Phenotypic Characterization and Determination of Gene expression of Genetically Modified Rice Strains Using CRISPR-CAS9 Technology With Sodium Chloride Effect

This study was conducted with the target of determine the role of OsHKT4 and OsHKT6 genes in rice plants under salt stress and observe its gene expression by GUS technology, as well as studying the Na+ and K+ accumulation in different tissues. The results obtained show that OsHKT4::GUS appeared strong GUS activity, expressed mainly in vascular tissues. In contrast, the GUS activity of the OsHKT6 promoters in NaCl-treated leaves was greater than that in water-treated leaves. Also in wild type plants, increasing the Na+ concentration has the effect of increasing the Na+ content of the tissues generally, the old leaves accumulating more Na+ which reduced the K+ content in roots and old leaves (Na+ levels are higher in the leaf lower parts). These results suggest that OsHKT4 and OsHKT6 genes plays a role in the accumulation of Na+ in old leaves, by adopting the mechanical exclusion of toxic ions in the old leaves of the plant.

Auxin response and transport during induction of pedicel abscission in tomato

Auxin plays a central role in control of organ abscission, and it is thought that changes in the auxin gradient across the abscission zone are the primary determinant of the onset of abscission. The nature of this gradient, whether in concentration, flow, or perhaps in the response system has not conclusively been determined. We employed a DR5::GUS auxin response reporter system to examine the temporal and spatial distribution of the auxin response activity in response to developmental and environmental cues during pedicel abscission in tomato. In pedicels of young and fully open flowers, auxin response, as indicated by GUS activity, was predominantly detected in the vascular tissues and was almost entirely confined to the abscission zone (AZ) and to the distal portion of the pedicel, with a striking reduction in the proximal tissues below the AZ—a ‘step’, rather than a gradient. Following pollination and during early fruit development, auxin response increased substantially throughout the pedicel. Changes in GUS activity following treatments that caused pedicel abscission (flower removal, high temperature, darkness, ethylene, or N-1-naphthylphthalamic acid (NPA) treatment) were relatively minor, with reduced auxin response in the AZ and some reduction above and below it. Expression of genes encoding some auxin efflux carriers (PIN) and influx carriers (AUX⁄LAX) was substantially reduced in the abscission zone of NPA-treated pedicels, and in pedicels stimulated to abscise by flower removal. Our results suggest that changes in auxin flow distribution through the abscission zone are likely more important than the auxin response system in the regulation of abscission.

VqMYB154 promotes polygene expression and enhances resistance to pathogens in Chinese wild grapevine

Resveratrol plays a crucial phytoalexin role in the grapevine and is beneficial to human health. However, the molecular mechanism of resveratrol accumulation in the enhancement of disease resistance is unclear. Here, we report that the transcription factor VqMYB154 from Vitis quinquangularis accession Danfeng-2 is strongly expressed under artificial inoculation with Uncinula necator and regulates resveratrol accumulation. Unlike its homolog, VqMYB154 has a pathogen-induced promoter and responds to stimulation by U. necator, Pseudomonas syringae, and other treatments. Yeast one-hybrid and GUS activity assays confirmed that VqMYB154 can activate the stilbene synthase genes VqSTS9, VqSTS32, and VqSTS42 by directly binding to their promoters. Overexpression of VqMYB154 in grape leaves resulted in activation of the stilbene pathway, upregulation of STS genes, and accumulation of stilbenoids. In addition, heterologous overexpression of VqMYB154 in Arabidopsis activated resistance-related genes and resulted in greater programmed cell death and accumulation of reactive oxygen species, which led to resistance against P. syringae. These results suggest that the transcription factor VqMYB154 from V. quinquangularis accession Danfeng-2 participates in the regulatory mechanism that improves the biosynthesis and accumulation of stilbenes and enhances resistance to disease in grapevine.

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

O27: MICROGLIAL CORRECTION AFTER FETAL THERAPY WITHOUT CONDITIONING IN MICE WITH MUCOPOLYSACCHARIDOSIS TYPE VII

Introduction Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder typically fatal in utero. Postnatal enzyme replacement therapy (ERT) to replace missing glucuronidase (GUS) does not penetrate the blood-brain barrier (BBB). We investigated whether in utero ERT (IUERT) specifically targets brain microglia (natural GUS storehouses and key brain inflammation mediators) and whether in utero hematopoietic stem cell transplantation (IUHSCT) results in microglial engraftment as a strategy for permanent correction. Methods We performed IUERT by injecting GUS into MPS7 fetuses mid-gestation, and analyzed tissue homogenates (via colorimetric substrate) and brain microglia (via flow cytometry) for enzyme activity after 4-7 days. We performed IUHSCT by transplanting HSCs mid-gestation from CX3CR1-GFP donors. We examined blood, bone marrow, and brain for engraftment. We assessed brain inflammation by staining for CD68. We performed RNA sequencing to characterize engrafted microglia. Results IUERT resulted in detectable brain GUS activity. Flow cytometry showed that GUS activity after IUERT was near wild-type levels, and brains harvested in adulthood had decreased inflammation via CD68 immunohistochemistry. IUHSCT resulted in multilineage engraftment of hematopoietic cells in blood and bone. Confocal microscopy revealed multifocal engraftment of donor-derived microglia. RNA sequencing indicated that engrafted microglia were nearly identical to endogenous microglia. MPS7 chimeras had evidence of reduced brain inflammation near donor microglia. Conclusion Both IUERT and IUHSCT are complementary treatment modalities that can penetrate the BBB and ameliorate neurologic manifestations of diseases such as MPS7. These results lay the foundation for future studies using in utero molecular therapies for MPS7 as well as other storage disorders.

Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii

Miltiradiene synthase (MS) genes, TwTPS27a and TwTPS27b, are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in Tripterygium wilfordii. However, the mechanism underlying the regulation of key genes TwTPS27a/b in triptolide biosynthesis remains unclear. In this study, the promoters of TwTPS27a (1496 bp) and TwTPS27b (1862 bp) were isolated and analyzed. Some hormone-/stress-responsive elements and transcription factor (TF) binding sites were predicted in both promoters, which might be responsible for the regulation mechanism of TwTPS27a/b. The β-glucuronidase (GUS) activity analysis in promoter deletion assays under normal and methyl jasmonate (MeJA) conditions showed that the sequence of −921 to −391 bp is the potential core region of the TwTPS27b promoter. And the TGACG-motif, a MeJA-responsive element found in this core region, might be responsible for MeJA-mediated stress induction of GUS activity. Moreover, the TGACG-motif is also known as the TGA TF-binding site. Yeast one-hybrid and GUS transactivation assays confirmed the interaction between the TwTPS27a/b promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in T. wilfordii), indicating that TwTPS27a/b are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, TwTPS27a and TwTPS27b, as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.

Molecular Cloning and Functional Characterization of Bisabolene Synthetase (SaBS) Promoter from Santalum album

Bisabolene-type sesquiterpenoids, which have multiple bioactivities, including anticancer activity, are one of the main groups of compounds in the essential oil extracted from Santalum album L. and other Santalum species. Bisabolene synthetase (SaBS) is a key enzyme for the synthesis of bisabolene in S. album, but the regulation of the SaBS gene’s expression is poorly understood. In this study, a 1390-bp promoter sequence of the SaBS gene was isolated from the leaves of six-year-old S. album. A bioinformatics analysis showed that certain environment stresses and phytohormone-activated cis-acting elements were distributed in different regions of the SaBS promoter (PSaBS). Transgenic Arabidopsis carrying full-length PSaBS had significantly higher β-glucuronidase (GUS) activity than the untreated control after treatment with salicylic acid (SA), suggesting that PSaBS is a SA-inducible promoter. Histochemical GUS staining and GUS fluorometric assays of transgenic Arabidopsis showed that the GUS activity directed by PSaBS was mainly expressed in stem tissue, followed by leaves and flowers. Moreover, different regions of PSaBS showed significantly different GUS activity. A 171-bp fragment upstream of the transcriptional initiation codon (ATG) is the core promoter region of PSaBS. Our results provide insight into and a greater understanding of the transcriptional regulation mechanism of the SaBS gene, which could serve as an alternative inducible promoter for transgenic plant breeding.

The expression of LjROP4 is required for rhizobial infection in Lotus japonicus

Increasing evidence suggests that Rho of plant (ROP) GTPases play important roles in the rhizobium–legume symbiotic nodulation, but the molecular mechanisms of their regulation in symbiosis remain poorly understood. In this study, we showed that ROP4 in Lotus japonicus (LjROP4) is involved in the symbiotic interaction between L. japonicus and Mesorhizobium loti. Tissue expression analysis showed that LjROP4 expressed highly in the root. Histochemical staining analysis showed that after rhizobia inoculation, GUS reporter activity increased in the root vascular bundle, root tip, and lateral root primordia. During nodule development, GUS activity was detected in the cortex of nodule primordia and young nodules. In the mature nodules, GUS activity was detected only in the vascular bundle. Compared with the control, the overexpression of ROP4 and ROP4-CA produced much more pronounced root hair swelling and curling induced by M. loti. The infection event and nodule number noticeably increased, which was consistent with this promotion of root hair deformation. Moreover, RNA interference of LjROP4 produced opposite phenotypes. These data suggest that LjROP4 is required for root hair deformation during rhizobial infection. Thus, our study provides important information about root hair deformation responses induced by nod factors in the early stages of symbiotic interaction.

A modular steroid-inducible gene expression system for use in rice

Background Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. Results Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). Conclusions Our results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Vancomycin relieves mycophenolate mofetil–induced gastrointestinal toxicity by eliminating gut bacterial β-glucuronidase activity

Mycophenolate mofetil (MMF) is commonly prescribed and has proven advantages over other immunosuppressive drugs. However, frequent gastrointestinal side effects through an unknown mechanism limit its use. We have found that consumption of MMF alters the composition of the gut microbiota, selecting for bacteria expressing the enzyme β-glucuronidase (GUS) and leading to an up-regulation of GUS activity in the gut of mice and symptomatic humans. In the mouse, vancomycin eliminated GUS-expressing bacteria and prevented MMF-induced weight loss and colonic inflammation. Our work provides a mechanism for the toxicity associated with MMF and a future direction for the development of therapeutics.