Isolation And Characterization Of Defense Responses Genes Involved In Leaf And Neck Blast Resistance Of Rice (Oryza Sativa L.)

There is a correlation between the leaf and neck resistance to blast in rice. Because of its difficult identification of neck blast resistance, it could be predicted according to its leaf resistance to certain degrees. However, it has been found that there were differences between resistances to leaf and neck blast in breeding practice. Some varieties have broad-spectrum resistances in all developmental stages, while others show different resistances not only to different isolates but also at different stages. In order to study the molecular mechanism of race-specific resistance and growth stage-specific defense responses to rice blast, we carried out the following: Two rice lines with different neck blast resistances were used to isolate defense response genes involved in neck blast by suppression subtractive hybridization (SSH). Isolation of differentially expressed genes involved in incompatible and compatible interactions between rice and Magnapothe grisea using a rice variety with different resistances to different isolates of the same pathogen. And expressions ofselected genes in different developmental stages were profiled by RT-PCR.Two SSH cDNA libraries enriched for transcripts differentially expressed in plants of two rice lines G9S/G2S8 with similar genetic backgrounds and same leaf blast resistance but different responses to neck blast after inoculation with isolate ZC15 of Magnapothe grisea by using SSH. After differential screening and sequence analysis of the selected clones, ninety unique cDNA clones were found. Expression differences of 17 out of the 26 selected cDNA clones in resistant and susceptible lines were confirmed by RT-PCR. Expression profilings of the 26 cDNA clones at the early stages after inoculation were also revealed by RT-PCR. The expressions of these cDNAs were either induced or repressed in resistant or susceptible plants after inoculation. But the time points and levels of their expression are different in resistance and susceptible plants after inoculation. According to biological information functions of some selected genes it is speculates that the Ca²⁺ signal transduction may play a important role in regulating defense response to neck blast. And there may be different paths of the Ca²⁺ signal transduction between resistance and susceptible plants.RNAs isolated from plants of a rice cultivar zhong 156 inoculated with an incompatible isolate ZC15 and a compatible isolate ZB1 of the blast pathogen were used to construct two SSH cDNA libraries enriched for transcripts differentially expressed in incompatible/compatible interactions with SSH. Twenty-five unique cDNA clones showing differentially expressions in incompatible and compatible interactions were isolated and identified combining the differential screen with sequencing of the selected clones and their differential expressions were further confirmed by RT-PCR. Expression profilings of the 25 cDNA clones at the seedling early stages after inoculation were also revealed by RT-PCR. All of their expressing levels were identical at Oh of inoculation, followed by inducing or suppressing in the two interactions, indicating that the expression changes of these genes were solely due to different races of the pathogen and the reliability of the selected genes in the present experiment. It is hence postulated that in rice-Magnapothe grisea interactions, the timepoints and levels of expressions of some defense-related genes might determine the race-special resistance of the host. Expression profilings of the 25 cDNA clones at the neck stages after inoculation were also revealed by RT-PCR. These differential expression genes relating to race-special defense response to blast at seedling stage also be induced or suppressed at neck stage after inoculation with same isolates. But their change trends of expression are difference from seedling stage. So we postulate that the defense response to the blast in cultivar zhong 156 may be controlled by both the race of pathogen and the development stage of rice.According to the putative functions of their homologous genes in the database, these genes selected from both neck and leaf stages might be involved in pathogen response, signal transduction, transcription, protein synthesis /subcellular localization, protein fate (folding, modification, destination, metabolism and transposable.The above results indicated that the expressions of most blast defence related genes were either induced or suppressed in all defence responses of both susceptible and resistance interactions at leaf and neck stages, as well as in host-specific or race-specific responses. These genes might participate in the defence responses as the fundmental resistance genes. Differences in susceptibility and resistance might be resulted from the differences of both the time at which the changes of gene expressions occurred and the degrees of these changes. A few genes showed major differences in expressions in incompatible/compatible interactions, even in different directions. These genes might play key roles in regulating the resistance. The expression changes at different developmental stages of these genes selected from the leaf stage were different. It could be further postulated that the expressions of some defence response genes regulated by both different races of the pathogen and developmental stages of the host plants resulted in the race-specific resistance and host developmental stage-specific resistance.Two full-length cDNA libraries were constructed using two rice varieties with different resistance to blast at different developmental stages after inoculation with different isolates of Magnapother grisea.