Science diplomacy in the 21st century.

Science diplomacy is the use of scientific collaborations among nations to address the common problems facing 21(st) century humanity and to build constructive international partnerships. There are many ways that scientists can contribute to this process.

The persistent threat of emerging plant disease pandemics to global food security.

Plant disease outbreaks are increasing and threaten food security for the vulnerable in many areas of the world. Now a global human pandemic is threatening the health of millions on our planet. A stable, nutritious food supply will be needed to lift people out of poverty and improve health outcomes. Plant diseases, both endemic and recently emerging, are spreading and exacerbated by climate change, transmission with global food trade networks, pathogen spillover, and evolution of new pathogen lineages. In order to tackle these grand challenges, a new set of tools that include disease surveillance and improved detection technologies including pathogen sensors and predictive modeling and data analytics are needed to prevent future outbreaks. Herein, we describe an integrated research agenda that could help mitigate future plant disease pandemics.

Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processing.

Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.

McClintock's challenge in the 21st century.

In 1950, Barbara McClintock published a Classic PNAS article, "The origin and behavior of mutable loci in maize," which summarized the evidence leading to her discovery of transposition. The article described a number of genome alterations revealed through her studies of the Dissociation locus, the first mobile genetic element she identified. McClintock described the suite of nuclear events, including transposon activation and various chromosome aberrations and rearrangements, that unfolded in the wake of genetic crosses that brought together two broken chromosomes 9. McClintock left future generations with the challenge of understanding how genomes respond to genetic and environmental stresses by mounting adaptive responses that frequently include genome restructuring.

SOS1 tonoplast neo-localization and the RGG protein SALTY are important in the extreme salinity tolerance of Salicornia bigelovii.

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during microRNA biogenesis.

Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher K(cat) and lower K(m) values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates.

The endophytic fungus Piriformospora indica enhances Arabidopsis thaliana growth and modulates Na+/K+ homeostasis under salt stress conditions.

The mutualistic, endophytic fungus Piriformospora indica has been shown to confer biotic and abiotic stress tolerance to host plants. In this study, we investigated the impact of P. indica on the growth of Arabidopsis plants under normal and salt stress conditions. Our results demonstrate that P. indica colonization increases plant biomass, lateral roots density, and chlorophyll content under both conditions. Colonization with P. indica under salt stress was accompanied by a lower Na+/K+ ratio and less pronounced accumulation of anthocyanin, compared to control plants. Moreover, P. indica colonized roots under salt stress showed enhanced transcript levels of the genes encoding the high Affinity Potassium Transporter 1 (HKT1) and the inward-rectifying K+ channels KAT1 and KAT2, which play key roles in regulating Na+ and K+ homeostasis. The effect of P. indica colonization on AtHKT1;1 expression was also confirmed in the Arabidopsis line gl1-HKT:AtHKT1;1 that expresses an additional AtHKT1;1 copy driven by the native promoter. Colonization of the gl1-HKT:AtHKT1;1 by P. indica also increased lateral roots density and led to a better Na+/K+ ratio, which may be attributed to the observed increase in KAT1 and KAT2 transcript levels. Our findings demonstrate that P. indica colonization promotes Arabidopsis growth under salt stress conditions and that this effect is likely caused by modulation of the expression levels of the major Na+ and K+ ion channels, which allows establishing a balanced ion homeostasis of Na+/K+ under salt stress conditions.

A case study in pathway knowledgebase verification.

BACKGROUND: Biological databases and pathway knowledge-bases are proliferating rapidly. We are developing software tools for computer-aided hypothesis design and evaluation, and we would like our tools to take advantage of the information stored in these repositories. But before we can reliably use a pathway knowledge-base as a data source, we need to proofread it to ensure that it can fully support computer-aided information integration and inference. RESULTS: We design a series of logical tests to detect potential problems we might encounter using a particular knowledge-base, the Reactome database, with a particular computer-aided hypothesis evaluation tool, HyBrow. We develop an explicit formal language from the language implicit in the Reactome data format and specify a logic to evaluate models expressed using this language. We use the formalism of finite model theory in this work. We then use this logic to formulate tests for desirable properties (such as completeness, consistency, and well-formedness) for pathways stored in Reactome. We apply these tests to the publicly available Reactome releases (releases 10 through 14) and compare the results, which highlight Reactome's steady improvement in terms of decreasing inconsistencies. We also investigate and discuss Reactome's potential for supporting computer-aided inference tools. CONCLUSION: The case study described in this work demonstrates that it is possible to use our model theory based approach to identify problems one might encounter using a knowledge-base to support hypothesis evaluation tools. The methodology we use is general and is in no way restricted to the specific knowledge-base employed in this case study. Future application of this methodology will enable us to compare pathway resources with respect to the generic properties such resources will need to possess if they are to support automated reasoning.

RNA-binding proteins in plants: the tip of an iceberg?

RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

Cross-talk in abscisic acid signaling.

"Cross-talk" in hormone signaling reflects an organism's ability to integrate different inputs and respond appropriately, a crucial function at the heart of signaling network operation. Abscisic acid (ABA) is a plant hormone involved in bud and seed dormancy, growth regulation, leaf senescence and abscission, stomatal opening, and a variety of plant stress responses. This review summarizes what is known about ABA signaling in the control of stomatal opening and seed dormancy and provides an overview of emerging knowledge about connections between ABA, ethylene, sugar, and auxin synthesis and signaling.

Molecular genetics and epigenetics of CACTA elements.

The CACTA transposons, so named for a highly conserved motif at element ends, comprise one of the most abundant superfamilies of Class 2 (cut-and-paste) plant transposons. CACTA transposons characteristically include subterminal sequences of several hundred nucleotides containing closely spaced direct and inverted repeats of a short, conserved sequence of 14-15 bp. The Supressor-mutator (Spm) transposon, identified and subjected to detailed genetic analysis by Barbara McClintock, remains the paradigmatic element of the CACTA family. The Spm transposon encodes two proteins required for transposition, the transposase (TnpD) and a regulatory protein (TnpA) that binds to the subterminal repeats. Spm expression is subject to both genetic and epigenetic regulation. The Spm-encoded TnpA serves as an activator of the epigenetically inactivated, methylated Spm, stimulating both transient and heritable activation of the transposon. TnpA also serves as a negative regulator of the demethylated active element promoter and is required, in addition to the TnpD, for transposition.

Identification and analysis of red sea mangrove (Avicennia marina) microRNAs by high-throughput sequencing and their association with stress responses.

Although RNA silencing has been studied primarily in model plants, advances in high-throughput sequencing technologies have enabled profiling of the small RNA components of many more plant species, providing insights into the ubiquity and conservatism of some miRNA-based regulatory mechanisms. Small RNAs of 20 to 24 nucleotides (nt) are important regulators of gene transcript levels by either transcriptional or by posttranscriptional gene silencing, contributing to genome maintenance and controlling a variety of developmental and physiological processes. Here, we used deep sequencing and molecular methods to create an inventory of the small RNAs in the mangrove species, Avicennia marina. We identified 26 novel mangrove miRNAs and 193 conserved miRNAs belonging to 36 families. We determined that 2 of the novel miRNAs were produced from known miRNA precursors and 4 were likely to be species-specific by the criterion that we found no homologs in other plant species. We used qRT-PCR to analyze the expression of miRNAs and their target genes in different tissue sets and some demonstrated tissue-specific expression. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and experimentally validated through endonucleolytic cleavage assays. Our results suggested that expression profiles of miRNAs and their predicted targets could be useful in exploring the significance of the conservation patterns of plants, particularly in response to abiotic stress. Because of their well-developed abilities in this regard, mangroves and other extremophiles are excellent models for such exploration.

Characterization and DNA-binding specificities of Ralstonia TAL-like effectors.

Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.

The past, present and future of crop genetic modification.

The introduction of science and technology into agriculture over the past two centuries has markedly increased agricultural productivity and decreased its labor-intensiveness. Chemical fertilization, mechanization, plant breeding and molecular genetic modification (GM) have contributed to unparalleled productivity increases. Future increases are far from assured because of underinvestment in agricultural research, growing population pressure, decreasing fresh water availability, increasing temperatures and societal rejection of GM crops in many countries.

RNA secondary structural determinants of miRNA precursor processing in Arabidopsis.

MicroRNAs (miRNAs) are excised from hairpin structures within primary miRNAs (pri-miRNAs). Most animal pri-miRNAs are processed by two cleavages, the first at a loop-distal site approximately 11 nucleotides (nt) from the end of the hairpin and the second approximately 22 nt beyond the first. To identify RNA structural determinants of miRNA processing in plants, we analyzed the functional consequences of changing the secondary structure of the lower (loop-distal), middle (miRNA:miRNA(*)), and upper (loop-proximal) stems of the hairpin in two different pri-miRNAs. Closing bulges immediately below the loop-distal cleavage sites increased the accumulation of accurately cleaved precursor miRNAs but decreased the abundance of the mature miRNAs. A pri-miRNA variant with an unpaired lower stem was not processed, and variants with a perfectly paired middle or upper stem were processed normally. Bioinformatic analysis of pri-miRNA structures, together with physical mapping of initial cleavage sites and in vitro processing of pri-miRNA, reveals that the first, loop-distal cleavage is often at a distance of approximately 15 nt from an unpaired region. Hence, a common determinant of the rate and location of the initial pri-miRNA cleavage is an imperfectly base-paired duplex of approximately 15 nt between the miRNA:miRNA(*) duplex and either a less structured region of the lower stem or its end.

Arabidopsis bZIP60 is a proteolysis-activated transcription factor involved in the endoplasmic reticulum stress response.

Proteins synthesized in the endoplasmic reticulum (ER) of eukaryotic cells must be folded correctly before translocation out of the ER. Disruption of protein folding results in the induction of genes for ER-resident chaperones, for example, BiP. This phenomenon is known as the ER stress response. We report here that bZIP60, an Arabidopsis thaliana basic leucine zipper (bZIP) transcription factor with a transmembrane domain, is involved in the ER stress response. When compared with wild-type Arabidopsis plants, homozygous bzip60 mutant plants show a markedly weaker induction of many ER stress-responsive genes. The bZIP60 protein resides in the ER membrane under unstressed condition and is cleaved in response to ER stress caused by either tunicamycin or DTT. The N-terminal fragment containing the bZIP domain is then translocated into the nucleus. Cleavage of bZIP60 is independent of the function of Arabidopsis homologs of mammalian S1P and S2P proteases, which mediate the proteolytic cleavage of the mammalian transcription factor ATF6. In Arabidopsis, expression of the bZIP60 gene and cleavage of the bZIP60 protein are observed in anthers in the absence of stress treatment, suggesting that the ER stress response functions in the normal development of active secretory cells.

Characterization of the Arabidopsis heterotrimeric G protein.

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the alpha, beta, and gamma1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed Ggamma1 localizes to protoplast membranes, but Gbeta exhibits membrane localization only when the Ggamma1 protein is co-overexpressed. Overexpressed Galpha shows membrane localization unaccompanied by overexpression of either Gbeta or Ggamma1. We detect fluorescence resonance energy transfer between Gbeta and Ggamma1 in the absence of Galpha overexpression and between Galpha and Ggamma1 but only when all three subunits are co-overexpressed. Both Galpha and Gbeta are associated with large macromolecular complexes of approximately 700 kDa in the plasma membrane. Galpha is present in both large complexes and as free Galpha in plasma membranes from wild type plants. In plants homozygous for a null allele of the Gbeta gene, Galpha is associated with smaller complexes in the 200-400-kDa range, indicating that its presence in the large complex depends on association with Gbetagamma. Activation of the Galpha subunit with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) results in partial dissociation of Galpha from the complex. Hydrogen peroxide (H2O2) promotes extensive dissociation of the Galpha complex but does not interfere with binding of GTPgammaS to purified recombinant Galpha, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of Galpha itself.