Figure 2-18: Symptoms on N. benthamiana leaves inoculated with SACMV-VIGS constructs at 14 dpi in SACMV-challenged and SACMV unchallenged plants [A], [B] SACMV-VIGS vector; [C], [D] SACMV::M15.1 plants; [E], [F] SACMV::M8.B plants; [G], [H] SACMV::M11.F plants; [I], [J] SACMV::M11.2 plants
Figure 2-19: Symptoms on N. benthamiana leaves inoculated with SACMV-VIGS constructs at 28 dpi in SACMV-challenged and SACMV unchallenged plants
[A], [B] SACMV-VIGS vector; [C], [D] SACMV::M15.1 plants; [E], [F] SACMV::M8.B plants; [G], [H] SACMV::M11.F plants; [I], [J] SACMV::M11.2 plants.
Figure 2-20: Symptoms on N. benthamiana leaves inoculated with TRV-VIGS constructs at 14 dpi in SACMV-challenged and SACMV unchallenged plants
[A], [B] TRV-VIGS vector; [C], [D] TRV::M15.1 plants; [E], [F] TRV::M8.B plants; [G], [H] TRV::M11.F plants; [I], [J] TRV::M11.2 plants.
Figure 2-21: Symptoms on N. benthamiana leaves inoculated with TRV-VIGS constructs at 28 dpi in SACMV-challenged and SACMV unchallenged plants
[A], [B] TRV-VIGS vector; [C], [D] TRV::M15.1 plants; [E], [F] TRV::M8.B plants; [G], [H] TRV::M11.F plants; [I], [J] TRV::M11.2 plants.
Typical geminiviral symptom such as curling, blistering and leaf area reduction was observed with varying degree in all infected plants (figure 2-18-figure 2-21). Plants were scored according to the 0 to 5 scale, with 0 indicating no symptoms and 5 severe leaf reduction (Allie and Rey 2013). Plant agroinfiltrated with VIGS constructs showed minimal to no symptoms. SACMV::M15.1 plants agroinfiltrated plants had a score of 2 with the rest of the plants scoring 1 or less and the SSS were not reported.
The SSS for SACMV-challenged/SACMV-VIGS plants revealed that at 14 dpi, SACMV- challenged/SACMV::M15.1 plants had a higher SSS (score 3) and SACMV- challenged/SACMV::M11.F plants infected plants had lower SSS (score 1.5) then the SACMV- challenged/SACMV-VIGS vector (score 2). The SSS of SACMV-challenged/SACMV::M8.B, SACMV-challenged/SACMV::M11.2 plants and SACMV-challenged/SACMV::M11.K plants weren’t significantly different to SACMV-challenged/SACMV-VIGS vector. At 28 dpi, SACMV- challenged/SACMV::M8.B plants and SACMV-challenged/SACMV::M11.K plants had a lower SSS (score 3 for both vs score 5 for the control) whilst the SSS revealed no difference for SACMV-challenged/SACMV::M15.1, SACMV-challenged/SACMV::M11.F plants and SACMV- challenged/SACMV::M11.2 plants relative to SACMV-challenged/SACMV-VIGS vector (figure 2-22a; appendix A1.2.11 ).
In SACMV-challenged/TRV-VIGS plants, at 14 dpi, SSS in SACMV-challenged/TRV::M15.1, SACMV-challenged/TRV::M8.B plants and SACMV-challenged/TRV::M11.F plants were higher (score 3) than in of SACMV-challenged/TRV-VIGS vector (score 2). No difference was observed in SSS for SACMV-challenged/TRV::M11.2 and SACMV-challenged/TRV::M11.K plants compared to SACMV-challenged/TRV-VIGS vector. At 28 dpi, no difference in SSS was observed between SACMV-challenged/TRV VIGS-silenced myosin constructs and SACMV- challenged/TRV-VIGS vector (figure 2-22b; appendix A1.2.11).
To assess for the worsening of symptoms, often likened to an increase in infection, the SSS between the 2 time points were compared and for SACMV-challenged/SACMV VIGS- silenced myosin plants, there was a significant increase in symptoms from 14 dpi to 28 dpi for all the plants except for SACMV-challenged/SACMV::M11.K, where the increase in symptom was not significant (figure 2-22a). Comparing the scores between 14 and 28 dpi
for SACMV-challenge TRV-VIGS myosin silencing plants, there was a significant increase in symptoms from 14 dpi to 28 dpi, across all plants (figure 2-22b; appendix A1.2.12).
A
B
Figure 2-22: SSS measured at 14 and 28 days post inoculation (dpi) in
N. benthamiana infected with SACMV-VIGS construct [A] and TRV-
VIGS constructs [B]
Symptomatic leaves of infected plants were scored at 14 and 28 dpi according to the 0 to 5 SSS, with 0 being the least symptomatic or uninfected and 5 being the most infected. [A] SACMV-challenged/SACMV::myosin (SM) and SACMV-challenged/SACMV-VIGS (SA-IC) and [B] TRV-SACMV- challenged/VIGS::myosin (TM) and SACMV-challenged/TRV-VIGS vector (TR- IC). The median value of three independent plants is plotted and the error bars represent the standard error of the mean.
Comparing the SSS in SACMV-challenged/SACMV::myosin plants to their corresponding SACMV-challenged/TRV::myosin plants, a 2-way ANOVA analysis revealed that on average,
SSS for SACMV-challenged/TRV-VIGS plants were higher compared to SACMV- challenged/SACMV-VIGS plants (2.57 vs 2.32 at 14 dpi and 3.81 p value 0.01 vs 4.17 at 28, p value 0.03; A1.3.4). A Pearson’s correlation analysis revealed at 14 dpi, a significant positive correlation between DNA-A viral load and SSS for SACMV-challenged/SACMV::M8.B (0.64), a negative correlation between viral load and SSS for SACMV-challenged/TRV::M11.F plants (- 0.68). At 28 dpi, there was a negative correlation between DNA viral load and SSS for SACMV-challenged/SACMV::M15.1 plants (-0.73) and SACMV-challenged/SACMV::M11.F plants (0.85). There were no significant correlations between and SSS for SACMV- challenged/TRV-VIGS (appendix A1.4.1).
Stunting is a known symptom that occurs with geminivirus infection, and the height of SACMV-challenged plants was measured to assess whether the presence of myosin VIGS constructs affected plant height. The height of SACMV-challenged/SACMV-VIGS vector plants at 14 dpi was not different to SACMV-challenged/SACMV::myosin, except for SACMV- challenged/SACMV::M8.B plants that was on average shorter. By 28 dpi however, plant height of SACMV-challenged/SACMV::M8.B plants was not different to SACMV-challenged/ SACMV-VIGS vector plants, while SACMV-challenged/SACMV::M15.1, SACMV- challenged/SACMV::M11.F, SACMV-challenged/SACMV::M11.2 plants and SACMV- challenged/SACMV::M11.K plants all grew taller (figure 2-23a; appendix A1.2.13). With SACMV-challenged/TRV-VIGS, at 14 dpi SACMV-challenged/TRV::M15.1 plants grew taller than the SACMV-challenged/TRV-VIGS, and at 28 dpi all SACMV-challenged/TRV::myosin grew taller than SACMV-challenged/TRV-VIGS vector plants and there was no statistical different in height between SACMV-challenged/TRV-VIGS vector and the other SACMV- challenged/TRV::myosin (figure 2-23b). At 28 dpi, all SACMV-challenged/TRV::myosin plants grew taller than SACMV-challenged/TRV-VIGS vector (figure 2-23b; appendix A1.2.13). All the SACMV-challenged plants grew significantly at 28 dpi, from 14 dpi (A1.2.14). A 2-way ANOVA study (appendix A1.3.5) revealed that at 14 and 28 dpi, infected TRV-VIGS plants were shorter than SACMV-VIGS plants (4.50 vs 4.86 at 14 dpi and 9.69 vs 13.56 at 28 dpi. p value 0.00). A Pearson’s correlation analysis revealed, a negative correlation between DNA- A viral load and height in at 14 dpi for SACMV-challenged/SACMV::M15.1 plants (-0.63), SACMV-challenged/SACMV::M8.B plants (-1) , SACMV-challenged/SACMV::M11.F (-0.83) and SACMV-challenged/SACMV::M11.2 plants (-0.61). At 28 dpi, there was a negative
correlation between SACMV-challenged/SACMV-VIGS vector (-0.79) and SACMV- challenged/TRV::M8.B (-0.60). There was a positive correlation between SACMV- challenged/SACMV::M15.1 (0.83). No correlation was observed for the other samples (appendix A1.4.2).
A
B
Figure 2-23: Plant height measured at 14 and 28 days post inoculation (dpi) in
N. benthamiana infected with SACMV
The height of infected plant was measured at 14 and 28 dpi. [A] SACMV- challenged/SACMV::myosin (SM) and SACMV-challenged/SACMV-VIGS (SA-IC) and [B] TRV-SACMV-challenged/VIGS::myosin (TM) and SACMV-challenged/TRV-VIGS vector (TR-IC). The median values of three independent plants are plotted and the error bars represent the standard error of the mean.