| TaGW2,a RING-finger type E3 ubiquitin ligase,is widely used in high-yield breeding of wheat as a key locus regulating grain size and grain weight.In recent years,although TaGW2 has been widely studied for its function of regulating grain size and grain weight in wheat,whether TaGW2 is involved in the regulation of stress response in wheat,and its role in the trade-off between stress response and wheat yield are still unknown.To this end,TaGW2 was used to study its function and mechanism in drought resistance and stripe rust resistance of wheat.The main results are as follows:Ⅰ.TaGW2 mediates the ubiquitination and degradation of TaARR12 to regulate drought resistance in wheat1.TaGW2 negatively regulates grain size and weight and positively regulates drought resistance of wheatThe tagw2 gene editing mutant in KN199 background and the TaGW2 overexpression（OE）and RNA interference（RNAi）transgenic wheat in Fielder background were obtained,respectively.Yield traits such as plant height,spike length,kernel length,kernel width,and thousand kernel weight were evaluated.It was confirmed that TaGW2 was a negative regulator of grain size and weight in wheat.To clarify the function of TaGW2 in drought resistance of wheat,the drought resistance of TaGW2 transgenic wheat and tagw2 mutant was evaluated respectively.The results showed that the water consumption and water loss rate of OE lines under drought stress were slower than those of the control plants,and the drought tolerance of OE lines was significantly better than that of the control plants.However,RNAi silencing lines and tagw2mutants lost water faster than control plants and were more sensitive to drought stress.under water deficit conditions,the stomatal apertures of OE lines were significantly smaller while those of RNAi lines larger compared to WT plants.OE plants had significantly higher water use efficiency（WUE）than Fielder,while RNAi plants had lower WUE than the control plants under water scarcity conditions.The carbon isotope discrimination(δ13C)analyses also revealed the reduced 13C fractionation in leaves of OE lines while increased 13C fractionation in RNAi lines than that in WT.Taken together,TaGW2 could enhance drought resistance of wheat by increasing water use efficiency.2.Transcription factor TaARR12 is the ubiquitination target of TaGW2A total of 68 proteins interacting with TaGW2 were identified by yeast two-hybrid（Y2H）screening library.Further combined with ubiquitinome and proteome mass spectrometry analysis and yeast two-hybrid screening library,9 candidate proteins with significantly down-regulated ubiquitination level and up-regulated protein expression level in tagw2 mutants were finally identified.Among them,the expression level of TaARR12protein was most significantly up-regulated in tagw2 mutant,indicating that it may be the TaGW2 interaction target protein.The interaction between TaGW2 and TaARR12 was further verified by yeast two-hybrid（Y2H）,Luciferase Complementation Imaging（LCI）,in vitro Pull-down assay and in vivo Co-IP in N.benthamiana.A series of experiments,including in vitro ubiquitination,cell-free protein degradation in vitro,and in vivo ubiquitination in N.benthamiana,verified that TaGW2 can mediate the ubiquitination and degradation of TaARR12 through ubiquitin-26S proteasome pathway.3.TaARR12 negatively regulates wheat drought resistanceIn order to identify the function of TaARR12 in drought tolerance,TaARR12overexpressed（OE）and silenced（RNAi）transgenic wheat lines were created and their drought tolerance was analyzed.The results showed that the drought resistance of TaARR12RNAi lines was higher than Fielder,and OE lines were more sensitive to drought.Comparation of the stomates revealed that the stomatal apertures of RNAi lines were significantly smaller than OE and control plants under well-watered conditions,while the stomatal apertures of OE and control plants were not differed.However,the stomatal apertures of RNAi lines were significantly smaller while those of OE lines larger compared to control plants under water deficit conditions.Taken together,TaARR12 was a negative regulator of drought stress response in wheat.4.TaARR12 binds to CRM cis-elements to inhibit the expression of drought response genesTo gain a deeper understanding of the regulatory mechanism of TaARR12 in wheat drought stress response,a total of 8,561 TaARR12 binding sites were identified in 5,485candidate target genes by DNA affinity purification sequencing（DAP-seq）.Further combined DAP-seq and RNA-seq analysis identified 2,108 differentially expressed genes that were transcriptionally regulated by TaARR12.Four TaARR12-binding motifs containing cytokine-response elements（CRM,5’-[A/G]GAT[T/C]-3’）were screened by MEME-Ch IP analysis.Furthermore,In vitro electrophoretic mobility shift assay（EMSA）confirmed that TaARR12 protein could directly bound to four motifs.In addition,several drought-induced b ZIP genes were found to be downregulated in TaARR12 overexpression lines.Among them,Ta ABF2 was verified as the direct target of TaARR12.By EMSA,Ch IP-q PCR and dual-LUC detection,it was further confirmed that TaARR12 inhibited the expression of Ta ABF2.In addition,Ta ABF2 OE lines can improve drought resistance of wheat.In conclusion,TaARR12 can directly binds to CRM cis-elements to inhibit the expression of Ta ABF2 and other drought response genes,thus reducing drought resistance of wheat.Ⅱ.TaGW2 mediates ubiquitination and degradation of Ta Sn RK1γto inhibit plant immunity1.TaGW2 negatively regulates resistance to Pst in wheatTaGW2 could be induced in both the incompatible and compatible interaction patterns of wheat and Pst,suggesting that TaGW2 might be involved in the interaction between wheat and Pst.When TaGW2 transgenic lines were inoculated with CYR23 and CYR31,the OE transgenic lines produced more spores than control Fielder,and the hypersensitivity necrosis reaction（HR）was significantly reduced.While the HR of RNAi transgenic lines was significantly higher than that of Fielder,and the sporulation was significantly less than that of Fielder.The biomass analysis,the H2O2area and the hypha length and area statistical analysis results were consistent with the phenotype.All these results suggested that TaGW2negatively regulated wheat resistance to Pst.After inoculation of various Pst races,it was found that the negative regulation of TaGW2 on stripe rust resistance was broad-spectrum.2.The protein kinase Ta Sn RK1γis the ubiquitination target of TaGW2In order to elucidate the mechanism of TaGW2 regulating stripe rust resistance of wheat,further analysis of TaGW2 candidate ubiquitination target protein showed that Ta Sn RK1γhad four significant down-regulated ubiquitination sites.The interaction between TaGW2and Ta Sn RK1γwas further verified by Y2H,LCI,Pull-down in vitro and Co-IP in N.benthamiana.The ubiquitination and degradation of Ta Sn RK1γmediated by TaGW2through ubiquitin-26S proteasome pathway were verified by in vitro ubiquitination,in vitro cell free protein degradation assay and in vivo ubiquitination assay in N.benthamiana.3.Ta Sn RK1γpositively regulates resistance to Pst in wheatSubcellular localization analysis showed that Ta Sn RK1γwas localized only in the cytoplasm.Both Ta Sn RK1γoverexpressed and silenced lines were inoculated with CYR23and CYR31,respectively.The spores of Pst in OE lines were significantly lower than that of Fielder,while the spores were evident in RNAi lines.Further biomass analysis and the H2O2area and the hypha length and area statistical analysis results of Ta Sn RK1γtransgenic wheat after inoculation also showed that Ta Sn RK1γplayed a positive role in regulating wheat resistance to Pst,and TaGW2 inhibited plant immunity by mediating Ta Sn RK1γubiquitination.In summary,the function of TaGW2 in the positive regulation of drought resistance and the negative regulation of grain size,yield and Pst resistance in wheat was identified by the investigation of yield traits,drought resistance and disease resistance.Furthermore,molecular biology,plant physiology,genetics and bioinformatics methods were used to reveal the molecular mechanism of TaGW2 enhancing drought resistance by promoting the ubiquitination and degradation of transcription factor TaARR12,and the molecular mechanism of TaGW2 regulating plant immunity by mediating the ubiquitination and degradation of Ta Sn RK1γ.These results provide an important theoretical basis for the trade-off between stress resistance and yield in wheat,and also provide important genetic resources for the improvement of stress resistance in wheat. |
