Pathogenicity and HR assays. The effect of hrcC deletion on
the ability of A. citrulli to induce an HR was evaluated using 6-
week-old tobacco plants (Nicotiana tabacum). Using a hypodermic
syringe without a needle, AAC00-1, AAC00-1ΔhrcC, AAC00-
1ΔhrcCcomp at approximately 1 × 108 CFU/ml, or PBS was infiltrated
into the intercellular spaces between the major veins of a
fully expanded tobacco leaf. Plants were incubated under conditions
of approximately 75% relative humidity (RH), 28°C, and 12
h of fluorescent light daily in a growth chamber (Percival, Perry,
IA) for 2 days. Plants were subsequently observed for cell death
associated with a typical HR. To determine the effect of the T3SS
mutation on A. citrulli pathogenicity, 2-week-old watermelon seedlings
(‘Sugar Baby’) were infiltrated as described above with
AAC00-1ΔhrcC, AAC00-1ΔhrcCcomp AAC00-1 (wild-type) at
approximately 1 × 106 CFU/ml, or PBS as a negative control. The
cotyledons of three seedlings were inoculated (28) and plants were
incubated for 2 days at 28°C with approximately 75% RH and 12 h
of fluorescent light daily in a growth chamber. Plants were then
visually observed for BFB symptoms (necrosis and water soaking).
This experiment was conducted three times.