Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 12/3/2012
Publication Date: 2/27/2013
Citation: Zhang, Z., Jia, Y., Wang, Y., Liu, F., Sun, G. 2013. Construction of a yeast two hybrid library in a compatible interaction of rice with an AVR-Pita1 containing isolate. Thirty-Fourth Rice Technical Working Group, Hot Springs, AR. Page 73. Interpretive Summary:
Technical Abstract: To control rice blast, the utility of host resistance has been proven to be the most effective and economical method. The resistance is mediated by the interaction of a single R gene from the host and the corresponding AVR gene from some races of M. oryzae after infection. Pita and AVR-Pita1 are a matched pair of R/AVR genes, and the Pita-mediated resistance, directly binding to the effector protein AVR-Pita1, has been used to manage rice blast in the Southern US. Recently, accumulation in the Biotrophic Interfacial Complex (BIC) and cell-to-cell movement of effectors proteins during invasive growth of the blast fungus have been demonstrated using fluorescent protein tag technology by others. This raises the possibility that these effectors proteins prepare rice cells for subsequent fungal entry and biotrophic growth. Our goal in this project is to identify proteins that interact with AVR-Pita1 using yeast two hybrid technology. As the first step toward this goal, we constructed a yeast two hybrid library. Rice cultivar Nipponbare without Pita and M. oryzae isolate ZN61 with AVR-Pita1, belonging to race IB49, were used. Firstly, 3 to 4 leaf stage seedlings of rice were grown in a greenhouse and the isolate ZN61 were grown on oatmeal agar at 25C for 7 days for spore production. The second youngest leaf of the seedlings were removed and cut into 5-cm segments for spot inoculation. Each detached leaf was inoculated 4 to 5 times with 500 spores each time. After 24h, 48h, 72h post-inoculation, leaf tissue surrounding the lesions were cut, immediately frozen with liquid nitrogen, and stored at -70C. Total RNA was extracted using Trizol reagents and mRNAs were purified by Qiagen Oligotex mRNA Midi Kit. Approximately 2ug mRNA were used to construct a library with CloneMiner**TM II cDNA Library Construction Kit. A primary library with bacteria of 1.1 × 10**7 colony forming unit carrying prey plasmid of the yeast two hybrid system was constructed. Plasmid DNAs were isolated from random 18 bacterial colonies and digested with restriction enzyme BsrGI, and results showed that the range of the insert size was from 0.3-2.2Kb, and the average insert size was 0.9Kb. Progress on the identification of AVR-Pita1 interacting proteins will be reported.