluorescent light after staining the tissue sections with calcofluor.