Inserts from each DNA clone were PCR-amplified directly from bacteria. Amplification reactions were performed in 96-well plates,
with each well carrying a 50-μl volume containing 0.2 μM of each primer (T7 and SP6), 200 μM of each dNTP, 1× PCR buffer, and 1.25 units of Taq polymerase (AmpliTaq® DNA polymerase, Promega Corporation). An MJ Research thermal cycler was used for 35 PCR cycles, as follows: 95°C for 45 s, 56°C for 45 s, and 72°C for 1 min. We also amplified a selected set of conserved effector and hrp genes (e.g. XopX, avrXa7, XopD, avrRxv, avrXv3, hpaF, and hrpx), housekeeping AZD5153 research buy genes, and other conserved bacterial genes from genomic DNA of Xoo MAI1. Random PCR samples were visualized on agarose gels. All PCR Rabusertib clinical trial products were transferred to a 384-well plate and a volume of 2× betaine solution was added. The PCR products were arrayed once on poly-L-lysine slides (TeleChem International, Inc., Sunnyvale, CA, USA), using an SPBIO™ Microarray Spotting Station (MiraiBio, Inc., Alameda, CA, USA). The microarray contained 4708 elements. Bacterial inoculation and quantification The Xoo strain MAI1 was grown on PSA medium (10 g l-1 peptone,
10 g l-1 sucrose, 1 g l-1 glutamic acid, 16 g l-1 agar, and pH 7.0) for 2 days at 30°C. The bacterial cells were re-suspended in sterilized water at an optical density of 600 nm (OD 600) (about 10-9 cfu ml-1). Bacterial blight inoculation was carried out on the two youngest, fully expanded leaves on each tiller of 6-week-old rice plants (var. Nipponbare), using Orotidine 5′-phosphate decarboxylase the leaf-clipping method . Experiments were conducted under greenhouse conditions at 26°C and 80% relative humidity. We determined Xoo MAI1 multiplication in planta at seven time points after infection by leaf clipping (0 and 12 h, and 1, 3, 6, 10, and 15 days after inoculation) in 8-week-old plants of the susceptible rice cultivar Nipponbare. The number of cells
in the leaves was determined at the top 10 cm of each leaf which was cut into five 2-cm sections, and labelled A, B, C, D, and E, with A being the inoculation point. The leaf pieces were then ground in 1 ml of sterilized water. Serial dilutions were made and spread onto PSA agar plates. The plates were incubated at 28°C until single colonies could be counted. The number of colony-forming units (cfu) per leaf (equivalent to about 2 cm2) was counted and standard deviations calculated. The experiment was repeated independently three times. RNA extraction To obtain RNA from cells growing in planta, 30 rice leaves were inoculated by the leaf-clipping method. At each time point, leaves extending 2 cm from the tip were collected and, to facilitate exudation of bacterial cells, vortexed for 30 s with RNAprotect EPZ015938 purchase bacteria Reagent (QIAGEN, Inc., Courtaboeuf, France). The leaves were removed and bacterial cells were collected in a 15-ml tube by centrifuging at 4000 rpm for 30 min at 4°C.