Title: Xylem-specific RNA isolation from Fusarium oxysporum infected chickpea (Cicer arietinum) roots using LCM approach

Abstract

Fusarium oxysporum f. sp. ciceris, the causal organism of chickpea vascular wilt disease reduces yield upto 90% annually. Xylem play prominent role in vascular wilt resistance in plants. Transcriptome profiling of root xylem from contrasting chickpea genotypes will be very useful to decipher genes involved in wilt resistance. RNA isolation from root xylem tissue is an arduous procedure that has not yet been standardized. In this study, we have described an efficient method to obtain RNA of high quality in a sufficient amount from LCM-derived chickpea root xylem tissue. For this, we have infected two contrasting chickpea genotypes WR315 (wilt resistant) and JG62 (wilt susceptible) with F. oxysporum f. sp. ciceris race 2 (Foc2) strain to understand molecular interactions. Control and Fusarium infected root tissue samples were collected post-inoculation of Foc2. Root tissue samples were processed by fixing, embedding, and cryosectioning. Root sections were collected on poly-L-lysine coated PEN membrane slides. These slides were processed for OCT removal and dehydration. Sections were dehydrated using ethanol and xylene and further utilized for microdissectioning under Laser Capture Microdissection (LCM) machine. Histological analysis using lactophenol cotton blue stain indicating the infestation of Foc2 in the vicinity of root xylem tissue. Dehydrated sections were imaged, marked around xylem cells, dissected using infrared rays, and finally catapulted in AdhesiveCaps through LCM (Figure 1). RNA was extracted from approx 7200 xylem cells of each sample and quantified using bio-analyzer that yielded intact RNA ranges from 92-114 ng µl-1 with RIN value up to 7 (Table 1). These RNA samples would be utilized for molecular studies i.e. transcriptome analysis to find out genes involved in Fusarium wilt resistance in chickpea. Our protocol was capable of extracting good quality RNA from Fusarium-infected chickpea root tissues, with good RIN value. This optimized LCM-based RNA extraction method from root xylem tissue is useful in the applications like gene expression studies in xylem cells during biotic and abiotic stresses.