Antagonism in Orthotospoviruses Is Reflected in Plant Small RNA Profile

Figure 1. Sample preparation for ELISA (a) and data showing antagonistic interaction of INSV and TSWV (b). The X-axis represents the percentage of virus inoculum, as shown in (a), and the Y-axis indicates the O.D.405nm values obtained from ELISA. Left panel (b): INSV titer was reduced in mixed infections (orange line) compared to single INSV infections (blue line). Right panel (b): similarly, the TSWV titer (orange line) in mixed infection was lower than in single TSWV infections (violet line). N. benthamiana plants were mechanically inoculated using five different dilutions (five plants per dilution) for each virus. ELISA results from single-virus infections were compared with those from mixed infections, where each dilution was spiked with an equal amount of the second virus. Statistical analysis using MANCOVA showed a significant decrease in viral titer for both viruses in mixed infections compared to single infections, with p-values confirming this effect across inoculum dilutions.

Figure 2. Sample preparation for ELISA (a), and data showing antagonistic interaction of INSV and TSWV when they were inoculated sequentially on different leaves, with a one-hour interval between inoculations (b). The X-axis represents the percentage of virus inoculum as shown in (a), and the Y-axis indicates the O.D.405nm values obtained from ELISA. Left panel (b): INSV titer was reduced in mixed infections (orange line) compared to single INSV infections (blue line). Right panel (b): Similarly, TSWV titer (orange line) in mixed infection was lower than in single TSWV infections (violet line). We used five different inoculum dilutions (X, 0.8X, 0.6X, 0.4X, and 0.2X), with five plants per dilution for each virus and analyzed the differences between the regression lines of single and mixed infections using MANCOVA. Although the O.Ds. were lower in the mixed infections than the single infections in both cases, it was not significant (p-values were higher than 0.05). However, the higher Pillai’s trace values (INSV: 0.78; TSWV: 0.79) indicate a strong group effect.

Figure 3. Summary of small RNA reads.

Figure 4. Hotspot distributions on the viral genome. Peaks represent multiple reads aligned to the genome in the same position. Y-axis: number of reads. X-axis: nucleotide position on the viral genome. (a) INSV single and mixed infection. (b) TSWV single and mixed infection. Blue: viral sense; (Red: viral antisense. Figures were generated using MISIS. L, M, and S segments are represented by the consecutive segments with color codes on the solid lines between the top and bottom panels. For INSV, L segment: 1–8774nt, M segment: 8775–13,751nt, and S segment: 13,752–16,761nt. For TSWV, L segment: 1–8914nt, M segment: 8915–13,679nt, and S segment: 13,689–16,663nt.

Figure 5. Summary of small RNA reads based on their size. NC: mock-inoculated negative control. Size distribution of host-endogenous sRNAs, vsRNAs from INSV, and vsRNAs from TSWV (a–c).

Figure 6. Host miRNAs and their differential regulation in different treatments (NC: mock-inoculated control, INSV, TSWV, and mixed infections. (a) PCA plot based on de novo identified host miRNA clusters. (b–d) Mean abundance plot of N. benthamiana miRNA loci comparing two different treatments. Significantly upregulated and downregulated miRNA loci are highlighted (alternative hypothesis: FDR ≤ 0.1, true difference > 2-fold after Benjamini–Hochberg correction for multiple testing).