Publications

Calmodulin as a Ca2+-Sensing Subunit of Arabidopsis Cyclic Nucleotide-Gated Channel Complexes

Posted on by biota in Publications

Fischer C, DeFalco TA, Karia P, Snedden WA, Moeder W, Yoshioka K, Dietrich P

Plant Cell Physiol. 2017 Jul;58(7):1208-1221

PMID: 28419310

Abstract

Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and the spatial and temporal patterns of Ca2+ concentration changes are determined by feedback and feed-forward regulation of the involved transport proteins. Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin (CaM). CNGCs interact with CaMs via diverse CaM-binding sites, including an IQ-motif, which has been identified in the C-termini of CNGC20 and CNGC12. Here we present a family-wide analysis of the IQ-motif from all 20 Arabidopsis CNGC isoforms. While most of their IQ-peptides interacted with conserved CaMs in yeast, some were unable to do so, despite high sequence conservation across the family. We showed that the CaM binding ability of the IQ-motif is highly dependent on its proximal and distal vicinity. We determined that two alanine residues positioned N-terminal to the core IQ-sequence play a significant role in CaM binding, and identified a polymorphism at this site that promoted or inhibited CaM binding in yeast. Through detailed biophysical analysis of the CNGC2 IQ-motif, we found that this polymorphism specifically affected the Ca2+-independent interactions with the C-lobe of CaM. This same polymorphism partially suppressed the induction of programmed cell death by CNGC11/12 in planta. Our work expands the model of CNGC regulation, and posits that the C-lobe of apo-CaM is permanently associated with the channel at the N-terminal part of the IQ-domain. This mode allows CaM to function as a Ca2+-sensing regulatory subunit of the channel complex, providing a mechanism by which Ca2+ signals may be fine-tuned.

Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species

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DeFalco TA, Toyota M, Phan V, Karia P, Moeder W, Gilroy S, Yoshioka K

Plant Cell Physiol. 2017 Jul;58(7):1173-1184

PMID: 28482045

Abstract

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.

Diversification of Pseudomonas aeruginosa within the CF lung

Posted on by biota in Featured Publications, Publications

Clark ST, Guttman DS, Hwang DM

FEMS Microbiol. Lett. 2018 Feb;

PMID: 29401362

Abstract

The evolution and diversification of bacterial pathogens within human hosts represent potential barriers to the diagnosis and treatment of life-threatening infections. Tremendous genetic and phenotypic diversity is characteristic of host adaptation in strains of Pseudomonas aeruginosa that infect the airways of individuals with chronic lung diseases and prove to be extremely difficult to eradicate. In this MiniReview, we examine recent advances in understanding within-host diversity and antimicrobial resistance in P. aeruginosa populations from the lower airways of individuals with the fatal genetic disease cystic fibrosis and the potential impacts that this diversity may have on detecting and interpreting antimicrobial susceptibility within these populations.

Assembly and ecological function of the root microbiome across angiosperm plant species

Posted on by biota in Publications

Fitzpatrick CR, Copeland J, Wang PW, Guttman DS, Kotanen PM, Johnson MTJ

Proc. Natl. Acad. Sci. U.S.A. 2018 Jan;

PMID: 29358405

Abstract

Across plants and animals, host-associated microbial communities play fundamental roles in host nutrition, development, and immunity. The factors that shape host-microbiome interactions are poorly understood, yet essential for understanding the evolution and ecology of these symbioses. Plant roots assemble two distinct microbial compartments from surrounding soil: the rhizosphere (microbes surrounding roots) and the endosphere (microbes within roots). Root-associated microbes were key for the evolution of land plants and underlie fundamental ecosystem processes. However, it is largely unknown how plant evolution has shaped root microbial communities, and in turn, how these microbes affect plant ecology, such as the ability to mitigate biotic and abiotic stressors. Here we show that variation among 30 angiosperm species, which have diverged for up to 140 million years, affects root bacterial diversity and composition. Greater similarity in root microbiomes between hosts leads to negative effects on plant performance through soil feedback, with specific microbial taxa in the endosphere and rhizosphere potentially affecting competitive interactions among plant species. Drought also shifts the composition of root microbiomes, most notably by increasing the relative abundance of the Actinobacteria. However, this drought response varies across host plant species, and host-specific changes in the relative abundance of endosphere Streptomyces are associated with host drought tolerance. Our results emphasize the causes of variation in root microbiomes and their ecological importance for plant performance in response to biotic and abiotic stressors.

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases

Posted on by biota in Featured Publications, Publications

Smakowska-Luzan E, Mott GA, Parys K, Stegmann M, Howton TC, Layeghifard M, Neuhold J, Lehner A, Kong J, Grünwald K, Weinberger N, Satbhai SB, Mayer D, Busch W, Madalinski M, Stolt-Bergner P, Provart NJ, Mukhtar MS, Zipfel C, Desveaux D, Guttman DS, Belkhadir Y

Nature 2018 01;553(7688):342-346

PMID: 29320478

Abstract

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSI) that consists of 567 interactions. To demonstrate the power of CSI for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSI operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

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Ung H, Karia P, Ebine K, Ueda T, Yoshioka K, Moeder W

Plant Physiol. 2017 Sep;175(1):473-485

PMID: 28733390

Abstract

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

A high-sensitivity, microtiter-based plate assay for plant MAMP-triggered immunity

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Mott GA, Desveaux D, Guttman DS

Mol. Plant Microbe Interact. 2017 Dec;

PMID: 29199888

Abstract

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant PTI that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMP required, and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species (ROS) assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens up the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.

Oh, the places they’ll go! A survey of phytopathogen effectors and their host targets

Posted on by biota in Publications

Khan M, Seto D, Subramaniam R, Desveaux D

Plant J. 2017 Nov;

PMID: 29160935

Abstract

Phytopathogens translocate effector proteins into plant cells where they sabotage the host cellular machinery to promote infection. An individual pathogen can translocate numerous distinct effectors during the infection process to target an array of host macromolecules (proteins, metabolites, DNA, etc…) and manipulate them using a variety of enzymatic activities. In this review, we have surveyed the literature for effector targets and curated them to convey the range of functions carried out by phytopathogenic proteins inside host cells. In particular, we have curated the locations of effector targets, as well as their biological and molecular functions and compared these properties across diverse phytopathogens. This analysis validates previous observations about effector functions (eg. immunosuppression), and also highlights some interesting features regarding effector specificity as well as functional diversification of phytopathogen virulence strategies. This article is protected by copyright. All rights reserved.

Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae

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Assis RAB, Polloni LC, Patané JSL, Thakur S, Felestrino ÉB, Diaz-Caballero J, Digiampietri LA, Goulart LR, Almeida NF, Nascimento R, Dandekar AM, Zaini PA, Setubal JC, Guttman DS, Moreira LM

Sci Rep. Published Nov 23, 2017.
https://doi.org/10.1038/s41598-017-16325-1

PMID: 29170530

Abstract

The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.

Convergent selection pressures drive the evolution of rhodopsin kinetics at high altitudes via non-parallel mechanisms

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Castiglione GM, Schott RK, Hauser FE, Chang BSW

Evolution. Published Nov 16, 2017.
https://doi.org/10.1111/evo.13396

PMID: 29143302

Abstract

Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via non-parallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems. This article is protected by copyright. All rights reserved.