Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys in planta Herbicide Resistance in Sugarcane

Abstract Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX. Like other heme biosynthetic proteins, PPOX is involved in synthesizing heme for red cells (erythroid-specific expression) and as a cofactor for the respiratory cytochromes (housekeeping expression). To date, little is known about transcriptional regulation of the human PPOX gene (PPOX). We established the molecular basis for erythroid-specific expression of PPOX. Using transient in vitro transfection assays in human erythroleukemic K562 cells we studied tissue-specific expression of PPOX. We found that reporter constructs lacking exon 1 showed a 75% reduction in promoter strength in K562 cells (Figure, no 1 and 2). Hence, in vitro high-level erythroid-specific expression of PPOX is dependent on the presence of exon 1. Examination of erythroid-specific regulatory elements in exon 1 revealed two GATA-1 sites, one consensus (A/T)GATA(A/G) site (GATA-1_II AGATAA) and one non-consensus site, deviating at the first nucleotide (GATA-1_I, CGATAG). To study the relative contribution of these two GATA-1 sites to erythroid-specific transcriptional regulation, we performed in vitro transfections of wild-type and mutant (GATA → GTTA) reporter plasmids in K562 cells. We found that the highest level of transcription depended on the integrity of both sites (Figure, no 5). The consensus GATA-1_II site contributed the most to promoter strength (Figure, no 4). Subsequent electrophoretic mobility shift assay and supershift experiments using K562 nuclear extracts demonstrated that both GATA sites were able to bind GATA-1 in vitro. Our experiments showed that exon 1 was dispensable for PPOX promoter activity in human hepatoma HepG2 cells. Interestingly, in HeLa human cervical carcinoma cells the presence of exon 1 decreased promoter activity. Conclusively, exon 1 of the human PPOX gene contains two GATA-1 binding sites, which are required for high level erythroid-specific expression of PPOX and, in addition, bind GATA-1 in vitro. Our results contribute to a better understanding of the molecular mechanisms involved in differential regulation of the human PPOX promoter in erythroid and non-erythroid cells. Figure Figure

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ptxD/Phi as alternative selectable marker system for genetic transformation for bio-safety concerns: a review

PeerJ ◽

10.7717/peerj.11809 ◽

2021 ◽

Vol 9 ◽

pp. e11809

Author(s):

Richard Dormatey ◽

Chao Sun ◽

Kazim Ali ◽

Sajid Fiaz ◽

Derong Xu ◽

...

Keyword(s):

Transgenic Plants ◽

Genetic Transformation ◽

Herbicide Resistance ◽

Selectable Marker ◽

Selection Marker ◽

Marker Genes ◽

Selection System ◽

Efficient System ◽

Marker System ◽

Selectable Marker Genes

Antibiotic and herbicide resistance genes are the most common marker genes for plant transformation to improve crop yield and food quality. However, there is public concern about the use of resistance marker genes in food crops due to the risk of potential gene flow from transgenic plants to compatible weedy relatives, leading to the possible development of “superweeds” and antibiotic resistance. Several selectable marker genes such as aph, nptII, aaC3, aadA, pat, bar, epsp and gat, which have been synthesized to generate transgenic plants by genetic transformation, have shown some limitations. These marker genes, which confer antibiotic or herbicide resistance and are introduced into crops along with economically valuable genes, have three main problems: selective agents have negative effects on plant cell proliferation and differentiation, uncertainty about the environmental effects of many selectable marker genes, and difficulty in performing recurrent transformations with the same selectable marker to pyramid desired genes. Recently, a simple, novel, and affordable method was presented for plant cells to convert non-metabolizable phosphite (Phi) to an important phosphate (Pi) for developing cells by gene expression encoding a phosphite oxidoreductase (PTXD) enzyme. The ptxD gene, in combination with a selection medium containing Phi as the sole phosphorus (P) source, can serve as an effective and efficient system for selecting transformed cells. The selection system adds nutrients to transgenic plants without potential risks to the environment. The ptxD/Phi system has been shown to be a promising transgenic selection system with several advantages in cost and safety compared to other antibiotic-based selection systems. In this review, we have summarized the development of selection markers for genetic transformation and the potential use of the ptxD/Phi scheme as an alternative selection marker system to minimize the future use of antibiotic and herbicide marker genes.

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A Laminarin-Based Formulation Protects Wheat Against Zymoseptoria tritici Via Direct Antifungal Activity and Elicitation of Host Defense-Related Genes

Plant Disease ◽

10.1094/pdis-08-21-1675-re ◽

2022 ◽

Author(s):

Marlon C. de Borba ◽

Aline Cristina Velho ◽

Mateus B. de Freitas ◽

Maxime Holvoet ◽

Alessandra Maia-Grondard ◽

...

Keyword(s):

Antifungal Activity ◽

Metabolic Cost ◽

Allene Oxide ◽

In Planta ◽

Zymoseptoria Tritici ◽

Oxidase Gene ◽

Induce Resistance ◽

Oxide Synthase ◽

Related Proteins

The present study aimed to evaluate the potential of the laminarin-based formulation Vacciplant® to protect and induce resistance in wheat against Zymoseptoria tritici, a major pathogen on this crop. Under greenhouse conditions, a single foliar spraying of the product two days before inoculation with Z. tritici reduced disease severity and pycnidium density by 42% and 45%, respectively. Vacciplant® exhibited a direct antifungal activity on Z. tritici conidial germination both in vitro and in planta. Moreover, it reduced in planta substomatal colonization as well as pycnidium formation on treated leaves. Molecular investigations revealed that Vacciplant® elicits but did not prime the expression of several wheat genes related to defense pathways, including phenylpropanoids (phenylalanine ammonia-lyase and chalcone synthase), octadecanoids (lipoxygenase and allene oxide synthase), and pathogenesis‐related proteins (β‐1,3‐endoglucanase and chitinase). By contrast, it did not modulate the expression of oxalate oxidase gene involved in the reactive oxygen species metabolism. UHPLC-MS analysis indicated limited changes in leaf metabolome after product application in both non-inoculated and inoculated conditions, suggesting a low metabolic cost associated with induction of plant resistance. This study provides evidence that the laminarin-based formulation confers protection to wheat against Z. tritici through direct antifungal activity and elicitation of plant defense-associated genes.

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RNA Interference of Dual Oxidase in the Plant Nematode Meloidogyne incognita

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-1099 ◽

2005 ◽

Vol 18 (10) ◽

pp. 1099-1106 ◽

Cited By ~ 75

Author(s):

Manjula Bakhetia ◽

Wayne Charlton ◽

Howard J. Atkinson ◽

Michael J. McPherson

Keyword(s):

Rna Interference ◽

Gene Function ◽

Meloidogyne Incognita ◽

Feeding Strategy ◽

Model Organisms ◽

Parasitic Nematodes ◽

In Planta ◽

Oxidase Gene ◽

Dual Oxidase

RNA interference (RNAi) is a powerful tool for the analysis of gene function in model organisms such as the nematode Caenorhabditis elegans. Recent demonstrations of RNAi in plant parasitic nematodes provide a stimulus to explore the potential of using RNAi to investigate disruption of gene function in Meloidogyne incognita, one of the most important nematode pests of global agriculture. We have used RNAi to examine the importance of dual oxidases (peroxidase and NADPH oxidase), a class of enzyme associated with extracellular matrix cross-linking in C. elegans. RNAi uptake by M. incognita juveniles is highly efficient. In planta infection data show that a single 4-h preinfection treatment with double-stranded RNA derived from the peroxidase region of a dual oxidase gene has effects on gene expression that are phenotypically observable 35 days postinfection. This RNAi effect results in a reduction in egg numbers at 35 days of up to 70%. The in vitro feeding strategy provides a powerful tool for identifying functionally important genes, including those that are potential targets for the development of new agrochemicals or transgenic resistance strategies.

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A peroxisomal β-oxidative pathway contributes to the formation of C6–C1 aromatic volatiles in poplar

Plant Physiology ◽

10.1093/plphys/kiab111 ◽

2021 ◽

Author(s):

Nathalie D Lackus ◽

Axel Schmidt ◽

Jonathan Gershenzon ◽

Tobias G Köllner

Keyword(s):

Cinnamic Acid ◽

Aromatic Compounds ◽

Populus Trichocarpa ◽

Alternative Pathway ◽

Gene Families ◽

Defense Responses ◽

In Planta ◽

Black Cottonwood ◽

Oxidative Pathway

AbstractBenzenoids (C6–C1 aromatic compounds) play important roles in plant defense and are often produced upon herbivory. Black cottonwood (Populus trichocarpa) produces a variety of volatile and nonvolatile benzenoids involved in various defense responses. However, their biosynthesis in poplar is mainly unresolved. We showed feeding of the poplar leaf beetle (Chrysomela populi) on P. trichocarpa leaves led to increased emission of the benzenoid volatiles benzaldehyde, benzylalcohol, and benzyl benzoate. The accumulation of salicinoids, a group of nonvolatile phenolic defense glycosides composed in part of benzenoid units, was hardly affected by beetle herbivory. In planta labeling experiments revealed that volatile and nonvolatile poplar benzenoids are produced from cinnamic acid (C6–C3). The biosynthesis of C6–C1 aromatic compounds from cinnamic acid has been described in petunia (Petunia hybrida) flowers where the pathway includes a peroxisomal-localized chain shortening sequence, involving cinnamate-CoA ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD), and 3-ketoacyl-CoA thiolase (KAT). Sequence and phylogenetic analysis enabled the identification of small CNL, CHD, and KAT gene families in P. trichocarpa. Heterologous expression of the candidate genes in Escherichia coli and characterization of purified proteins in vitro revealed enzymatic activities similar to those described in petunia flowers. RNA interference-mediated knockdown of the CNL subfamily in gray poplar (Populus x canescens) resulted in decreased emission of C6–C1 aromatic volatiles upon herbivory, while constitutively accumulating salicinoids were not affected. This indicates the peroxisomal β-oxidative pathway participates in the formation of volatile benzenoids. The chain shortening steps for salicinoids, however, likely employ an alternative pathway.

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Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽

10.3390/plants10061102 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1102

Author(s):

Galina N. Raldugina ◽

Sergey V. Evsukov ◽

Liliya R. Bogoutdinova ◽

Alexander A. Gulevich ◽

Ekaterina N. Baranova

Keyword(s):

Choline Oxidase ◽

Culture Methods ◽

Gene Encoding ◽

Oxidase Gene ◽

Bacterial Enzyme ◽

Transgenic Lines ◽

High Regeneration ◽

Whole Plants ◽

Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

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Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00444-z ◽

2021 ◽

Author(s):

Mara Quaglia ◽

Marika Bocchini ◽

Benedetta Orfei ◽

Roberto D’Amato ◽

Franco Famiani ◽

...

Keyword(s):

Disease Severity ◽

Pseudomonas Syringae ◽

Zinc Phosphate ◽

Plant Defence ◽

In Planta ◽

Tomato Plants ◽

Pathogenesis Related Protein ◽

Defence Responses ◽

Plant Defence Responses

AbstractThe purpose of this study was to determine whether zinc phosphate treatments of tomato plants (Solanum lycopersicum L.) can attenuate bacterial speck disease severity through reduction of Pseudomonas syringae pv. tomato (Pst) growth in planta and induce morphological and biochemical plant defence responses. Tomato plants were treated with 10 ppm (25.90 µM) zinc phosphate and then spray inoculated with strain DAPP-PG 215, race 0 of Pst. Disease symptoms were recorded as chlorosis and/or necrosis per leaf (%) and as numbers of necrotic spots. Soil treatments with zinc phosphate protected susceptible tomato plants against Pst, with reductions in both disease severity and pathogen growth in planta. The reduction of Pst growth in planta combined with significantly higher zinc levels in zinc-phosphate-treated plants indicated direct antimicrobial toxicity of this microelement, as also confirmed by in vitro assays. Morphological (i.e. callose apposition) and biochemical (i.e., expression of salicylic-acid-dependent pathogenesis-related protein PR1b1 gene) defence responses were induced by the zinc phosphate treatment, as demonstrated by histochemical and qPCR analyses, respectively. In conclusion, soil treatments with zinc phosphate can protect tomato plants against Pst attacks through direct antimicrobial activity and induction of morphological and biochemical plant defence responses.

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HopA1 Effector from Pseudomonas syringae pv syringae Strain 61 Affects NMD Processes and Elicits Effector-Triggered Immunity

International Journal of Molecular Sciences ◽

10.3390/ijms22147440 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7440

Author(s):

Shraddha K. Dahale ◽

Daipayan Ghosh ◽

Kishor D. Ingole ◽

Anup Chugani ◽

Sang Hee Kim ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Disease Susceptibility ◽

Null Mutant ◽

In Planta ◽

Host Range Expansion ◽

Unique System ◽

Effector Triggered Immunity ◽

Transcriptomic Changes ◽

Immune Detection

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.

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Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice

Nature Plants ◽

10.1038/s41477-021-00942-w ◽

2021 ◽

Author(s):

Rongfang Xu ◽

Xiaoshuang Liu ◽

Juan Li ◽

Ruiying Qin ◽

Pengcheng Wei

Keyword(s):

Herbicide Resistance ◽

Library Screening ◽

In Planta

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Further In Vitro Assessment and Mid-Term Evaluation of Control Strategy of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy)

Pathogens ◽

10.3390/pathogens10010085 ◽

2021 ◽

Vol 10 (1) ◽

pp. 85

Author(s):

Giuseppe Tatulli ◽

Vanessa Modesti ◽

Nicoletta Pucci ◽

Valeria Scala ◽

Alessia L’Aurora ◽

...

Keyword(s):

Control Strategy ◽

Xylella Fastidiosa ◽

In Planta ◽

Bacterial Concentration ◽

Infected Area ◽

Olive Groves ◽

Term Evaluation ◽

In Vitro Assessment ◽

Olive Trees

During recent years; Xylella fastidiosa subsp. pauca (Xfp) has spread in Salento causing relevant damage to the olive groves. Measures to contain the spreading of the pathogen include the monitoring of the areas bordering the so-called “infected” zone and the tree eradication in case of positive detection. In order to provide a control strategy aimed to maintain the tree productivity in the infected areas, we further evaluated the in vitro and in planta mid-term effectiveness of a zinc-copper-citric acid biocomplex. The compound showed an in vitro bactericidal activity and inhibited the biofilm formation in representative strains of X. fastidiosa subspecies, including Xfp isolated in Apulia from olive trees. The field mid-term evaluation of the control strategy assessed by quantitative real-time PCR in 41 trees of two olive groves of the “infected” area revealed a low concentration of Xfp over the seasons upon the regular spraying of the biocomplex over 3 or 4 consecutive years. In particular, the bacterial concentration lowered in July and October with respect to March, after six consecutive treatments. The trend was not affected by the cultivar and it was similar either in the Xfp-sensitive cultivars Ogliarola salentina and Cellina di Nardò or in the Xfp-resistant Leccino. Moreover, the scoring of the number of wilted twigs over the seasons confirmed the trend. The efficacy of the treatment in the management of olive groves subjected to a high pathogen pressure is highlighted by the yielded a good oil production

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