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. 2000 Oct;12(10):1975-86.
doi: 10.1105/tpc.12.10.1975.

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons

Z Havelda  1 , A J Maule
Affiliations

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons

Z Havelda et al. Plant Cell. 2000 Oct.

Abstract

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP(+)-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.

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Figures

Figure 1.
Figure 1.
Gel Blot Analysis of Host mRNAs from CMV-Infected and Mock-Inoculated Tissues. Total mRNAs, prepared from CMV-infected (lanes 1) or mock-inoculated (lanes 2) tissues, were separated under denaturing conditions, blotted, and hybridized with probes (top) for HSP70, GAP-DH-A, GAP-DH-B, NADP+-ME, NAD+-ME, or G6P-DH mRNAs. Relative gel loadings are shown from ethidium bromide staining of the rRNAs (bottom).
Figure 2.
Figure 2.
Spatial Analysis of the Expression of HSP70 Associated with CMV Infection. (A) to (C) Consecutive sections of CMV-infected tissues analyzed by in situ hybridization using a negative-sense (A) or a positive-sense (C) RNA probe to detect HSP70 mRNAs or by immunocytochemistry using antiserum to CMV CP (B). The alignment is shown relative to the edge of the CMV lesions (dotted lines). Irrespective of the probe used, in situ hybridization resulted in the formation of artifactual dark spots within the infected areas, as shown by (e.g.) the arrow in (C). (D) and (E) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The direction of virus movement from infected to uninfected tissues is identified (white arrowhead) between (D) and (E). (F) to (H) Consecutive sections of control mock-inoculated tissues analyzed by in situ hybridization using a negative-sense (F) or a positive-sense (H) RNA probe to detect HSP70 mRNAs or by immunocytochemistry using antiserum to CMV CP (G). Comparing (A) with (B) and (F) shows that infection increased HSP70 mRNA to more than that seen in mock-inoculated tissues. This increase occurred at the edge of the lesion (arrowheads), which on closer examination ([D] and [E]) was seen to span the edge of the infected zone to include approximately five cells in the apparently uninfected area. PM, palisade mesophyll layer; SM, spongy mesophyll layer. [Formula: see text]; [Formula: see text].
Figure 3.
Figure 3.
Spatial Analysis of the Expression of GAP-DH-A Associated with CMV Infection. (A) to (C) Consecutive sections of CMV-infected tissues analyzed by in situ hybridization using a negative-sense (A) or a positive-sense (C) RNA probe to detect GAP-DH-A mRNAs or by immunocytochemistry using antiserum to CMV CP (B). The alignment is shown relative to the edge of the CMV lesions (dotted lines). (D) and (E) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The direction of virus movement from infected to uninfected tissues is identified (white arrowhead) between (D) and (E). (F) to (H) Consecutive sections of control mock-inoculated tissues analyzed by in situ hybridization using a negative-sense (F) or a positive-sense (H) RNA probe to detect GAP-DH-A mRNAs or by immunocytochemistry using antiserum to CMV CP (G). (I) Relative GAP-DH-A enzyme activity across the lesion (redrawn from Técsi et al., 1996). In mock-inoculated tissues, GAP-DH-A was expressed predominantly in the palisade mesophyll layer (F). This was also true in infected tissues except within the zone of infection, which was markedly depleted in transcript accumulation, as shown in (A) and (D). This decrease correlates with a decrease in GAP-DH enzyme activity within the lesion (I). PM, palisade mesophyll layer; SM, spongy mesophyll layer. [Formula: see text]; [Formula: see text].
Figure 4.
Figure 4.
Spatial Analysis of the Expression of GAP-DH-B Associated with CMV Infection. (A) to (C) Consecutive sections of CMV-infected tissues analyzed by in situ hybridization using a negative-sense (A) or a positive-sense (C) RNA probe to detect GAP-DH-B mRNAs or by immunocytochemistry using antiserum to CMV CP (B). The alignment is shown relative to the edge of the CMV lesions (dotted lines). (D) and (E) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The direction of virus movement from infected to uninfected tissues is identified (white arrowhead) between (D) and (E). (F) to (H) Consecutive sections of control mock-inoculated tissues analyzed by in situ hybridization using a negative-sense (F) or a positive-sense (H) RNA probe to detect GAP-DH-B mRNAs or by immunocytochemistry using antiserum to CMV CP (G). (I) Relative GAP-DH-B enzyme activity across the lesion (redrawn from Técsi et al., 1996). In mock-inoculated tissues, GAP-DH-B was expressed predominantly in the palisade mesophyll layer (F). This was also true in infected tissues except within the zone of infection; see (A) and (D). As with GAP-DH-A, there was a marked depletion in transcript accumulation although with some delay, as seen by comparing (D) and (E) with Figures 3D and 3E. Again, this decrease correlates with a decrease in GAP-DH activity within the lesion (I). PM, palisade mesophyll layer; SM, spongy mesophyll layer. [Formula: see text]; [Formula: see text].
Figure 5.
Figure 5.
Spatial Analysis of the Expression of NADP+-ME Associated with CMV Infection. (A) to (C) Consecutive sections of CMV-infected tissues analyzed by in situ hybridization using a negative-sense (A) or a positive-sense (C) RNA probe to detect NADP+-ME mRNAs or by immunocytochemistry using antiserum to CMV CP (B). The alignment is shown relative to the edge of the CMV lesions (dotted lines). (D) and (E) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The direction of virus movement from infected to uninfected tissues is identified (white arrowhead) between (D) and (E). (F) to (H) Shown are consecutive sections of control mock-inoculated tissues analyzed by in situ hybridization using a negative-sense (F) or a positive-sense (H) RNA probe to detect NADP+-ME mRNAs or by immunocytochemistry using antiserum to CMV CP (G). (I) Relative NADP+-ME enzyme activity across the lesion (redrawn from Técsi et al., 1996). In mock-inoculated tissues (F), NADP+-ME was expressed predominantly in the vascular tissues and in the palisade and the upper spongy mesophyll layers. In infected tissues (A), a complex pattern of expression was seen. Within the lesion, NADP+-ME transcripts were markedly depleted from the mesophyll layers but not from the vascular tissues (white arrow). Outside the lesion, an increase in NADP+-ME expression (arrowheads) extended for ∼0.5 mm from the edge of the lesion; beyond this point, expression was equivalent to that seen in uninfected tissues (black arrow). This expression profile correlated with the activity profile across the lesion (I). PM, palisade mesophyll layer; SM, spongy mesophyll layer; V, vascular tissues. [Formula: see text]; [Formula: see text].
Figure 6.
Figure 6.
Colocalization of the CMV CP and MP in Infected Tissues. (A) and (B) Consecutive sections of CMV-infected cotyledonary tissues subjected to immunocytochemical analysis with antiserum specific for the viral movement (A) or CP (B), respectively. The alignment is shown relative to the edge of the CMV lesion (dotted lines between arrowheads). (C) and (D) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The locations of the two proteins correspond across the lesion, although the distribution of the MP (C) extends by one to two cells beyond that of the CP (D). (E) and (F) Consecutive sections of control, mock-inoculated tissues treated with antisera specific for the MP and CP, respectively. PM, palisade mesophyll layer; SM, spongy mesophyll layer. [Formula: see text]; [Formula: see text].
Figure 7.
Figure 7.
Spatial Analysis of the Expression of Catalase Associated with CMV Infection. (A) to (C) Consecutive sections of CMV-infected tissues analyzed by in situ hybridization using a negative-sense (A) or a positive-sense (C) RNA probe to detect catalase mRNA or by immunocytochemistry using antiserum to CMV CP (B). The alignment is shown relative to the edge of the CMV lesions (dotted lines). (D) and (E) Magnification (×2.5) of the alignment at one edge of a lesion, as marked by the triangles in (A) and (B). The direction of virus movement from infected to uninfected tissues is identified (white arrowhead) between (D) and (E). (F) to (H) Consecutive sections of control mock-inoculated tissues analyzed by in situ hybridization using a negative-sense (F) or a positive-sense (H) RNA probe to detect catalase mRNAs or by immunocytochemistry using antiserum to CMV CP (G). (I) Gel blot analysis of catalase mRNA (top) extracted from infected (lanes 1) or mock-inoculated (lanes 2) cotyledons. Relative gel loadings are shown from ethidium bromide staining of the rRNAs (bottom). In mock-inoculated tissues, catalase was expressed predominantly in the vascular tissues (F). In infected tissues, expression extended to the upper layers of the spongy mesophyll (A), where it accumulated uniformly in the uninfected areas in the cotyledon lamina. The catalase transcripts were markedly depleted within the lesion. This downregulation abruptly coincided with the edge of lesion (i.e., the zone of maximal virus accumulation). Despite the combination of upregulation and downregulation, an increase in the average steady state amounts of catalase mRNA was detected by gel blot analysis of total RNA (I). PM, palisade mesophyll layer; SM, spongy mesophyll layer; V, vascular tissues. [Formula: see text]; [Formula: see text].
Figure 8.
Figure 8.
Summary of Changes in Host Gene Expression in Response to CMV Infection. For all the host genes studied by in situ hybridization, there was a shutoff of expression within the central region of the CMV-infected area. Three types of upregulation were observed: HSP70 mRNA showed increased accumulation in a narrow band of cells crossing the infection front, NADP+-ME mRNA showed increased accumulation at and in advance of the infection front, and catalase mRNA showed a uniform increase across the uninfected tissues. Except for the catalase mRNA (asterisk), all of the host mRNAs showed a basal level of accumulation equivalent to that in mock-inoculated tissues beyond 0.5 mm from the edge of the lesion.
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