Preliminary Study On The Mechanism Of Rice MiR439 Regulating Disease Resistance And Yield

Rice blast is one of the most serious rice diseases leading to a yield reduction or even no grain harvest,and seriously threatens the safety of rice production in China.Plant endogenous micro RNAs(miRNAs)is a kind of 20～24 nt,single small RNAs,which is involved in the regulation of plant growth and stress responses.In our previous experiments,we found that rice miR439 negatively regulate rice blast disease resistance.However,whether miR439 co-regulated rice disease resistance and yield traits,as well as the related downstream signaling pathway was unclear.In this study,we systematically analyzed the phenotypes and agronomical characters of the transgenic lines blocking miR439 function(MIM439)and miR439-overexpressing transgenic lines(OX439a)in the field.We also screened and identified the downstream target genes of miR439.We constructed the target gene knockout mutants,the target site-mutated overexpressing lines,and the MIR439 knockout mutants.We analyzed the disease resistance of the above materials,and investigated their phenotypes and agronomic traits in the field.In summary,we obtained the following research results:1.Blocking miR439 function improves rice yield.OX439a lines displayed shortened plant height,smaller panicle size with reduced grain number per panicle,and decreased seed setting rate resulting in reduced grain yield per plant compared to the NPB control.Further study revealed that OX439 a grains showed increased grain length and width but decreased grain thickness,leading to unchange grain weight.MIM439 lines showed higher plants,but more panicles per plant resulting in enhanced grain yield per plant.2.Blocking miR439 function enhances rice blast disease resistance and reduce yield loss in field experiments.Compared with the control field,MIM439 lines showed a yield loss less than the NPB control,whereas OX439 a exhibited a yield loss more than the NPB control.3.Mutant MIR439 enhanced rice disease resistance and affected some agronomic characters.The mutant material was obtained by mutating MIR439 in the background of elite restorer line YH2115.Analysis of the disease resistance of the mutant material found that mutant of MIR439 enhanced rice resistance against the causative agents of blast(Magnaporthe oryzae)and sheath blight(Rhizoctonia solani),but had no significant effect on the resistance to the causative agents of leaf blight(Xanthomonas oryzae pv oryzae).Mutant of MIR439 reduced grain width and thousand grain weight,but a slight increase in the number of grains per spike,with no significant increase in yield per plant.4.miR439 targets and suppresses the expression of OsSTB1.4,OsWAKL14,and OsPR5.Screen miR439’s potential target genes from target gene prediction websites and validate them.OsSTB1.4 encodes a subtilase,OsWAKL14 encodes a cell wall associated receptor kinase,and OsPR5 encoding a sweet protein of PR5 protein family.RT-q PCR results showed that OsSTB1.4 and OsWAKL14 were down-regulated in OX439 a lines.Moreover,transient expression experiments showed that the expression of the intensity of the target site-fused YFP was repressed by MIR439 a.These results indicate that miR439 suppresses the expression of these three target genes.5.Target genes regulate rice blast resistance and yield traits.OsSTB1.4 and OsWAKL14 is differently responsive to M.oryzae.The results showed that the mutants of the three target genes were more susceptible to rice blast to M.oryzae.On the contrary,the transgenic lines overexpressing these target genes were more resistant to M.oryzae.In addition,mutations of OsMAKL14 lead to enhanced susceptibility to Xoo,while mutations of OsSTB1.4 or OsPR5 had no significant effect on rice leaf blight resistance.Mutations of OsSTB1.4 or OsPR5 showed smaller panicles than the ZH11 control.6 "miR439-target gene" module regulates PTI.GO analysis of the transcriptomic profiles of MIM439 and the NPB control revealed that the upregulated genes are most enriched in three pathways: up-regulated genes in MIM439 were enriched in three pathways: “response to chitin”,“defense response to bacterium”,and “defense response”,indicating that miR439 may negatively regulate the basal disease resistance or PTI.Reactive oxygen species(ROS)burst assay showed that miR439 a inhibited ROS burst and MIM439 enhanced ROS burst,indicating that blocking miR439 could enhance PAMP-triggered ROS.The target genes OsSTB1.4 and OsWAKL14 enhanced ROS burst and restored the ROS inhibited by miR439.In conclusion,miR439 negatively regulates blast resistance and rice yield.On the contrary,Blocking miR439 function could enhance blast resistance and rice yield simultaneously.Mutant of MIR439 in the YH2115 background can enhance rice resistance against the causative agents of blast and sheath blight.miR439’s target genes OsPR5,OsSTB1.4 and OsWAKL14 positively regulate blast resistance and enhance PTI signaling.The mutations of target genes OsPR5 or OsSTB1.4 resulting in reduced panicle length.These results indicate that miR439 regulates rice disease resistance and agronomic straits through the target genes OsPR5,OsSTB1.4 and OsWAKL14.Our results provide new mechanism that a miRNA ordinates disease resistance and yield traits,and provide gene resources for rice breeding.