Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (3): 482-490.doi: 10.3864/j.issn.0578-1752.2016.03.007

• PLANT PROTECTION • Previous Articles     Next Articles

Screening of Putative Proteins That are Interacted with NBS-LRR Protein Pik-h by the Yeast Two-Hybrid System

WANG Jia-feng, LIU Hao, WANG Hui, CHEN Zhi-qiang   

  1. National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou 510642
  • Received:2015-09-14 Online:2016-02-01 Published:2016-02-01

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Abstract: 【Objective】 To further investigate the signaling pathways activated directly by the resistance proteins Pikh-1 and Pikh-2 from the resistance gene Pik-h, yeast two-hybrid assay was performed to screen a rice leaf cDNA library using Pikh-1 and Pikh-2 as bait, respectively. 【Method】 All rice leaves of IRBL8 (Pik-h NIL) from the time points (12 and 24 h after inoculation with GD0193) were harvested for total RNA preparation. The rice leaf cDNA library was further constructed with the Make Your Own Mate&Plate Library System kit (Clontech). Bait plasmids pGBKT7-Pikh1 and pGBKT7-Pikh2 were constructed using the rapid homologous recombination method. The expression of the two proteins in the Y2H Gold strain was further detected with Western blot and the self-activation and cytotoxicity were also tested. Proteins interacting with the baits Pikh1 and Pikh2 were screened on SD/-Ade/-His/-Leu/-Trp/X-α-Gal plates from the rice leaf cDNA library with the mating strategy. Then the plasmids were prepared from the blue interacting clones and retransformed back into Y2H Gold strain with the corresponding bait. The plasmids that passed the reconfirmation were sequenced and the DNA sequences were further used to blast rice genome annotation project database. The functions of the interaction proteins were further analyzed with gene ontology (GO) annotation. 【Result】 The rice leaf cDNA library contained 2.2×106 independent clones. The sizes of most inserts were above 400 bp in the cDNA library. Bait plasmids (pGBKT7-Pikh1 and pGBKT7-Pikh2) expressed the corresponding proteins (BD-Pikh-1 and BD-Pikh-2) well and showed no self-activation and cytotoxicity, and suited to this screen. Thirteen candidate interacting proteins of pGBKT7-Pikh1 and five candidate interacting proteins of pGBKT7-Pikh2 were verified. Function annotation showed that four putative proteins were transcription factors involved in stress response and hormone signaling pathways, three were signaling proteins, four were involved in photosynthesis, one U-BOX containing protein was involved in ubiquitin mediated proteolysis, and four were unknown function proteins. 【Conclusion】 A high quality cDNA library was constructed and 16 proteins interacting with Pik-h proteins were identified, and these results provided good clues for elucidating the Pik-h- or other R-gene-mediated disease-resistant mechanisms in rice.

Key words: rice (Oryza sativa), Magnaporthe oryzae, yeast two-hybrid, interacting protein, Pik-h, screening

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