Sweet potato plantlets with 3-4 leaves were agroinoculated with the infectious SPLCV-JS clone by vacuum infiltration. Details of the protocol for vacuum infiltration with the SPLCV-JS clone are shown in Figure 1. At 20 dpi, all the infected sweet potato plants remained asymptomatic. At a late infection stage (35 dpi), mild symptoms (yellowing) were observed in newly emerged leaves (Figure 2B, arrows); mock-inoculated plants did not show any symptoms (Figure 2A). Symptom evaluation, together with PCR detection, identified five infection-positive plants among the 20 that were inoculated with SPLCV-JS (Figure 3A; Table 1). Only symptomatic plants were PCR-positive. In addition, Southern blot analysis using a probe specific for the SPLCV-JS genome revealed the presence of the typical viral DNA forms, i.e., single-stranded genomic DNA (ssDNA) and double-stranded replicative DNA (Lin) of begomovirus, in agroinoculated Xushu 22 sweet potato plants (Figure 3B, lanes 2 and 3). The experiment was repeated twice independently with similar results. These data support the successful infection of the sweet potato cultivar Xushu 22 by the infectious SPLCV-JS clone using vacuum infiltration.
Figure 1. Protocol for vacuum agroinfiltration of sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone.
Figure 2. Symptoms of sweet potato cultivar Xushu 22 infected with the SPLCV-JS infectious clone by vacuum agroinfilitration. A, Mock control; B, SPLCV-JS; C, SPLCV-JS and TYLCCNV-Y10 DNA-β; and D, TYLCCNV-Y10 DNA-A and DNA β. Symptoms in the leaves are indicated by red arrows.
Figure 3. SPLCV viral DNA accumulation in sweet potato plants infected with the SPLCV-JS infectious clone as determined by PCR detection and Southern blot analysis. Panel A, PCR amplification of the SPLCV-JS C1 gene; Panel B, Southern blot analysis of SPLCV accumulation using a DIG-labeled probe specific to the entire SPLCVJS genome. Geminiviral DNA forms are marked as open circular double-stranded (OC), linear double-stranded (Lin), supercoiled double-stranded (SC), or single-stranded (SS). Panel C, PCR detection of TYLCCNV-Y10 DNA-β. M, Molecular marker; Lane 1, Mock infected (MI); Lanes 2 and 3, SPLCV-JS; Lanes 4 and 5, SPLCV-JS and TYLCCNV-Y10 DNA-β; Lane 6, Field-collected sweet potato Xu 35-5 infected by SPLCV-JS as positive control in panels A and B or the Xushu 22 plants infected by TYLCCNV-Y10 DNA-A and DNA-β as positive controls in panel C.
Inoculum Infected/inoculated plants (n) Symptoms SPLCV-JS 5/20 Mild yellowing in newly emerged leaves SPLCV-JS + TYLCCNV-Y10 DNA-β 6/20 Obvious leaf yellowing and vein clearing TYLCCNV-Y10 DNA-A + DNA-β 2/5 Severe leaf yellowing Empty vector 0/5 Symptomless Empty vector + TYLCCNV-Y10 DNA-β 0/5 Symptomless
Table 1. Infectivity analysis of the SPLCV-JS infectious clone in sweet potato cultivar Xushu 22 plants.
Our previous study reported a synergistic effect of that SPLCV-JS and a heterologous betasatellite DNA(TYLCCNV-Y10 DNA-β)on enhanced symptom severity and viral DNA accumulation in N. benthamiana (Bi H, et al., 2012). We further tested that effect in sweet potato plants agroinoculated concomitantly with both SPLCVJS and TYLCCNV-Y10 DNA-β infectious clones. The enhancement of symptom severity by TYLCCNV-Y10 DNA-β was shown at 35 dpi by increased severity of obvious leaf yellowing and vein clearing in six of the 20 plants that were inoculated(Figure 2C, arrows). In addition, two of the five plants agroinoculated with TYLCCNV-Y10 DNA-A and DNA-β developed severe leaf yellowing in the new leaves, somewhat similar the appearance of an albino seedling(Figure 2D, the arrows). The experiment was repeated twice independently with similar results.
In Southern blot analysis, the accumulation of replicative DNA forms including open circular double-stranded (OC), supercoiled double-stranded (SC), and single-stranded (ss) DNA were observed in the plants inoculated with SPLCV-JS and TYLCCNV-Y10 DNA-β (Figure 3B, lanes 4 and 5). The accumulation of SPLCVJS viral DNA was more obvious in these mix-infected plants than that seen with SPLCV-JS infection alone. No signal was detected in the mock-infected control plants (Figure 3B, lane 1) using a probe specific for the entire SPLCV-JS genome. Additionally, a field-collected, diseased Xu 35-5 s weet potato sample plant that was used to amplify the SPLCV-JS genome and served as a positive control showed a potent SPLCV signal (Figure 3B, lane 6). PCR assays confirmed the presence of DNA-β in sweet potato plants coinoculated with SPLCV-JS and TYLCCNV-Y10 DNA-β (Figure 3C, lanes 4-5) and in plants infected with TYLCCNV-Y10 DNA-A and DNA-β (Figure 3C, lane 6). Thus, we showed that SPLCV-JS interacts synergistically with the heterologous betasatellite in sweet potato, much like the results seen in N. benthamiana (Bi H, et al., 2012).