VAPA, an Innovative “Virus-Acquisition Phenotyping Assay” Opens New Horizons in Research into the Vector-Transmission of Plant Viruses
Figure 2
Transmission of CaMV from infected protoplasts.
A. Influence of acquisition time on transmission of CaMV from infected protoplasts. After a fasting period of 60 min, aphids were allowed access to protoplasts for 15–45 min (<60 min), 60 min, or 75–180 min (>60 min) before transfer to healthy test plants. Acquisition times shorter than 60 min diminished somewhat, but not significantly transmission (P = 0.142; df = 2; Kruskal-Wallis test). Also acquisition times longer than 60 min were not associated with any significant difference in transmission rate compared to a 60-min acquisition. The graph presents data from 57 assays of 24 test plants each (9 lots for acquisition times shorter than 60 min, 17 lots for an acquisition time of 60 min, and 31 lots for acquisition times longer than 60 min). B. Effect of fasting on CaMV transmission from infected protoplasts. Aphids were starved for between 15 and 45 min (<60 min), for 60 min, or between 90 and 240 min (>60 min). They were then allowed a 60-min acquisition access period on protoplasts before transfer to test plants for inoculation. Under our conditions, no prominent effect of starvation on transmission was observed, although starving times shorter than 60 min seemed to reduce transmission. However, the effect was not significant (P = 0.083; df = 2; Kruskal-Wallis test). Data are from 59 tests using 24 plants each (9 assays for fasting of less then 60 min, 19 assays for a fasting time of 60 min, and 31 assays with fasting times longer then 60 min. Note that the data for 60 min fasting contains data from (A). C. Transmission of CaMV from protoplasts absolutely requires living cells. Intact (live) or ultrasonicated (dead) infected protoplasts were offered to aphids in acquisition assays. Only living protoplasts supported transmission. Data are from 11 assays of 24 test plants for each condition. The difference between the two acquisition conditions was highly significant (P<0.001; Mann-Whitney test). D. Ultrasonication does not inactivate CaMV. Infected protoplasts were disrupted by ultrasonication and then transmitted by rub-inoculation. The graph shows that ultrasonicated protoplasts performed at least as well as intact protoplasts in mechanical transmission. Three lots of 24 plants were tested per condition. E. Oryzalin inhibits transmission of CaMV. Protoplasts were incubated for 60 min with DMSO (Ctl) or 10–50 µM oryzalin (Ory) before being offered to aphids for virus acquisition. After a 60-min access period, aphids were transferred to test plants for inoculation. The data shown are from 11 sets of 24 plants for each condition. One set was excluded from analysis because it was an outlier. The difference between the two conditions is significant (P = 0.014; Mann-Whitney test). F. Oryzalin does not impair in vitro acquisition of CaMV. Purified CaMV particles were mixed with helper component P2 and P3 and offered with or without 50 µM oryzalin to aphids for a 30-min acquisition access feeding period, before the insects were used for inoculation of test plants (10 aphids per plant). Three 24 plant lots were used per condition. All graphs present mean values ± standard deviation.
