Abstract: 【Objective】The occurrence of rice false smut disease is increasing in rice production worldwide, but the underlying mechanism of Ustilaginoidea virens-rice interaction is still unclear. The objective of this study is to construct a yeast two-hybrid library from rice spikelets infected with U. virens, and apply it for screening interacting proteins of a U. virens effector Uv_1261, so as to provide valuable resource for studying the mechanism of U. virens-rice interaction. 【Method】A susceptible indica rice Chuannong H2S was challenged with the inoculum of a GFP-tagged U. virens strain P4. Infected rice spikelets were collected after confirmation by a confocal laser scanning miscroscope at 13 days post inoculation (dpi), when pathogen hyphae extended into inner floral organs of rice spikelets. The total RNA was isolated from U. virens-infected spikelets, and used for synthesizing the first-strand cDNA by Oligo (dT) primer. ds cDNA was amplified by PCR and purified by agarose gel electrophoresis before mixed with vector pGADT7-Rec. The yeast two-hybrid cDNA library was constructed by homologous recombination technology in yeast line Y187. Then Uv_1261 bait vector was constructed and subjected to self-activation verification. Then the library was screened, and the blue colonies that grew well in medium containing SD/-Ade/-His/-Leu/-Trp/X-α-Gal were selected as candidate interacting proteins of Uv_1261. After sequencing confirmation, these candidates were annotated and classified by Blast and gene ontology (GO) analyses. 【Result】The cDNA library titer was 5.7×10⁸ cfu/mL and the average length of the insertions was 750 bp, indicating that this library is of high quality. Yeast transformation analysis showed that Uv_1261 had no self-activation. Screening for the library with Uv_1261 revealed 56 candidate interacting proteins including 28 proteins in U.virens, 16 protiens in rice and 12 unknown proteins. The candidate interacting proteins from U. virens were involved in 17 biological processes including translation, metabolic process, oxidation-reduction process, cellular amino acid biosynthetic process, etc; or possessed molecular functions such as metal ion binding, hydrolase activity, ATP binding, etc; or were associated with cell components like ribosome, cytoplasm, mitochondrion, etc. Uv_1261-interacting proteins from rice were involved in 11 biological processes including protein dephosphorylation, carbohydrate metabolic process, translation, etc; or possessed molecular functions such as metal ion binding, nucleotide binding, transferase activity; or were associated with cell components like ribosome, membrane, nucleus, etc. 【Conclusion】The cDNA library has good quality, and is suitable for identifying interacting proteins of U. virens effectors. This work can provide a valuable resource for studying molecular interaction between U. virens and rice.

Key words: Ustilaginoidea virens, rice, effector, cDNA library, yeast two-hybrid