Label-free nanoUPLC-MSE based quantification of antimicrobial peptides from the leaf apoplast of Nicotiana attenuata

Abstract

BackgroundOverexpressing novel antimicrobial peptides (AMPs) in plants is a promising approach for crop disease resistance engineering. However, the in planta stability and subcellular localization of each AMP should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pI ranges of AMPs which limits the utility of standard proteomic gel-based methods. Despite recent advances in quantitative shotgun proteomics, its potential for AMP analysis has not been utilized and high throughput methods are still lacking.ResultsWe created transgenic Nicotiana attenuata plants that independently express 10 different AMPs under a constitutive 35S promoter and compared the extracellular accumulation of each AMP using a universal and versatile protein quantification method. We coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanoUPLC-MSE analysis using Hi3 method and identified/quantified 7 of 10 expressed AMPs in the transgenic plants ranging from 37 to 91 amino acids in length. The quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of 91 to 254 pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in N. attenuata. The chosen mass spectrometric approach proved to be highly sensitive in the detection of different AMP types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation.ConclusionsHeterologous expression of AMPs in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. The method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0398-9) contains supplementary material, which is available to authorized users.

Flower-specific jasmonate signaling regulates constitutive floral defenses in wild tobacco

“…S10A). NaDEF2 ectopic overexpression lines of N. attenuata (OvDEF2) have been previously characterized (66) and these accumulate high levels of the DEF2 peptide in their leaves. To evaluate the insecticidal activity of NaDEF2, the performance of M. sexta larvae was quantified on leaves of OvDEF2 plants.…”

“…EV transformed plants were used as transgenic control plants (67). N. attenuata plants overexpressing NaDEF2 were screened as previously described (66). The single insertion lines A-09-230 (C 230) and A-09-278 (F 278) were used in this study.…”

Engineering plants to secrete affinity‐tagged pathogen elicitors for deciphering immune receptor complex or inducing enhanced immunity

“…These SP‐GFP reporters were transiently expressed in Arabidopsis protoplasts for 6 h. For cells expressing each SP‐GFP reporter, total proteins in the protoplast culture medium were harvested and concentrated by trichloroacetic acid precipitation, where the secreted GFP was assayed by immunoblotting using the anti‐GFP antibody. It was found that the SP AnAXE1a from Aspergillus niger (Pawar et al 2016), SP LjLEA from Leonurus japonicus (Weinhold et al 2015), and SP NtPR1b from Nicotiana tabacum (Lund and Dunsmuir 1992) could lead to more efficient secretion of GFP (Figure 1C). The CERK1‐FLAG construct encoding the plasma membrane‐localized CERK1 proteins was cotransfected as an internal control to indicate comparable transfection efficiencies between different SP‐GFP constructs (Figure 1C).…”

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“…This “Hi3” approach has been applied for the analysis of different mutants of Staphylococcus aureus SCV [14] and the probing of the exoproteome of Corynebacterium pseudotuberculosis [15]. Recently, Weinhold and coworkers quantified the overexpressed novel antimicrobial peptides (AMPs) from the leaf apoplast of Nicotiana attenuate using the “Hi3” technique [16]. Norais et al applied the “Hi3” methodology to qualitatively and quantitatively characterize the outer-membrane-vesicle (DOMV) proteins of the Bexsero vaccine for the study of Meningococcal disease [17].…”