Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

Raman, Vidhyavathi; Rojas, Clemencia M.; Balaji, Vasudevan; Dunning, Kevin; Kolape, Jaydeep; Oh, Sunhee; Yun, Jianfei; Yang, Lishan; Li, Guangming; Pant, Bikram Datt; Jiang, Qingzhen; Mysore, Kirankumar S.

doi:10.1038/s41467-022-30180-3

Cited by 36 publications

(17 citation statements)

References 87 publications

(106 reference statements)

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“…We also optimized specific parameters to enhance the transformation, especially for recalcitrant varieties. It is well known that A. tumefaciens triggers basal plant immune responses ( Zipfel et al., 2006 ; Tsuda et al., 2012 ; Raman et al., 2022 ), which can be a two-edged sword. It may reduce the transformation frequency of the infected cells and necrotize the explant tissues due to the production of excessive ROS and secondary metabolites.…”

Section: Resultsmentioning

confidence: 99%

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars

Dominguez¹,

Padilla²,

Mandadi³

2022

Front. Plant Sci.

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Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens-based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., β-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes (virG and virE), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications.

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Section: Resultsmentioning

confidence: 99%

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars

Dominguez¹,

Padilla²,

Mandadi³

2022

Front. Plant Sci.

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“…Plant defense responses against Agrobacterium significantly contribute to recalcitrance. 42 Peptides have been regarded as outstanding delivery tools with good biocompatibility and various functions. 43 Our data showed that there was no significant upregulation of NbrbohB in the peptide-based delivery system, providing evidence that the peptide transportation pathway did not trigger the production of reactive oxygen species (ROS) and thus did not impose stress on mature plants.…”

Section: ■ Discussionmentioning

confidence: 99%

“…tumefaciens abuses components of the host immune system, mimics plant protein functions, and manipulates hormone levels to bypass or override plant defenses, and the application of Agrobacterium -mediated plant transformation is limited to particular plant species/varieties. Plant defense responses against Agrobacterium significantly contribute to recalcitrance . Peptides have been regarded as outstanding delivery tools with good biocompatibility and various functions .…”

Section: Discussionmentioning

confidence: 99%

Nuclear Delivery of Exogenous Gene in Mature Plants Using Nuclear Location Signal and Cell-Penetrating Peptide Nanocomplex

Song

et al. 2022

ACS Appl. Nano Mater.

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Delivery and expression of exogenous plasmid DNA (pDNA) into mature plants for plant genetic engineering mainly rely on Agrobacterium-mediated transformation and biolistic bombardment. Meanwhile, the process of pDNA entering the nucleus via traditional methods is random and there is no nuclear targeting effect. Here, we reported a dual-peptide-based gene delivery system to achieve nuclear delivery of exogenous pDNA in mature plants. This system is a combination of engineered peptides composed of nuclear location signal (NLS) fusion with the DNA binding domain (KH) 9 and cell-penetrating peptides (CPPs), which could bind pDNA to form a sphere-like CPP-NLS (KH) -pDNA nanocomplex. We found that this system could overcome barriers of the nuclear pore complexes (NPCs) and deliver exogenous pDNA entering the nucleus efficiently. Besides, we demonstrated that the CPP-NLS (KH) -pDNA nanocomplex could efficiently cross the rigid plant cell wall into the nucleus at 24 h after treatment and bring about a transient expression in plant suspension cells and living plants 3 days after treatment. This strategy has obvious advantages of good biocompatibility, nongenomic integration, and nonspecies limitation and shows the potential of NLS peptides as gene carriers, which would provide a delivery tool to complement preset methods serving plant genetic engineering.

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“…More recently, Agrobacterium transformation efficiency and host range was improved by modifying its genome to express Pseudomonas syringae type III secretion system (T3SS). Using the engineered Agrobacterium, a 250%-400% increase in wheat, alfalfa, and switchgrass transformation is observed (Raman et al, 2022). Moreover, bacteria and fungi that can deliver proteins to plant cells have been identified, and their use as vectors is discussed in the later sections of this review.…”

Section: Agrobacterium-mediated Gene Delivery Methods and Its Modific...mentioning

confidence: 99%

Plant biomacromolecule delivery methods in the 21st century

et al. 2022

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The 21st century witnessed a boom in plant genomics and gene characterization studies through RNA interference and site-directed mutagenesis. Specifically, the last 15 years marked a rapid increase in discovering and implementing different genome editing techniques. Methods to deliver gene editing reagents have also attempted to keep pace with the discovery and implementation of gene editing tools in plants. As a result, various transient/stable, quick/lengthy, expensive (requiring specialized equipment)/inexpensive, and versatile/specific (species, developmental stage, or tissue) methods were developed. A brief account of these methods with emphasis on recent developments is provided in this review article. Additionally, the strengths and limitations of each method are listed to allow the reader to select the most appropriate method for their specific studies. Finally, a perspective for future developments and needs in this research area is presented.

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Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

Cited by 36 publications

References 87 publications

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars

Nuclear Delivery of Exogenous Gene in Mature Plants Using Nuclear Location Signal and Cell-Penetrating Peptide Nanocomplex

Plant biomacromolecule delivery methods in the 21st century

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