The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., 2015). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm (Michard et al., 2015). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

Barley is a diploid inbreeding crop with a genome of 5 GB organized into seven chromosomes. The relatively low chromosome number and their large size make barley an excellent model for chromosome cytogenetic studies of large genome cereal crops. Chromatin can be defined as euchromatin or heterochromatin. Euchromatin is gene-rich, less condensed, and transcriptionally active while the heterochromatin is gene-poor, remains highly condensed and has low transcriptional activity (Bartova et al., 2008; Sharakhov and Sharakhova, 2015). However, the mapping of nine Histone modifications has shown that this simple description is not accurate in barley. Instead, it has been shown that combinations of histones carrying different covalent modifications reveal 10 chromatin states partitioning barley chromosomes into three global environments (Baker et al., 2015). Briefly, in this protocol, barley roots (cv Morex) were collected, fixed in paraformaldehyde and squashed onto slides. Chromosome spreads were immunostained using antibodies against specific histone modifications, in particular H3K27me3, K3K27me1 and H3K9me2. We used confocal imaging to acquire stacked images and confirm the locations of these histone modifications on barley chromosomes.

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 10⁶ sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

Rhizosphere bacterial communities have become a major focal point of research in recent years, especially regarding how they affect plants and vice versa (Philippot et al., 2013). Changes in microbial density and diversity within the rhizosphere occur in a spatial temporal manner. The soil zone closest to the plant roots has the most density and diversity of microbes (Clark, 1940). The lack of methods to consistently isolate rhizosphere samples in a spatially defined manner is a major bottleneck in rhizosphere microbiology. We hypothesized that microbes with increasing affinities to and distance from the plant root can be isolated using increasing strengths of physical disruption. Sonication is an excellent choice due to the ability to gently remove rhizosphere soil and bacterial biofilms without damaging plant roots (Doi T et al., 2007; Bulgarelli et al., 2012; Lundberg et al., 2012). In addition, simply increasing the time of sonication can increase the amount of physical force. We used such an approach to consistently isolate microbial communities with different affinities to the soybean roots (White et al., 2014). This article describes the use of successive sonication to isolate distal, middle, and proximal soil from the rhizosphere of soybean roots.

Water is considered perhaps the most limiting factor for plant growth and productivity (Boyer, 1982), and climate change predicts more frequent, more severe and longer drought periods for a significant portion of the world in coming years. Unfortunately, drought resistance is particularly difficult to measure due in part to the complexity of the underlying biology that contributes to a plant’s ability to cope with water limitations. For example, water deficit is frequently examined by detaching leaves or withholding water for a set period of time prior to tissue collection. Such approaches may elucidate the early stages of drought response but are generally not physiologically relevant for maintenance of drought resistance over a longer period. A more realistic approach is to impose a gradual water limitation with a sustained soil moisture level, particularly in the case of woody perennials. We describe here a protocol that imposes a long-term water deficit under controlled laboratory conditions that allow a molecular biology approach to understanding how woody plants survive severe water limitations. Representative data can be found in Artlip et al. (1997) and Bassett et al. (2014).

One of the major goals of plant research is to improve crop yield, by for instance increasing seed oil or protein content. Besides this, extensive research is done to change seed fatty acid (FA) composition in order to make vegetable oils more suitable for specific purposes. To determine the effect of genetic changes on seed FA composition, oil, protein and sugar content it’s important to use standardised protocols to compare results between different research groups. Here we describe standardised methods for the analysis of seed FA composition, oil, protein and sugar content.