Evaluation of BG, NPR1, and PAL in cotton plants through Virus Induced gene silencing reveals their role in whitefly stress.

Whitefly is one of the most hazardous insect pests that infests a wide range of host plants and causes huge damage to crop worldwide. In order to engineer plants resilient to whitefly stress, it is important to identify and validate the responsive genes by exploring the molecular dynamics of plants under stress conditions. In this study three genes BG, NPR1, and PAL genes have been studied in cotton for elucidating their role in whitefly stress response. Initially, insilico approach was utilized to investigate the domains and phylogeny of BG, NPR1 and PAL genes and found out that these genes showed remarkable resemblance in four cotton species Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. In BG proteins the main functional domain was X8 belonging to glycohydro superfamily, in NPR1 two main functional domains were BTB_POZ at N terminal and NPR1_like_C at C terminal. In PAL functional domain PLN was found which belongs to Lyase class I superfamily. The promoter analysis of these genes displayed enrichment of hormone, stress and stimuli responsive cis elements. Through Virus Induced Gene Silencing (VIGS), these genes were targeted and kept under whitefly infestation. Overall, the whitefly egg and nymph production were observed 60-70% less on gene down regulated plants as compared to control plants. The qPCR-based expression analysis of certain stress-responsive genes showed that in BG down regulated plants the elevated expression of these whitefly responsive genes was detected, in NPR1 down regulated plants JAZ1 and HSP were found up regulated, ERF1 and WRKY40 didn't show significant differential expression, while MAPK6 was slightly down regulated. In PAL down regulated plants ERF1 and JAZ1 showed elevated expression while others didn't show significant alternation. Differential expression in gene down-regulated plants showed that whitefly responsive genes act in a complex inter signaling pathway and their expression impact each other. This study provides valuable insight into the structural and functional analysis of important whitefly responsive genes BG, NPR1, and PAL. The results will pave a path to future development of whitefly resilient crops.