Meredith L. Biedrzycki

Root exudates mediate kin recognition in plants

Though recent work has demonstrated that plants can recognize species, kin versus strangers, and self/non-self roots, no mechanism for identity recognition in plants has yet been found. Here we examined the role of soluble chemicals in signaling among roots. Utilizing Arabidopsis thaliana, we exposed young seedlings to liquid media containing exudates from siblings, strangers (non-siblings), or only their own exudates. In one experiment, root secretions were inhibited by sodium orthovanadate and root length and number of lateral roots were measured. In a second experiment, responses to siblings, strangers, and their own exudates were measured for several accessions (genotypes), and the traits of length of the longest lateral root and hypocotyl length were also measured. The exposure of plants to the root exudates of strangers induced greater lateral root formation than exposure of plants to sibling exudates. Stranger recognition was abolished upon treatment with the secretion inhibitor. In one experiment, plants exposed to sibling or stranger exudates have shorter roots than plants only exposed to their own exudates. This self/non-self recognition response was not affected by the secretion inhibitor. The results demonstrate that that kin recognition and self/non-self are two separate identity recognition systems involving soluble chemicals. Kin recognition requires active secretion by roots.

The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana.

Majority of plant growth promoting rhizobacteria (PGPR) confer plant immunity against a wide range of foliar diseases by activating plant defences that reduce a plant’s susceptibility to pathogen attack. Here we show that Arabidopsis thaliana (Col-0) plants exposed to Bacillus subtilis strain FB17 (hereafter FB17), results in reduced disease severity against Pseudomonas syringae pv. tomato DC3000 (hereafter DC3000) compared to plants without FB17 treatment. Exogenous application of the B. subtilis derived elicitor, acetoin (3-hydroxy-2-butanone), was found to trigger induced systemic resistance (ISR) and protect plants against DC3000 pathogenesis. Moreover, B. subtilis acetoin biosynthetic mutants that emitted reduced levels of acetoin conferred reduced protection to A. thaliana against pathogen infection. Further analysis using FB17 and defense-compromised mutants of A. thaliana indicated that resistance to DC3000 occurs via NPR1 and requires salicylic acid (SA)/ethylene (ET) whereas jasmonic acid (JA) is not essential. This study provides new insight into the role of rhizo-bacterial volatile components as elicitors of defense responses in plants.

Causes and consequences of plant-associated biofilms

The rhizosphere is the critical interface between plant roots and soil where beneficial and harmful interactions between plants and microorganisms occur. Although microorganisms have historically been studied as planktonic (or free-swimming) cells, most are found attached to surfaces, in multicellular assemblies known as biofilms. When found in association with plants, certain bacteria such as plant growth promoting rhizobacteria not only induce plant growth but also protect plants from soil-borne pathogens in a process known as biocontrol. Contrastingly, other rhizobacteria in a biofilm matrix may cause pathogenesis in plants. Although research suggests that biofilm formation on plants is associated with biological control and pathogenic response, little is known about how plants regulate this association. Here, we assess the biological importance of biofilm association on plants.

Cyanogenic Pseudomonads Influence Multitrophic Interactions in the Rhizosphere

In the rhizosphere, plant roots cope with both pathogenic and beneficial bacterial interactions. The exometabolite production in certain bacterial species may regulate root growth and other root-microbe interactions in the rhizosphere. Here, we elucidated the role of cyanide production in pseudomonad virulence affecting plant root growth and other rhizospheric processes. Exposure of Arabidopsis thaliana Col-0 seedlings to both direct (with KCN) and indirect forms of cyanide from different pseudomonad strains caused significant inhibition of primary root growth. Further, we report that this growth inhibition was caused by the suppression of an auxin responsive gene, specifically at the root tip region by pseudomonad cyanogenesis. Additionally, pseudomonad cyanogenesis also affected other beneficial rhizospheric processes such as Bacillus subtilis colonization by biofilm formation on A. thaliana Col-0 roots. The effect of cyanogenesis on B. subtilis biofilm formation was further established by the down regulation of important B. subtilis biofilm operons epsA and yqxM. Our results show, the functional significance of pseudomonad cyanogenesis in regulating multitrophic rhizospheric interactions.

An Optical Clearing Technique for Plant Tissues Allowing Deep Imaging and Compatible with Fluorescence Microscopy

An optical clearing technique complements common fluorescent microscopic techniques and enables deep imaging in a wide range of plant tissues. We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process.

Root secretions: from genes and molecules to microbial associations

As the field of root biology continues to gain momentum, researchers have begun to recognize the importance of root secretions in innumerable plant–plant and plant–microbe interactions hidden beneath the ground. It is well documented that diverse plant species form both beneficial and harmful associations with soil bacterial communities. Understanding all of the factors involved in these rhizosphere communications is the daunting task that has been laid before root biologists. However, Micallef et al. (2009) have made progress in this field by describing the influence of genetic background on root secretions in Arabidopsis thaliana and the implications of the unique root secretion patterns on associated bacterial communities. In the past, root secretion patterns of rice, wheat, and barley have been analysed and compared, revealing variations between species (Mazzola et al., 2004). In addition, root secretion profiles of the model plant A. thaliana (Columbia-0) have been analysed after treatment with biological elicitors and signalling molecules to reveal 289 possible different secondary metabolites present in the secretions (Walker et al., 2003; Narasimhan et al. 2003). These studies point to the countless combinations of secondary metabolites that may be present in the rhizosphere at any given time. Micallef et al. (2009) took the current research one step further and were interested in determining whether genetic influences within a species can produce unique root secretion cocktails. In their recent paper, they compared the differences between root secretion profiles, through HPLC, of eight different A. thaliana accessions (ecotypes) and determined that root secretion profiles did indeed differ in the compounds present and in the relative abundance of many of these compounds (Micallef et al., 2009). As all of the plants from different accessions were grown under the same conditions, natural genetic variation is attributed to cause differences in the secreted compounds. These data support the research of Clark et al. (2007) where it was demonstrated that when 20 A. thaliana accessions were compared, approximately 4% of the genome differed or were deleted with reference to the control (Columbia-0). As the eight different accessions tested by Micallef et al. (2009) were originally collected from a wide geographical range, it supports the suggestion that allelic variations between these plants may confer a selective advantage in certain environments, as has been demonstrated for traits such as flowering time and plant growth (Koornneef et al., 2004). Along these lines, Micallef et al. (2009) further investigated as to whether these unique secretion cocktails resulted in variations in bacterial community associations. Previous research by Broeckling et al. (2008) demonstrated that, in both model species, Medicago truncatula and A. thaliana, plants were able to maintain resident soil fungal populations but were not able to maintain non-resident populations, demonstrating that root exudates are able to regulate some soil microbe populations. Micallef et al. (2009) continued this line of research and demonstrated that bacterial populations are also influenced by root secretion compounds as they found that each of the eight accessions tested has distinct and reproducible bacterial community associations. These bacterial community associations differed in the species present and in the abundance of the species associated with the A. thaliana roots. Although a direct link was not examined, strong evidence has been presented to support that differences in bacterial community assemblages is a result, in part, of root secretions compounds. Other root traits that differ between accessions, such as root architecture are likely to play a role as well. It has long been known that microbial communities in the soil contribute valuable nutrients to plants (e.g. rhizobia and Azolla-associated cyanobacteria provide nitrogen to legumes and to rice, respectively). In addition, beneficial microbes can also protect plants from disease, as shown by the recognized ‘suppressive soil’’ effect (Schroth and Hancock, 1982). Yet relatively little is known about the diversity of microbes that associate with plants, i.e. the microbiome, and their combinatorial interactions and effects on performance and plant yields. Root-derived microbial colonization initiation and development is complex and not well understood due to the dynamic nature of plant root surfaces and microbial diversity. However, it remains unclear whether specific plant derived compounds influence colonization structures and if microbial colonization patterns affect the plant genomic and metabolic responses.

Transcriptome analysis of Arabidopsis thaliana plants in response to kin and stranger recognition

Recent reports have demonstrated that Arabidopsis thaliana has the ability to alter its growth differentially when grown in the presence of secretions from other A. thaliana plants that are kin or strangers, however, little knowledge has been gained as to the physiological processes involved in these plant-plant interactions. Therefore, we examined the root transcriptome of A. thaliana plants exposed to stranger versus kin secretions to determine genes involved in these processes. We conducted a whole transcriptome analysis on root tissues and categorized genes with significant changes in expression. Genes from four categories of interest based on significant changes in expression were identified as ATP/GST transporter, auxin/auxin related, secondary metabolite and pathogen response genes. Multiple genes in each category were tested and results indicated that pathogen response genes were involved in the kin recognition response. Plants were then infected with Pseudomonas syringe pv. Tomato DC3000 to further examine the role of these genes in plants exposed to own, kin and stranger secretions in pathogen resistance. This study concluded that multiple physiological pathways are involved in the kin recognition. The possible implication of this study opens up a new dialogue in terms of how plant-plant interactions change under a biotic stress.

Catechin is a phytototoxin and a pro-oxidant secreted from the roots of Centaurea stoebe.

When applied to the roots of Arabidopsis thaliana, the phytotoxin (±)-catechin triggers a wave of reactive oxygen species (ROS), leading to a cascade of genome-wide changes in gene expression and, ultimately, death of the root system. Biochemical links describing the root secreted phytotoxin, (±)-catechin, represent one of most well studied systems to describe biochemically based negative plant-plant interactions, but of late have also sparked controversies on phytotoxicity and pro-oxidant behavior of (±)-catechin. The studies originating from two labs 1- 3 maintained that (±)-catechin is not at all phytotoxic but has strong antioxidant activity. The step-wise experiments performed and the highly correlative results reported in the present study clearly indicate that (±)-catechin indeed is phytotoxic against A. thaliana and Festuca idahoensis. Our results show that catechin dissolved in both organic and aqueous phase inflict phytotoxic activity against both A. thaliana and F. idahoensis. We show that the deviation in results highlighted by the two labs1- 3 could be due to different media conditions and a group effect in catechin treated seedlings. We also determined the presence of catechin in the growth medium of C. stoebe to support the previous studies. One of the largest functional categories observed for catechin-responsive genes corresponded to gene families known to participate in cell death and oxidative stress. Our results showed that (±)-catechin treatment to A. thaliana plants resulted in activation of signature cell death genes such as accelerated cell death (acd2) and constitutively activated cell death 1 (cad1). Further, we confirmed our earlier observation of (±)-catechin induced ROS mediated phytotoxicity in A. thaliana. We also provide evidence that (±)-catechin induced ROS could be aggravated in the presence of divalent transition metals. These observations have significant impact on our understanding regarding catechin phytotoxicity and pro-oxidant activity. Our data also illustrates that precise conditions are needed to evaluate the effect of catechin phytotoxicity.

Kin recognition: another biological function for root secretions.

In the past, studies have shown that plants do indeed have the ability to recognize other plants in their surroundings based on relatedness and identity.1-5 Although, mechanisms for these recognitions have been proposed, to date, one has not been supported. In our recent work6, we showed that Arabidopsis plants distinguish self/non-self or kin/stranger plants through secretion and by recognition of root secretions. Additionally, we show that this kin response can be eliminated through treatment with a root secretion inhibitor and that the kin recognition response is robust through several Arabidopsis accessions. We hope that this study can promote the understanding of root secretion and its numerous roles in rhizosphere communications.

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms—from sample processing to image analysis—to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence‐specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize–fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae‐maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species‐specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141–152, 2018.