An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

Buddhini Ranawaka,Milos Tanurdzic,Peter Waterhouse,Fatima Naim

doi:10.1007/s11033-020-06013-1

Abstract

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

Highlights

Nicotiana benthamiana is a plant species endemic to Australia first discovered by Benjamin Bynoe in 1839 [1]
The majority of published plant ChIP protocols are developed for seedlings and young plant tissues [12, 36, 37, 39–41] and we found that the methods developed for Arabidopsis [39] and tomato leaves [41] were unsuitable for ChIP of mature N. benthamiana leaves
Prepared 1% formaldehyde is used for crosslinking and this was found to be appropriate for mature leaves of N. benthamiana (Fig. 1a, b)

Summary

Introduction

Nicotiana benthamiana is a plant species endemic to Australia first discovered by Benjamin Bynoe in 1839 [1]. The N. benthamiana ecotype used in laboratories all over the world is referred to as the ‘Lab’ isolate and another five wild isolates were collected from climaticaly diverse parts of Australia [2, 3]. N. benthamiana may have successfully passed the initial stage of genome instability and entered the prolonged phase of genome evolution referred to as diploidisation. In this phase, duplicated genes, chromosomes or chromosome fragments are progressively lost or retained due to the combined modifications in genetic and epigenetic structures [4–6]. The species has an allotetraploid genome (~3.1 Gb)

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