DIP1 Interacts With DST To Coordinate DST-regulated Yield Formation In Rice(Oryza Sativa L.)

Rice is one of the world's leading food crops and serves as a staple food source for more than half of the world's population.Grain yield of rice is a complex agronomic trait and it is mainly determined by three components,including number of panicles per plant,grain number per panicle and grain weight.Enhanced grain yield is a major focus of rice breeding programs worldwide.It's recognized that reduced expression of OsCKX2 causes CK accumulation in inflorescence meristems and increases the number of reproductive organs,which results in enhanced grain production.Prominently,the mutation alleles of OsCKX2 have been successfully used in rice breeding practice to improve seed production.DST?DROUGHT AND SALT TOLERANCE?encodes a zinc finger transcription factor regulates the grain yield through controlling the expression of OsCKX2.In addition,DST also regulates drought and salt tolerance in rice.Although the importance of DST in yield formation and abiotic stress are well recognized in rice,the exact mechanisms by which DST regulates the expression of target genes remain elusive.To elucidated the exact mechanisms that DST regulates the expression of target genes,the forward and reverse genetics approach were exploited to screen the interacting proteins of DST.In this research,we identified DIP1,which is an interacting partner of DST,using yeast two hybrid library screening.To evaluate the physical interaction between DIP1 and DST,we performed Yeast two-hybrid?Y2H?,Luciferase?LUC?complementation imaging?LCI?and Co-Immunoprecipitation?Co-IP?assays.Then genetic analysis indicated that DIP1 play an important role in DST-mediated yield formation.Eventually,the results of chromatin immunoprecipitation assay and transient expression assay of Nicotiana benthamiana leaves confirm that DIP1 takes part in DST-mediated regulation of OsCKX2 expression.The main results were described as follows:1.RNA-seq results showed that NAL1 controls leaf width,at least partially,through its effect on cell expansion,probably via the acid growth mechanism.The genetic analysis showed that DST and NAL1 work in the same pathway to regulate the leaf width,but the physical interaction was not detected between DST and NAL1 by biochemical experiment.These results indicated that NAL1-DST-mediated mechanism in regulating leaf width is complicated.To identify the interacting protein of DST,we performed yeast two-hybrid?Y2H?screening with Zf802 young panicle cDNA library.Three DST interacting proteins?DIPs?were found and confirmed to interact with DST through additional yeast two-hybrid?Y2H?assays.Interesting,the function of three DIPs was related to the formation of the transcriptional pre-initiation complex and acetylation of histones.These indicated that DST-mediated transcriptional regulation is subtly.2.To further verify the interaction between DIP1 and DST occur in planta,Luciferase?LUC?complementation imaging?LCI?and Co-Immunoprecipitation?Co-IP?assays in Nicotiana benthamiana leaves were performed.Next,we tried to map the domains involved in the DIP1/DST interaction using yeast two-hybrid?Y2H?assays.Our analyses revealed that DIP1 is able to interact with DST in vivo and the conserved activator-interacting domain?ACID?of DIP1 is sufficient for its interaction with DST;meanwhile,the separate middle region of DST(DST^73-188)is able to interact physically with DIP1.3.To further characterize the function of DIP1,we first use amino acids sequence of DIP1as query sequence to blast the Arabidopsis survey amino acids sequences.The result showed that DIP1 and AtMED25 are homologous with a sequence identity of 47.63%.To further test whether AtMED25 is the homologous protein of DIP1,the 35S_pro:DIP1 construct was introduced into the Atmed25 mutant using Agrobacterium tumefaciens-mediated transformation.The result showed that the DIP1 cDNA of rice complemented the phenotype of Atmed25 mutant.Taken together,our results indicated that DIP1 and AtMED25 are indeed homologous.4.We determined the spatial expression pattern and the subcellular localization of DIP1.The results show that DIP1-GFP fluorescence was observed at the cell nucleus and DIP1 was richly expressed in young panicle and vascular tissues of leaf and culm using RNA in situ hybridization.To further elucidate the biological significance of DIP1,we obtained T-DNA insertion mutant line,dip1,from RISD DB.At the mature stage,dip1 mutant showed increased panicle branching and produced more grains per panicle in comparison with wild-type?WT?.The results indicated that DIP1 plays role in yield formation.To further determine the effect of DIP1 on grain production,we generated DIP1-RNAi plants and DIP1-OE plants.At the mature stage,DIP1-RNAi plants showed increased panicle branching and produced more grains per panicle in comparison with WT,in contrast,DIP1-OE plants showed decreased panicle branching and produced less grains per panicle in comparison with WT.The results indicated that DIP1 indeed plays role in yield formation.5.Considering that DIP1 acts as a DST interacting proteins plays role in yield formation,we hypothesized that DIP1 plays a key role in DST-mediated yield formation.To test this hypothesis,we set out to identify a dip1 dst double mutant line from a cross between the T-DNA insertion mutant dip1 mutant and T-DNA insertion mutant dst mutant.At the mature stage,the panicle branching and grains per panicle of dip1 dst double mutant had no significant changes in comparison with dip1 mutant.These results support the notion that DIP1 acts genetically in the DST pathway to regulate yield formation.6.Taken into account of the function of DST in regulating grain yield through controlling the expression of OsCKX2,we hypothesized that DIP1,acts as DST interacting protein,also regulated grain yield through controlling the expression of OsCKX2.To test this hypothesis,we examined the expression of OsCKX2 and other OsCKXs and found that the expression of these genes was significantly reduced in dip1 mutant compared with WT.These results were also confirmed by RNA in situ hybridization.In addition,we investigated whether DIP1 participates in DST-mediated transcription of OsCKX2 using chromatin immunoprecipitation?ChIP?-qPCR assays.We found that DIP1 and DST were highly enriched on the same site of OsCKX2promoter.Taken together,our results indicated that DIP1 regulates grain yield through controlling the expression of OsCKX2.7.Next,we used a well-established dual-luciferase?LUC?reporter system to verify the relationship between DIP1 and DST.The result showed that DIP1 acts as a coactivator of DST.8.Considering that AtMED25,acts as the Arabidopsis homolog of DIP1,involved in RNA Poll II-dependent transcription,we hypothesized that DIP1 also performed the same function.To test this hypothesis,we examined whether DIP1 interact with the conserved C-terminal domain?CTD?of RNA Poll II,an interaction platform between RNA Poll II and other proteins involved in transcription.The results confirmed that the DIP1-CTD interaction occurs in vivo.Taken together,our findings have identified DIP1 as a coactivator of DST for yield formation.These results will shed light on the role of DST and provide a new clue on elucidating a new mechanism underlying rice yield formation.