Symbiont-host interactome mapping reveals effector-targeted modulation of hormone networks and activation of growth promotion.

Plants have benefited from interactions with symbionts for coping with challenging environments since the colonisation of land. The mechanisms of symbiont-mediated beneficial effects and similarities and differences to pathogen strategies are mostly unknown. Here, we use 106 (effector-) proteins, secreted by the symbiont Serendipita indica (Si) to modulate host physiology, to map interactions with Arabidopsis thaliana host proteins. Using integrative network analysis, we show significant convergence on target-proteins shared with pathogens and exclusive targeting of Arabidopsis proteins in the phytohormone signalling network. Functional in planta screening and phenotyping of Si effectors and interacting proteins reveals previously unknown hormone functions of Arabidopsis proteins and direct beneficial activities mediated by effectors in Arabidopsis. Thus, symbionts and pathogens target a shared molecular microbe-host interface. At the same time Si effectors specifically target the plant hormone network and constitute a powerful resource for elucidating the signalling network function and boosting plant productivity.

Structural and functional insights into the mechanism of action of plant borate transporters.

Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causing mutations of hAE1 (S466R, A500R) and (ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of borate efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease-causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3, lacking the 30 C-terminal amino acid residues. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

Mediator Subunits MED16, MED14, and MED2 Are Required for Activation of ABRE-Dependent Transcription in Arabidopsis.

The Mediator complex controls transcription of most eukaryotic genes with individual subunits required for the control of particular gene regulons in response to various perturbations. In this study, we reveal the roles of the plant Mediator subunits MED16, MED14, and MED2 in regulating transcription in response to the phytohormone abscisic acid (ABA) and we determine which cis elements are under their control. Using synthetic promoter reporters we established an effective system for testing relationships between subunits and specific cis-acting motifs in protoplasts. Our results demonstrate that MED16, MED14, and MED2 are required for the full transcriptional activation by ABA of promoters containing both the ABRE (ABA-responsive element) and DRE (drought-responsive element). Using synthetic promoter motif concatamers, we showed that ABA-responsive activation of the ABRE but not the DRE motif was dependent on these three Mediator subunits. Furthermore, the three subunits were required for the control of water loss from leaves but played no role in ABA-dependent growth inhibition, highlighting specificity in their functions. Our results identify new roles for three Mediator subunits, provide a direct demonstration of their function and highlight that our experimental approach can be utilized to identify the function of subunits of plant transcriptional regulators.

Novel markers for high-throughput protoplast-based analyses of phytohormone signaling.

Phytohormones mediate most diverse processes in plants, ranging from organ development to immune responses. Receptor protein complexes perceive changes in intracellular phytohormone levels and trigger a signaling cascade to effectuate downstream responses. The in planta analysis of elements involved in phytohormone signaling can be achieved through transient expression in mesophyll protoplasts, which are a fast and versatile alternative to generating plant lines that stably express a transgene. While promoter-reporter constructs have been used successfully to identify internal or external factors that change phytohormone signaling, the range of available marker constructs does not meet the potential of the protoplast technique for large scale approaches. The aim of our study was to provide novel markers for phytohormone signaling in the Arabidopsis mesophyll protoplast system. We validated 18 promoter::luciferase constructs towards their phytohormone responsiveness and specificity and suggest an experimental setup for high-throughput analyses. We recommend novel markers for the analysis of auxin, abscisic acid, cytokinin, salicylic acid and jasmonic acid responses that will facilitate future screens for biological elements and environmental stimuli affecting phytohormone signaling.

The Tetrazole Analogue of the Auxin Indole-3-acetic Acid Binds Preferentially to TIR1 and Not AFB5.

Indole-3-acetic acid (auxin) is considered one of the cardinal hormones in plant growth and development. It regulates a wide range of processes throughout the plant. Synthetic auxins exploit the auxin-signaling pathway and are valuable as herbicidal agrochemicals. Currently, despite a diversity of chemical scaffolds all synthetic auxins have a carboxylic acid as the active core group. By applying bio-isosteric replacement we discovered that indole-3-tetrazole was active by surface plasmon resonance spectrometry, showing that the tetrazole could initiate assembly of the Transport Inhibitor Resistant 1 (TIR1) auxin coreceptor complex. We then tested the tetrazole's efficacy in a range of whole plant physiological assays and in protoplast reporter assays, which all confirmed auxin activity, albeit rather weak. We then tested indole-3-tetrazole against the AFB5 homologue of TIR1, finding that binding was selective against TIR1, absent with AFB5. The kinetics of binding to TIR1 are contrasted to those for the herbicide picloram, which shows the opposite receptor preference, as it binds to AFB5 with far greater affinity than to TIR1. The basis of the preference of indole-3-tetrazole for TIR1 was revealed to be a single residue substitution using molecular docking, and assays using tir1 and afb5 mutant lines confirmed selectivity in vivo. Given the potential that a TIR1-selective auxin might have for unmasking receptor-specific actions, we followed a rational design, lead optimization campaign, and a set of chlorinated indole-3-tetrazoles was synthesized. Improved affinity for TIR1 and the preference for binding to TIR1 was maintained for 4- and 6-chloroindole-3-tetrazoles, coupled with improved efficacy in vivo. This work expands the range of auxin chemistry for the design of receptor-selective synthetic auxins.

In roots of Arabidopsis thaliana, the damage-associated molecular pattern AtPep1 is a stronger elicitor of immune signalling than flg22 or the chitin heptamer.

Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer.

The Innate Immune Signaling System as a Regulator of Disease Resistance and Induced Systemic Resistance Activity Against Verticillium dahliae.

In the last decades, the plant innate immune responses against pathogens have been extensively studied, while biocontrol interactions between soilborne fungal pathogens and their hosts have received much less attention. Treatment of Arabidopsis thaliana with the nonpathogenic bacterium Paenibacillus alvei K165 was shown previously to protect against Verticillium dahliae by triggering induced systemic resistance (ISR). In the present study, we evaluated the involvement of the innate immune response in the K165-mediated protection of Arabidopsis against V. dahliae. Tests with Arabidopsis mutants impaired in several regulators of the early steps of the innate immune responses, including fls2, efr-1, bak1-4, mpk3, mpk6, wrky22, and wrky29 showed that FLS2 and WRKY22 have a central role in the K165-triggered ISR, while EFR1, MPK3, and MPK6 are possible susceptibility factors for V. dahliae and bak1 shows a tolerance phenomenon. The resistance induced by strain K165 is dependent on both salicylate and jasmonate-dependent defense pathways, as evidenced by an increased transient accumulation of PR1 and PDF1.2 transcripts in the aerial parts of infected plants treated with strain K165.

Reactive oxygen species and plant resistance to fungal pathogens.

Reactive oxygen species (ROS) have been studied for their role in plant development as well as in plant immunity. ROS were consistently observed to accumulate in the plant after the perception of pathogens and microbes and over the years, ROS were postulated to be an integral part of the defence response of the plant. In this article we will focus on recent findings about ROS involved in the interaction of plants with pathogenic fungi. We will describe the ways to detect ROS, their modes of action and their importance in relation to resistance to fungal pathogens. In addition we include some results from works focussing on the fungal interactor and from studies investigating roots during pathogen attack.

Perception of soft mechanical stress in Arabidopsis leaves activates disease resistance.

BACKGROUND: In a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound-induced resistance to B. cinerea. RESULTS: In this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves. CONCLUSION: Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.

In planta function of compatible solute transporters of the AtProT family.

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.

Proline metabolism and transport in plant development.

Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plants.