Plant basal resistance - Universiteit Utrecht

Chapter 3
leaves deposited callose in a dose-dependent manner (Figure S4), confirming that chitosan-
induce defence of maize is marked by callose deposition. In Arabidopsis, PAMP-induced
callose requires endogenous production of indolic GSs (Clay et al., 2009), suggesting a
signalling function by these secondary metabolites. Since monocots do not produce indolic
GSs, we tested whether BXs fulfil a similar role in maize innate immunity. To this end, we
treated leaves with non-saturating concentrations of chitosan (0.05%) and assayed callose
deposition in leaves of BX-producing Bx1 igl lines and BX-deficient bx1 igl lines revealed that
the BX-deficient lines deposit significantly lower amounts of chitosan-induced callose than
the BX-producing lines (Figure 7). Hence, PAMP-induced callose requires regulation by one
or more Bx1-dependent metabolites. Since chitosan increases DIMBOA and HDMBOA-glc
in the apoplast (Figure 5), we examined which of these two compounds are responsible for
Bx1-dependent callose. To this end, we quantified callose intensities at 24 h after apoplast
infiltration with either of both compounds. As observed for Bx gene repression (Figure 6),
DIMBOA was active and triggered callose deposition in a dose-dependent manner, whereas
HDMBOA-glc was inactive and failed to boost callose deposition (Figure 8). Hence, DIMBOA
functions as an extracellular signal for PAMP-induced callose.
Figure 7: Bx1 regulates chitosan-induced callose deposition. Leaf segments from igl single mutant
lines and bx1 igl double mutant lines were infiltrated with chitosan (0.05%) or mock solution. At 24 h
after infiltration, leaf segments were collected for aniline-blue staining, UV-epifluoresence microscopy,
and digital quantification of callose intensity. Shown are fold-induction values of callose (± SEM; n =
15), relative to the average callose intensity in mock-treated Bx1 igl lines from each cross. Photographs
show representative differences in fluorescent callose signals under UV-epifluoresence microscopy.
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