LCM-based xylem-specific RNA extraction from Fusarium oxysporum infected Cicer arietinum roots

Abstract

Fusarium oxysporum f. sp ciceris, the causal organism of chickpea vascular wilt disease reduces yield up to 90% annually. Xylem plays a prominent role during Fusarium wilt. In resistant hosts, vascular wilt pathogens are presumably recognized by both extra and intracellular plant receptors that elicit defense responses in the xylem vessels. However, in susceptible hosts, fungus colonized in the xylem causing vessel clogging leading to wilting of the plant. In this study, we have described an efficient method to obtain RNA of high quality in sufficient amount from LCM-derived chickpea root xylem tissue. This includes sample preparation by tissue fixation, OCT embedding, cryo-sectioning, dissecting the target cells, and finally capturing them using infrared rays. RNA was extracted from these captured xylem cells, quantified using bioanalyzer to yield quantity ranging from 92–114 ng µl⁻¹ with RIN value up to 7. These RNA samples would be utilized for molecular studies like in our case transcriptome analysis to find out genes involved in wilt resistance in chickpea. We were able to successfully isolate RNA using the above procedure from fungus-infected chickpea root samples of susceptible, resistant, and control plants. Histological observation indicating the infestation of Foc2 in the vicinity of xylem tissue of root was done.