Caracterización del contexto escolar.

2.4.1. Reactive oxygen species (ROS) production

To measure the production of reactive oxygen species in different genotypes a previously described method was used (Boutrot et al., 2010). Briefly, approximately 12 leaf discs per genotype were taken from leaves 5 and 7 from adult A. thaliana plants using a No. 1 cork borer, placed into a white 96-well plate and kept overnight on distilled water. Assay solution with luminol (100μM final), horseradish peroxidase (10μg/mL) and flg22

Antibody

Name Target Protein/Epitope Company Concentration

Primary Antibody

α-FLAG (Mouse) FLAG tag Sigma 1:10,000

α-H3 (Rabbit) Histone H3 (no PTM) Millipore 1:10,000

α-H4 (Rabbit) Histone H4 (no PTM) Millipore 1:4,000

α-H3Ac (Rabbit) Histone 3 Lys 9/14 _(Acetylated) Millipore 1:5,000

α-H3K9Ac

(Rabbit) Histone 3 Lys 9 (Acetylated) Millipore 1:5,000

α-H3K14Ac

(Rabbit) Histone 3 Lys 14 (Acetylated) Millipore 1:5,000

α-p44-ERK/p42 Phosphorylated MPK6,3,4 Cell _Signalling 1:5,000

α-His (Mouse) His-tag Sigma 1:10,000

α-HA HA-tag Sigma 1:2,000

α-GFP GFP-tag ChromoTek 1:2,000

Secondary Antibody

α-Mouse-HRP

(Goat) Mouse IgG Sigma 1:10,000

α-Rabbit-HRP Rabbit IgG Sigma 1:10,000

TrueBlot™ Non-denatured proteins Sigma 1:10,000

Affinity Resin

GFP Beads GFP-tag ChromoTek

HA Beads HA-tag ChromoTek

(100nM) was freshly prepared the next day. Luminescence was recorded using a High Resolution Photon Counting System (Photek) for 1 hour af- ter treatment.

2.4.3. Mitogen-Activated Protein Kinase (MAPK) activation assay

Seedlings were grown on solid MS for two weeks and then trans- ferred on liquid MS. Seedlings were then treated with 100 nM flg22 solu- tion and flash frozen in liquid nitrogen at different time points. Frozen tissue was ground and proteins extracted in MAPK extraction buffer (Tris- HCl, EDTA, NaCl, Sodium Fluoride, Sodium Molybdate, Sodium Ortho- vanadate, 1% IGEPAL® _{CA-630) for 10-15 minutes. The lysates were} cleared by centrifugation at 16,000g for 10 minutes. After protein quan- tification (Bradford), proteins were boiled in 1x SDS loading dye contain- ing 50mM DTT for 10 minutes at 90o_{C. Approximately 160μg of total} protein were separated on a 12% SDS-PAGE. The semi-dry method was used for transferring the proteins onto a PVDF membrane (GE Health- care). Primary antibody α-phospho-p44/42 MAPK (Erk1/2) (Thr202/ Tyr204) from Cell Signalling was used according to manufacturer’s in- structions to identify the double phosphorylation of the activation loop of MAPK3,4 and 6 (pTEpY). Primary antibody was added to 5% BSA in TBS- Tween 20 at 1:5000 using a peroxidase-conjugated goat anti-rabbit IgG (Sigma) as a secondary antibody. The PVDF membranes were stained with Coomassie Brilliant Blue (CBB) to assess equal loading.

2.4.4. Bacterial infection

Overnight cultures of appropriate strains were washed and resus- pended in 10mM MgCl2. For the infiltration method, cells were diluted serially to an OD600 0.001 with 10mM MgCl2. Hand-infiltration took place immediately into leaves from 4-5-week old Arabidopsis plants using a 1

of

mL syringe. Samples were collected 2-3 dpi. For the spraying method the OD600 was adjusted to 0.1 and 0.02% Silwet-L77 was added. After ho- mogenous spraying of 4-5-week old Arabidopsis plants (30mL culture per 24 plants), lids were added to the trays to maintain high humidity. Sam- ples were harvested 2-3 dpi.

2.4.5. Quantification of bacteria in infected leaves

Infected tissue was collected as leaf disc using No. 4 cork borer such that 2 leaf discs equalled 1cm2_{. Two leaf discs were placed into a} 1.5mL tube containing 200mL 10mM MgCl2 and ground using a tissue lyser (brand) at frequency of 28MHz for 30 seconds twice. An additional 800μL of MgCl2 solution was added. Serial dilutions were made in a 96 well plate first at a 1:1 dilution (10-1_{) and then 1:9 serial dilutions (10}-2_, 10-3_{, 10}-4_{, 10}-5_{, 10}-6_{, 10}-7_{) were made. 10μL of these were spotted onto} Kings B plates with appropriate antibiotic selection. Two days later single colonies were counted.

2.4.6. PTI and ETI elicitation prior to mass spectrometry

A. thaliana hag1-6 plants complemented with HAG1-FLAG con-

struct downstream a native promoter (np::HAG1-FLAG) were grown to adult stage. For PTI induction, plants were sprayed with 1μM flg22 solu- tion and leaf tissue was harvested after 1 hour, while for ETI induction, plants were sprayed with Pst DC3000 AvrRpt2 at OD600 0.1 and leaf tissue was harvested after 4 hours. Mock treatment included spraying with wa- ter and harvesting after 1 hour. In all cases, 0.02% Silwet L-77 was used. Harvested tissue was placed into a 1% formaldehyde solution and were vacuum-infiltrated 3-5 times 5 minutes to crosslink protein-protein and DNA-protein interactions. The reaction was quenched using 0.125 M

glycine. Tissue was thoroughly dried on kitchen paper and flash frozen in liquid nitrogen.

2.4.7. Chlorophyll fluorescence imaging

Photosystem II chlorophyll fluorescence imaging of Arabidopsis rosettes was performed as described in de Torres Zabala et al (2015) using a CF Imager (Technologica Ltd., Colchester, UK). P. syringae strains DC3000 WT and DC3000 ΔHopO1-1 were used to infect Col-0 adult plants. Infection was made by hand-infiltration of a OD600 0.15 culture. Plants were immediately placed in the chamber for 40 min post inocula- tion and then dark adapted for 20 min. This was followed by a saturating light pulse (6349 μmol m2 _s-1 _{for 0.8 s) to maximum obtain dark-adapt-} ed fluorescence (Fm). Actinic light was applied at the same light intensity as plant growth (120 μmol m2 _s-1_{) for 15 min, followed by a saturating} pulse to obtain maximum light adapted fluorescence (Fm’). The plants were left for a further 24 min in actinic light before returning to the dark for 20 min. At this point the cycle of measurements (59 min duration) was repeated 23 times. Fm, Fm’ and Fo (minimal fluorescence with fully oxidised PSII centres) were used to calculate chlorophyll fluorescence pa- rameters related to photosystem II photochemistry, specifically Fv/Fm (maximum dark adapted quantum efficiency). The temperature was kept constant at 20 °C.