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

The open reading regions of ZmPHT1s (inorganic phosphate [Pi] transporters) in maize 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 lncRNA 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 Pi homeostasis by interfering with miRNA399-guided cleavage of ZmPHT1 mRNAs.