scholarly journals An improved Nicotiana benthamiana strain for aphid and whitefly research

2020 ◽  
Author(s):  
Honglin Feng ◽  
Lucia Acosta-Gamboa ◽  
Lars H. Kruse ◽  
Alba Ruth Nava Fereira ◽  
Sara Shakir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nicotiana Benthamiana ◽  
Plant Virus ◽  
Transient Gene Expression ◽  
Model System ◽  
Plant Insect Interactions ◽  
Transient Overexpression ◽  
Insect Interactions ◽  
Mutant Lines ◽  
Virus Interactions

AbstractNicotiana benthamiana is used extensively as a platform for transient gene expression and as a model system for studying plant-virus interactions. However, many tobacco-feeding insects, e.g. Myzus persicae (green peach aphids) and Bemisia tabaci (whiteflies), grow poorly on N. benthamiana, limiting its utility for research on plant-insect interactions. Using CRISPR/Cas9, we generated knockout mutations in two N. benthamiana acylsugar acyltransferase genes, ASAT1 and ASAT2, which contribute to the biosynthesis of insect-deterrent acylsucroses. Whereas ASAT1 mutations reduced the abundance of two predominant acylsucroses, ASAT2 mutations caused almost complete depletion of foliar acylsucroses. Both M. persicae and B. tabaci survived and reproduced significantly better on asat2 mutant plants than on wildtype N. benthamiana. Furthermore, ASAT1 and ASAT2 mutations reduced the water content and increased the temperature of leaves, indicating that foliar acylsucroses can protect against desiccation. Improved aphid and whitefly performance on ASAT2 mutants will make it possible to use the efficient transient overexpression and gene expression silencing systems that are available for N. benthamiana to study plant-insect interactions. Additionally, the absence of acylsugars in ASAT2 mutant lines will simplify transient expression assays for the functional analysis of acylsugar biosynthesis genes from other Solanaceae.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

2020 ◽  
Vol 11 ◽  
Author(s):  
Chao Zhang ◽  
Peixiu Du ◽  
Hailin Yan ◽  
Zongcai Zhu ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Transcriptome Profiling ◽  
Secretory Protein ◽  
Hypersensitive Cell Death ◽  
Challenge Inoculation ◽  
Transient Overexpression ◽  
Shock Proteins

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.

scholarly journals Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana.

2006 ◽  
Vol 53 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Mateusz Wydro ◽  
Edward Kozubek ◽  
Przemysław Lehmann
Keyword(s):  
Gene Expression ◽  
Industrial Production ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Expression System ◽  
Transient Gene Expression ◽  
Maximum Level ◽  
Gene Expression System ◽  
Gfp Gene ◽  
Viral Suppressors

Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.

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