Mobile Host mRNAs Are Translated to Protein in the Associated Parasitic Plant Cuscuta campestris

Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a β-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions.

CsGSTU8, a Glutathione S-Transferase From Camellia sinensis, Is Regulated by CsWRKY48 and Plays a Positive Role in Drought Tolerance

Glutathione S-transferases (GSTs) constitute a large family of enzymes with a wide range of cellular functions. Recently, plant GSTs have gained a great deal of attention due to their involvement in the detoxification of electrophilic xenobiotics and peroxides under adverse environmental conditions, such as salt, cold, UV-B and drought stress. A previous study reported that a GST gene (CsGSTU8) in tea plant was distinctly induced in response to drought, suggesting this gene plays a critical role in the drought stress response. In this study, by using quantitative real-time PCR (qRT-PCR) and β-glucuronidase (GUS) reporter lines, we further demonstrated that CsGSTU8 was upregulated in response to drought stress and exogenous abscisic acid (ABA) treatments. Overexpression of CsGSTU8 in Arabidopsis resulted in enhanced drought tolerance as indicated by the improved scavenging of excess amounts of reactive oxygen species (ROS) under drought conditions. Furthermore, we found that CsWRKY48 acts as a transcriptional activator and that its expression is induced in response to drought stress and ABA treatment. Electrophoretic mobility shift assays (EMSAs), dual-luciferase (LUC) assays and transient expression assays in tea plant leaves revealed that CsWRKY48 directly binds to the W-box elements in the promoter of CsGSTU8 and activates its expression. Taken together, our results provide additional knowledge of drought stress responses in tea plant.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

A Cotton Lignin Biosynthesis Gene, GhLAC4, Fine-Tuned by ghr-miR397 Modulates Plant Resistance Against Verticillium dahliae

Plant lignin is a component of the cell wall, and plays important roles in the transport potential of water and mineral nutrition and plant defence against biotic stresses. Therefore, it is necessary to identify lignin biosynthesis-related genes and dissect their functions and underlying mechanisms. Here, we characterised a cotton LAC, GhLAC4, which participates in lignin biosynthesis and plant resistance against Verticillium dahliae. According to degradome sequencing and GUS reporter analysis, ghr-miR397 was identified to directedly cleave the GhLAC4 transcript through base complementary. GhLAC4 knockdown and ghr-miR397 overexpression significantly reduced basal lignin content compared to the control, whereas ghr-miR397 silencing significantly increased basal lignin levels. Based on staining patterns and GC/MS analysis, GhLAC4 acted in G-lignin biosynthesis. Under V. dahliae infection, we found that G-lignin content in ghr-miR397-knockdowned plants significantly increased, compared to these plants under the mock treatment, while G-lignin contents in GhLAC4-silenced plants and ghr-miR397-overexpressed plants treated with pathogen were comparable with these plants treated with mock, indicating that GhLAC4 participates in defence-induced G-lignin biosynthesis in the cell wall. Knockdown of ghr-miR397 in plants inoculated with V. dahliae promoted lignin accumulation and increased plant resistance. The overexpression of ghr-miR397 and knockdown of GhLAC4 reduced lignin content and showed higher susceptibility of plants to the fungal infection compared to the control. The extract-free stems of ghr-miR397-knockdowned plants lost significantly less weight when treated with commercial cellulase and V. dahliae secretion compared to the control, while the stems of ghr-miR397-overexpressed and GhLAC4-silenced plants showed significantly higher loss of weight. These results suggest that lignin protects plant cell walls from degradation mediated by cellulase or fungal secretions. In summary, the ghr-miR397-GhLAC4 module regulates both basal lignin and defence-induced lignin biosynthesis and increases plant resistance against infection by V. dahliae.

CHARACTERISATION OF HORDEUM VULGARE CELLULOSE SYNTHASE-LIKE F6 PROMOTER VIA TRANSGENE EXPRESSION IN RICE

Beta-glucan in cereal crops is known as a functional food, which can reduce cardiovascular diseases by lowering blood cholesterol levels. However, beta-glucan content is relatively low in rice grains, despite being relatively abundant in barley and oat grains. Taking advantage of rice as the staple food for Asians, increasing beta-glucan content in rice for their consumption may help to reduce cardiovascular-related diseases among them. Previous attempts in increasing beta-glucan content in rice via transgene expression of beta-glucan synthase genes from barley into rice were unsuccessful due to the use of non-tissue specific as well as constitutively expressing promoter. The current transgenic expression study was performed to characterise the promoter of beta-glucan synthase gene in barley using beta-glucuronidase (GUS) reporter gene. Two fragments of HvCslF6 promoter (2771 bp and 1257 bp) were successfully fused with GUS reporter gene and integrated into rice plants, demonstrated that the promoter was functional in the heterologous plant system. The presence of blue GUS staining was observed on the leaf, root, stem, and grain of the transgenic rice regardless of the promoter length used and stayed functional up to the next generation. GUS qualitative analysis confirmed that the shorter promoter length generated a stronger GUS activity in comparison to the longer one. This indicated that the presence of repressor elements in between the -2771 bp and -1257 bp regions. The preliminary results shed light on the strong promoter activity in the rice endosperm tissue. It can become an alternative to the collection of plant promoters that can be used for grain quality improvement and biofortification.

A Procedure for Dex-induced Gene Transactivation in Arabidopsis Ovules

Background: Elucidating the genetic and molecular control of plant reproduction requires the deployment of functional approaches based, for instance, on reverse or forward genetic screens. The loss-of-function of essential genes, however, may lead to plant lethality prior to reproductive developmentt or to the formation of sterile structures before the organ-of-interest can be analysed. In these cases, inducible approaches that enable a spatial and temporal control of the genetic perturbation are extremely valuable. Genetic induction in reproductive organs, such as the ovule, deeply embedded in the flower, is a delicate procedure that requires both optimization and validation. Results: Here we report on a streamlined procedure enabling reliable induction of gene expression in Arabidopsis ovule and anther tissues using the popular pOP/LhGR Dex-inducible system. We demonstrate its efficiency and reliability using fluorescent reporter proteins and histochemical detection of the GUS reporter gene. Conclusion: The pOP/LhGR system allows for a rapid, efficient and reliable induction of transgenes in developing ovules without compromising developmental progression. This approach opens new possibilities for the functional analysis of candidate regulators in sporogenesis and gametogenesis, which are otherwise affected by early lethality of conventional, stable mutants.

Comparison and Characterization of a Cell Wall Invertase Promoter from Cu-Tolerant and Non-Tolerant Populations of Elsholtzia haichowensis

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β-glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher (p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5′-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis-elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.

Genetic transformation of Paulownia elongata S. Y. Hu., mediated by Agrobacterium tumefaciens and biolistic system

Objective: The most appropriate conditions for genetic transformation through direct (bioballistic) and indirect (Agrobacterium tumefaciens) transformation systems in Paulownia elongata were established. Design/methodology/approach: Starting from in vitro propagation through both direct and indirect organogenesis, internodal stem segments with 0.5 to 1 cm length were determined as the best explant. The optimum dose for selection media was determined to be 15 mg L-1 of kanamycin. It was possible to obtain transgenic plants under both transformation systems. In the case of Agrobacterium tumefaciens, two hours of incubation, 48 h of co-cultivation, and optical density of 0.9 were used; while for bioballistics, the best conditions were 120 PSI of shot pressure, shot height at level 6 (16 cm), and vacuum pressure of 22 Hg mm, with particle inflow gun system (PIG). Results: Both systems produced complete transformants, chimeras, as well as those confirmed by histochemical X-GLUC and PCR analysis, producing a total of 14 positive plants by A. tumefaciens transformation from 26 trials and ten positive plants by the bioballistic system from 30 trials; a construction with chitinase and glucanase, NPT II selection gene and the GUS reporter gene were used. Findings/conclusions: So far, this has been the first report including integration of chitinase and glucanase genes.

Coordination between MIDASIN 1-mediated ribosome biogenesis and auxin for modulating plant development

Ribosomes are required for plant growth and development, and ribosome-biogenesis-deficiency mutants generally display auxin-related phenotypes. Although the relationship between ribosome dysfunction and auxin has been raised, many aspects of this subject remain to be understood. We previously reported that MIDASIN 1 (MDN1) is an essential pre-60S ribosome biogenesis factor (RBF) in Arabidopsis. In this study, we further characterized the aberrant auxin-related phenotypes of mdn1-1, a weak mutant allele of MDN1. Auxin response is disturbed in both shoots and roots of mdn1-1 indicated by the DR5:GUS reporter. Through combining transcriptomic profiling analysis and reporter detection, we found that expression of genes involved in auxin biosynthesis, transport, and signaling is changed. Furthermore, the MDN1 deficiency affects both the posttranscriptional regulation and protein distribution of PIN2 (an auxin efflux facilitator) in mdn1-1 roots. These results indicate that MDN1 is required for maintaining the auxin system. More interestingly, MDN1 is an auxin-responsive gene, and its promoter can be targeted by multiple AUXIN RESPONSE FACTORs (ARFs), including ARF7 and ARF19, in vitro. Indeed, in arf7 arf19, the auxin sensitivity of MDN1 expression is significantly reduced. Together, our results reveal a coordination mechanism between the auxin system with the MDN1-dependent ribosome biogenesis for regulating plant development.

Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency

Phosphorus (P) deficiency is one of the major nutrient stresses restricting plant growth. The uptake of P by plants from soil is mainly mediated by the phosphate (Pi) transporters belonging to the PHT1 family. Multiple PHT1 genes from diverse plant species have been shown to be strongly up-regulated upon Pi starvation, however, the underlying mechanisms for the Pi-starvation-induced (PSI) up-regulation have not been well deciphered for most Pi transporter genes. Here, we reported a detailed dissection of the promoter activity of a PSI rice Pi transporter gene OsPT6, using the β-glucuronidase (GUS) reporter gene. OsPT6 promoter could drive GUS expression strongly in both roots and blades of rice plants grown under low P, but not high P. Cis-acting element analysis identified one copy of the P1BS motif and two copies of the W-box motif in OsPT6 promoter. Targeted deletion of the P1BS motif caused almost complete abolition of GUS induction in response to Pi starvation, irrespective of the presence or absence of the W-box motif, Four repeats of the P1BS motif fused to the CaMV35S minimal promoter was sufficient to induce GUS expression responsive to Pi starvation. Targeted deletion of the upstream W-box motif (W1) did not affect the GUS expression activity compared with the full-length OsPT6 promoter, while targeted deletion of the downstream W-box motif (W2) or both of the W-box motifs remarkably reduced the GUS induction rate upon Pi starvation. Our results proposed that the PSI response of OsPT6 was positively regulated by at least two elements, the sole P1BS and the downstream W-box, in its promoter, and the W-box-mediated up-regulation of OsPT6 might be highly dependent on the P1BS motif.