Persistent Southern Tomato Virus (STV) Interacts with Cucumber Mosaic and/or Pepino Mosaic Virus in Mixed- Infections Modifying Plant Symptoms, Viral Titer and Small RNA Accumulation

Abstract

Southern tomato virus (STV) is a persistent virus that was, at the beginning, associated with some tomato fruit disorders. Subsequent studies showed that the virus did not induce apparent symptoms in single infections. Accordingly, the reported symptoms could be induced by the interaction of STV with other viruses, which frequently infect tomato. Here, we studied the effect of STV in co- and triple-infections with Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). Our results showed complex interactions among these viruses. Co-infections leaded to a synergism between STV and CMV or PepMV: STV increased CMV titer and plant symptoms at early infection stages, whereas PepMV only exacerbated the plant symptoms. CMV and PepMV co-infection showed an antagonistic interaction with a strong decrease of CMV titer and a modification of the plant symptoms with respect to the single infections. However, the presence of STV in a triple-infection abolished this antagonism, restoring the CMV titer and plant symptoms. The siRNAs analysis showed a total of 78 miRNAs, with 47 corresponding to novel miRNAs in tomato, which were expressed differentially in the plants that were infected with these viruses with respect to the control mock-inoculated plants. These miRNAs were involved in the regulation of important functions and their number and expression level varied, depending on the virus combination. The number of vsiRNAs in STV single-infected tomato plants was very small, but STV vsiRNAs increased with the presence of CMV and PepMV. Additionally, the rates of CMV and PepMV vsiRNAs varied depending on the virus combination. The frequencies of vsiRNAs in the viral genomes were not uniform, but they were not influenced by other viruses.

Keywords: Amalgaviridae; antagonism; miRNAs; mixed-infections; persistent virus; synergism; vsiRNAs.

Figure 1

Mosaic and deformation leaf symptoms showed by tomato plants infected with Southern tomato, Pepino mosaic, and Cucumber mosaic viruses (STV, PepMV, and CMV, respectively) in single and mixed infections. Symptoms were considered as mild, moderate or severe (Panels B, C, and D, respectively). The right part of (Panel D) shows a strong leaf deformation in the plant shoots in tomato plants with severe symptoms. Panel A shows a symptomless leaf corresponding to a mock-inoculated tomato plant. (Panel E) shows three tomato leaves showing mild, moderate, or severe symptoms (from left to the right).

Figure 2

Graphic representation (mean values) of leaf symptoms severity (ordinate axis) of tomato plants infected with STV, PepMV, and CMV in single and mixed infections at 5, 10, 15, and 20 dpi (abscise axis). Leaf symptoms intensity was scored from 0 to 3, where 0 corresponds to symptomless, and 1, 2, and 3 to mild, moderate, and severe symptoms, respectively. Bars and letters up to the columns correspond to standard errors (from 0 to 0.23) and different plant groups (p-value ≤ 0.05), respectively.

Figure 3

Graphical representation (mean values) of height (Panel A) and weight (Panel B) measured in cm and g, respectively, of tomato plants infected with STV, PepMV, and CMV in single and mixed infections at 20 dpi. Bars and letters up to the columns correspond to standard errors and different plant groups (p-value ≤ 0.05), respectively. At the bottom of (Panel A), we show the height of tomato plants infected with different virus combinations in comparison with mock-inoculated plants.

Figure 4

Virus accumulation (mean values) in tomato plants (ordinated axis) shown as log of concentration (no. RNA copies/ng of total RNA) of STV, PepMV, and CMV (Panel A, B, C, respectively) in single and mixed infections at 5, 10, 15, and 20 dpi (abscise axis). Bars and letters up to the columns correspond to standard errors and plant groups (in each dpi), respectively, showing differences (p-value ≤ 0.05). In each panel, virus accumulation is represented by columns (right) and in lineal representation (left).

Figure 5

Graphic representation of the percentages (%) of vsiRNAs respect to the useful reads obtained by small RNA high throughput sequencing of the different STV, CMV, and PepMV virus combinations. The percentages of vsiRNAs were obtained from the useful reads mean values of the three biological replicates.

Figure 6

Graphical representation of plus (+) (blue) and minus (−) (orange) vsiRNAs frequencies along STV (Panel A), CMV (Panel B), and PepMV (Panel C) genomes for the different virus combinations. Virus genome organization is showed in the bottom of each graph. STV and CMV vsiRNAs frequencies were not represented, since amounts of vsiRNAs were so low in STV-single infected and CMV + PepMV double- infected tomato plants.