Functional Annotation of Hypothetical Proteins Derived from Suppressive Subtraction Hybridization (SSH) Analysis Shows NPR1 (Non-Pathogenesis Related)-Like Activity

Abstract

Fusarium wilt is considered the most devastating banana disease incited by Fusarium oxysporum f. sp. cubense (FOC). The present study addresses suppressive subtraction hybridization (SSH) analysis for differential gene expression in banana plant, mediated through FOC and its interaction with biocontrol agent Trichoderma asperellum (prr2). SSH analysis yielded a total of 300 clones. The resultant clones were sequenced and processed to obtain 22 contigs and 87 singleton sequences. BLAST2GO (Basic Local Alignment Search Tool 2 Gene Ontology) analysis was performed to assign known protein function. Initial functional annotation showed that contig 21 possesses p38-like endoribonuclease activity and duality in subcellular localization. To gain insights into its additional roles and precise functions, a sequential docking protocol was done to affirm its role in the defense pathway. Atomic contact energies revealed binding affinities in the order of miRNA > phytoalexins > polyubiquitin, emphasizing their role in the Musa defense pathway. Contig 21 and polyubiquitin showed an atomic contact energy value of −479.60 kJ/mol, and even higher atomic contact energies were observed for miRNA (−804.86, −482.28, −494.75 kJ/mol), demonstrating its high RNA-binding properties. Phytoalexin contig 21-interacting interfacial residues were identified as rigid (10)/non-rigid (2) based on Bi, N values, and B-factor per residue. Hence, based on these results, contig 21 was characterized as a NPR1 (non-pathogenesis-related protein) homolog that is involved in plant defense and systemic induced resistance.