The exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new ß-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.

A maize polygalacturonase functions as a suppressor of programmed cell death in plants.

BACKGROUND: The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7. RESULTS: Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field. CONCLUSIONS: ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.

Molecular evolution and expression divergence of the Populus polygalacturonase supergene family shed light on the evolution of increasingly complex organs in plants.

Plant polygalacturonases (PGs) are involved in cell separation processes during many stages of plant development. Investigation into the diversification of this large gene family in land plants could shed light on the evolution of structural development. We conducted whole-genome annotation, molecular evolution and gene expression analyses of PG genes in five species of land plant: Populus, Arabidopsis, rice, Selaginella and Physcomitrella. We identified 75, 44, 16 and 11 PG genes from Populus, rice, Selaginella and Physcomitrella genomes, respectively, which were divided into three classes. We inferred rapid expansion of class I PG genes in Populus, Arabidopsis and rice, while copy numbers of classes II and III PG genes were relatively conserved in all five species. Populus, Arabidopsis and rice class I PG genes were under more relaxed selection constraints than class II PG genes, while this selective pressure divergence was not observed in Selaginella and Physcomitrella PG families. In addition, class I PG genes underwent marked expression divergence in Populus, rice and Selaginella. Our results suggest that PG gene expansion occurred after the divergence of the lycophytes and euphyllophytes, and this expansion was likely paralleled by the evolution of increasingly complex organs in land plants.

The ThPG1 endopolygalacturonase is required for the trichoderma harzianum-plant beneficial interaction.

Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol agent at the plant rhizosphere, proteomic, genomic, and transcriptomic approaches were used to study a novel Trichoderma gene coding for a plant cell wall (PCW)-degrading enzyme. A proteome analysis, using a three-component (Trichoderma spp.-tomato plantlets-pathogen) system, allowed us to identify a differentially expressed Trichoderma harzianum endopolygalacturonase (endoPG). Spot 0303 remarkably increased only in the presence of the soilborne pathogens Rhizoctonia solani and Pythium ultimum, and corresponded to an expressed sequence tag from a T. harzianum T34 cDNA library that was constructed in the presence of PCW polymers and used to isolate the Thpg1 gene. Compared with the wild-type strain, Thpg1-silenced transformants showed lower PG activity, less growth on pectin medium, and reduced capability to colonize tomato roots. These results were combined with microarray comparative data from the transcriptome of Arabidopsis plants inoculated with the wild type or a Thpg1-silenced transformant (ePG5). The endoPG-encoding gene was found to be required for active root colonization and plant defense induction by T. harzianum T34. In vivo assays showed that Botrytis cinerea leaf necrotic lesions were slightly smaller in plants colonized by ePG5, although no statistically significant differences were observed.

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper.

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata.

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.

Structure and function of pectic enzymes: virulence factors of plant pathogens.

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Perspectives in the biological function and the technological application of polygalacturonases.

Polygalacturonases (PG) have evolved in the past years from a pectinase "simply" being used for food processing to an important parameter in plant-fungal interaction. PG-inhibiting proteins (PGIP) that are synthesised in plants as a specific response to PGs of pathogenic fungi, have become a focus as a possible target in resistance breeding, and PGIPs are also a concern as an inhibiting factor in food processing. Plant PGs have been identified as a major factor in fruit ripening, and PG-deficient transgenic plants have been bred. Mainly fungal PGs are used in industrial processes for juice clarification and the range of enzymes is being extended through new recombinant and non-recombinant fungal strains. Finally, novel fields of application can be envisaged for PGs in the production of oligogalacturonides as functional food components. Here we aim to highlight the various fields where PGs are encountered and where they are of biological or technological importance.

Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti-repressor against KdgR.

Isolation, characterization, and sugar chain structure of endoPG Ia, Ib and Ic from Stereum purpureum.

Three endopolygalacturonases (endoPG Ia, Ib, and Ic) were isolated from the culture filtrate of Stereum purpureum, the causative fungus of apple silver-leaf disease. Their properties, including specific activities, optimum pHs, thermal stabilities, and kinetic parameters (K(m) and Vmax) were compared. Their properties were very similar to one another except for the substrate specificity and relative molecular mass. The sugar chains of endoPG Is were released by hydrazinolysis, and one major sugar chain common to endoPG Is was isolated. The pyridylamino sugar was characterized by a two-dimensional mapping method using HPLC, and identified as a high mannose type N-linked sugar chain, Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3) Man beta 1-4 GlcNAc beta 1-4 GlcNAc (designated as M5.1). Observation of the course of Western blot analysis for the proteins from the culture filtrate with endoPG I antibodies showed that the fungus secreted three endoPG Is into the culture broth during the growing period.

Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora.

Plant calcium can modulate a particular plant-pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora, the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora, the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant-pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.

Molecular genetic evidence for the involvement of a specific polygalacturonase, P2c, in the invasion and spread of Aspergillus flavus in cotton bolls.

Isolates of Aspergillus flavus can be differentiated based on production of the polygalacturonase P2c. One group of isolates produces P2c, whereas the other group does not. In general, the group that produces P2c causes more damage and spreads to a greater extent in cotton bolls than those isolates that do not produce P2c. To determine whether P2c contributes to disease, the expression of pecA, the gene previously determined to encode P2c, was genetically altered. Adding the pecA gene to a strain previously lacking the gene resulted in the ability to cause significantly more damage to the intercarpellary membrane and the ability spread to a greater extent within the adjacent locule compared to the abilities of a control transformant. Conversely, eliminating the expression of pecA by targeted disruption caused a significant reduction in aggressiveness compared to that of a nondisrupted control transformant. These results provide direct evidence that P2c contributes to the invasion and spread of A. flavus during infection of cotton bolls. However, other factors not evaluated in this study also contribute to aggressiveness.

Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pglA and its involvement in phytopathogenicity.

A major endopolygalacturonase excreted by Pseudomonas solanacearum was purified to greater than 95% homogeneity and shown to have an isoelectric point of 9.0 and a subunit molecular mass of 52 kilodaltons (kDa). The gene encoding this enzyme (pglA) was isolated from a genomic library of P. solanacearum DNA based on its expression in Escherichia coli and shown to be contained on a 1.8-kilobase DNA fragment. The identity of the pglA gene product and the 52-kDa polygalacturonase was demonstrated by immunoadsorption and isoelectric focusing experiments. The cloned pglA gene was apparently expressed from its own promoter in E. coli and its product was partially secreted into the periplasm. The pglA gene was insertionally inactivated in vitro and used to mutate the chromosomal pglA gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant strain was deficient in production of the 52-kDa polygalacturonase and took twice as long to wilt and kill tomato plants as the wild-type parent in plant bioassay experiments. Complementation in trans with the wild-type cloned pglA gene restored virulence to near wild-type levels. The data indicate that the pglA gene is important, but not absolutely necessary, for pathogenesis.