Avirulent Population Research Articles

Comparative histopathology of virulent and avirulent Meloidogyne javanica populations on susceptible and resistant tomato plants.

The Mi-1.2 gene confers resistance to a wide range of Meloidogyne species, being the most important resistance factor employed in tomato breeding so far. However, many aspects related to the interaction of Mi-1.2-carrying tomato cultivars and virulent/avirulent Meloidogyne populations have not yet been clarified. Herein, comparative histopathological analyses were carried after inoculation of the homozygous (Mi-1.2/Mi-1.2) tomato rootstock 'Guardião' and the susceptible cultivar 'Santa Clara' (mi-1.2/mi-1.2) with virulent and avirulent populations of M. javanica. In the susceptible control, it was possible to visualize second stage juveniles (J2) of avirulent population and feeding sites from 2 to 30 days after infection (DAI) with females reaching maturity at 24-34 DAI. In the resistant rootstock, the Mi-1.2 gene-mediated resistance was related mainly to early defense responses (pre-infection and hypersensitive reaction), which led to an immunity-like phenotype that completely prevented the reproduction of the avirulent Meloidogyne population. On the other hand, J2s of the virulent M. javanica population were able to penetrate roots much more than the avirulent population, migrated and developed normally, showing intense and similar pattern of penetration from 4 to 34 DAI in the root tissues of both resistant and susceptible tomato genotypes. The total numbers of J2, J3, J4, and females counted in 'Santa Clara' for the virulent population of M. javanica were higher than in 'Guardião'.

Transcriptome-wide N6-methyladenosine (m6A) profiling of compatible and incompatible responses reveals a nonhost resistance–specific m6A modification involved in soybean–soybean cyst nematode interaction

Soybean cyst nematode (SCN, Heterodera glycines) is a devastating pathogen that infects soybean (Glycine max L. Merrill) and disrupts soybean production worldwide. SCN infection upregulates or downregulates the expression of multiple genes in soybean. However, the regulatory mechanisms that underlie these changes in gene expression remain largely unexplored. N6-methyladenosine (m6A) methylation, one of the most prevalent mRNA modifications, contributes to transcriptional reprogramming during plant responses to pathogen infection. Nevertheless, the role of m6A methylation in establishing compatible and incompatible soybean responses to SCN has not previously been studied. Here, we performed transcriptome-wide m6A profiling of soybean roots infected with virulent and avirulent populations of SCN. Compared with the compatible response, the incompatible response was associated with higher global m6A methylation levels, as well as more differentially modified m6A peaks (DMPs) and differentially expressed genes (DEGs). A total of 133 and 194 genes showed significant differences in both expression and m6A methylation levels in compatible and incompatible interactions; the most significantly enriched gene ontology terms associated with these genes were plant–pathogen interaction (compatible) and folate biosynthesis (incompatible). Our findings demonstrate that the m6A methylation profiles of compatible and incompatible soybean responses are distinct and provide new insights into the regulatory mechanism underlying soybean response to SCN at the post-transcriptional modification level, which will be valuable for improving the SCN resistance breeding.

Comparative Transcriptome Analysis Reveals the Specific Activation of Defense Pathways Against Globodera pallida in Gpa2 Resistant Potato Roots

Cyst nematodes are considered a dominant threat to yield for a wide range of major food crops. Current control strategies are mainly dependent on crop rotation and the use of resistant cultivars. Various crops exhibit single dominant resistance (R) genes that are able to activate effective host-specific resistance to certain cyst nematode species and/or populations. An example is the potato R gene Gpa2, which confers resistance against the potato cyst nematode (PCN), Globodera pallida population D383. Activation of Gpa2 results in a delayed resistance response, which is characterized by a layer of necrotic cells formed around the developing nematode feeding structure. However, knowledge about the Gpa2-induced defense pathways is still lacking. Here, we uncover the transcriptional changes and gene expression network induced upon Gpa2 activation in potato roots infected with G. pallida. To this end, in vitro-grown Gpa2-resistant potato roots were infected with the avirulent population D383 and virulent population Rookmaker. Infected root segments were harvested at 3 and 6 dpi and sent for RNA sequencing. Comparative transcriptomics revealed a total of 1,743 differentially expressed genes (DEGs) upon nematode infection, of which 559 DEGs were specifically regulated in response to D383 infection. D383-specific DEGs associated with Gpa2-mediated defense mainly relates to calcium-binding activity, salicylic acid (SA) biosynthesis, and systemic acquired resistance (SAR). These data reveal that cyst nematode resistance in potato roots depends on conserved downstream signaling pathways involved in plant immunity, which are also known to contribute to R genes-mediated resistance against other pathogens with different lifestyles.

Impact of horizontal gene transfer on emergence and stability of cooperative virulence in Salmonella Typhimurium

Intestinal inflammation fuels the transmission of Salmonella Typhimurium (S.Tm). However, a substantial fitness cost is associated with virulence expression. Mutations inactivating transcriptional virulence regulators generate attenuated variants profiting from inflammation without enduring virulence cost. Such variants interfere with the transmission of fully virulent clones. Horizontal transfer of functional regulatory genes (HGT) into attenuated variants could nevertheless favor virulence evolution. To address this hypothesis, we cloned hilD, coding for the master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. The resulting mobile hilD allele allows virulence to emerge from avirulent populations, and to be restored in attenuated mutants competing against virulent clones within-host. However, mutations inactivating the mobile hilD allele quickly arise. The stability of virulence mediated by HGT is strongly limited by its cost, which depends on the hilD expression level, and by the timing of transmission. We conclude that robust evolution of costly virulence expression requires additional selective forces such as narrow population bottlenecks during transmission.

Identification of Four Populations of Meloidogyne incognita in Georgia, United States, Capable of Parasitizing Tomato-Bearing Mi-1.2 Gene

Meloidogyne incognita, the southern root-knot nematode (RKN), is the most predominant plant-parasitic nematode species of tomato and causes significant yield loss. The Mi-1.2 gene confers resistance in tomatoes to M. incognita; however, virulent RKN populations capable of parasitizing resistant tomato cultivars have been reported from different regions in the world. Four naturally occurring virulent populations of M. incognita were found in vegetable fields from four counties in Georgia with no history of tomato cultivation of the Mi gene. Two consecutive greenhouse trials showed that all four virulent RKN populations reproduced on tomato cultivars, including Amelia, Skyway, and Myrtle, with the Mi-1 gene, while an avirulent population of M. incognita race 3 was unable to overcome host resistance. Virulent RKN populations varied in reproduction among resistant cultivars, with Ma6 population having the greatest reproduction potential. No difference in penetration potential of the virulent (Ma6) and avirulent populations was found on susceptible and resistant tomato cultivars. However, virulent Ma6 population females were successful at egg-laying, whereas avirulent female development was arrested in the resistant cultivars. The virulent Ma6 population also induced feeding sites in the roots of resistant cultivars, whereas the avirulent population did not. To our knowledge, this is the first report of resistance-breaking populations of M. incognita in Georgia and the second state in the United States after California.

Genome-wide Analysis of Alternative Gene Splicing Associated with Virulence in the Brown Planthopper Nilaparvata lugens (Hemiptera: Delphacidae).

Alternative splicing of protein coding genes plays a profound role in phenotypic variation for many eukaryotic organisms. The development of high-throughput sequencing and bioinformatics algorithms provides the possibility of genome-wide identification of alternative splicing events in eukaryotes. However, for the brown planthopper Nilaparvata lugens, a destructive pest of rice crops, whole-genome distribution of alternative splicing events and the role of alternative splicing in the phenotypic plasticity of virulence have not previously been estimated. Here, we developed an analysis pipeline to identify alternative splicing events in the genome of N. lugens. Differential expression analysis and functional annotation were performed on datasets related to different virulence phenotypes. In total, 27,880 alternative splicing events corresponding to 9,787 multi-exon genes were detected in N. lugens. Among them, specifically expressed alternative splicing transcripts in the virulent Mudgo population were enriched in metabolic process categories, while transcripts in the avirulent TN1 population were enriched in regulator activity categories. In addition, genes encoding odorant receptor, secreted saliva protein and xenobiotic metabolic P450 monooxygenase showed different splicing patterns between Mudgo population and TN1 population. Host change experiment also revealed that an isoform of a P450 gene could be specially induced by the stimulation of resistant rice variety Mudgo. This research pioneered a genome-wide study of alternative gene splicing in the rice brown planthopper. Differences in alternative splicing between virulent and avirulent populations indicated that alternative splicing might play an important role in the formation of virulence phenotypes in N. lugens.

Molecular variation among virulent and avirulent strains of the quarantine nematode Bursaphelenchus xylophilus

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD.

The efficacy of molecular markers associated with virulence in root-knot nematodes

Summary Root-knot nematodes (Meloidogyne spp.) are major crop pests that cause heavy economic losses. The use of resistant cultivars is one of the most important management methods. The Mi-1 gene in tomato confers effective resistance against several species of Meloidogyne. However, Mi-1-virulent root-knot nematodes restrict the use of resistant cultivars. Molecular markers associated with virulence have been developed in previous studies but not validated in large number of Meloidogyne populations so far. In this study, molecular markers were screened in a total of 69 (a)virulent Meloidogyne incognita and M. javanica isolates. The results showed these markers did not separate virulent and avirulent populations from Turkey. In addition, polymerase chain reaction products obtained from primers HM1.F2/HM1.R2 were digested with 30 restriction enzymes but no differences among populations were found. To the best of our knowledge, this is the first report on screening large root-knot nematode populations with these markers.

Soybean aphid (Hemiptera: Aphididae) response to lambda-cyhalothrin varies with its virulence status to aphid-resistant soybean.

Soybean aphid, Aphis glycines, is an invasive insect in North America, considered one of the most important pests of soybean. Their management relies heavily on foliar insecticides, but there is growing effort to expand these tools to include aphid-resistant varieties. We explored if the LC50 and LC25 of lambda-cyhalothrin varied between virulent (resistant to Aphis glycines (Rag) soybeans) and avirulent (susceptible to Rag-genes soybeans) populations of soybean aphid with a leaf-dip bioassay. We also investigated the response to the LC25 of lambda-cyhalothrin on adults (F0) and their progeny (F1) for both avirulent and virulent soybean aphid. The LC50 of the virulent aphid population was significantly higher compared with the LC50 of the avirulent population. The LC25 significantly reduced fecundity of the F0 generation of avirulent soybean aphid, but no significant effect was observed for virulent aphids. In addition, the LC25 significantly shortened the adult pre-oviposition period (APOP) and lengthened the total pre-oviposition period (TPOP) of avirulent aphids, while the mean generation time (T) was significantly increased. For the virulent aphid, sublethal exposure significantly lengthened development time of first and third instars, TPOP, and adult longevity. In addition, all demographic parameters of virulent soybean aphid were significantly affected when they were exposed to the LC25 of lambda-cyhalothrin. Our results demonstrate lambda-cyhalothrin is less toxic to virulent aphids and exposure to the LC25 can trigger hormesis, which may have implications for the long-term management of this pest with this insecticide as well as with aphid-resistant varieties of soybean. © 2019 Society of Chemical Industry.

Reproduction and life history traits of a resistance breaking Globodera pallida population

Abstract The main and most efficient measure to control potato cyst nematode (PCN) is the use of resistant cultivars. German and Dutch National Plant Protection Organizations (NPPOs) recently reported the emergence of Globodera pallida populations virulent on potato cultivars carrying resistance against pathotype 2/3. The development and virulence of the virulent population Oberlangen from Germany in comparison to the reference population G. pallida Pa3 Chavornay were investigated on resistant and susceptible cultivars in glasshouse experiments. Various life history traits associated with change in virulence were also assessed. Hatching of second-stage juveniles (J2s) was similar for both populations but incubation of cysts in potato root diffusate resulted in higher hatching rates compared to 3 mM Zinc Chloride and tap water. Both populations showed high penetration rates in the roots of the resistant and susceptible cultivars. However, only the population Oberlangen was able to complete the life cycle in the roots of the resistant potato cultivar. In ‘Seresta’, the resistance response restricted the formation of females by avirulent individuals in favor of males. Oberlangen was virulent on all cultivars tested. No difference in cyst size, number of eggs per cyst, length of juveniles, and males was found for Oberlangen and Chavornay on the susceptible cv. ‘Desiree’. However, cysts of virulent populations from the same region Oberlangen was obtained from had a significantly larger diameter compared to avirulent populations. The population Oberlangen showed a higher reproduction and fitness than the reference population Chavornay on susceptible cultivars and could serve as a future reference population in testing of new potato cultivars for resistance against this new virulence type in Europe.

12-Oxo-Phytodienoic Acid Enhances Wheat Resistance to Hessian Fly (Diptera: Cecidomyiidae) Under Heat Stress

12-Oxo-phytodienoic acid (OPDA) plays unique roles in plant defenses against biotic and abiotic stresses. In the current study, we infested two resistant wheat (Triticum aestivum L.) cultivars, 'Molly' and 'Iris', with an avirulent Hessian fly population and determined the impact of exogenous OPDA application on wheat resistance to the insect under heat stress. We observed that Molly and Iris treated with OPDA solution prior to the heat treatment exhibited significantly enhanced insect resistance. We also measured OPDA concentrations at Hessian fly feeding sites in Molly infested with Hessian flies. We found that exogenous application of OPDA resulted in increased abundance of endogenous OPDA in Molly seedlings and that OPDA abundance in plants treated with the combination of heat and OPDA was similar to that of plants in the incompatible interaction. Our results suggest that high abundance of endogenous OPDA may be necessary for wheat under heat stress to resist to Hessian fly infestation.

Resistance-breaking population of Meloidogyne incognita utilizes plant peroxidase to scavenge reactive oxygen species, thereby promoting parasitism on tomato carrying Mi-1 gene

Resistance conferred by the Mi-1 gene from Solanum peruvianum is effective and widely used for controlling root-knot nematodes (RKNs, Meloidogyne spp.). However, breakdown of resistance by RKNs seriously threatens the durable application of the resistance resource. Here, a resistance-breaking population of M. incognita was selected from an avirulent population by continuously inoculating on Mi-1-carrying tomato. Histological observations showed the resistance-breaking population would not induce hypersensitive response (HR) when infecting Mi-1-carrying tomato, while avirulent population did. A total of 308 differentially expressed genes (DEGs) were identified from Mi-1-carrying tomato upon infection with resistance-breaking versus avirulent populations by RNA-seq. The expression patterns of 23 selected DEGs were validated by quantitative real-time PCR (qRT-PCR). Subsequently, seven out of nine highly up-regulated DEGs were successfully knocked down in Mi-1-carrying tomato by tobacco rattle virus (TRV) mediated RNAi. The TRV line targeting a peroxidase gene showed a much higher magnitude of reactive oxygen species (ROS) and distinct reduction of pathogenicity upon infection of the resistance-breaking population compared with that of TRV::gfp line. Our results suggested that plant peroxidase might be exploited by resistance-breaking population of M. incognita to scavenge ROS, so as to overcome Mi-1-mediated resistance.

Determining the duration of Aphis glycines (Hemiptera: Aphididae) induced susceptibility effect in soybean

Insect herbivores can increase the suitability of host plants for conspecifics by inducing susceptibility. Induced susceptibility can be separated into feeding facilitation, whereby herbivore feeding increases performance of conspecifics regardless of the genotype of the herbivore or plant, and obviation of resistance, whereby feeding by a virulent herbivore increases performance of avirulent conspecifics on resistant plants. Both forms occur between Aphis glycines (Hemiptera: Aphididae) and soybean. In natural and agricultural settings, A. glycines populations can colonize plants for brief periods before emigrating or being removed due to predation or insecticides. It is unclear if induced susceptibility lasts beyond the period when A. glycines are present on the plant. We measured the duration of induced susceptibility in the A. glycines-soybean system within a growth chamber by removing inducer populations after 24 h. We used an A. glycines-resistant soybean infested with an inducer population of either virulent, avirulent, or no aphids. Response populations of either virulent or avirulent aphids were added at three post-infestation times (24, 120, 216 h) and their densities measured 11 days after infestation. Feeding facilitation was lost within 24 h of the removal of avirulent inducer populations, and obviation of resistance diminished over time and was completely lost within 216 h of the removal of the virulent inducer populations. We discuss how these results support a hypothesis that virulence in A. glycines is due to effector proteins secreted by feeding aphids. We suggest that the duration of induced susceptibility may impact the durability of A. glycines resistance in soybean.

An Induced Susceptibility Response in Soybean Promotes Avirulent Aphis glycines (Hemiptera: Aphididae) Populations on Resistant Soybean.

Observations of virulent Aphis glycines Matsumura populations on resistant soybean in North America occurred prior to the commercial release of Rag genes. Laboratory assays confirmed the presence of four A. glycines biotypes in North America defined by their virulence to the Rag1 and Rag2 genes. Avirulent and virulent biotypes can co-occur and potentially interact on soybean, which may result in induced susceptibility. We conducted a series of experiments to determine if the survival of avirulent biotypes on susceptible and resistant soybean containing the Rag1 or Rag1 + Rag2 genes was affected by the presence of either avirulent or virulent conspecifics. Regardless of virulence to Rag genes, initial feeding by conspecifics increased the survival of subsequent A. glycines populations on both susceptible and resistant soybean. Avirulent populations increased at the same rate as virulent populations if the resistant plants were initially colonized with virulent aphids. These results are the first to demonstrate that virulent A. glycines increase the suitability of resistant soybean for avirulent conspecifics, thus explaining the lack of genetic differentiation observed in North America between A. glycines populations on resistant and susceptible soybean. These results suggest the occurrence of virulence toward Rag genes in North America may be overestimated. In addition this may alter the selection pressure for virulence genes to increase in a population. Therefore, insect resistance management models for A. glycines may need to incorporate induced susceptibility factors to determine the relative durability of resistance genes.

ITS-RFLP pattern of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) does not reflect nematode virulence

Analysis of the restriction fragment length polymorphism of the internal transcribed spacer region of ribosomal DNA (ITS-RFLP) is an efficient tool for classifying Bursaphelenchus nematodes at or below the species level. In B. xylophilus, it has been shown that the ITS-RFLP pattern of Japanese avirulent (nonpathogenic) isolates is different from that of Japanese virulent (pathogenic) isolates. To clarify the relationship between the virulence and the ITS-RFLP pattern of B. xylophilus, an experiment involving inoculation of the nematodes was performed. Two types of nematode subculture that showed the same RFLP pattern as the virulent isolate or the avirulent isolate were produced from a new nematode population obtained in the field and inoculated into healthy pine seedlings. All seedlings inoculated with the nematode subcultures died within six months, irrespective of whether the subculture had the virulent or avirulent RFLP pattern; this indicated that the nematode virulence does not correspond with the ITS-RFLP pattern. This study shows that the ITS-RFLP pattern is not always available to discriminate between avirulent and virulent populations of B. xylophilus.

Virulence response to the Mi.1 gene of Meloidogyne populations from tomato in greenhouses

In a comparison of reproductive traits of 29 field populations of Meloidogyne spp. on resistant and susceptible tomatoes, 48% of the populations were virulent against the Mi.1 gene. Virulent populations produced more (P<0.05) egg masses/plant, eggs/plant, eggs/g root, and showed higher infection frequencies and multiplication rates than the avirulent populations. The fecundity of females did not differ between the resistant and susceptible genotype, and did not change with the virulence status of the populations. On the resistant genotype, the populations collected from the susceptible tomato showed lower (P<0.05) reproductive traits than those collected from rootstocks followed by those from resistant tomato. Meloidogyne javanica produced more (P<0.05) egg masses/plant and eggs/g root than Meloidogyne incognita on the resistant genotype. A time course experiment was conducted to determine if root penetration by M. javanica populations with different Mi.1 virulence status is conditioned by the expression of resistance on tomato. Significant differences between populations were observed as early as of 2 days post-inoculation on the susceptible genotype. On the resistant genotype, low juvenile numbers from populations with acquired virulence penetrated the roots up to 4 dpi, but their numbers significantly increased 7 dpi. Juveniles of the natural virulent population followed similar penetration pattern on the resistant and susceptible genotypes. Peroxidases are involved in the oxidative burst typically associated with the hypersensitive response in incompatible host–pathogen interactions. Isoelectrofocusing isozyme analysis revealed an increase in the peroxidase activity on the resistant genotype after inoculation with the avirulent population and to a lesser degree with the populations with acquired virulence in comparison with the natural virulent population.

An association between host acceptance and virulence status of different populations of Cotesia sesamiae, a braconid larval parasitoid of lepidopteran cereal stemborers in Kenya

Cotesia sesamiae, an endoparasitoid of lepidopteran cereal stem borers in East and Southern Africa, possesses a locally adapted immune suppressive ability. Parasitoid populations are virulent to the noctuid Busseola fusca in the highlands, where the species predominates, and avirulent in the mid-altitudes and lowlands, where it is of minor importance or absent. All C. sesamiae populations are virulent to the noctuid Sesamia calamistis, which is the predominant species in the mid-altitudes and lowlands. The ability of a parasitoid to select a suitable host has not been studied for parasitoid species that vary in both their host range and virulence. In this study, host acceptance behavior of C. sesamiae was examined in relation to the parasitoid virulence status. We assessed the sequence of events leading to oviposition in B. fusca and S. calamistis larvae. In contrast to virulent C. sesamiae, avirulent females avoided B. fusca larvae at the early steps of oviposition, except for the avirulent population from the lowlands, where B. fusca is absent. However, none of the avirulent population oviposited in B. fusca within the duration of the experiment. Both virulent and avirulent populations readily accepted S. calamistis. Apparently C. sesamiae is able to discriminate between hosts thereby limiting wastage of eggs in unsuitable hosts and the risk of being killed by the host larvae. The findings also indicate that non-volatile cues play an important role in host discrimination and acceptance. The insight gathered will help to choose the appropriate parasitoid populations for release in areas with multi-species infestations in West and Central Africa.

Selection of virulent populations of Meloidogyne javanica by repeated cultivation of Mi resistance gene tomato rootstocks under field conditions

Field trials were conducted in a plastic house artificially infested with an avirulent population of Meloidogyne javanica to determine the durability of the resistance mediated by the Mi gene in tomato rootstocks after repeated cultivation for three consecutive years. Treatments included an experimental rootstock cv. PG76 (Solanum lycopersicum × Solanum sp.), a commercial rootstock cv. Brigeor (S. lycopersicum × S. habrochaites), a resistant tomato cv. Monika (S. lycopersicum), and a susceptible cv. Durinta (S. lycopersicum). Based on the reproduction index (RI: number of eggs per g root on the resistant cultivar divided by number of eggs per g root on the susceptible cultivar × 100), rootstock cv. PG76 responded as highly resistant (RI = 7%) after the first cropping cycle (3·4 nematode generations), showed intermediated resistance (RI = 33%) after the second cropping cycle (3·3 generations), and was fully susceptible (RI = 94%) after the third cycle (3·3 generations). In contrast, rootstock cv. Brigeor and resistant cv. Monika retained intermediate resistance levels (RI = 41 and 25%, respectively) after the third cropping cycle. Virulent nematode populations were rapidly selected from an avirulent one after repeated cultivation of resistant tomatoes under field conditions. Bioassays conducted under controlled conditions confirmed that selection for virulence occurred more rapidly in plots with cv. PG76 followed by Brigeor and Monika. The nematode population in the field not exposed to Mi resistance remained avirulent to Mi genotypes. The genetic background of the resistant rootstocks and the frequency of cropping were critical factors for the appearance of virulent nematode populations. Irrespective of nematode infection, all resistant tomatoes yielded more than the susceptible cultivar.

The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.

Antioxidant enzymes in (a)virulent populations of root-knot nematodes

AbstractAssays of antioxidant enzymes, including catalase, peroxidase and superoxide dismutase (SOD), have been carried out on extracts of females and second-stage juveniles (J2) of a pair of Meloidogyne incognita isolates, one virulent and one avirulent, selected on tomato, and an avirulent field population of M. incognita. Catalase and SOD activity were found to be higher in extracts of the virulent isolate SM1 when compared with the avirulent counterparts. Peroxidase activity, assayed with o-dianisidine as the substrate, was enhanced in SM1 J2 compared with the avirulent avr1 J2. Catalase isozymes were separated by isoelectrofocusing into a very acidic and a basic isoform; this latter isoform was found to be responsible for the enhancement of catalase activity in virulent populations. SOD isozyme electrophoresis patterns (IEP) of root-knot nematodes, obtained by native PAGE, showed the presence of slow- and fast-migrating bands. SOD IEP of virulent females contained a slow-migrating band with a relative mobility (Rm) on the gels slightly higher (0.52) than the corresponding band from avirulent populations (0.50). This change was confirmed with native PAGE gels loaded with extracts from J2. To check how widespread this change is in field populations of RKN, a survey of SOD IEP from 12 RKN field (a)virulent populations was carried out. The specificity of the 0.52 Rm band for virulent populations was confirmed. Separation by native PAGE of peroxidases, stained either by o-dianisidine or diamino-benzidine, showed two isoforms with no apparent differences between the populations tested.