Oligogalacturonide treatments
Oligogalacturonides (DP 10-15) were prepared as previously described
(Benedetti et al. 2017): A PGA solution [2% (w/v; Alfa Aesar)] was
incubated with endo-polygalacturonase II (0.1 RGU/mL), purified fromAspergillus niger Pectinase (Sigma), for 180 min at 30 °C in a
water bath under gentle shaking. The digest was boiled for 10 min in a
water bath to inactivate the enzyme and cooled at 4 °C on ice.
Oligoalacturonides were precipitated by diluting the solution with cold
50 mM sodium acetate and ice-cold ethanol to a final concentration of
0.5% (w/v) PGA and 17% (v/v) ethanol. The solution was incubated
overnight at 4 °C and centrifuged at 30,000 × g for 30 min to recover
the pellet. This was solubilized and centrifuged at 30,000 × g for 30
min. The supernatant containing the oligogalacturonides was recovered,
dialyzed against ultrapure water in a dialysis tube with a molecular
mass cut-off of 1000 Da (Spectra/Por®) and lyophilized.
Four-week-old tomato plants were treated with aqueous oligogalacturonide
solution (50 µg/mL in milliQ water) either in leaves or roots. A time
course analysis of the response was performed by harvesting leaf
material at 1, 6 and 24h after treatments. For leaf treatments (LT-),
the fourth true leaf of each plant was sprayed with the
oligogalacturonide solution using an aerograph until running off.
Control treatments were carried out with water (CT-). Plastic was used
to cover the rest of the plant during spraying to avoid contact of the
solution with other plant parts. Water was applied similarly for the
control treatment. Treated leaves were harvested at the different time
points after treatment for the study of local responses (LT-TL). The
sixth fully developed untreated leaf of each plant was also harvested to
study systemic leaf responses (LT-SL), and the untreated roots were
harvested to study root systemic responses to leaf treatments (LT-Root).
For the root treatments (RT-), roots were incubated in a 50 µg/mL
oligogalacturonide solution for one hour, water was used as control
treatment. As for leaf treatments, different plant parts were harvested
at 1,6 and 24h following the incubation. Treated roots were harvested
for local responses (RT-Root). The sixth fully developed untreated
leaves were also harvested to study systemic responses in shoots upon OG
root treatments (RT-SL). (Sup. Fig. 1)