HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

Abstract

Background: RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent).

Results: Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were also decreased in these plants, apparently leading to decreased transmethylation capacity. The proteome analysis using 2D-PAGE indicated significantly altered proteome profile, which may have been both due to altered transcript levels, decreased translation, and increased proteosomal/protease activity.

Conclusion: Expression of the HC-Pro RSS mimics transcriptional changes previously shown to occur in plants infected with intact viruses (e.g. Tobacco etch virus, TEV). The results indicate that the HC-Pro RSS contributes a significant part of virus-plant interactions by changing the levels of multiple cellular RNAs and proteins.

Figure 1

Phenotypes observed in Nicotiana tabacum plants expressing HC-Pro transgene. A typical morphology of flowers is indicated in the upper part of the figure (A-C). A wild type tobacco flower is presented in A, a vector control flower (pBIN61) in B and a transgenic HC-Pro expressing flower in C. Phenotypes of two wild type tobacco plants at the flowering state (on the left) and one vector control plant (pBIN61, in between of these wild type plants) and four transgenic HC-Pro expressing plants are presented in D. One representative of one-month old wild type tobacco plant (E) and one transgenic HC-Pro expressing plant (F) demonstrating differences in growth and leaf morphology. A growing pattern of 10 one-month old wild type tobacco plants (G) and 10 transgenic HC-Pro expressing plants (H) are presented at the bottom of the figure

Figure 2

Starch granules at the bottom of Eppenforf tube pelleted during thylakoid preparation. For each of thylakoid isolation, 1 g of wild type (WT) or transgenic HC-Pro (HC-Pro) leaves (fresh weight, FW) was used. Three biological replicates are presented in the figure. The amount of starch was also quantified after removing the soluble sugars (on the right). The quantification of starch indicated about four-times less starch in HC-Pro expressing leaf samples than in wild type tobacco leaf samples (n = 4).

Figure 3

Light-responsive O₂-evolution of photosystem II was measured of wild type (WT) and HC-Pro expressing plants. O₂-evolution was measured of freshly isolated thylakoid membranes using DCBQ as an electron acceptor. Standard error of mean is presented as bars abobe the columns (n = 6, consisting of three biological and two technical replicates).

Figure 4

Proteome analysis of two biological replicates of wild type (WT) and HC-Pro expressing plants (HC-Pro). Proteins isolated from leaves were separated by using 2D-polyacrylamide gel electroforesis (2D-PAGE). Proteins in two isoelectric focused strips (WT and HC-Pro) were separated the second dimension in a large SDS-polyacrylamide gel. Upper gels (A and B) are stained using colloidal coomassie blue and the lower gel (C) using silver staining. White circles indicate control protein spots, whose intensity was not changed and black circles indicate protein spots that were either increased (1, RBCL and 4, PsaN, CP12) or decreased (2, OEE33 and 3, CYP2) in HC-Pro expressing plants. The identity of numbered protein spots was analysed using LC-ESI MS/MS mass spectrometry.