The Plastid-Localized AtFtsHi3 Pseudo-Protease of Arabidopsis thaliana Has an Impact on Plant Growth and Drought Tolerance.

While drought severely affects plant growth and crop production, the molecular mechanisms of the drought response of plants remain unclear. In this study, we demonstrated for the first time the effect of the pseudo-protease AtFtsHi3 of Arabidopsis thaliana on overall plant growth and in drought tolerance. An AtFTSHi3 knock-down mutant [ftshi3-1(kd)] displayed a pale-green phenotype with lower photosynthetic efficiency and Darwinian fitness compared to wild type (Wt). An observed delay in seed germination of ftshi3-1(kd) was attributed to overaccumulation of abscisic acid (ABA); ftshi3-1(kd) seedlings showed partial sensitivity to exogenous ABA. Being exposed to similar severity of soil drying, ftshi3-1(kd) was drought-tolerant up to 20 days after the last irrigation, while wild type plants wilted after 12 days. Leaves of ftshi3-1(kd) contained reduced stomata size, density, and a smaller stomatic aperture. During drought stress, ftshi3-1(kd) showed lowered stomatal conductance, increased intrinsic water-use efficiency (WUEi), and slower stress acclimation. Expression levels of ABA-responsive genes were higher in leaves of ftshi3-1(kd) than Wt; DREB1A, but not DREB2A, was significantly upregulated during drought. However, although ftshi3-1(kd) displayed a drought-tolerant phenotype in aboveground tissue, the root-associated bacterial community responded to drought.

Genome wide association study reveals plant loci controlling heritability of the rhizosphere microbiome.

Host genetics has recently been shown to be a driver of plant microbiome composition. However, identifying the underlying genetic loci controlling microbial selection remains challenging. Genome-wide association studies (GWAS) represent a potentially powerful, unbiased method to identify microbes sensitive to the host genotype and to connect them with the genetic loci that influence their colonization. Here, we conducted a population-level microbiome analysis of the rhizospheres of 200 sorghum genotypes. Using 16S rRNA amplicon sequencing, we identify rhizosphere-associated bacteria exhibiting heritable associations with plant genotype, and identify significant overlap between these lineages and heritable taxa recently identified in maize. Furthermore, we demonstrate that GWAS can identify host loci that correlate with the abundance of specific subsets of the rhizosphere microbiome. Finally, we demonstrate that these results can be used to predict rhizosphere microbiome structure for an independent panel of sorghum genotypes based solely on knowledge of host genotypic information.

Loss of Arabidopsis matrix metalloproteinase-5 affects root development and root bacterial communities during drought stress.

Matrix metalloproteinases (MMPs) are zinc-dependent endo-peptidases that in mammals are known to be involved in remodeling the extracellular matrix (ECM) in developmental and pathological processes. In this study, we report At5-MMP of Arabidopsis thaliana to be important for root development and root bacterial communities. At5-MMP is mainly localized in the root vasculature and lateral root, an At5-MMP T-DNA insertion mutant (mmp5 KO) showed reduced root growth and a lower number of root apexes, causing reduced water uptake from the soil. Subsequently, mmp5 KO is sensitive to drought stress. Inhibited auxin transport was accompanied with resistance to indole-3-acetic acid (IAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D), and 1-naphthaleneacetic acid (NAA). The content of endogenous abscisic acid (ABA) was lower in roots of mmp5 KO than in wild type. Genes responsive to ABA as well as genes encoding enzymes of the proline biosynthesis were expressed to a lower extent in mmp5 KO than in wild type. Moreover, drought stress modulated root-associated bacterial communities of mmp5 KO: the number of Actinobacteria increased. Therefore, At5-MMP modulates auxin/ABA signaling rendering the plant sensitive to drought stress and recruiting differential root bacterial communities.

Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae.

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli-expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Silencing of the Rice Gene LRR1 Compromises Rice Xa21 Transcript Accumulation and XA21-Mediated Immunity.

BACKGROUND: The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. We previously demonstrated that an auxilin-like protein, XA21 BINDING PROTEIN 21 (XB21), positively regulates resistance to Xoo. RESULTS: To further investigate the function of XB21, we performed a yeast two-hybrid screen. We identified 22 unique XB21 interacting proteins, including LEUCINE-RICH REPEAT PROTEIN 1 (LRR1), which we selected for further analysis. Silencing of LRR1 in the XA21 genetic background (XA21-LRR1Ri) compromises resistance to Xoo compared with control XA21 plants. XA21-LRR1Ri plants have reduced Xa21 transcript levels and reduced expression of genes that serve as markers of XA21-mediated activation. Overexpression of LRR1 is insufficient to alter resistance to Xoo in rice lines lacking XA21. CONCLUSIONS: Taken together, our results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity.

The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity.

Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.

Protein phosphorylation in plant immunity: insights into the regulation of pattern recognition receptor-mediated signaling.

Plants are continuously challenged by pathogens including viruses, bacteria, and fungi. The plant immune system recognizes invading pathogens and responds by activating an immune response. These responses occur rapidly and often involve post-translational modifications (PTMs) within the proteome. Protein phosphorylation is a common and intensively studied form of these PTMs and regulates many plant processes including plant growth, development, and immunity. Most well-characterized pattern recognition receptors (PRRs), including Xanthomonas resistance 21, flagellin sensitive 2, and elongation factor-Tu receptor, possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation events. Here, we focus on the phosphorylation events of plant PRRs that play important roles in the immune response. We also discuss the role of phosphorylation in regulating mitogen-associated protein kinase cascades and transcription factors in plant immune signaling.

PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis.

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1-PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1-ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling.