The WRKY family of transcription factors orchestrate the reprogrammed expression of the complex network of defense genes at various biotic and abiotic stresses. Within the last 96 million years, three rounds of Musa polyploidization events had occurred from selective pressure causing duplication of MusaWRKYs with new activities. Here, we identified a total of 153 WRKY transcription factors available from the DH Pahang genome. Based on their phylogenetic relationship, the MusaWRKYs available with complete gene sequence were classified into the seven common WRKY sub-groups. Synteny analyses data revealed paralogous relationships, with 17 MusaWRKY gene pairs originating from the duplication events that had occurred within the Musa lineage. We also found 15 other MusaWRKY gene pairs originating from much older duplication events that had occurred along Arecales and Poales lineage of commelinids. Based on the synonymous and nonsynonymous substitution rates, the fate of duplicated MusaWRKY genes was predicted to have undergone sub-functionalization in which the duplicated gene copies retain a subset of the ancestral gene function. Also, to understand the regulatory roles of MusaWRKY during a biotic stress, Illumina sequencing was performed on resistant and susceptible cultivars during the infection of root lesion nematode, Pratylenchus coffeae. The differential WRKY gene expression analysis in nematode resistant and susceptible cultivars during challenged and unchallenged conditions had distinguished: 1) MusaWRKYs participating in general banana defense mechanism against P.coffeae common to both susceptible and resistant cultivars, 2) MusaWRKYs that may aid in the pathogen survival as suppressors of plant triggered immunity, 3) MusaWRKYs that may aid in the host defense as activators of plant triggered immunity and 4) cultivar specific MusaWRKY regulation. Mainly, MusaWRKY52, -69 and -92 are found to be P.coffeae specific and can act as activators or repressors in a defense pathway. Overall, this preliminary study in Musa provides the basis for understanding the evolution and regulatory mechanism of MusaWRKY during nematode stress.

Eumusae leaf spot disease, caused by Mycosphaerella eumusae, is widely distributed in different banana growing countries in Asia and Africa, causing severe losses in yield and quality of banana. The aim of this study was to identify the differentially expressed defense responsive genes through subtractive suppression hybridization (SSH) in the Musa-M. eumusae interaction system. In this method, leaf samples collected at different intervals between 0 and 72 h postinoculation (hpi) of M. eumusae from resistant (Manoranjitham-AAA) and susceptible (Grand Naine-AAA) cultivars were subjected to cDNA-SSH library construction. From the SSH library, 832 clones having the insert were selected and sequenced. All these sequences were assembled using CAP3, which resulted in 498 unigenes (59.85%), consisting of 78 contigs and 420 singletons. Furthermore, the BLAST2GO analysis performed showed that, out of the 498 unigenes, only 161 (32.32%) were involved in molecular functions, cellular component and biological processes. However, among these 161 unigenes, 57 were found to have top BLAST hits. Six genes were selected based on the function and subjected to validation through qPCR. Four genes, namely ethylene responsive factor, flavin containing monoxygenase, serine glyoxylate, and metallothionein, were found to have the highest-fold regulation in the resistant cultivar at 48 hpi, followed by lipoxygenase at 36 hpi. The main findings are that resistant plants deploy HR activity as a strategy to evade the fungus followed by the activation of various ROS scavengers to maintain homeostasis and the role of phytohormones may be induced as a part of plant defense against M. eumusae.