Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

Florent Weiller,Lorenz Gerber,Jonatan U Fangel,Johan Trygg,William G T Willats,Azeddine Driouich,John P Moore,Melané A Vivier

doi:10.3390/vaccines8030388

Abstract

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC–MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

Highlights

Pathogens such as necrotrophic or biotrophic plant pathogenic fungi have to break through/disrupt the host cell wall before colonising and feeding can begin
Nine genotypes were tested: eight transgenic tobacco lines including four plants overexpressing the NtCAD14 gene
The overexpression of cinnamyl alcohol dehydrogenase (CAD) and PGIP1 tobacco plants led to modifications of their cell wall structure in uninfected plants

Summary

Introduction

Pathogens such as necrotrophic or biotrophic plant pathogenic fungi have to break through/disrupt the host cell wall before colonising and feeding can begin. The plant cell wall matrix of fruits is a co-evolved pectin-protein-phenolic matrix, which aids in both the protection and dispersal of encapsulated seeds. Lignification, and pectin esterification alterations are common responses at local points of contact between the pathogen and plant [1,3]. This must mean that the plant pathogen (e.g., fungus) must develop a molecular toolkit of weapons, such as enzymes, which are able to overcome and degrade the cell wall to access the host organism [1,4]. Plant pathogens can use mechanical force via an appressorium [7], or use a wide range of plant cell wall degrading enzymes (CWDEs) to a good effect [8,9] to overcome the plant cell wall barrier

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Conclusion