Map-based Cloning And Functional Analysis Of DHT1,a Plant Architecture Regulator In Rice(Oryza Sativa L.)

Rice（Oryza sativa L.）is one of the most important food crops in the world.In recent years,the research of improving plant architecture to increase rice yield has become the focus.The proposal of“ideal plant architecture”,which consists of less tiller number,stout stalk and large panicle,provides a target of improving rice plant architecture for breeders.Therefore,it is of great significance to continue exploring the genes that regulate plant architecture to investigate their application value for cultivating elite rice varieties with ideal plant architecture to increase rice yield.In this study,we identified a dwarf and high tillering mutant dht1.By map-based cloning,we cloned the mutation-causing gene and performed detailed functional analyses.The main findings of this paper are as follows:1.Compared to the wild type,the dht1 mutant has significantly reduced plant height at the seedling and mature stage,and has more tillers（with 1.5 times increase）and smaller leaves,florets,and grains.In addition,agronomic traits such as panicle length,number of grains per panicle,number of panicle branches and 1000-grain weight were also significantly reduced.2.By resin slicing analysis and microscope observation,we found that the length,width and area of the mutant parenchyma cells were severely reduced while the number of parenchyma cells were unchange.Therefore,the short stem is likely caused by the reduction of cell size.3.The target gene was finely mapped to the 46 kb region between the molecular marker L-9 and L-49 of chromosome 4 using the F₂ mapping population derived from the cross of dht1 mutant x Japonica variety IRAT129.In this interval,there are 7 open reading frames（ORFs）.By sequencing analyses,we found that there is only a single base mutation in the exon of ORF4,which resulted in the conversion of the encoded isoleucine to phenylalanine.By genetic complementation,RNA interference and CRISPR knockout analyses,we verified that ORF4 was the mutation-causing gene.4.The sequence analysis revealed that DHT1 encodes an RNA-binding protein containing two RRM,belonging to a member of the hn RNP superfamily.Subcellular localization analysis showed that DHT1was localized on the nuclear speckle of the nucleus.The m RNA expression analysis showed that the DHT1 gene is widely expressed in various tissues of rice,and its expression is high especially in young panicle and axillary buds.5.Western blot analysis showed that DHT1 protein content was significantly reduced in the mutant compared to the wild type.Both the Cell-free Degradation assay and the LUC assay showed that the mutant protein dht1 is unstable.RNA-EMSA experiments showed that though both DHT1 and dht1proteins specifically bind to single-stranded poly（U）,dht1 protein has reduced binding ability to RNA.6.Transient expression assays in tobacco leaves revealed that DHT1 can co-localize on nuclear speckle with many SR proteins and U1 sn RNP subunit U1-70K.Luciferase complementary imaging analysis showed that DHT1 proteins can physically interact with these proteins.7.By a RIP-seq analysis,we found that the transcripts of some Strigolactones（SLs）synthesis and signaling pathway genes D17,D3,D53 and D14 can be bound by DHT1.Further analysis revealed that the intron splicing efficiency of the D14 gene and the D14 m RNA level were downregulated in the dht1mutant,and the content of endogenous D53 proteins was upregulated.These results suggest that DHT1affects the morphogenesis of rice plants by regulating the post-transcriptional modification of SLs-related genes.