Molecular Cloning And Functional Analysis Of A Homeodomain Type Transcriptional Factor Gene OsBIHD1 Involved In Rice Disease Resistance

The previous studies of our lab have demonstrated that benzothiadiazole (BTH) could induce rice resistance to blast, bacterial leaf blight and sheath blight, and got hundreds of differentially expressed cDNAs associated with the BTH-induction through suppression subtractive hybridization. Of them, HIHN-w5 was inferred to be a fragment of rice transcription factor gene encoding homeodomain protein based on the similarity search against the database. This study cloned and identified a full-length cDNA of a rice gene, OsBIHD1 (GenBank AY524972), encoding a homeodomain type BELL transcriptional factor. Its putative functions in disease resistance as well as in abiotic stress tolerance were studied by OsBIHDl overexpression in tobacco.The differentially expressed cDNA clone, HIHN-w5, contained a 733 bp insert. A sequence similarity search against the GenBank database revealed that the insert in the HIHN-w5 clone showed significant similarity to Arabidopsis thaliana BELLI protein. To obtain the full-length cDNA of this putative BELL gene, the 5'- sequence flanking the sequence in the clone HIHN-w5 was amplified by PCR using phage DNA prepared from a rice cDNA library as a template. According to the result of 5'-sequence, the full-length cDNA of HIHN-w5 was obtained and designated as OsBIHD1. The full-length cDNA of the OsBIHD1 gene is 2524 bp with a predicted 1929 bp open reading frame (ORF), and has 257 bp and 339 bp non-coding sequences at 5'- and 3'-ends, respectively. OsBIHD1 encodes a protein of 642 amino acids and the deduced OsBIHDl protein contains all conserved domains characteristic of the BELL type of homedomain proteins. The OsBIHD1 gene locates on chromosome 3 ofthe rice genome as a single copy gene and consists of four exons and three introns. Recombinant OsBIHD1 protein expressed in and purified from E. coli was bound to the TGTCA core motif of DNA sequence characteristic of cw-element sequence for the homeodomain transcriptional factors. Subcellular localization analysis revealed that the OsBIHD1 protein was localized in the nucleus in the plant cells. Take together, we concluded that OsBIHD1 encodes a homedomain protein beloning to the BELL subfamily of the homeodomain family.To illuminate the function of OsBIHD1 in rice disease resistance, the expression patterns of OsBIHD1 in BTH-induced disease resistance response and in the compatible/incompatible interactions between rice and the blast fungus Magnaporthe grisea were analyzed. Northern blotting analysis showed that the OsBIHD1 gene has a relatively high level of basal expression in rice leaf tissue. However, expression of OsBIHD1 was activated upon induction of BTH treatment, which is capable of inducing disease resistance. Expression of OsBIHD1 was up-regulated in BTH-pretreated rice seedlings during the first 6 h after inoculation with M. grisea and also in the incompatible interaction between M. grisea and a resistant genotype during the first 6 h after inoculation. These results suggest that the inducible expression of OsBIHD1 is associated with disease resistance response in rice.To better understand function of OsBIHD1 in disease resistance response, we performed a functional analysis of OsBIHD1 in transgenic tobacco plants. The OsBIHD1 coding region was cloned into a plant transformation binary vector CHF3pp2p212 under control of the CaMV 35S promoter. A total of 21 independent transgenic tobacco lines were obtained through the Agrobacterium-mediated leaf disc transformation and confirmed by PCR. Southern analysis of the transgenic lines showed that the OsBIHD1 gene was integrated into the tobacco genome with 1-2 copies; and Northern blotting indicated that the OsBIHD1 gene is expressed normally in most of the transgenic tobacco lines. The transgenic lines overexpressing OsBIHD1 showed some morphological abnormalities in the top buds, roots and reduced fertility, suggesting a role for OsBIHD1 in development process. Interestingly, the defense-related PR-1 gene showed an enhanced constitutive expression in thetransgenic...