Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

Reduced photosynthesis in Arabidopsis thaliana atpme17.2 and atpae11.1 mutants is associated to altered cell wall composition.

The cell wall is a complex and dynamic structure that determines plants' performance by constant remodeling of its compounds. Although cellulose is its major load-bearing component, pectins are crucial to determine wall characteristics. Changes in pectin physicochemical properties, due to pectin remodeling enzymes (PRE), induce the rearrangement of cell wall compounds, thus, modifying wall architecture. In this work, we tested for the first time how cell wall dynamics affect photosynthetic properties in Arabidopsis thaliana pectin methylesterase atpme17.2 and pectin acetylesterase atpae11.1 mutants in comparison to wild-type Col-0. Our results showed maintained PRE activities comparing mutants with wild-type and no significant differences in cellulose, but cell wall non-cellulosic neutral sugars contents changed. Particularly, the amount of galacturonic acid (GalA) - which represents to some extent the pectin cell wall proportion - was reduced in the two mutants. Additionally, physiological characterization revealed that mutants presented a decreased net CO2 assimilation (AN ) because of reductions in both stomatal (gs ) and mesophyll conductances (gm ). Thus, our results suggest that atpme17.2 and atpae11.1 cell wall modifications due to genetic alterations could play a significant role in determining photosynthesis.

Substrate specificity of plant and fungi pectin methylesterases: Identification of novel inhibitors of PMEs.

Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of ∼3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Structural alteration of cell wall pectins accompanies pea development in response to cold.

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype 'Terese' and a frost-tolerant genotype 'Champagne'. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-ß-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-ß-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease.

BACKGROUND AND AIMS: In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. METHODS: Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. KEY RESULTS: A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. CONCLUSIONS: By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Major changes in the cell wall during silique development in Arabidopsis thaliana.

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.