Functioanl Analysis Of Receptor-like Kinases DIS1 And DIS2 Rice

Rice inflorescence development, flower organ differentiation, and successful pollination and fertilization are key factors that determine seeds production and therefore yield potential. Those processes involve frequent cell-to-cell communication. Many genes identified up to date that control these processes are largely transcriptional factors, transporters and enzymes that participate in metabolism, which usually function downstream of signal transduction pathways or not directly participating in cell-to-cell signal transduction. To look for the genes acting upstream of the signal casecade, particularly those cell surface molecules being able to perceive and transduce the signals during reproductive stage, we performed reverse genetic mutants screen for the receptor-like kinases (RLKs) genes with higher expression before or after flowering via RNA interference (RNAi), in which gene-specific segments in extracellular domain or intracellular domain were as targets. We characterized the transgenic rice lines with severe developmental defects in inflorescence and spikelet after knocking down of two closely related RLK genes simultaneously, and then named the two genes as DIS1 and DIS2 for defective inflorescence and spikelet 1 and 2.DIS1 and DIS2 RNAi plants display short panicle, reduced panicle branches, aberrant spikelets, pooly developed male and female reproductive organs, etc. The severity of phenotype corelated reduced mRNA level of DIS1 and DIS2, and all the phenotypes list above are stably inherited from TO to T2 generation. However, loss-of-function of DIS1 and DIS2 individually cause weak phenotype, indicating their function redundancy. In agreement with it, their expression by promoter::GUS reporter gene in reproductive organs are very similar such as in infloresecce, spikelet, filament, vascular bundles of anther, stigma, style, lodicule and the regions near micropyle, etc. Microscope analysis shows that both male and female reproductive tissues are affected in dis1 and dis2, with pollen grains more severer, especially in disⅠ. Reciprocal cross of DIS1 and DIS2 with DJ (Donjing, WT) also show that male and female transmission efficiency are affected in dis1 and dis2 to different extent, especially in disⅠ, although the expression of DIS1 and DIS2 in pollen is not detectable. This discrepancy implies that the pollen developmental defects are indirectly caused by adjacent cells possibly from the tapetal through cell-to-cell communication.DIS1 and DIS2 are two members of CrRLK1-L family, without any known functional domains or motifs in extracellular region. However, both have the in vitro kinase activity, and DIS1 kinase activity (probably also DIS2) is essential in rescuing the homozygous lethal phenotype of fer-1, its Arabidopsis counterpart. The cell surface localization of DIS1 and DIS2 strongly suggest that both are prabably the cell surface receptors in sensing developmental cues using the extracellular domain and relay signals within cytosol using the kinase domain by initiating a phosphorylation cascade.In addition to the phenotypes list above, other defects such as dwarfism and decreased tiller number are also displayed in RNAi-disl and dis2 mutants, with a little weak for disl and dis2 compared to RNAi. Howerve, overexpression of DIS1 and DIS2 resulted in more tiller number than that of WT. The expression of DIS1 and DIS2 are all induced by IAA and GA. DIS1 is also induced by 6-BA while DIS2 is down-regulated by 6-BA. These indicate that the two RLK genes are IAA, GA and cytokinin responsive genes and may participate in the downstream gene expression control via these phytohormore-mediated signal transduction passways. Consistently, the induced expression of some downstream responsive genes of IAA and 6-BA is delayed and the expression level is decreased in RNAi plants. Taken together, DIS1 and DIS2 are important for controlling plant height and tiller formation, and possibly function via IAA or cytokinin passway. DIS1 and DIS2 may have independent signal passways and cross-talk where with these known phytohormore-mediated signaling passways.