Generation And Fuctional Analysis Of CRISPR/Cas9-Mediated Clathrin Mutants In Rice

Clathrin is a triskelion-shaped complex composed of three heavy chains(CHC)and three light chains(CLC).Clathrin-mediated endocytosis(CME)is an evolutionarily conserved endocytic pathway,which is one of the main pathways for endocytosis of plasma membrane proteins in eukaryotic cells.Previous studies have shown that clathrin plays a key role in growth and development of dicot model plant Arabidopsis,such as organ development,polar auxin transport,tropic growth,and nutrient absorption.However,functional roles of clathrin in growth and development of monocot plants are largely unknown.CRISPR technology is an emerging gene editing technology which possesses some advantages,including low cost,easy to operate and high efficiency,and thereby is a preferred method to generate loss-of-function mutants,which is important to deeply dissect gene function.To explore functional roles of clathrin in rice,monocot model plant,this study used CRISPR/Cas9 technology to generate rice chc and clc mutants and meanwhile used cell biology,molecular genetics,rice hybridization,rice transformation and physiological and biochemical methods or strategies to analyze genetic functions of clathrin in rice.The detailed results are as follows:(1)Generated the chc,clc and Hub transgenic mutants in rice.Crossing and sequencing analysis showed that chc single and double mutants,and clc single,double and triple mutants were successfully recovered.Hub transgenic plants were obtained but not identified.Furthermore,fluorescent molecular markers have been crossed into chc and clc mutants,which will be used for cell biology analysis of clathrin in rice.(2)The agronomic traits of chc and clc mutants were analyzed.Analysis of agronomic traits in rice showed that,compared to the wild type,multiple agronomic traits(including primary root length,root size,plant height,tiller,flag leaf length,leaf width,main panicle length,primary branch,secondary branch,seed setting rate and 1000-grain weight)in chc and clc mutants displayed a significant reduction,suggesting that clathrin plays a regulatory role in rice yield trait.(3)Due to early aging of leaves in chc mutants,multiple senescence indices(including dark treatment of leaves,chlorophyll contents,peroxide contents,senescence-related gene expression and photosynthesis activity).The results showed that chc mutant senescence rate was slower,peroxide content and senescence-related genes expression levels were up-regulated,chlorophyll content and photosynthetic rate were decreased compared to the wild type.These results suggest that CHC plays a regulatory role in leaf senescence.(4)Quantitative analyses of the flag leaf angles revealed that the chc and clc mutants exhibited significantly increased leaf angles relative to the wild type.Analysis of qRT-PCR showed that the expression levels of BR-related genes in chc and clc mutants were elevated compared to the wild type,indicating that clathrin is involved in the regulation of leaf angle formation in rice.(5)The analyses of flower organs and seed phenotypes showed that pollen activities of chc1 chc2/+ and clc1/+ clc2 clc3 were reduced relative to the wild type.The chc1 chc2/+ mutants displayed multiple stigmas,whereas clc1/+ clc2 clc3 exhibited small seeds.These results indicate that clathrin may regulate rice fertility and seed development.(6)Live-cell imaging analysis showed the fluorescence signal intensities of DR5-GFP were significantly reduced in chc1 chc2/+ root tip elongation epidermal cells relative to the wild type,indicating that clathrin may regulate auxin biosynthesis and/or transport;and that CLC1-GFP fluorescence signal intensities in chc1 chc2/+ were reduced relative to the wild type,suggesting that CHC is necessary for CLC recruitment to the plasma membrane.In summary,these results showed that clathrin regulates rice yield through modulating yield-related agronomic traits(including leaf angles,flower organs,and seed size),leaf aging,and auxin biosynthesis and/or transport which provide a new insight to dissect the molecular action mechanisms of rice clathrin and are important for rice genetic breeding.