Phenotype screens

2.1.4.1 B. cinerea

Twelve leaf 7 leaves of 28-day-old plants of the test lines plus one leaf seven of both Col0 and bos1 were detached and placed on a bed of 0.8% agar in three propagator trays. A scale measure was placed between every other line. Ten leaves from each line was spotted with 5μl of 104 _{spores mL}-1 _{spore suspension and the remaining two}

leaves with 5μl of 50% filtered grape juice 9 hours after subjective dawn. Trays were covered with lids, sealed and placed in a growth chamber with the same conditions as for plant growth. Photos of the duplicate screen trays were taken at 34, 48, 54 and 72 hours for At4g39270 screen and 34, 48 and 72 hpi for the MAC screen using Nikon D50 Camera. The area of the lesion was determined using ImageJ.

2.1.4.2 B. cinerea growth

B. cinerea β-tubulin gene expression levels were used as a measure of the growth of

B. cinerea in leaf 7 of four-week-old Arabidopsis plants. This was determined using

RQ-RT PCR. RNA was extracted from individual leaves (1 leaf per biological replicate) using TRIzol (Invitrogen) and purified using the Qiagen RNeasy Mini Kit. The RNA quality and purity was evaluated using NanoDrop; the integrity of the RNA was determined using the Agilent 2100 BioAnalyser. All samples required a RNA Integrity Number (RIN) higher greater than five, this was used because that is the minimum RNA quality value recommendation by Schroeder et al (2006) for use with RQ-RT PCR. RNA exceeding this threshold then underwent DNase treatment using the Ambion Turbo DNase™ kit as per manufacturers instructions. 1 μg of RNA was converted into cDNA using SUPERSCRIPT™ II RNase H Reverse Transcriptase

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(Invitrogen) as per manufacturer’s instructions. The cDNA was used as templates in a RQ-RT PCR.

Samples were diluted 1 in 10 with DEPC-treated water before being run in triplicate on a Roche LightCycler 480 using PrecisionPlus™by PrimerDesign as per

manufactures instructions. β-tubulin gene expression levels were compared using the primers 5ʹ-TTCCATGAAGGAGGTTGAGG-3ʹ and 5ʹ-TACCAACGAAGGTGGAGGAC-3ʹ, to PUX1 expression using primers 5ʹ-TTTTTACCGCCTTTTGGCTA-3ʹ and 5ʹ-

ATGTTGCCTCCAATGTGTGA-3ʹ.

2.1.4.3 Chitosan screen

Chitosan (obtained from SIGMA) was dissolved in 400 µl of 5% acetic acid and diluted to 150mg/l, 100mg/l and 50mg/l in 20 ml volumes with sterile H2O. Mock

inoculum consisted of 20ml H2O and 400 µl of acetic acid. 28-day-old plants were

spray-inoculated using an airbursh system attached to a compressor (GS, model AS18). 20ml of Chitosan solution was used per 24 plants (one tray) at a pressure of 1012 psi, the plants were allowed to dry for 30 minutes before being replaced in growth cabinet. Leaf 8 leaves were harvested 2hpi and snap frozen in liquid nitrogen.

2.1.4.4 Pseudomonas Syringe pv tomato DC3000

Pseudomonas syringae pv. Tomato (Pst) DC3000 was streaked onto Kings B agar

plates supplemented with rifampicin (100 µg/ml) from frozen glycerol stocks, using a sterile loop and incubated at 28°C for 2 days. Single colonies were transferred to liquid Kings B medium supplemented with rifampicin (100 µg/ml) and incubated overnight in a shaking incubator (28°C, 220rpm). 20ml of inoculum was prepared per 24 plants at an OD600 = 0.1 and siluet L-77 (a silicone surfactant) was added to create a final concentration of 0.05%. Plants were spray-inoculated using an airbursh system attached to a compressor (GS, model AS18) at a pressure of 10-12 psi. 1cm2 leaf disc were obtained from separate plants (at 1, 2 and 3 dpi) and immediately placed into 1.5ml Eppendorf tubes containing 200 µl of MgCl2. Sterile glass beads

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seconds, 28 Hertz). 800 µl of MgCl2 was added and the samples were serially diluted.

10µl of each dilution was plated onto Kings B agar and incubated (24 hours at 28°C) followed by a second incubation step (24 hours, room temperature). Colonies were manually counted and the number of colony forming units (CFU) calculated. Nahg

((Delaney et al., 1994)) was included in each screen as positive control.

2.1.4.5 Hyaloperonospora parasitica

Arabidopsis seeds were sown directly onto p40 trays containing Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1)

containing intercept, and then stratified for 3 days in 0.1% agarose at 4°C in the dark. With each outside module of the tray containing ws-eds1 (a mutant with enhanced disease susceptibility) to create a border to aid with infection, test lines were randomly assigned an inside module. Plants were grown at 20°C, 350 ppm CO2, 70%

humidity, and light intensity of 100 µmol photons-m-2._s-1 _{in 10:14 light:dark cycle.}

Six modules of stock seedling inoculated with Hyaloperonospora parasitica (Hpa)

Nok1 were harvested into 50 ml falcon tubes and 20 ml of cold sterile H2O added,

this was filtered using microcloth to remove seedlings, and the concentrations adjusted to 30000 conidiospores per ml.

Two-week-old seedlings were spray-inoculated with a suspension of 30000 conidiospores per ml of Hpa isolate noks1 using an airbursh system attached to a compressor (GS, model AS18) at a pressure of 10-12 psi. Trays were covered with lids, sealed and placed in a growth chamber with the same conditions as for plant growth. At four days post infection the number of conidiophores per 15 seedlings was counted. NahG was included in each screen as a positive control.

2.1.4.6 Morphological

2.1.4.6.1 Soil based

Five-week-old plants were photographed using Nikon D50 Camera and the leaf area determined using ImageJ.

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2.1.4.6.2 Agar based

Ten-day-old seedlings were photographed using Nikon D50 Camera and the root length determined using ImageJ. Seedling were then completely removed from agar and weighed to give dry weight.