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Not AvailableResistance against the fungal pathogen Colletotrichum falcatum causing red rot is one of the most desirable traits for sustainable crop cultivation in sugarcane. To gain new insight into the host defense mechanism against C. falcatum, we studied the role of sugarcane microRNAs during compatible and incompatible interactions by adopting the NGS platform. We have sequenced a total of 80 miRNA families that comprised 980 miRNAs, and the putative targets of the miRNAs include transcription factors, membrane-bound proteins, glutamate receptor proteins, lignin biosynthesis proteins, signaling cascade proteins, transporter proteins, mitochondrial proteins, ER proteins, defense-related, stress response proteins, translational regulation proteins, cell proliferation, and ubiquitination proteins. Further, qRT-PCR analyses of 8 differentially regulated miRNAs and 26 gene transcript targets expression indicated that these miRNAs have a regulatory effect on the expression of respective target genes in most of the cases. Also, the results suggest that certain miRNA regulates many target genes that are involved in inciting early responses to the pathogen infection, signaling pathways, endoplasmic reticulum stress, and resistance gene activation through feedback response from various cellular processes during the compatible and incompatible interaction with the red rot pathogen C. falcatum. The present study revealed the role of sugarcane miRNAs and their target genes during sugarcane - C. falcatum interaction and provided new insight into the miRNA mediated defense mechanism in sugarcane for the first timeNot Availabl