Pathogen induced suppression of ABA enhancesFv/Fm reduction.
ABA biosynthesis and signalling is hijacked by DC3000 to suppress immunity (de Torres-Zabala et al. , 2007; De Torres Zabalaet al. , 2009). The impact of ABA mutants on virulence is reflected in Fv/Fm signatures (de Torres Zabala et al. , 2015). As ABA is made predominately in the chloroplasts, we investigated whether high light induced susceptibility was underpinned by ABA signalling. We monitored the impact of an ABA hypersusceptible signalling mutant (triple mutant) or the ABA insensitive biosynthetic mutant Arabidopsis aldehyde oxidase 3(aao3 ) on infection under normal and high light, monitoring bothFv/Fm and non-photochemical quenching (NPQ), the latter measuring the energy released as heat. Theaao3 mutant exhibited less suppression ofFv/Fm during DC3000 infection compared to Col-0 plants, reflected also in slightly lower levels of NPQ in comparison to Col-0 (Figure 8A, B, Sup Fig 4a, b). By contrast the hypersensitive triple PP2C mutant (abi1/abi2/hab1 ) shows a faster decrease in Fv/Fm and a stronger increase in NPQ compared to Col-0 (Figure 8A, B, Supp Figure 4a, b). As previously reported (de Torres-Zabala et al. , 2007; De Torres Zabala et al. , 2009; Rubio et al. , 2009) under normal light conditions aao3 plants are more resistant to DC3000 while the triple PP2C mutant is more susceptible (Figure 8C). However, under high light (450 µmol m-2s-1) Col-0 and aao3 plants are more susceptible however there was no enhanced susceptibility evident in the triple PP2C mutant (Figure 8C) implying either ABA signalling is important for high light enhanced susceptibility or the abi1/abi2/hab1 plants cannot support further bacterial multiplication. In addition, Col-0 plants grown under high light show accumulation of ABA after 5 days of high light growth and 9 days of high light with subsequent DC3000 infection compared to no increase in ABA under normal light or bacterial infection (Supp Figure 3c). In contrast the aao3 plants do not show an increase in ABA under normal or high light (Supp Figure 3c).
To next assess the interaction of ABA and light on chloroplast function during pathogen infection 10 µM ABA was co-infiltrated with DC3000 into Col-0 leaves. Under normal light, 10 µM ABA co-infiltration enhances the decrease in Fv/Fm levels as previously reported (de Torres Zabala et al. , 2015)(Figure 8D, E). Under high light conditions infiltration with 10 µM and 100 µM ABA treatments show a faster decrease ofFv/Fm levels from 3.5 hours onwards (Figure 8F, G). To ensure that 10 and 100 µM ABA treatment was not toxic to P. syringae , DC3000 was plated on Kings B agar containing 0, 10, 50 and 100 µM ABA. Bacterial growth showed that there is a small reduction in growth of bacteria in the presence of ABA, notably being significant (p<0.0005)with increased ABA concentration, (Supp Figure 4c).