A novel dominant selection system for plant transgenics based on phosphite metabolism catalyzed by bacterial alkaline phosphatase

Selective markers are generally indispensable in plant genetic transformation, of which the frequently used are of antibiotic or herbicide resistance. However, the increasing concerns on transgenic biosafety have encouraged many new and safe selective markers emerging, with an eminent representative as phosphite (Phi) in combination to its dehydrogenase (PTDH, e.g. PtxD). As bacterial alkaline phosphatase (BAP) can resemble PtxD to oxidatively convert toxic Phi into metabolizable phosphate (Pi), herein we harnessed it as the substitute of PtxD to develop an alternative Phi-based selection system. We first validated the Escherichia coli BAP (EcBAP) did own an extra enzymatic activity of oxidizing Phi to Pi. We further revealed EcBAP could be used as a dominant selective marker for Agrobacterium-mediated tobacco transformation. Although the involved Phi selection for transformed tobacco cells surprisingly required the presence of Pi, it showed a considerable transformation efficiency and dramatically accelerated transformation procedure, as compared to the routine kanamycin selection and the well-known PtxD/Phi system. Moreover, the EcBAP transgenic tobaccos could metabolize toxic Phi as a phosphorus (P) fertilizer thus underlying Phi-resistance, and competitively possess a dominant growth over wild-type tobacco and weeds under Phi stress. Therefore, this novel BAP/Phi-coupled system, integrating multiple advantages covering biosafe dominant selective marker, plant P utilization and weed management, can provide a PTDH-bypass technological choice to engineer transgenic plant species, especially those of great importance for sustainable agriculture.

Identification of a novel ZmSBEIIb-interacting protein involved in apparent amylose synthesis

Inhibition of the maize starch branching enzyme IIb (ZmSBEIIb) significantly alters the amylose content and grain yield in maize. Only a few ZmSBEIIb-interacting proteins involved in the starch branching process have been identified, and the understanding of this mechanism is limited. We report here the successful isolation of an Anoctamin protein, ZmSIP, that binds directly to ZmSBEIIb. In our study, the detection of interaction between ZmSIP and different truncated ZmSBEIIb baits in yeast showed that the full-length ZmSBEIIb is essential for binding. The direct protein–protein interaction could also be found in co-transformed tobacco protoplasts. Moreover, knocking out the ZmSIP gene using the CRISPR/Cas9 system could effectively increase the apparent amylose content but maintain the maize kernel weight. Thus, our results suggest that ZmSIP may be involved in the ZmSBEIIb-mediated amylose synthesis pathway through its physical interaction with ZmSBEIIb.

Characterization of a Drought-Inducible Dehydrin Promoter from Sugarcane (Saccharum officinarum L.) in Tobacco (Nicotiana tabacum L.)

Dehydrin (DHN) is known to play an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Previous research reported the increased expression of DHN in sugarcane stems exposed to drought stress for 15 days which may be controlled by its corresponding stress inducible promoter. The DHN promoter was succesfully isolated from sugarcane variety PSJT 941 (Pr-1DHNSo) and was cloned to pBI121 expression vector fused to a β-glucuronidase (GUS) reporter gene. The aim of this research was the functional testing of the Pr-1DHNSo promoter through transformation into tobacco plant treated with in vitro drought stress. Genetic transformation of Pr-1DHNSo construct was conducted by Agrobacterium tumefaciens. The transformed tobacco was then subjected to drought stress treatment using 40% PEG 6000 for five sequential incubations (0, 12, 24, 48 and 72 hours). The GUS assay reveal that the transformed tobacco treated with drought stress showed a blue color denoting GUS activity in leaf, stem and root tissues and this expression increased along with the length of the drought treatment. The analysis of gusA gene using real time-qPCR normalized to the L25 reference gene also showed that the expression increased in line with the length of time of drought stress. The results presented in this study indicated that the Pr-1DHNSo promoter from sugarcane was expressed and induced by drought stress treatment in tobacco.

Identification of plant enhancers and their constituent elements by STARR-seq in tobacco leaves

ABSTRACTGenetic engineering of cis-regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted STARR-seq — a technology for the high-throughput identification of enhancers — for its use in transiently transformed tobacco leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea and wheat was just upstream of a minimal promoter, and that none of these four known enhancers was active in the 3′-UTR of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome.One-sentence summaryWe developed a high-throughput assay in transiently transformed tobacco leaves that can identify enhancers, characterize their functional elements and detect condition-specific enhancer activity.

Identification and Functional Characterization of an Effector Secreted by Cronartium ribicola

Cri-9402 was identified as a protein effector from Cronartium ribicola, based on the effect of its expression on growth of Pseudomonas syringae Psm ES4326 introduced into transiently transformed tobacco leaves and stably transformed Arabidopsis seedlings. In tobacco leaves transiently expressing its coding sequence, growth of P. syringae Psm ES4326 was inhibited. Expression of pathogenesis-related (PR) protein 2 (PR2), PR4a, endochitinase B, hypersensitive-related 201 (HSR201), HSR203J, and proteinase inhibitor 1 was upregulated but expression of PR1, coronatine insensitive 1, and abscisic acid 1 was significantly suppressed. In transformed Arabidopsis seedlings, the effector stimulated growth of P. syringae Psm ES4326; significantly suppressed expression of PR1, PR2, nonexpresser of pathogenesis-related genes 1 (NPR1), NPR3, NPR4, phytoalexin deficient 4, and salicylic acid induction deficient 2; and enhanced expression of plant defensin 1.2 (PDF1.2). The above results showed that the majority of responses to this effector in tobacco leaves were converse to those in transformed Arabidopsis. We could conclude that Cri-9402 promoted disease resistance in tobacco leaves and disease susceptibility in Arabidopsis seedlings. Its transcript was mainly expressed in aeciospores of C. ribicola and was probably involved in production or germination of aeciospores, and it was an effector potentially functioning in white pine–blister rust interactions.

High Level Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Co-expression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Tobacco BY-2 Cells

Two genes encoding δ-guaiene synthase ( GS) and farnesyl diphosphate synthase ( FPS) involved in δ-guaiene biosynthesis in Aquilaria microcarpa were co-expressed in tobacco ( Nicotiana tabacum) BY-2 cells by Agrobacterium -mediated transformation. GC-MS analysis revealed that the transformed tobacco cells liberated δ-guaiene, and the compound was found in the headspace of the culture but not accumulated either in the medium or in the cells. Tobacco cells transformed by solely GS produced 0.2 mg δ-guaiene /L culture, however, concentration of the compound elevated to 0.6 – 5.9 mg/L when GS and FPS were co-expressed in the cells. The stirring efficiency of the cell suspension was improved by the reduction of the culture volume in the vials, and this resulted in an appreciable increase in δ-guaiene content to the level of 102 mg/L culture. Addition of mevalonolactone as the precursor markedly activated δ-guaiene production, and content of the compound elevated to more than 400 mg/L culture. These results strongly suggested that tobacco BY-2 is a suitable host to construct the bio-production system of sesquiterpene compounds, and co-expression of FPS and appropriate sesquiterpene synthase genes in the cells should be the good strategy to establish the highly productive platform of this class of compounds.

Osmotic stress on genetically transformed tobacco plant seeds

Salinity and water deficit limit the productivity of several crops; thus, studies related to the genetic transformation of seeds in a model plant, such as tobacco, can be an alternative to minimize negative impacts caused by environmental conditions. The purpose of this work was to evaluate the tolerance to osmotic stress of seeds from genetically transformed tobacco plants, with the introduction of the proline-synthesizer gene (p5csf129a), under salinity and water deficit conditions. To do so, five events with differences in proline content were selected, ranging from 0.70 to 10.47 µmoles.g-1 of fresh mass. The used saline concentrations were: zero (distilled water); 50; 100; 150 and 200 mmol.L-1 of NaCl, whereas for the water deficit, simulated with PEG 6000, the following osmotic potentials were used: zero (distilled water); -0.2; -0.4; -0.6 and -0.8 MPa. Each tested treatment was evaluated through germination, first germination count and germination speed index tests. It is possible to conclude that seeds from genetically transformed tobacco plants with overexpression of the gene p5csf129a, a proline synthesizer, are more tolerant to osmotic stresses. Tabacco seeds with a proline content of 10.47 µmol.g-1 showed a better perfomance, revealing higher physiological potential.

Early Detection of the Orchid Flowering Gene PaFT1 in Tobacco Cells Using a GFP Reporter

Here we describe a novel method of using green fluorescence protein (GFP) as a reporter gene for early detection of an integrated T­DNA containing the orchid flowering gene, PaFT1 (Phalaenopsis aphrodite Flowering locus T1) in the tobacco genome. Functional assays that report the presence of exogenous DNA early in development are especially useful in plants where the desired phenotype is only apparent after long periods of vegetative growth. The objective of this study is to establish a method for detecting an inserted Phalaenopsis orchid flowering gene and examining its function in tobacco. The p35S::PaFT1­ 35S::GFP construct was introduced into Agrobacterium tumefaciens strain EHA101. Transformed tobacco leaves were cultured on MS medium with addition of 1 mgL-1 NAA+3 mgL-1 BAP+50 mgL-1 Kanamycin+300 mgL-1 timentin for selection. Results showed bright green GFP fluorescent signals in 11 out of 15 (73%) tobacco leaf cells at a 2­month time point after transformation. GFP and PaFT1 fragments were amplified in genomic PCR using GFP and PaFT1 specific primers. The accumulated PaFT1 transcripts were observed in 3 month­old transgenic tobacco plants containing p35S::PaFT1­35S::GFP. Green florescence was observed only in the transgenic plants at the 5 month­old stage but not in the wild type controls.