The long non-coding RNA PILNCR2 increases low phosphate tolerance in maize by interfering with miRNA399-guided cleavage of ZmPHT1s

Theopen reading regions of ZmPHT1s(inorganic phosphate[Pij transporters)inmaize possess target sites of microRNA399(miR399).However,the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored.We show here that ZmPHT1;1,ZmPHT1;3,and ZmPHT1;13 are the targets of ZmmiRNA399.We found that a long non-coding RNA,PILNCR2(Pi-deficiency-induced IncRNA 2),is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm.A ribonuclease protection assay and an RNA-RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro.A co-expression assay in N.benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs.Overexpression of PILNCR2 increased low-Pi tolerance in maize,whereas its knockout and knockdown decreased low-Pi tolerance in maize.Consistently,ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants,suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize.Collectively,these results indicate that PILNCR2 plays an important role in maize Pihomeostasisby interfering with miRNA399-guided cleavageof ZmPHT1mRNAs.