Molecular Mechanism And Breeding Application Of MiR156-IPA1 In Rice Resistance To Bacterial Blight Disease

Pests and diseases cause enormous loss of crop yield and threaten global food security.Breeding crops with disease resistance is the most efficient and safe way to control diseases.However,increasing disease resistance in crop often has a negative effect on growth and development,resulting in yield penalty.Therefore,it is a fundamental challenge to simultaneously improve disease resistance and yield in crop breeding.Cloning disease resistance genes without adverse effect on yield-related traits is an efficient way to solve this problem.However,due to the scarcity of such kind of genes,it is urgent to explore new breeding strategies to achieve high yield and high disease resistance.In rice,as one target gene of miR156,IPA1 encodes a transcription factor with SBP domain.Slightly increased IPA1 in ipal-1D and ipa1WFP reduces inefficient tillers,but increases panicle branches and stem strength,which result in yield enhancement.As an ideal plant architecture gene,IPA1 is an important breeding target for increasing yield in rice.In this thesis,we found that IPA1 functions in disease resistance against bacterial blight,and the underlying mechanism was elucidated.Based on these knowledges,we created new germplasm with high yield and high resistance through inducible overexpression of IPA1.The research results are summarized as follows:1.miR156-IPA1regulate disease resistance in rice.Under Xoo infection,miR156 was up-regulated,while its target gene OsSPLs were down-regulated at the beginning,and then recovered,suggesting that miR156-OsSPLs might be involved in disease resistance against bacterial blight.However,the two elite alleles of IPA1,ipa1-1D and ipa1WFP,in which IPA1 was slightly increased,did not enhance disease resistance against bacterial blight,demonstrating that a slight increase in IPA1 expression can not enhance disease resistance against bacterial blight.Meanwhile,we found that downregulation of miR156(MIM156)and overexpression of target genes IPA1(IPA1-OE)and OsSPL7(OsSPL7-OE)significantly enhanced disease resistance against bacterial blight.In contrast,overexpression of miR156(miR156-OE)compromised disease resistance,demonstrating that miR156 negatively regulates disease resistance,while IPA1 and OsSPL7 positively regulate disease resistance.Although MIM156,IPA1-OE and OsSPL7-OE greatly enhanced disease resistance,these plants showed delayed germination,shorter plant height,fewer tillers and lower fertility compared to wild type(WT).Even the number of panicle branches in those plants increased,the number of tillers and panicle grains significantly decreased,resulting in a significant decrease in yield.The yield-related traits hindered the application of IPA1 and OsSPL7 in rice breeding.2.miR156-IPA1 enhanced disease resistance and inhibited growth through modulating gibberellin pathway.The protein-protein interaction assays including yeast two hybrid,split-LUC and BiFC demonstrated that IPA1 and OsSPL7 directly interact with gibberellin repressor protein,SLR1.Western Blot assays showed that SLR1 proteins were over-accumulated in MIM156,IPA1-OE and OsSPL7-OE plant.In addition,GA-mediated SLR1 degradation in MIM156,IPA1-OE and OsSPL7-OE was significantly delayed than in the WT,indicating that IPA1 and OsSPL7 increased the protein stability of SLR1.Further studies found that MIM156,IPA1OE and OsSPL7-OE were less sensitive to gibberellin in seed germination,plant growth and disease susceptibility against bacterial blight than WT.All the results suggested the possibility that MIM156,IPA1-OE and OsSPL7-OE enhance disease resistance and inhibit growth through modulating gibberellin pathway.Furthermore,the gibberellin-inactivated enzyme encoding gene,EUI1,was upregulated in MIM156,IPA1-OE and OsSPL7-OE than in WT.Consistently,the levels of several GA molecules,including GA4 in IPA1-OE were significantly lower than in the WT.To further determine the relationship between EUI1 and IPA1,we conducted genetic analysis.We introduced EUI1-RNAi into MIM156 and IPA1-OE to get EUI1-RNAi/MIM156 and EUI1-RNAi/IPA1-OE double transgenic rice.SLR1 protein accumulation was reduced in these plants.Consequently,plant stature was rescued and disease resistance was compromised.Taken together,IPA1 regulate disease resistance and plant growth via gibberellin.The slr1-1/MIM156 and slr1-1/IPA1-OE were constructed by crossing MIM156,IPA1-OE with slr 1-1 mutant.The plant stature in slr1-1/MIM156 and slr1-1/IPA1-OE were significantly increased,while disease resistance were significantly reduced than in MIM156 and IPA1-OE,respectively,indicating that the regulatory role of IPA1 and OsSPL7 on disease resistance and plant growth depends on SLR1.Overall,IPA1 and OsSPL7 increased SLR1 protein stability through physically interacting with SLR1.In addition,the upregulation of EUI1 in MIM156,IPA1-OE and OsSPL7-OE plants decreased gibberellin levels,which also contribute to the SLRl accumulation.The elevated SLRl in those plants enhanced disease resistance and inhibited growth.3.Inducible overexpression of IPA1 improved both yield and disease resistance in rice.The rice varieties with ipa1-1D and ipa1WFF significantly enhanced yield,but did not increase disease resistance.Whereas,MIM156,IPA1-OE and OsSPL7-OE transgenic rice increased disease resistance,but adversely affect yield-related traits.This antagonism between yield and disease resistance in rice limited the application of IPA1 in rice breeding.In order to coordinate disease resistance and yield,we used the promoter of OsHEN1,which contains TALE binding site in its promoter and is up-regulated by Xoo infection,to express IPA1.In OsHEN1::IPA1(HIP)transgenic rice plants,the transcriptional level of IPA1 was slightly up-regulated in the absence of pathogen,resulting ideal plant architecture,including large panicle,strong stem,and less tillers.In the presence of pathogens,the expression of IPA1 was induced rapidly and locally,which significantly enhanced disease resistance.Therefore,the strategy of inducible overexpression of IPA1 achieved high yield and high disease resistance.Together,we found that the down-regulation of miR1 56 and overexpression of IPA1 and OsSPL7 enhanced disease resistance against Xoo,but reduced yield.IPA1 regulated disease resistance and growth through regulating gibberellin metabolism and signaling.We utilized the pathogen inducible promoter to drive IPA1 to create the transgenic rice HIP.The HIP plants exhibited high yield and high disease resistance.