Synchronization of Developmental Processes and Defense Signaling by Growth Regulating Transcription Factors

Growth regulating factors (GRFs) are a conserved class of transcription factor in seed plants. GRFs are involved in various aspects of tissue differentiation and organ development. The implication of GRFs in biotic stress response has also been recently reported, suggesting a role of these transcription factors in coordinating the interaction between developmental processes and defense dynamics. However, the molecular mechanisms by which GRFs mediate the overlaps between defense signaling and developmental pathways are elusive. Here, we report large scale identification of putative target candidates of Arabidopsis GRF1 and GRF3 by comparing mRNA profiles of the grf1/grf2/grf3 triple mutant and those of the transgenic plants overexpressing miR396-resistant version of GRF1 or GRF3. We identified 1,098 and 600 genes as putative targets of GRF1 and GRF3, respectively. Functional classification of the potential target candidates revealed that GRF1 and GRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance. GRF1 and GRF3 participate specifically in the regulation of defense-related transcription factors, cell-wall modifications, cytokinin biosynthesis and signaling, and secondary metabolites accumulation. GRF1 and GRF3 seem to fine-tune the crosstalk between miRNA signaling networks by regulating the expression of several miRNA target genes. In addition, our data suggest that GRF1 and GRF3 may function as negative regulators of gene expression through their association with other transcription factors. Collectively, our data provide new insights into how GRF1 and GRF3 might coordinate the interactions between defense signaling and plant growth and developmental pathways

Cooperative Regulatory Functions of miR858 and MYB83 during Cyst Nematode Parasitism

MicroRNAs (miRNAs) recently have been established as key regulators of transcriptome reprogramming that define cell function and identity. Nevertheless, the molecular functions of the greatest number of miRNA genes remain to be determined. Here, we report cooperative regulatory functions of miR858 and its MYB83 transcription factor target gene in transcriptome reprogramming during Heterodera cyst nematode parasitism of Arabidopsis (Arabidopsis thaliana). Gene expression analyses and promoter-GUS fusion assays documented a role of miR858 in posttranscriptional regulation of MYB83 in the Heterodera schachtii-induced feeding sites, the syncytia. Constitutive overexpression of miR858 interfered with H. schachtii parasitism of Arabidopsis, leading to reduced susceptibility, while reduced miR858 abundance enhanced plant susceptibility. Similarly, MYB83 expression increases were conducive to nematode infection because overexpression of a noncleavable coding sequence of MYB83 significantly increased plant susceptibility, whereas a myb83 mutation rendered the plants less susceptible. In addition, RNA-seq analysis revealed that genes involved in hormone signaling pathways, defense response, glucosinolate biosynthesis, cell wall modification, sugar transport, and transcriptional control are the key etiological factors by which MYB83 facilitates nematode parasitism of Arabidopsis. Furthermore, we discovered that miR858-mediated silencing of MYB83 is tightly regulated through a feedback loop that might contribute to fine-tuning the expression of more than a thousand of MYB83-regulated genes in the H. schachtii-induced syncytium. Together, our results suggest a role of the miR858-MYB83 regulatory system in finely balancing gene expression patterns during H. schachtii parasitism of Arabidopsis to ensure optimal cellular function

Arabidopsis miR827 mediates post‐transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility

MicroRNAs (miRNAs) are a major class of small non-coding RNAs with emerging functions in biotic and abiotic interactions. Here, we report on a new functional role of Arabidopsis miR827 and its NITROGEN LIMITATION ADAPTATION (NLA) target gene in mediating plant susceptibility to the beet cyst nematode Heterodera schachtii. Cyst nematodes are sedentary endoparasites that induce the formation of multinucleated feeding structures termed syncytia in the roots of host plants. Using promoter:GUS fusion assays we established that miR827 was activated in the initial feeding cells and this activation was maintained in the syncytium during all sedentary stages of nematode development. Meanwhile, the NLA target gene, which encodes an ubiquitin E3 ligase enzyme, was post-transcriptionally silenced in the syncytium, to permanently suppress its activity during all nematode parasitic stages. Overexpression of miR827 in Arabidopsis resulted in hyper-susceptibility to H. schachtii. In contrast, inactivation of miR827 activity through target mimicry or by overexpression a miR827-resistant cDNA of NLA produced the opposite phenotype of reduced plant susceptibility to H. schachtii. Gene expression analysis of several pathogenesis-related genes together with Agrobacterium–mediated transient expression in Nicotiana benthamiana provided strong evidence that miR827-mediated downregulation of NLA to suppress basal defense pathways. In addition, using yeast two-hybrid screens we identified several candidates of NLA-interacting proteins that are involved in a wide range of biological processes and molecular functions, including three pathogenesis-related proteins. Taken together, we conclude that nematode-activated miR827 in the syncytium is necessary to suppress immune responses in order to establish infection and cause disease

An orange fluorescent protein tagging system for real-time pollen tracking

BACKGROUND: Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum x Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. RESULTS: We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum \u27TN 90\u27 and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. CONCLUSION: The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring

The methylome of soybean roots during the compatible interaction with the soybean cyst nematode, Heterodera glycines

Soybean cyst nematode (SCN, Heterodera glycines) induces the formation of a multinucleated feeding site, or syncytium, whose etiology includes massive gene expression changes. Nevertheless, the genetic networks underlying gene expression control in the syncytium are poorly understood. DNA methylation is a critical epigenetic mark that plays a key role in regulating gene expression. To determine the extent to which DNA methylation is altered in soybean roots during the susceptible interaction with SCN, we generated whole-genome cytosine methylation maps at single nucleotide resolution. The methylome analysis revealed that SCN induces hypo-methylation to a much higher extent than hyper-methylation. We identified 2,465 differentially hyper-methylated regions and 4,692 hypo-methylated regions in the infected roots compared with the non-infected control. In addition, a total number of 703 and 1346 unique genes were identified as overlapping with hyper- or hypo-methylated regions, respectively. The differential methylation in genes apparently occurs independently of gene size and GC content but exhibits strong preference for recently duplicated paralogs. Furthermore, a set of 278 genes was identified as specifically syncytium differentially methylated genes. Of these, we found genes associated with epigenetic regulation, phytohormone signaling, cell wall architecture, signal transduction and ubiquitination. This study provides new evidence that differential methylation is part of the regulatory mechanisms controlling gene expression changes in the nematode-induced syncytium, which seems to be heavily influenced by the traditional well-known transcription factor-based regulatory mechanisms

The Cyst Nematode Effector Protein 10A07 Targets and Recruits Host Posttranslational Machinery to Mediate Its Nuclear Trafficking and to Promote Parasitism in Arabidopsis

Plant-parasitic cyst nematodes synthesize and secrete effector proteins that are essential for parasitism. One such protein is the 10A07 effector from the sugar beet cyst nematode, Heterodera schachtii, which is exclusively expressed in the nematode dorsal gland cell during all nematode parasitic stages. Overexpression of H. schachtii 10A07 in Arabidopsis thaliana produced a hypersusceptible phenotype in response to H. schachtii infection along with developmental changes reminiscent of auxin effects. The 10A07 protein physically associates with a plant kinase and the IAA16 transcription factor in the cytoplasm and nucleus, respectively. The interacting plant kinase (IPK) phosphorylates 10A07 at Ser-144 and Ser-231 and mediates its trafficking from the cytoplasm to the nucleus. Translocation to the nucleus is phosphorylation dependent since substitution of Ser-144 and Ser-231 by alanine resulted in exclusive cytoplasmic accumulation of 10A07. IPK and IAA16 are highly upregulated in the nematode-induced syncytium (feeding cells), and deliberate manipulations of their expression significantly alter plant susceptibility to H. schachtii in an additive fashion. An inactive variant of IPK functioned antagonistically to the wild-type IPK and caused a dominant-negative phenotype of reduced plant susceptibility. Thus, exploitation of host processes to the advantage of the parasites is one mechanism by which cyst nematodes promote parasitism of host plants

Age and geochemistry of the Charlestown Group, Ireland:Implications for the Grampian orogeny, its mineral potential and the Ordovician timescale

Accurately reconstructing the growth of continental margins during episodes of ocean closure has important implications for understanding the formation, preservation and location of mineral deposits in ancient orogens. The Charlestown Group of county Mayo, Ireland, forms an important yet understudied link in the Caledonian-Appalachian orogenic belt located between the well documented sectors of western Ireland and Northern Ireland. We have reassessed its role in the Ordovician Grampian orogeny, based on new fieldwork, high-resolution airborne geophysics, graptolite biostratigraphy, U–Pb zircon dating, whole rock geochemistry, and an examination of historic drillcore from across the volcanic inlier. The Charlestown Group can be divided into three formations: Horan, Carracastle, and Tawnyinah. The Horan Formation comprises a mixed sequence of tholeiitic to calc-alkaline basalt, crystal tuff and sedimentary rocks (e.g. black shale, chert), forming within an evolving peri-Laurentian affinity island arc. The presence of graptolites Pseudisograptus of the manubriatus group and the discovery of Exigraptus uniformis and Skiagraptus gnomonicus favour a latest Dapingian (i.e. Yapeenian Ya 2/late Arenig) age for the Horan Formation (equivalent to c. 471.2–470.5 Ma according to the timescale of Sadler et al., 2009). Together with three new U–Pb zircon ages of 471.95–470.82 Ma from enclosing felsic tuffs and volcanic breccias, this fauna provides an important new constraint for calibrating the Middle Ordovician timescale. Overlying deposits of the Carracastle and Tawnyinah formations are dominated by LILE- and LREE-enriched calc-alkaline andesitic tuffs and flows, coarse volcanic breccias and quartz-feldspar porphyritic intrusive rocks, overlain by more silicic tuffs and volcanic breccias with rare occurrences of sedimentary rocks. The relatively young age for the Charlestown Group in the Grampian orogeny, coupled with high Th/Yb and zircon inheritance (c. 2.7 Ga) in intrusive rocks indicate that the arc was founded upon continental crust (either composite Laurentian margin or microcontinental block). Regional correlation is best fitted to an association with the post-subduction flip volcanic/intrusive rocks of the Irish Caledonides, specifically the late-stage development of the Tyrone Igneous Complex, intrusive rocks of Connemara (western Ireland) and the Slishwood Division (Co. Sligo). Examination of breccia textures and mineralization across the volcanic inlier questions the previous porphyry hypothesis for the genesis of the Charlestown Cu deposit, which are more consistent with a volcanogenic massive sulfide (VMS) deposit.</p

J-Integral Calculation by Finite Element Processing of Measured Full-Field Surface Displacements

© 2017 The Author(s)A novel method has been developed based on the conjoint use of digital image correlation to measure full field displacements and finite element simulations to extract the strain energy release rate of surface cracks. In this approach, a finite element model with imported full-field displacements measured by DIC is solved and the J-integral is calculated, without knowledge of the specimen geometry and applied loads. This can be done even in a specimen that develops crack tip plasticity, if the elastic and yield behaviour of the material are known. The application of the method is demonstrated in an analysis of a fatigue crack, introduced to an aluminium alloy compact tension specimen (Al 2024, T351 heat condition)

Role of Organic Cation Transporter 1, OCT1 in the Pharmacokinetics and Toxicity of cis-Diammine(pyridine)chloroplatinum(II) and Oxaliplatin in Mice

PurposeThe goal of this study was to test the hypothesis that by controlling intracellular uptake, organic cation transporter 1, Oct1 is a key determinant of the disposition and toxicity of cis-diammine(pyridine)chloroplatinum(II)(CDPCP) and oxaliplatin.MethodsPharmacokinetics, tissue accumulation and toxicity of CDPCP and oxaliplatin were compared between Oct1-/- and wild-type mice.ResultsAfter intravenous administration, hepatic and intestinal accumulation of CDPCP was 2.7-fold and 3.9-fold greater in Oct1 wild-type mice (p &lt; 0.001). Deletion of Oct1 resulted in a significantly decreased clearance (0.444 ± 0.0391 ml/min*kg versus 0.649 ± 0.0807 ml/min*kg in wild-type mice, p &lt; 0.05) and volume distribution (1.90 ± 0.161 L/kg versus 3.37 ± 0.196 L/kg in wild-type mice, p &lt; 0.001). Moreover, Oct1 deletion resulted in more severe off-target toxicities in CDPCP-treated mice. Histologic examination of the liver and measurements of liver function indicated that the level of hepatic toxicity was mild and reversible, but was more apparent in the wild-type mice. In contrast, the effect of Oct1 on the pharmacokinetics and toxicity of oxaliplatin in the mice was minimal.ConclusionsOur study suggests that Oct1 plays an important role in the pharmacokinetics, tissue distribution and toxicity of CDPCP, but not oxaliplatin