Pasqua Veronico

Nematode chitin synthases: gene structure, expression and function in Caenorhabditis elegans and the plant parasitic nematode Meloidogyne artiellia

Abstract. Although the presence of chitin in nematodes is well documented little is known about its synthesis in this phyletic group. The recently completed genome sequence of Caenorhabditis elegans predicts two sequences with homology to chitin synthases (chitin-UDP acetyl-glucosaminyl transferase; EC 2.4.1.16). We show that these genes are differentially expressed in a pattern that may reflect different functional roles. One gene is expressed predominantly in the adult hermaphrodite (the main egg-producing stage in the nematode) and later larval stages, which is consistent with a role in production of chitin for the eggshell. The other gene, however, is expressed in the cells that form the pharynx, and only in the period directly preceding a moult. These data suggest that the product of this gene is involved in synthesis of the feeding apparatus, which is replaced during each moult. We have also isolated a full-length genomic sequence of a chitin synthase orthologue from the plant parasitic nematode Meloidogyne artiellia. The single gene present in M. artiellia shows an expression pattern that is consistent with a role for the protein in production of the eggshell.

A Novel Lipoxygenase in Pea Roots. Its Function in Wounding and Biotic Stress

The genome of pea (Pisum sativum) contains genes encoding a family of distinct lipoxygenases (LOX). Among these, LOXN2 showed eight exons encoding a 93.7-kD enzyme, harboring two C-terminal deletions and an unusual arginine/threonine-tyrosine motif in the domain considered to control the substrate specificity. LOXN2, when overexpressed in yeast, exhibited normal enzyme activity with an optimum at pH 4.5, and a dual positional specificity by releasing a 3:1 ratio of C-9 and C-13 oxidized products. The predicted LOXN2 structure lacked a loop present in soybean (Glycine max) LOX1, in a position consistent with control of the degree of substrate access to the catalytic site and for LOXN2s dual positional specificity. The LOXN2 gene was tightly conserved in the Progress 9 and MG103738 genotypes, respectively, susceptible and resistant to the root cyst nematode Heterodera goettingiana. LOXN2 transcription was monitored in roots after mechanical injury and during nematode infection. The message peaked at 3 and 24 h after wounding in both genotypes and was more abundant in the resistant than in the susceptible pea. In nematode-infected roots, transcription of several LOX genes was triggered except LOXN2, which was repressed in both genotypes. In situ hybridization revealed that LOXN2 message was widespread in the cortex and endodermis of healthy roots, but specifically localized at high level in the cells bordering the nematode-induced syncytia of infected roots. However, LOXN2 transcript signal was particularly intense in collapsing syncytia of MG103738 roots, suggesting LOXN2 involvement in late mechanisms of host resistance.

Analysis of Class III Peroxidase Genes Expressed in Roots of Resistant and Susceptible Wheat Lines Infected by Heterodera avenae

The response of resistant wheat-Aegilops ventricosa introgression line H-93-8 and its susceptible parent, Triticum aestivum H-10-15, to Ha71 Spanish population of Heterodera avenae was studied to determine the changes in peroxidase gene expression during incompatible and compatible wheat-nematode interactions. Twenty peroxidase genes were characterized from both 211 expressed sequence tags and 259 genomic DNA clones. Alignment of deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species showed that these enzymes fall into seven different groups (designated TaPrx108 to TaPrx114) which represent peroxidases secreted to the apoplast by a putative N-terminal peptide signal. TaPrx111, TaPrx112, and TaPrx113 were induced by nematode infection in both genotypes but with differing magnitude and timing. TaPrx112 and TaPrx113 groups increased more in resistant than in susceptible infected lines. In addition, in situ hybridization analyses of genes belonging to TaPrx111, TaPrx112, and TaPrx113 groups revealed a more intense signal in cells close to the vascular cylinder and parenchyma vascular cells of resistant than susceptible wheat when challenged by nematodes. These data seem to suggest that wheat apoplastic peroxidases, because of their different expression in quantity and timing, play different roles in the plant response to nematode infection.

Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant-parasitic nematode Meloidogyne artiellia

Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.

Characterization of the (GAAA) microsatellite region in the plant parasitic nematode Meloidogyne artiellia.

Microsatellites have become one of the most powerful genetic markers in biology. We have used DNA sequencing to characterize a highly variable microsatellite (GAAA) locus in the root-knot nematode Meloidogyne artiellia. The use of microsatellite flanking primers produced four amplification products that are defined as electromorphs, based on conventional length criteria. The sequencing of these four amplification products revealed the presence of new variants in the population due to sequence variability. The sum of electromorphs and sequence polymorphisms resulted in a total of six variants. The high degree of variability in the microsatellite containing region is due not only to variation in the number of tetranucleotide repeats but also to variation (length and site variation) in the flanking regions of the microsatellite. These investigations show that, in spite of the size homoplasy, the variability of the microsatellite flanking sequences of M. artiellia could be used as informative markers for phylogenetic reconstructions.

A polygalacturonase-inhibiting protein with a role in pea defence against the cyst nematode Heterodera goettingiana

A cDNA of 312 bp, similar to polygalacturonase-inhibiting proteins (PGIPs), was isolated by cDNA-amplified fragment length polymorphism (cDNA-AFLP) from pea roots infected with the cyst nematode Heterodera goettingiana. The deduced amino acid sequence obtained from the complete Pspgip1 coding sequence was very similar to PGIPs described from several other plant species, and was identical in both MG103738 and Progress 9 genotypes, resistant and susceptible to H. goettingiana, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) expression analysis revealed the differential regulation of the Pspgip1 gene in the two genotypes in response to wounding and nematode challenge. Mechanical wounding induced Pspgip1 expression in MG103738 within 8 h, but this response was delayed in Progress 9. In contrast, the response to nematode infection was more complex. The transcription of Pspgip1 was triggered rapidly in both genotypes, but the expression level returned to levels observed in uninfected plants more quickly in susceptible than in resistant roots. In addition, in situ hybridization showed that Pspgip1 was expressed in the cortical cells damaged as a result of nematode invasion in both genotypes. However, it was specifically localized in the cells bordering the nematode-induced syncytia in resistant roots. This suggests a role for this gene in counteracting nematode establishment inside the root.

Benzothiadiazole effect in the compatible tomato-Meloidogyne incognita interaction: changes in giant cell development and priming of two root anionic peroxidases.

AbstractMain conclusionBTH application is effective in root-knot nematode-tomato interaction in a way that involves a delay in the formation of nematode feeding site and triggers molecular responses at several levels. The compatible interaction between root-knot nematodes and their hosts requires the nematode to overcome plant defense systems so that a sophisticated permanent feeding site (giant cells) can be produced within the host roots. It has been suggested that activators of plant defenses may provide a novel management strategy for controlling root-knot nematodes but little is known about the molecular basis by which these elicitors operate. The role of pre-treatment with Benzothiadiazole (BTH), a salicylic acid analog, in inducing resistance against Meloidogyne incognita infection was investigated in tomato roots. A decrease in galling in roots and feeding site numbers was observed following BTH treatment. Histological investigations showed a delay in formation of feeding sites in treated plants. BTH-treated galls had higher H2O2 production, lignin accumulation, and increased peroxidase activity than untreated galls. The expression of two tomato genes, Tap1 and Tap2, coding for anionic peroxidases, was examined by qRT-PCR and in situ hybridization in response to BTH. Tap1 was induced at all infection points, reaching the highest level at 15 dpi. Tap2 expression, although slightly delayed in untreated galls, increased during infection in both treated and untreated galls. The expression of Tap1 and Tap2 was observed in giant cells of untreated roots, whereas the transcripts were localized in both giant cells and in parenchyma cells surrounding the developing feeding sites in treated plants. These results show that BTH applied to tomato plants makes them more resistant to infection by nematodes, which become less effective in overcoming root defense pathway.

Changes in lignin biosynthesis and monomer composition in response to benzothiadiazole and root-knot nematode Meloidogyne incognita infection in tomato

Benzothiadiazole (BTH) acts as a priming agent in plant defence leading to a reduction in penetration and development of the root-knot nematode Meloidogyne incognita in susceptible tomato roots. Changes in lignin biosynthesis in the susceptible tomato cv. Roma following nematode infection and/or BTH treatment were investigated in comparison to the resistant cv. Rossol. Both untreated and BTH-treated susceptible infected roots (galls) showed an increased level of expression of lignin synthesis-related genes (PAL, C4H, HCT and F5H) at early times during infection (2-4 days post inoculation). Peroxidase (soluble and cell-wall bound, POX) enzyme activities increased after inoculation with M. incognita and the priming effect of BTH treatment was evident at later stages of infection (7 days post inoculation). As expected, the induction of PAL and POXs and lignin synthesis-related genes was faster and greater in resistant roots after infection. Histochemical analysis revealed accumulation of higher lignin levels at later infection stages in BTH-treated galls compared to untreated ones. Furthermore, the monomer composition of lignin indicated a different composition in guaiacyl (G) and syringyl (S) units in BTH-treated galls compared to untreated galls. The increase in G units made G/S ratio similar to that in the resistant genotype. Overall, lignin played a critical role in tomato defence to M. incognita in response to BTH.