Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking ß-1,2-xylose and core α-1,3-fucose.

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of ß-1,2-xylose and core α-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking ß-1,2-xylose and core α-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific α-1,3-fucosyltransferase and ß-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.

N-cadherin relocalization during cardiac trabeculation.

During cardiac trabeculation, cardiomyocytes delaminate from the outermost (compact) layer to form complex muscular structures known as trabeculae. As these cardiomyocytes delaminate, the remodeling of adhesion junctions must be tightly coordinated so cells can extrude from the compact layer while remaining in tight contact with their neighbors. In this study, we examined the distribution of N-cadherin (Cdh2) during cardiac trabeculation in zebrafish. By analyzing the localization of a Cdh2-EGFP fusion protein expressed under the control of the zebrafish cdh2 promoter, we initially observed Cdh2-EGFP expression along the lateral sides of embryonic cardiomyocytes, in an evenly distributed pattern, and with the occasional appearance of punctae. Within a few hours, Cdh2-EGFP distribution on the lateral sides of cardiomyocytes evolves into a clear punctate pattern as Cdh2-EGFP molecules outside the punctae cluster to increase the size of these aggregates. In addition, Cdh2-EGFP molecules also appear on the basal side of cardiomyocytes that remain in the compact layer. Delaminating cardiomyocytes accumulate Cdh2-EGFP on the surface facing the basal side of compact layer cardiomyocytes, thereby allowing tight adhesion between these layers. Importantly, we find that blood flow/cardiac contractility is required for the transition from an even distribution of Cdh2-EGFP to the formation of punctae. Furthermore, using time-lapse imaging of beating hearts in conjunction with a Cdh2 tandem fluorescent protein timer transgenic line, we observed that Cdh2-EGFP molecules appear to move from the lateral to the basal side of cardiomyocytes along the cell membrane, and that Erb-b2 receptor tyrosine kinase 2 (Erbb2) function is required for this relocalization.

A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems.

In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5' untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5' UTR) in tobacco, rice and sugarcane using ß-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.

Erianthus arundinaceus HSP70 (EaHSP70) Acts as a Key Regulator in the Formation of Anisotropic Interdigitation in Sugarcane (Saccharum spp. hybrid) in Response to Drought Stress.

Plant growth during abiotic stress is a long sought-after trait especially in crop plants in the context of global warming and climate change. Previous studies on leaf epidermal cells have revealed that during normal growth and development, adjacent cells interdigitate anisotropically to form cell morphological patterns known as interlocking marginal lobes (IMLs), involving the cell wall-cell membrane-cortical actin continuum. IMLs are growth-associated cell morphological changes in which auxin-binding protein (ABP), Rho GTPases and actin are known to play important roles. In the present study, we investigated the formation of IMLs under drought stress and found that Erianthus arundinaceus, a drought-tolerant wild relative of sugarcane, develops such growth-related cell morphological patterns under drought stress. Using confocal microscopy, we showed an increasing trend in cortical F-actin intensity in drought-tolerant plants with increasing soil moisture stress. In order to check the role of drought tolerance-related genes in IML formation under soil moisture stress, we adopted a structural data mining strategy and identified indirect connections between the ABPs and heat shock proteins (HSPs). Initial experimental evidence for this connection comes from the high transcript levels of HSP70 observed in drought-stressed Erianthus, which developed anisotropic interdigitation, i.e. IMLs. Subsequently, by overexpressing the E. arundinaceus HSP70 gene (EaHSP70) in sugarcane (Saccharum spp. hybrid), we confirm the role of HSP70 in the formation of anisotropic interdigitation under drought stress. Taken together, our results suggest that EaHSP70 acts as a key regulator in the formation of anisotropic interdigitation in drought-tolerant plants (Erianthus and HSP70 transgenic sugarcane) under moisture stress in an actin-mediated pathway. The possible biological significance of the formation of drought-associated interlocking marginal lobes (DaIMLs) in sugarcane plants upon drought stress is discussed.

Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

KEY MESSAGE: EaDREB2 overexpressed in sugarcane enhanced tolerance to drought and salinity. When co-transformed with plant DNA helicase gene, DREB2 showed greater level of salinity tolerance than in single-gene transgenics. Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and can potentially cause up to 50 % yield loss. DREB proteins play a vital regulatory role in abiotic stress tolerance in plants. We previously reported that expression of EaDREB2 is enhanced by drought stress in Erianthus arundinaceus. In this study, we have isolated the DREB2 gene from E. arundinaceus, transformed one of the most popular sugarcane variety Co 86032 in tropical India with EaDREB2 through Agrobacterium-mediated transformation, pyramided the EaDREB2 gene with the gene coding for PDH45 driven by Port Ubi 2.3 promoter through particle bombardment and evaluated the V1 transgenics for soil deficit moisture and salinity stresses. Soil moisture stress was imposed at the tillering phase by withholding irrigation. Physiological, molecular and morphological parameters were used to assess drought tolerance. Salinity tolerance was assessed through leaf disc senescence and bud sprout assays under salinity stress. Our results indicate that overexpression of EaDREB2 in sugarcane enhances drought and salinity tolerance to a greater extent than the untransformed control plants. This is the first report of the co-transformation of EaDREB2 and PDH45 which shows higher salinity tolerance but lower drought tolerance than EaDREB2 alone. The present study seems to suggest that, for combining drought and salinity tolerance together, co-transformation is a better approach.

Heat-Killed Tobacco Mosaic Virus Mitigates Plant Abiotic Stress Symptoms.

Since the discovery of the tobacco mosaic virus in the 1890s, awareness has grown in regard to how viruses affect the environment. Viral infections are now known to cause various effects besides pathogenicity, with some viruses in fact having a beneficial impact on plants. Although research has focused on disease-causing viruses that can infect plants, many wild plants are also infected with non-pathogenic viral agents. Traditionally, abiotic, and biotic stresses have been studied as isolated stimuli that trigger signaling pathways within the plant. However, both biotic and abiotic stress can trigger complex molecular interactions within plants, which in turn drive interconnected response pathways. Here, we demonstrate that heat-killed tobacco mosaic virus (TMV) can increase abiotic stress tolerance in plants, an effect that could potentially be implemented in challenging growth environments. To our knowledge, this is the first report of plant abiotic stress tolerance following treatment with heat-killed viral particles.

Introduction of Pea DNA Helicase 45 Into Sugarcane (Saccharum spp. Hybrid) Enhances Cell Membrane Thermostability And Upregulation Of Stress-responsive Genes Leads To Abiotic Stress Tolerance.

DNA helicases are motor proteins that play an essential role in nucleic acid metabolism, by providing a duplex-unwinding function. To improve the drought and salinity tolerance of sugarcane, a DEAD-box helicase gene isolated from pea with a constitutive promoter, Port Ubi 2.3 was transformed into the commercial sugarcane variety Co 86032 through Agrobacterium-mediated transformation, and the transgenics were screened for tolerance to soil moisture stress and salinity. The transgene integration was confirmed through polymerase chain reaction, and the V 0 transgenic events showed significantly higher cell membrane thermostability under normal irrigated conditions. The V 1 transgenic events were screened for tolerance to soil moisture stress and exhibited significantly higher cell membrane thermostability, transgene expression, relative water content, gas exchange parameters, chlorophyll content, and photosynthetic efficiency under soil moisture stress compared to wild-type (WT). The overexpression of PDH45 transgenic sugarcane also led to the upregulation of DREB2-induced downstream stress-related genes. The transgenic events demonstrated higher germination ability and better chlorophyll retention than WT under salinity stress. Our results suggest the possibility for development of increased abiotic stress tolerant sugarcane cultivars through overexpression of PDH45 gene. Perhaps this is the first report, which provides evidence for increased drought and salinity tolerance in sugarcane through overexpression of PDH45.

Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

Heat shock proteins (HSPs) have a major role in stress tolerance mechanisms in plants. Our studies have shown that the expression of HSP70 is enhanced under water stress in Erianthus arundinaceus. In this paper, we evaluate the effects of overexpression of EaHSP70 driven by Port Ubi 2.3 promoter in sugarcane. The transgenic events exhibit significantly higher gene expression, cell membrane thermostability, relative water content, gas exchange parameters, chlorophyll content and photosynthetic efficiency. The overexpression of EaHSP70 transgenic sugarcane led to the upregulation of stress-related genes. The transformed sugarcane plants had better chlorophyll retention and higher germination ability than control plants under salinity stress. Our results suggest that EaHSP70 plays an important role in sugarcane acclimation to drought and salinity stresses and its potential for genetic engineering of sugarcane for drought and salt tolerance.