The Self-Defense Skill Behind AZD3759

Extended Experimental Procedures Plant Material and Growth Condition All seeds were surface sterilized by treating with 30% bleach and 0.02% Triton X-100 for 5 min, then rinsed 4 times with sterile water, and stratified at 4��C for 2 days before being planted on media. Seedlings were germinated on MS agar SIS3 clinical trial plates supplemented with 1% sucrose in a Percival incubator at 22��C with 16 hr light and 8 hr dark. Plasmids were constructed using the Gateway cloning technology (Invitrogen). Genomic DNA from Arabidopsis ecotype Col-0 was used as the template for amplification of the 3002 bp upstream region of UPB1. This promoter region was cloned into pDONR-P4P1R. UPB1 and Per57 open reading frames were cloned into pDONR221 with the primers listed in Table S3. The multisite gateway cloning technology was used to place these fragment into pdpGreenBar-T along with cGFP-NOS. For the pUPB1::UPB1-3YFP construct the GFP was replaced with 3YFP. For the 35S::UPB1-3YFP ATP12A construct, the native promoter region and GFP were replaced with CaMV35S and 3YFP. For the 35S::Per57-GFP construct, CaMV35S was used for promoter and cGFP-NOS was used for 3�� clone. For the transcriptional fusion (pUPB1::GFP) a similar approach was taken, the UPB1 promoter region was cloned, along with ER-GFP-NOS, and pmpENTR into dpGreenBar-T. Meristem- and elongation zones were micro-dissected from 6 day after imbibition (dai) roots and transferred immediately into liquid nitrogen. The RNeasy micro kit (QIAGEN) was used to isolate total RNA from these samples. First strand cDNA AZD3759 chemical structure was synthesized with oligo(dT)20 primers using SUPERSCRIPT III First-Strand synthesis system for RT-PCR (Invitrogen). Quantitative real-time PCR was carried out using the POWER SYBR GREEN PCR master MIX (Applied Biosystems) and gene specific primers (Table S3) in a StepOnePlus Real-Time PCR System (Applied Biosystems). The gcRMA implementation in the Bioconductor and R software packages was used for background correction, quantile normalization and expression estimate computation (http://www.bioconductor.org) (Gentleman et?al., 2004). For global analysis of the transcriptome responses the false discovery rates were calculated by the Significance Analysis of Microarray (SAM) algorithm (Tusher et?al., 2001). Differentially expressed genes were identified based on fold change in expression and a significant p value threshold (two fold change and p value?