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Agro Infiltration to N.Benthamiana leaves 1. Grow N.Benthamiana 2-4 weeks, preferably leaves should be fully open but not yet mature. 2.Grow agro (preferably from an up to 1 week old plate) in 10ml LB or YEP in a 50ml conical tube with 200uM of acetosyringone O.N or till O.D ~2 with antibiotics. 3.Measure O.D of the O.N agro culture, centrifuge (6K 5min) and re-suspended in infiltration media to O.D 600 of 0.1, incubate for 4hrs in a shaker at 28c (usually re-suspend in the morning and infiltrate in the evening). 4. At this stage combine equal volumes of agro culture that you want to co-infiltrate. Using an agro strain with 35S-P19 should make signals last several days longer by slowing down insert inactivation. 5.Use a 1 ml syringe to infiltrate the leaves on their bottom/ abaxial side while pushing the syringe gently against your gloved finger on the other side. 4. After infiltration plants should be kept in the dark for 36 hrs (it will work without the dark period as well), whole plants can then be left for 2 more days for entrainment or leaf discs from the area around the infiltration sites can be excised (sterilized for 5 min in 10% bleach, washed 3 times in sterile water for 5 min) placed on 1xMSS plates and entrained. The plates can be put into the CEF chamber for 12:12, 16:8 L:D or any other entrainment regime. 5. After entrainment spray the plates with Luciferine as specified in the LUC assay protocol and place into the CCD camera.

Infiltration media: 10mM MES, 10mM MgSO4 and 200uM Acetosyringone pH5.6 Make 10mM MES, 10mM MgSO4 pH5.6 (titrate with 1M NaOH) and freeze in aliquots, add Acetosyringone before use. Stocks: 100mM Acetosyringone in DMSO 1M MgSO4

Adapted by Akiva from Reetika, Hagop and the Sinha lab’s protocols.