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Metabolic effects of agro-infiltration on N. benthamiana accessions.

ABSTRACT: Over the recent years, Nicotiana benthamiana has gained great importance as a chassis for the production of high value, low volume pharmaceuticals and/or active pharmaceutical ingredients (APIs). The process involving infiltration of the N. benthamiana leaves with Agrobacterium spp, harbouring vectors with the gene of interest, facilitates transient expression. To date, little information is available on the effect of the agro-infiltration process on the metabolome of N. benthamiana, which is necessary to improve the process for large-scale, renewable manufacturing of high value compounds and medical products. Hence, the objective of the present study was to assess metabolic adaptation of N. benthamiana as a response to the presence of Agrobacterium. The present study elucidated changes of the steady-state metabolism in the agroinfiltrated leaf area, the area around the infection and the rest of the plant. Furthermore, the study discusses the phenotypic advantages of the N. benthamiana lab strain, optimised for agro-infiltration, compared to three other wild accessions. Results showed that the lab strain has a different metabolic composition and showed less alterations of the phenylpropanoid pathway and cell wall remodelling in the agroinfiltrated leaf areas, for example chlorogenic acid, cadaverine and C18:0-2-glycerol ester. In conclusion, both of these alterations present potential candidates to improve the phenotype of the N. benthamiana lab strain for a more efficient transient expression process.

SUBMITTER: Drapal M

PROVIDER: S-EPMC8080481 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundVirus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase (GmPDS1) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems.ResultsIn this report, the ALSV VIGS vector was modified by mobilizing ALSV cDNAs into a binary vector compatible with Agrobacterium tumefaciens-mediated delivery, so that VIGS-triggering ALSV variants could be propagated in agro-infiltrated Nicotiana benthamiana leaves. Homogenate of these N. benthamiana leaves was then applied directly onto the unifoliate of young soybean seedlings to initiate systemic gene silencing. This rapid inoculation method bypassed the need for a particle bombardment apparatus. Among the 19 soybean genotypes evaluated with this new method, photo-bleaching indicative of GmPDS1 silencing was observed in nine, with two exhibiting photo-bleaching in 100% of the inoculated individuals. ALSV RNA was detected in pods, embryos, stems, leaves, and roots in symptomatic plants of four genotypes.ConclusionsThis modified protocol allowed for inoculation of soybean plants via simple mechanical rubbing with the homogenate of N. benthamiana leaves agro-infiltrated with ALSV VIGS constructs. More importantly, inoculated plants showed no apparent virus disease symptoms which could otherwise interfere with VIGS phenotypes. This streamlined procedure expanded this functional genomics tool to nine soybean genotypes.

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Metabolic effects of agro-infiltration on N. benthamiana accessions.

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