Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants

Abstract

Temperature is an important environmental factor influencing plant development in natural and diseased conditions. The growth rate of plants grown at C27°C is more rapid than for plants grown at 21°C. Thus, temperature affects the rate of pathogenesis progression in individual plants. We have analyzed the effect of temperature conditions (either 21°C or 27°C during the day) on the accumulation rate of the virus and satellite RNA (satRNA) in Nicotiana benthamiana plants infected by peanut stunt virus (PSV) with and without its satRNA, at four time points. In addition, we extracted proteins from PSV and PSV plus satRNA-infected plants harvested at 21 dpi, when disease symptoms began to appear on plants grown at 21°C and were well developed on those grown at 27°C, to assess the proteome profile in infected plants compared to mock-inoculated plants grown at these two temperatures, using 2D-gel electrophoresis and mass spectrometry approaches. The accumulation rate of the viral RNAs and satRNA was more rapid at 27°C at the beginning of the infection and then rapidly decreased in PSV-infected plants. At 21 dpi, PSV and satRNA accumulation was higher at 21°C and had a tendency to increase further. In all studied plants grown at 27°C, we observed a significant drop in the identified proteins participating in photosynthesis and carbohydrate metabolism at the proteome level, in comparison to plants maintained at 21°C. On the other hand, the proteins involved in protein metabolic processes were all more abundant in plants grown at 27°C. This was especially evident when PSV-infected plants were analyzed, where increase in abundance of proteins involved in protein synthesis, degradation, and folding was revealed. In mock-inoculated and PSV-infected plants we found an increase in abundance of the majority of stress-related differently-regulated proteins and those associated with protein metabolism. In contrast, in PSV plus satRNA-infected plants the shift in the temperature barely increased the level of stress-related proteins.

Keywords: DIGE; cucumovirus; leaf proteome; plant defense; plant proteomics; plant-virus interactions; satellite RNA; temperature change.

Figure 1

Real-time (quantitative) PCR (RT-qPCR) analysis of the changes in the levels of PSV RNAs and satRNA in Nicotiana benthamiana plants infected with biologically infectious transcripts of PSV-P plus satRNA (A), or PSV RNAs alone (B) at 21 dpi, comparing differences in accumulation between plants maintained at 27°C vs. 21°C. In (A), the change in 2a-encoding RNA was statistically insignificant and in the (B) the change in 1a-endoding RNA was statistically insignificant. The error bars represent standard errors.

Figure 2

Quantification of the number of copies of ORF1 (A), ORF3b (CP; B), and satRNA (C) assessed on the basis of accumulation of all relative to reference gene. The inoculum was wild-type PSV-P naturally containing satRNA. X axis shows the time points analyzed and Y axis shows the number of copies (in thousands) of analyzed RNAs. At 21 dpi, the level of ORF1a, CP, and satRNA tended toward zero. The experiment was repeated twice with similar results. The results derived from a single experiment are shown.

Figure 3

The KEGG analysis of the pathways most affected at the experimental temperature for control (mock-inoculated) plants—blue, PSV-infected plants—yellow, and PSV plus satRNA-infected plants—green circles. Green and red lines indicate the pathways joined by the proteins which were found to be more abundant or less abundant (respectively). The middle panel shows the general view of the studied pathways. (A,B) focus on carbohydrate metabolism and (C,D) focus on amino acid metabolism.