Expression Analysis Of Rice Defence-related Genes After Infected By Rhizoctonia Solani

Rice sheath blight, caused by Rhizoctonia solani is an economically important disease of rice. To investigate the pathogenesis-related genes (PR) expression profile during the infection process of rice sheath blight disease, rice cultivar 9311 was inoculated with the fungal pathogen, Rhizoctonia solani which was isolated and purified from the breeding field of Fujian Agriculture and Forestry University in Fuzhou, China. Rice samples were harvested at 0, 4, 12, 24, 36, 48, 72hrs post-inoculation for total RNA extraction and observation of the disease development. Two PR genes, PRl and PBZl, were used as probes for Northern blot analysis. The results showed that expression of both PRl and PBZl genes were induced at 12 hrs post-inoculation, however PRl was increased gradually from 12 hours post-inoculation to 72hrs post-inoculation, while PBZl drastically increased at 48hrs post-inoculation to almost the maximum level of 72 hrs post-inoculation. Analysis of infection process showed that mycelium grew randomly on the surface of tissue at 12 hrs post-inoculation, few lesions started to come out at 36 hrs post-inoculation, and typical lesions were found in all plants at 48 hrs post-inoculation. Combining the results of molecular analysis and infection progress observation, we concluded that expression of PRl and PBZl may be related with disease development of rice sheath blight, which may suggest that SA signal is involved in the defense pathway.To understand the molecular mechanism of rice resistance to sheath blight, Subtractive Suppression Hybridization (SSH) was performed using the PCR Select cDNA Subtraction Kit from Clontech. Rrice plants of 9311 cultivar were inoculated by Rhizoctonia solani fungus and the mRNA samples from inoculated plants was used as a "tester" and no-inoculated ricesamples as a "driver" .The PCR products were cloned into pGEMT-T easy vector and transformed by electroporation to construct the forward subtractive cDNA library in which 2784 clones were selected. After two clycles of colony hybridization, 256 were PCR-amplif ied individually with M13 univerisal primers to transfer to Hybond membranes for reverse-northern analysis. 64 differertial expression clones were selected to sequence and 53 ESTs gave good sequence results. Blastn and blastx searching in gene databases showed that all 53 ESTs belonged to rice genome sequence, including 33 ESTs hit to known gene sequences in databases and 20 ESTs cannot hit to any known gene sequences in the databases. The 33 ESTs were cataloged according to the possible function of the homology proteins. Compaired with ESTs from SSH and/or cDNA array libraries induced by Magnaporthe grisea, Xanthomonas oryzae pv. oryze, 5 ESTs induced by both pathogens, Magnaporthe grisea and Xanthomonas oryzae pv. oryze, including two ESTs induced Magnaporthe grisea, Xanthomonas oryzae pv. oryze and Rhizoctonia solani, these were useful to broad-spectrum resistance breeding.