The soybean gene GmHsp22.4 is involved in the resistance response to Meloidogyne javanica in Arabidopsis thaliana.

BACKGROUND: Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. A previous study has showed strong induction of the gene GmHsp22.4 in response to the nematode Meloidogyne javanica in a resistant soybean genotype, while repression in a susceptible one. This study aimed to investigate the functional involvement of this small chaperone in response to M. javanica in Arabidopsis thaliana. First, it was evaluated the activation of the promoter region after the nematode inoculation, and the occurrence of polymorphisms between resistant and susceptible re-sequenced soybean accessions. Then functional analysis using A. thaliana lines overexpressing the soybean GmHsp22.4 gene, and knocked-out mutants were challenged with M. javanica infestation. RESULTS: High expression levels of the GFP gene marker in transformed A. thaliana plants revealed that the promoter region of GmHsp22.4 was strongly activated after nematode inoculation. Moreover, the multiplication of the nematode was significantly reduced in plants overexpressing GmHsp22.4 gene in A. thaliana compared to the wild type. Additionally, the multiplication of M. javanica in the A. thaliana mutants was significantly increased mainly in the event athsp22.0-2. This increase was not that evident in the event athsp22.0-1, the one that preserved a portion of the promoter region, including the HSEs in the region around - 83 bp. However, structural analysis at sequence level among soybean resistant and susceptible genotypes did not detect any polymorphisms in the whole gene model. CONCLUSIONS: The soybean chaperone GmHsp22.4 is involved in the defense response to root-knot nematode M. javanica in A. thaliana. Specifically, the promoter region covering until - 191 from the transcriptional start site (TSS) is necessary to promoter activation after nematode infection in Arabidopsis. No polymorphisms that could explain these differences in the defense response were detected in the GmHsp22.4 gene between resistant and susceptible soybean genotypes. Therefore, further investigation is needed to elucidate the triggering factor of the plant's defense mechanism, both at the sequence level of the soybean genotypes presenting contrasting reaction to root-knot nematode and by detecting cis-elements that are essential for the activation of the GmHsp22.4 gene promoter.

Sting nematodes modify metabolomic profiles of host plants.

Plant-parasitic nematodes are devastating pathogens of many important agricultural crops. They have been successful in large part due to their ability to modify host plant metabolomes to their benefit. Both root-knot and cyst nematodes are endoparasites that have co-evolved to modify host plants to create sophisticated feeding cells and suppress plant defenses. In contrast, the ability of migratory ectoparasitic nematodes to modify host plants is unknown. Based on global metabolomic profiling of sting nematodes in African bermudagrass, ectoparasites can modify the global metabolome of host plants. Specifically, sting nematodes suppress amino acids in susceptible cultivars. Upregulation of compounds linked to plant defense have negative impacts on sting nematode population densities. Pipecolic acid, linked to systemic acquired resistance induction, seems to play a large role in protecting tolerant cultivars from sting nematode feeding and could be targeted in breeding programs.

Tomato Natural Resistance Genes in Controlling the Root-Knot Nematode.

The root-knot nematode (RKN) is one of the most dangerous and widespread types of nematodes affecting tomatoes. There are few methods for controlling nematodes in tomatoes. Nature resistance genes (R-genes) are important in conferring resistance against nematodes. These genes that confer resistance to the RKN have already been identified as Mi-1, Mi-2, Mi-3, Mi-4, Mi-5, Mi-6, Mi-7, Mi-8, Mi-9, and Mi-HT. Only five of these genes have been mapped. The major problem is that their resistance breaks down at high temperatures. Some of these genes still work at high temperatures. In this paper, the mechanism and characteristics of these natural resistance genes are summarized. Other difficulties in using these genes in the resistance and how to improve them are also mentioned.

Bacteria isolated from the cuticle of plant-parasitic nematodes attached to and antagonized the root-knot nematode Meloidogyne hapla.

Plant-parasitic nematodes are associated with specifically attached soil bacteria. To investigate these bacteria, we employed culture-dependent methods to isolate a representative set of strains from the cuticle of the infective stage (J2) of the root-knot nematode Meloidogyne hapla in different soils. The bacteria with the highest affinity to attach to J2 belonged to the genera Microbacterium, Sphingopyxis, Brevundimonas, Acinetobacter, and Micrococcus as revealed by 16S rRNA gene sequencing. Dynamics of the attachment of two strains showed fast adhesion in less than two hours, and interspecific competition for attachment sites. Isolates from the cuticle of M. hapla J2 attached to the lesion nematode Pratylenchus penetrans, and vice versa, suggesting similar attachment sites on both species. Removal of the surface coat by treatment of J2 with the cationic detergent CTAB reduced bacterial attachment, but did not prevent it. Some of the best attaching bacteria impaired M. hapla performance in vitro by significantly affecting J2 mortality, J2 motility and egg hatch. Most of the tested bacterial attachers significantly reduced the invasion of J2 into tomato roots, suggesting their beneficial role in soil suppressiveness against M. hapla.

New Data Completing the Spectrum of the Ma, RMia, and RMja Genes for Resistance to Root-Knot Nematodes (Meloidogyne spp.) in Prunus.

Root-knot nematodes (RKN) (Meloidogyne spp.) are worldwide pests that affect a considerable number of plants, among which stone fruit (Prunus spp.) are severely attacked. Prevalent RKN species are Meloidogyne arenaria, M. incognita, and M. javanica in stone fruit but the emergent M. ethiopica and M. enterolobii are also reported to challenge perennial crops. In Prunus spp., the complete-spectrum resistance (R) gene Ma from plum and the more restricted-spectrum R genes RMia from peach and RMja from almond completely inhibit nematode multiplication and gall formation of the RKN species that they control. This study aimed to update the resistance spectra of these three major genes by evaluating their activity toward one isolate of the yet-untested RKN species mentioned above. To state whether a given gene controls a particular species, the principle of our experiment was to genotype with appropriate markers a number of individuals segregating for this gene and then to phenotype these individuals. A perfect matching of the genotype and the phenotype of individuals indicates that the gene of interest is active against and, thus, controls the corresponding isolate of this RKN species. Segregating materials used were an Ma F1 plum progeny, an RMia F2 peach progeny, and an RMja F2 almond progeny. In addition to previous data, our results establish a clear spectrum for each of the three genes toward isolates from both the three prevalent species and the two emerging species. Ultimately, our results reveal that (i) Ma controls all of them, (ii) RMja controls all species except M. incognita and M. floridensis, and (iii) RMia controls M. arenaria, M. incognita, and M. ethiopica but not M. javanica or M. enterolobii. Our data should have wide implications for RKN resistance management and breeding and for deciphering the molecular mechanisms of the spectrum of RKN R genes.

A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism.

Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.

Tomato progeny inherit resistance to the nematode Meloidogyne javanica linked to plant growth induced by the biocontrol fungus Trichoderma atroviride.

Root-knot nematodes (RKN) are major crop pathogens worldwide. Trichoderma genus fungi are recognized biocontrol agents and a direct activity of Trichoderma atroviride (Ta) against the RKN Meloidogyne javanica (Mj), in terms of 42% reduction of number of galls (NG), 60% of number of egg masses and 90% of number of adult nematodes inside the roots, has been observed in tomato grown under greenhouse conditions. An in vivo split-root designed experiment served to demonstrate that Ta induces systemic resistance towards Mj, without the need for the organisms to be in direct contact, and significantly reduces NG (20%) and adult nematodes inside tomato roots (87%). The first generation (F1) of Ta-primed tomato plants inherited resistance to RKN; although, the induction of defenses occurred through different mechanisms, and in varying degrees, depending on the Ta-Mj interaction. Plant growth promotion induced by Ta was inherited without compromising the level of resistance to Mj, as the progeny of Ta-primed plants displayed increased size and resistance to Mj without fitness costs. Gene expression results from the defense inductions in the offspring of Ta-primed plants, suggested that an auxin-induced reactive oxygen species production promoted by Ta may act as a major defense strategy during plant growth.

Root Transcriptome Analysis of Wild Peanut Reveals Candidate Genes for Nematode Resistance.

Wild peanut relatives (Arachis spp.) are genetically diverse and were adapted to a range of environments during the evolution course, constituting an important source of allele diversity for resistance to biotic and abiotic stresses. The wild diploid A. stenosperma harbors high levels of resistance to a variety of pathogens, including the root-knot nematode (RKN) Meloidogyne arenaria, through the onset of the Hypersensitive Response (HR). In order to identify genes and regulators triggering this defense response, a comprehensive root transcriptome analysis during the first stages of this incompatible interaction was conducted using Illumina Hi-Seq. Overall, eight cDNA libraries were produced generating 28.2 GB, which were de novo assembled into 44,132 contigs and 37,882 loci. Differentially expressed genes (DEGs) were identified and clustered according to their expression profile, with the majority being downregulated at 6 DAI, which coincides with the onset of the HR. Amongst these DEGs, 27 were selected for further qRT-PCR validation allowing the identification of nematode-responsive candidate genes that are putatively related to the resistance response. Those candidates are engaged in the salycilic (NBS-LRR, lipocalins, resveratrol synthase) and jasmonic (patatin, allene oxidase cyclase) acids pathways, and also related to hormonal balance (auxin responsive protein, GH3) and cellular plasticity and signaling (tetraspanin, integrin, expansin), with some of them showing contrasting expression behavior between Arachis RKN-resistant and susceptible genotypes. As these candidate genes activate different defensive signaling systems, the genetic (HR) and the induced resistance (IR), their pyramidding in one genotype via molecular breeding or transgenic strategy might contribute to a more durable resistance, thus improving the long-term control of RKN in peanut.

Calcium is involved in the R Mc1 (blb)-mediated hypersensitive response against Meloidogyne chitwoodi in potato.

KEY MESSAGE: Functional characterization of the Columbia root-knot nematode resistance gene R Mc1 ( blb ) in potato revealed the R gene-mediated resistance is dependent on a hypersensitive response and involves calcium. The resistance (R) gene R Mc1(blb) confers resistance against the plant-parasitic nematode, Meloidogyne chitwoodi. Avirulent and virulent nematodes were used to functionally characterize the R Mc1(blb)-mediated resistance mechanism in potato (Solanum tuberosum). Histological observations indicated a hypersensitive response (HR) occurred during avirulent nematode infection. This was confirmed by quantifying reactive oxygen species activity in response to avirulent and virulent M. chitwoodi. To gain an insight into the signal transduction pathways mediating the R Mc1(blb)-induced HR, chemical inhibitors were utilized. Inhibiting Ca(2+) channels caused a significant reduction in electrolyte leakage, an indicator of cell death. Labeling with a Ca(2+)-sensitive dye revealed high Ca(2+) levels in the root cells surrounding avirulent nematodes. Furthermore, the calcium-dependent protein kinase (CDPK), StCDPK4 had a higher transcript level in R Mc1(blb) potato roots infected with avirulent nematodes in comparison to roots infected with virulent M. chitwoodi. The results of this study indicate Ca(2+) plays a role in the R Mc1(blb)-mediated resistance against M. chitwoodi in potato.

Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

BACKGROUND: The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. RESULTS: We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. CONCLUSIONS: Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene appears to evolve under purifying selection. Its variability does not seem to influence the specificity of GPA2 recognition but is able to modulate this activity by enhancing the defence response. It seems therefore that the interaction with the plant resistance protein GPA2 (and/or Rx) rather than with the nematode effector was the major force in the evolution of the RanGAP2 locus in potato. From a mechanistic point of view these results are in accordance with a physical interaction of RanGAP2 with GPA2 and suggest that RBP-1 would rather bind the RanGAP2-GPA2 complex than the RanGAP2 protein alone.

Differential expression of antioxidant enzymes and PR-proteins in compatible and incompatible interactions of cowpea (Vigna unguiculata) and the root-knot nematode Meloidogyne incognita.

This study aimed to evaluated the resistance and susceptibility of 10 cowpea cultivars to Meloidogyne incognita in field studies and to analyze the kinetics of the enzymes superoxide dismutase, catalase, peroxidase, chitinase, ß-1,3-glucanases and cystein proteinase inhibitors in the root system of two contrasting cowpea cultivars after inoculation with M. incognita. The cultivars CE-31 and Frade Preto were highly resistant; CE-28, CE-01, CE-315, CE-237, were very resistant; CE-70 and CE-216 were moderately resistant, whereas Vita-3 and CE-109 were slightly resistant. In the roots of the highly resistant cultivar CE-31 the activity of the antioxidant enzyme superoxide dismutase increased and catalase decreased and those of the pathogenesis-related proteins chitinase, ß-1,3-glucanase, peroxidase and cystein proteinase inhibitor increased in comparison with the root system of the slightly resistant CE-109, during the course of M. incognita infestation. Thus the changes in the activities of these enzymes might be related to the smaller final population of M. incognita in CE-31 and may contribute to the high resistance of this cowpea cultivar against infection and colonization by this nematode species.

The receptor-like kinase SlSERK1 is required for Mi-1-mediated resistance to potato aphids in tomato.

The plant receptor-like kinase somatic embryogenesis receptor kinase 3 (SERK3)/brassinosteroid insensitive 1-associated kinase 1 (BAK1) is required for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Here we show that a distinct member of the SERK family, SERK1, is required for the full functioning of Mi-1, a nucleotide binding leucine-rich repeat (NB-LRR) resistance protein. Mi-1 confers resistance to Meloidogyne spp. (root-knot nematodes, RKNs) and three phloem-feeding insects, including Macrosiphum euphorbiae (potato aphid). SERK1 was identified in a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) screen in Nicotiana benthamiana. The screen was based on the suppression of a pest-independent hypersensitive response triggered by a constitutively active form of Mi-1, Mi-DS4. To assess the role of SERK1 in Mi-1-mediated resistance, Solanum lycopersicum (tomato) SlSERK genes were cloned. Three SlSERK members were identified with homologies to Arabidopsis AtSERK1 or AtSERK3/BAK1, and were named SlSERK1, SlSERK3A and SlSERK3B. SlSERK1 is ubiquitously expressed in tomato. Reducing SlSERK1 transcript levels in resistant plants, using gene-specific TRV-SERK1 VIGS, revealed a role for SlSERK1 in Mi-1-mediated resistance to potato aphids, but not to RKNs. In addition, Mi-1-dependent SlWRKY72 gene regulation was compromised in SlSERK1-silenced plants, placing SlSERK1 in the Mi-1 signaling pathway. Silencing SlSERK1 in a susceptible tomato background did not reduce the susceptibility to aphids, indicating that SlSERK1 is unlikely to be an essential virulence target. SlSERK1 is an active kinase, mainly localized at the plasma membrane. This work identifies a critical early component of Mi-1 signaling, and demonstrates a role for SlSERK1 in NB-LRR-mediated immunity.

Histological mechanisms of the resistance conferred by the Ma gene against Meloidogyne incognita in Prunus spp.

The Ma gene from Myrobalan plum is a TNL gene that confers a high-level resistance to all root-knot nematodes of major economic importance, including Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The nematode behavior in the roots and the corresponding histological mechanisms of the Ma resistance to M. incognita in the resistant (R) accessions of the plum 'P.2175' and the interspecific hybrid P.2175×almond-peach '35', carrying the Ma1 allele (Ma1/ma), were characterized in comparison with the susceptible (S) accessions in the plum 'P.2032' and the interspecific hybrid P.2175×almond-peach '253' (ma/ma). Second-stage juveniles (J2s) were inoculated in micropropagated plantlets grown in soil substrate under controlled conditions at 25°C. Nematodes penetrated both R and S plants preferentially along the apical zone or close to the young lateral buds and moved via similar routes. Then they migrated into the cortex downward in the direction of the apex and turned up in the meristematic apical region to colonize the differentiating stele. In R accessions, motile J2s neither swelled nor developed into J3s, and initiation of feeding sites was never observed. This complete absence of gall symptoms is associated with cell necroses and corresponding hypersensitive-like reaction (HLR) phenotypes occurring either in the stele or in the meristematic apical region or in the cortex. Nematode attacks often disorganized the meristematic apical tissues of R accessions, which induced the development of subterminal lateral roots replacing primary terminal apices and, thus, provided an active resistance reaction to HLR damage.

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate.

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.

The Arabidopsis bHLH25 and bHLH27 transcription factors contribute to susceptibility to the cyst nematode Heterodera schachtii.

Successful cyst nematode parasitism depends on the formation and maintenance of feeding sites (syncytia) in host roots, and these processes are highly regulated by the interaction between the cyst nematode and the host. Using an integrated research approach and the Arabidopsis-Beta vulgaris (sugar beet) cyst nematode (Heterodera schachtii) pathosystem, we have determined that the two Arabidopsis basic helix-loop-helix transcription factors bHLH25 and bHLH27 positively influence cyst nematode parasitism. Promoter studies indicated that as early as 1 day post-inoculation, both transcription factor genes were upregulated in developing syncytia, whereas in non-infected plants, these two promoters were not found to be active in the same cells. By using yeast two-hybrid analyses and bimolecular fluorescence complementation assays, we documented that the two bHLH transcription factors can dimerize in planta. Transgenic Arabidopsis plants overexpressing either one or both of the bHLH genes exhibited altered morphology of roots and shoots, as well as an increased susceptibility to H. schachtii. bhlh25 or bhlh27 single mutants were without strong phenotypes, presumably because of functional redundancies in this gene family. However, the bhlh25 bhlh27 double mutant was less susceptible to H. schachtii, confirming an important conducive role of the co-expression of both transcription factor genes for cyst nematode parasitism. Our results document an example of pathogen-induced ectopic co-expression of two regulatory genes to enhance pathogen success, although these transcription factors apparently do not function in concert in non-infected plants. This is an intriguing biological phenomenon that highlights the complexity of obligate biotrophic plant-pathogen interactions, like those of cyst nematodes.

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in Gossypium barbadense L. accession GB713.

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P(1), P(2), F(1), F(2), BC(1)P(1), and BC(1)P(2) generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F(2) population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren (barb1) and Ren (barb2) to designate these QTLs on chromosome 21 and Ren (barb3) on chromosome 18.

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes.

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans).

Specific host-parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.

Inducible antibacterial defence in the plant parasitic nematode Meloidogyne artiellia.

Animals and plants both respond rapidly to pathogens by inducing the expression of defence-related genes. Within this context, a prominent role has been assigned to the lysozyme. In the present study we isolated and carried out detailed analysis of the lysozyme gene in the plant nematode Meloidogyne artiellia. The expression of lysozyme was up-regulated following exposure of M. artiellia juveniles to the Gram-negative bacterium Serratia marcescens. On the other hand, when isolated eggs containing embryos at various developmental stages were challenged with bacteria, no increase in lysozyme expression was detected. Evidence of lysozyme expression regulation was obtained in the case of adult male and females worms collected from soil. The lysozyme gene was expressed solely in the nematode intestine and, as it is predicted to be secreted, may protect the nematode from microbial infections originating in the intestinal lumen or in the pseudocoelom. This paper demonstrates, to our knowledge for the first time, the immune response to infection in a plant parasitic nematode.

Molecular cloning and analysis of a new venom allergen-like protein gene from the root-knot nematode Meloidogyne incognita.

A new venom allergen-like protein gene isolated from Meloidogyne incognita (designated Mi-vap-2) was cloned and analysed. The genomic clone of Mi-vap-2 is 1917-bp long, contains three introns, which range in size from 39 to 797 bp, and four exons ranging in size from 37 to 361 bp. The cDNA of Mi-vap-2 contains an open reading frame encoding 294 amino acids, being the first 16 residues a putative secretion signal. Southern blot analysis suggested that Mi-vp-2 is probably a member of a small multigene family. In situ hybridization analysis showed that the transcripts of Mi-vap-2 accumulated exclusively within the subventral oesophageal gland cells of M. incognita. RT-PCR analyses confirmed that Mi-vap-2 was transcribed mainly in the pre-parasitic second-stage and early post-inoculated juveniles. Results indicated that this venom allergen-like protein gene may play an important role in establishment of the parasitic relationship between plants and nematodes.