Agrobacterium tumefaciens mediated transient expression of plant cell wall‐degrading enzymes in detached sunflower leaves

Abstract

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower.

Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein

“…Unlike Jung et al . () recently reporting increased expression of two plant cell wall‐degrading enzymes in detached sunflower leaves infiltrated with a MeJA‐supplemented agrobacterial solution, no yield increase was observed when applying MeJA at the moment of infiltration (not shown). Similarly, no yield increase was observed when spraying the same elicitor 24 h postinfiltration (Figure d) or when treating leaves 24 h before infiltration with arachidonic acid, a functional analogue of salicylic acid (Girard et al ., ) (Figure d).…”

RXLR and CRN Effectors from the Sunflower Downy Mildew Pathogen Plasmopara halstedii Induce Hypersensitive-Like Responses in Resistant Sunflower Lines

“…Stable transformation of sunflower by A. tumefaciens is still difficult, with poor efficiency and is restricted to specific sunflower lines (Knittel et al, 1994; Malone-Schoneberg et al, 1994; Burrus et al, 1996; Lucas et al, 2000). Transient transformation of detached sunflower leaves infiltrated under vacuo with A. tumefaciens was proposed in order to overcome structural leaf problems and low bacterial transformation efficiencies (Manavella and Chan, 2009; Jung et al, 2014). The tedious but successful adaptation of these protocols to sunflower leaves of entire plants was, for us, a prerequisite for functional studies of P. halstedii effectors.…”

Effect of leaf incubation temperature profiles on agrobacterium tumefaciens‐mediated transient expression

“…Endoglucanase E1 and endoxylanase gene constructs were developed as described . Briefly, the original amino acid sequences of the thermostable endo‐1,4‐β‐glucanase (Accession #P54583) and the thermostable endo‐1,4‐β‐xylanase (Accession #A0LRT6, Acel_0372) were obtained from the NCBI database and then codon‐optimized using GeneDesigner (DNA 2.0, Menlo Park, CA) and Visual Gene Developer software, respectively.…”

“…As model systems, CaMV 35S‐driven endoglucanase E1 and endoxylanase Xyn10A which are key enzymes in biofuel applications were transiently expressed in detached whole sunflower leaves. Our unique approach is based on the hypothesis that transient expression may consist of two sequential phases (early infection phase and late biosynthetic phase) and they can be separately optimized using chemical and environmental stimuli as initially demonstrated in our previous report . To support the idea, in this article we conduct a more in‐depth investigation of the effect of variable temperature profiling on transient expression.…”