Daniela Bellincampi

Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea.

Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.

Structural Basis for the Interaction between Pectin Methylesterase and a Specific Inhibitor Protein

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-Å resolution. The enzyme folds into a right-handed parallel β-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long α-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel β-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.

Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA+B+C) and by rolB alone provided an extended segment beyond its 5′ noncoding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB+C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13+14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13+14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA+B+C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA+B+C roots by the concomitant presence of ORF13+14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.

Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions

The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground.

Methyl esterification of pectin plays a role during plant–pathogen interactions and affects plant resistance to diseases

The cell wall is a complex structure mainly composed by a cellulose-hemicellulose network embedded in a cohesive pectin matrix. Pectin is synthesized in a highly methyl esterified form and is de-esterified in muro by pectin methyl esterases (PMEs). The degree and pattern of methyl esterification affect the cell wall structure and properties with consequences on both the physiological processes of the plants and their resistance to pathogens. PME activity displays a crucial role in the outcome of the plant-pathogen interactions by making pectin more susceptible to the action of the enzymes produced by the pathogens. This review focuses on the impact of pectin methyl esterification in plant-pathogen interactions and on the dynamic role of its alteration during pathogenesis.

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors1

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI‐1 and AtPMEI‐2) encoding functional inhibitors of pectin methylesterases. AtPMEI‐1 and AtPMEI‐2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant‐derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

The Ectopic Expression of a Pectin Methyl Esterase Inhibitor Increases Pectin Methyl Esterification and Limits Fungal Diseases in Wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonization by necrotrophic pathogens.

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.

Oligogalacturonides Prevent Rhizogenesis in rolB-Transformed Tobacco Explants by Inhibiting Auxin-Induced Expression of the rolB Gene.

Oligogalacturonides elicit several defense responses and regulate different aspects of growth and development in plants. Many of the development-related effects of oligogalacturonides appear to be amenable to an auxin antagonist activity of these oligosaccharins. To clarify the role of oligogalacturonides in antagonizing auxin, we analyzed their effect on root formation in leaf explants of tobacco harboring the plant oncogene rolB. We show here that oligogalacturonides are capable of inhibiting root morphogenesis driven by rolB in transgenic leaf explants when this process requires exogenous auxin. Because rolB expression is induced by auxin and dramatically alters the response to this hormone in transformed plant cells, the inhibiting effect of oligogalacturonides could be exerted on the induction of rolB and/or at some other auxin-requiring step(s) in rhizogenesis. We show that oligogalacturonides antagonize auxin primarily because they strongly inhibit auxin-regulated transcriptional activation of a rolB-[beta]-glucuronidase gene fusion in both leaf explants and cultured leaf protoplasts. In contrast, oligogalacturonides do not inhibit rhizogenesis when rolB transcriptional activation is made independent of auxin, as shown by the lack of inhibition of root formation in leaf explants containing rolB driven by a tetracycline-inducible promoter.