Molecular Mechanism Of Rice MiR156 And MiR535 To Coordinate Resistance To Rice Blast And Agronomic Traits

Rice blast is one of the important rice diseases.When the disease occurs,it reduces the rice grain yield and threatens the safety of agriculture food production.Micro RNAs（miRNAs）are one kind of 20-to 24-nucleotide（nt）noncoding RNA,and play essential roles in the regulation of plant growth and defense responses.Here,we found that over-expression of a target mimic of miR156fhl-3p affects the abundance of miR156-5p,which in turn regulates a target gene of miR156-5p to fine-tune the tradeoffs between blast disease resistance and yield traits.First,we found that miR156fhl-3p is responsive to the fungal pathogen in LTH and IRBLkm-Ts accessions.Then we constructed transgenic lines to over-expression of a target mimic of miR156fhl-3p（MIM156-3p）,which significantly decreased miR156fhl-3p accumulation.MIM156-3p plants showed much smaller disease lesions and less fungal mass than control via punch/spray-inoculation of the M.oryzae strain GZ8 and 97-27-2.To understand why blocking miR156fhl-3p leads to compromised susceptibility to blast disease,we examined the expression of two defense-related response marker genes,PR1a and NAC4,upon M.oryzae strain GZ8 infection.The expression of both genes were induced to significantly higher levels by GZ8 in MIM156-3p than that in control at 48 hpi.Then we compared the infection process in leaf sheath of control and MIM156-3p plants,and found that the fungal growth was inhibited at 24 and 36 hpi compared with WT.These observations suggested that blocking miR156fhl-3p promoted the induction of defense-related genes and delayed the infection process of M.oryzae.Activation of blast resistance often penalizes growth and agronomic traits.The results of statistical analysis show that several agronomic traits are slightly but not significantly decrease in MIM156-3p in comparison with those in WT,including plant height,number of tillers per plant,panicle length,and number of primary rachis branches per panicle（No.of PBs）.The number of secondary rachis branches per panicle（No.of SBs）is significantly decreased in MIM156-3p plants,leading to a reduced number of grains per panicle.In contrast,the seed length and width in MIM156-3p were increased,resulting in an increased 1000-grain weight.As a result,the grain yield per plant was unchanged in MIM156-3p in comparison with that in WT.To further investigate the mechanism of how miR156fhl-3p enhances rice resistance,we screened the target genes of miR156fhl-3p and try to find another mechanism to uncovered its function.We found that miR156-5p was down-regulated in MIM156-3p,and the pre-miR156fh was also reduced.It showed that miR156fhl-3p may exercise its function by affecting the corresponding miRNA-5p.miR156 target SPL genes to regulate plant growth and development,and SPL14 positively regulates the rice blast resistance via binding WRKY45 promoter to activate its expression.The m RNA amounts of SPL14 and WRKY45were significantly higher in MIM156-3p than in control,and the expression of WRKY45 was further significantly increased compared to that in control at 48 hpi upon M.oryzae induction.To confirm the interference of miR156fhl-3p with miR156-5p,we established a Yellow Fluorescent protein-based（YFP-based）reporter system.MIM156-3p could release the suppression of SPL14 by miR156 via interference with miR156-5p,leading to activation of SPL14.As MIM156-3p plants enhanced blast resistance,over-expression of miR156h（OX156）enhanced the susceptibility to rice blast disease.In OX156 plants,SPL14 and WRKY45 were down-regulated.We also focused on another differentially expressed miRNA,miR535.miR535 was expressed higher in the susceptibility accession LTH than in the resistance accession IRBLkm-Ts upon M.oryzae inoculation.Suppressing miR535 via s target mimic（MIM535）enhanced rice resistance to M.oryzae,increased expression of defense-related genes,delayed M.oryzae infection process and less H₂O₂ accumulation.In contrast,overexpression of miR535（OX535）compromised rice resistance to M.oryzae.To further clarify the mechanism of miR535,we confirmed that SPL14 was targeted by miR535 via using RT-q PCR and 5’RLM-RACE experiments.Taken together,these results indicate that miR156fhl-3p mounts a regulatory role on miR156-5p,which subsequently regulates the expression of SPL14 and WRKY45 to improve rice blast disease resistance and without yield penalty.miR535 cooperates with miR156 to regulate the expression of SPL14.Our results provided a potential tool to improve blast resistance without yield penalty and a new phenomenon in miRNA synthesis/accumulation.