Identification and characterization of microRNAs in turmeric (Curcuma longa L.) responsive to infection with the pathogenic fungus Pythium aphanidermatum

• 28 miRNAs responsive to Pythium aphanidermatum were identified from infected turmeric rhizomes.
• Stem-loop reverse transcription PCR validated the expression of representative miRNAs from different families.
• RLM-5′-RACE analyses identified and validated distinct miRNA targets.
• qPCR revealed simultaneous reciprocal changes in the expression pattern of miRNAs and the target transcripts.
• Turmeric miR164, miR393, miR482 and miRx2 were pried by P. aphanidermatum to facilitate pathogenesis.

Soft rot fungus, Pythium aphanidermatum, is the most devastating pathogen posing a serious constraint to turmeric (Curcuma longa L.) production worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important modulators of gene expression related to several stress responses. However, to the best of our knowledge, there is no report on miRNA expression profiling in turmeric and miRNA functions in response to P. aphanidermatum remains unclear. In the present study, we sequenced a small RNA library developed from P. aphanidermatum infected turmeric rhizomes and employed a de novo assembled turmeric genome for miRNA prediction and target characterization. In total, 28 miRNAs representing 25 conserved and three novel sequences responsive to P. aphanidermatum were identified. Stem-loop reverse transcription PCR validated the expression of representative miRNAs from different families. Further, we computationally predicted the miRNA targets, many of which were experimentally confirmed using ligation mediated 5′ rapid amplification of cDNA ends analysis. Quantitative RT-PCR analysis of 12 selected miRNAs revealed simultaneous reciprocal changes in the expression patterns of six miRNAs (miR159, miR160, miR164, miR393, miR482 and miRx2) and their target genes in the infected rhizomes, thus suggesting their critical involvement in the modulation of a compatible interaction between turmeric and P. aphanidermatum. Our results suggest that P. aphanidermatum may have developed a virulence mechanism by interfering with plant miRNAs like miR164, miR393, miR482 and miRx2 to reprogram the defense gene expression in turmeric and facilitate pathogenesis.