Specifications table br Value of data br Experimental design

Specifications table
Value of data
1. Experimental design, materials and methods
2. Direct link to deposited data Deposited data can be found here: http://www.ncbi.nlm.nih.gov.eleen.top/GBRH01000000.
3. Nucleotide sequence accession number The assembled and annotated A. donax USA KB-R7943 mesylate cost Rio Grande RNA transcriptome has been deposited at DDBJ/EMBL/GenBank under the project accession PRJNA256910. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GBRH00000000. The version described in this paper is the first version, GBRH01000000.
Conflict of interest
Acknowledgements This work was supported in part by funding provided by the USDA-ARS Knipling Bushland US Livestock Insects Research Laboratory CRIS Project no. 6205-32000-031-00, and by computational resources on the iVEC at Murdoch and Pawsey Nodes through the National Computational Merit Allocation Scheme. We acknowledge the Australian Government Super Science Initiative and the infrastructure support by the Education Investment Fund. We acknowledge the National Collaborative Research Infrastructure and Bioplatforms Australia initiative for access to their computational genomics pipelines. M. Bellgard, initiated this research via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems. This article reports the results of research only. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.
Value of the data Data, experimental design, materials and methods A time-course phosphoproteomic analysis of rice leaves was performed after M. oryzae inoculation, including venn diagram analysis of the phosphoproteins overlapped between two cultivars (Supplementary Material, Fig. 1), close-up views of the phosphoprotein spots in 2-DE gels (Supplementary Material, Fig. 2), relative intensities of all phosphoprotein spots (Supplementary Material, Fig. 3), the MS spectra of representative phosphoprotein (Supplementary Material, Fig. 4), functional category (Supplementary Material, Fig. 5), and the MS/MS spectra of representative phosphorylated peptides (Supplementary Material, Fig. 6). A detailed experimental procedure was generated for “identification of phosphorylation sites by NanoLC–MS/MS”. Furthermore, gene-specific primer sequences were designed for qRT-PCR (Supplementary Material, Table 1).
Materials Two near isogenic lines of rice (Oryza sativa indica), C101LAC and CO39, were provided by the International Rice Research Institute IRRI, Philippines. C101LAC contains the resistance gene Pi-1 in the CO39 background. The Magnaporthe oryzae race ZC13, one of the primary M. oryzae races in Guangdong Province, is incompatible with C101LAC but compatible with CO39 [2]. Maintenance of fungal cultures, spore solution preparation, and growth condition of rice plants were as previously [3]. Rice seedlings at the four-leaf stage were spray-inoculated with freshly prepared M. oryzae spores (1×105conidia/mL, containing 0.02% v/v Tween 20). The fourth leaves were harvested at 8, 12, and 24h post-inoculation for protein extraction and 2-DE analysis. The leaf samples were frozen in liquid nitrogen and stored at −80°C. Control plants were sprayed with sterilized water including 0.02% v/v Tween 20.
Preparation of samples for proteomics analysis Total proteins were extracted essentially as previously described with some modifications [4]. Briefly, a 5-g quantity of leaf tissue of each sample was extracted with Mg/NP-40 buffer (containing 0.5M Tris–HCl, pH 8.8, 2% v/v NP-40, 20mM MgCl2, 2% v/v 2-mercaptoethanol, 1% w/v PVPP, 1mM PMSF, 40mM NaF, and 15μL of cocktail) and fractionated with PEG 4000. Protein was precipitated with four volumes of ice-cold acetone for 2h at −20°C and dissolved in incubation buffer (IB) (30mM MES, 30mM imidazole, 8M urea, 0.1M potassium aspartate, 0.1M sodium glutamate, and 0.25% w/v CHAPS). Protein concentration was determined using the procedure as described previously, with BSA as the standard [5]. Each experiment was repeated three times with different pools of leaf samples.