The heterologous expression of a Glycine max homolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogen Meloidogyne incognita in Gossypium hirsutum

Experiments in Glycine max (soybean) identified the expression of the salicylic acid signaling and defense gene NONEXPRESSOR OF PR1 (NPR1) in root cells (i.e., syncytium) parasitized by the plant parasitic nematode Heterodera glycines undergoing the process of resistance. Gm-NPR1-2 overexpression in G. max effectively suppresses parasitism by H. glycines. The heterologous expression of Gm-NPR1-2 in Gossypium hirsutum impairs the ability of the parasitic nematode Meloidogyne incognita to form root galls, egg sacs, eggs and second-stage juvenile (J2) nematodes. In related experiments, a G. max β-glycosidase (Gm-βg-4) related to Lotus japonicus secreted defense gene α-hydroxynitrile glucosidase LjBGD7 suppresses M. incognita parasitism. The results identify a cumulative negative effect that the transgenes have on M. incognita parasitism and demonstrate that the G. max–H. glycines pathosystem is a useful tool to identify defense genes that function in other agriculturally relevant plant species to plant parasitic nematodes with different strategies of parasitism.     

Syncytium gene expression in Glycine max[PI 88788] roots undergoing a resistant reaction to the parasitic nematode Heterodera glycines

The plant parasitic nematode, Heterodera glycines is the major pathogen of Glycine max (soybean). H. glycines accomplish parasitism by creating a nurse cell known as the syncytium from which it feeds. The syncytium undergoes two developmental phases. The first is a parasitism phase where feeding sites are selected, initiating the development of the syncytium. During this earlier phase (1–4 days post infection), syncytia undergoing resistant and susceptible reactions appear the same. The second phase is when the resistance response becomes evident (between 4 and 6 dpi) and is completed by 9 dpi. Analysis of the resistant reaction of G. max genotype PI 88788 (G. max_{[PI 88788]}) to H. glycines population NL1-RHg/HG-type 7 (H. glycines_{[NL1-RHg/HG-type 7]}) is accomplished by laser microdissection of syncytia at 3, 6 and 9 dpi. Comparative analyses are made to pericycle and their neighboring cells isolated from mock-inoculated roots. These analyses reveal induced levels of the jasmonic acid biosynthesis and 13-lipoxygenase pathways. Direct comparative analyses were also made of syncytia at 6 days post infection to those at 3 dpi (base line). The comparative analyses were done to identify localized gene expression that characterizes the resistance phase of the resistant reaction. The most highly induced pathways include components of jasmonic acid biosynthesis, 13-lipoxygenase pathway, S-adenosyl methionine pathway, phenylpropanoid biosynthesis, suberin biosynthesis, adenosylmethionine biosynthesis, ethylene biosynthesis from methionine, flavonoid biosynthesis and the methionine salvage pathway. In comparative analyses of 9 dpi to 6 dpi (base line), these pathways, along with coumarin biosynthesis, cellulose biosynthesis and homogalacturonan degradation are induced. The experiments presented here strongly implicate the jasmonic acid defense pathway as a factor involved in the localized resistant reaction of G. max_{[PI 88788]} to H. glycines_{[NL1-RHg/HG-type 7]}.     

The use of laser capture microdissection to study the infection of Glycine max (soybean) by Heterodera glycines (soybean cyst nematode)

The soybean cyst nematode (Heterodera glycines) is an obligate parasite of soybean (Glycine max). It is the most destructive pathogen of G. max, accounting for approximately 0.46–0.82 billion dollars in crop losses, annually, in the U.S. Part of the infection process involves H. glycines establishing feeding sites (syncytia) that it derives its nourishment from throughout its lifecycle. Microscopic methods (i.e., laser capture microdissection [LCM]) that faithfully dissect out those feeding sites are important improvements to the study of this significant plant pathogen. Our isolation of developing feeding sites during an incompatible or a compatible reaction is providing new ways by which this important plant-pathogen interaction can be studied. We have used these methods to create cDNA libraries, clone genes and perform microarray analyses. Importantly, it is providing insight not only into how the root is responding at the organ level to H. glycines, but also how the syncytium is responding during its maturation into a functional feeding site.Key words: soybean, Glycine max, soybean cyst nematode, SCN, Heterodera glycines, microarray, gene expression, plant pathogen, parasite, laser capture microdissection     

A gene expression analysis of syncytia laser microdissected from the roots of the <Emphasis Type="Italic">Glycine max</Emphasis> (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by <Emphasis Type="Italic">Heterodera glycines</Emphasis> (soybean cyst nematode)

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix^® soybean GeneChip^® directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.     

The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.     

Segregation at the SCN resistance locus rhg1 in soybean is distorted by an association between the resistance allele and reduced field emergence

Segregation distortion has been reported repeatedly in soybean (Glycine max [L.] Merr.) inbred line populations segregating for the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) resistance gene rhg1. In each reported case, the frequency of the SCN resistance allele at the rhg1 locus was lower than expected. Segregation distortion was studied in 51 F₄ populations by counting the number of plants predicted to be homozygous resistant, susceptible, and heterozygous for rhg1 based on the genetic markers Satt309, CTA, or TMA5. Significant (P<0.05) segregation distortion was observed in 44 out of the 51 F₄ populations. When the heterozygotes were ignored, there were significantly fewer homozygous-resistant plants than expected in 33 populations. To study whether differential field emergence was a cause of the segregation distortion, three near isogenic line (NIL) populations segregating at the rhg1 locus for SCN resistance from plant introduction 88788 were tested. Population sizes ranged from 32 to 44 NILs and emergence was determined in field experiments in three environments. In each population, SCN-resistant NILs had significantly (P<0.05) less field emergence than susceptible NILs. In the population with the greatest effect, field emergence of resistant NILs was 6% less than susceptible NILs, with the entire population having an average emergence rate of 46%. Equations were derived to describe the effect of selection on segregation ratios over generations of population development and the observed emergence rates were transformed into fitness factors. Depending on assumptions of gene action, it was predicted from these fitness factors that segregation distortions were in the range of those reported previously for the rhg1 locus and were similar to what was observed on average across the 51 F₄ populations. While other factors might also be involved, the results suggest that reduced field emergence associated with the SCN resistance allele contributes to previously reported segregation distortion at the rhg1 locus.     

Stimulation of NSF ATPase Activity by α-SNAP Is Required for SNARE Complex Disassembly and Exocytosis

N-ethylmaleimide–sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH₂- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH₂-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH₂-terminal (1–160) and COOH-terminal (160–295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH₂- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1–285) and α-SNAP (L294A) were unable to stimulate Ca²⁺-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1–285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1–285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α-SNAP stimulation of exocytosis.     

<Emphasis Type="Italic">N</Emphasis>-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane

N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with β-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABA_A) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/β-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/β-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane.     

Rhg1 alleles from soybean PI 437654 and PI 88788 respond differentially to isolates of Heterodera glycines in the greenhouse

The production of resistant soybean [Glycine max (L.) Merr.] cultivars is the most effective means for controlling losses from soybean cyst nematode (SCN) (Heterodera glycines Ichinohe). The major resistance gene in most SCN resistance sources is rhg1, which has been mapped as a quantitative trait locus onto linkage group G. Our objective was to determine whether the SCN resistance sources PI 437654 and PI 88788 have different functional alleles at rhg1 based on resistance phenotypes. Populations segregating for resistance alleles at rhg1 from both PI 88788 and PI 437654 and at Rhg4, a second SCN resistance gene from PI 437654, were developed. These populations were screened for resistance to the H. glycines inbred isolates PA3 (HG type 7) and TN14 (HG type 1.2.5.7) in the greenhouse and evaluated with molecular markers linked to both rhg1 and Rhg4. Each isolate test was repeated, and the evaluations were done on a single-plant and a line-mean basis in Test 1, and solely on a single-plant basis in Test 2. Across two tests with the TN14 isolate, plants with the PI 437654 allele for a marker linked to rhg1 had significantly (P<0.0001) less SCN reproduction than plants carrying the PI 88788 allele. A marker linked to Rhg4, however, was not significantly associated with resistance to TN14. Across two tests with the PA3 isolate, alleles of rhg1 from both sources gave a resistant reaction, although plants homozygous for the PI 88788 allele had significantly (P<0.05) greater resistance than plants with the PI 437654 allele. The marker allele from PI 437654 linked to Rhg4 was significantly (P<0.0005) associated with greater resistance than the PI 88788 allele in both PA3 tests, and resistance was dominant. There was a significant interaction between alleles at rhg1 and Rhg4 in both PA3 tests. These results suggest that PI 437654 and PI 88788 each have a different functional SCN resistance allele at or close to rhg1. These allelic differences have implications that breeders should consider before incorporation into cultivars.     

Influence of Heterodera glycines on Interspecific and Intraspecific Competition Associated with Glycine max and Chenopodium album

The influence of Heterodera glycines (soybean cyst nematode) on the interspecific and intraspecific competition associated with Glycine max (soybean) and Chenopodium album (common lambsquarters) was studied in 1988 and 1989 in three de Wit replacement series experiments in growth chambers and microplots. Glycine max was grown alone (1 plant/experimental unit), in intraspecific competition (2 plants/experimental unit), in interspecific competition with C. album, and in presence or absence of H. glycines. No significant effects of H. glycines and C. album on G. max growth were observed 14 days after planting. By 42 days after planting, both H. glycines and C. album had a negative (P = 0.05) influence on the growth of G. max. Relative crowding coefficients for G. max were lower and deviated (P = 0.05 and P = 0.001) from 1.0 in the presence of H. glycines, compared to that of C. album and early emerged C. album in the absence of the nematode, respectively. Glycine max, therefore, became less competitive than C. album. There was a trend that the presence of H. glycines decreased the competitiveness of G. max on measures of the aggressivity and relative mixture response. Heterodera glycines decreased the aggressivity of G. max (ca. 150-350%) and increased the relative effects of intraspecific interference on G. max (ca. 10-50%) and interspecific interference (ca. 60-350%) after 42 days of plant growth, compared with plants grown in the absence of H. glycines. No H. glycines x C. album interactions were detected. Observations showed that H. glycines and early emerged C. album inhibited the growth of G. max 5-13%, as measured by plant dry weight.     

Recombination suppression at the dominant <Emphasis Type="Italic">Rhg1/Rfs2</Emphasis> locus underlying soybean resistance to the cyst nematode

Host resistance to “yellow dwarf” or “moonlight” disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24–48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred.     

Location of Heterodera glycines-induced Syncytia in Soybean as Affected by Soil Water Regimes

Locations of syncytia induced by the soybean cyst nematode (SCN), Heterodera glycines race 3, were compared in roots of ''Essex'', a susceptible soybean (Glycine max (L.) Merr.) cultivar, at three soil water regimes. The plants were grown in wet (-5 to -20 kPa), moderately wet (-30 to -50 kPa), and moderately dry (-60 to -80kPa) autoclaved Captina silt loam soil (Typic Fragiudult). In the moderately dry soil, syncytia were found only in the stele, but in moderately wet and wet soils, syncytia occurred primarily in the cortex and occasionally in the stele. The location of syncytia in the cortical tissue of roots growing in wet and moderately wet soils may account for the tolerance of susceptible soybean cultivars grown under well-irrigated conditions where there is less interference with water transport through roots. Cell-wall perforations and dense cytoplasm were characteristic of syncytial cells observed in root tissues of all treatments.     

Effect of the rhg1 Gene on Population Development of Heterodera glycines

The effect of the rhg1 gene on equilibrium population densities (E) and reproduction factors (Rf) of Heterodera glycines was studied by comparing the nematode population development on two near-isogenic soybean lines (NIL), differing at the rhg1 locus. The NIL were inoculated with a series of initial egg densities (Pi) in the greenhouse. The relationships between final population densities (Pf = females per plant or eggs per plant) or Rf (final egg density/Pi) on both NIL and Pi were adequately described by quadratic models. The rhg1 gene suppressed Pf and Rf at all Pi of a population of H. glycines race 3 (HG Type 0-); E and maximum Rf were higher on the NIL-S line than on the NIL-R line. After two generations of culture of the race 3 population on the NIL-R line, the population selected by the rhg1 gene (R-eggs) had higher Pf and Rf on the NIL-R line than the population cultured on the NIL-S line (S-eggs) at all Pi. Both R-eggs and S-eggs produced similar egg numbers on the NIL-S line, which was higher than the egg number of either population on the NIL-R line at all Pi. The ratio of E in female numbers on the NIL-R line to E on the NIL-S line increased from 29% for the original race 3 population (S-eggs) to 46% for the rhg1-selected population (R-eggs). Regardless of different egg sources, a trend of increase in the number of eggs per female with the rise of Pi was observed on the NIL-S line. In contrast, female fecundity of both populations declined with the increase of Pi on the NIL-R line. At most inoculum densities, the highest number of eggs per female was observed on the NIL-S line inoculated with the R-eggs, whereas the lowest number of eggs per female was detected on the NIL-R line inoculated with the S-eggs. This study demonstrated that the E and maximum Rf determined by the quadratic models are useful measurements of plant resistance to nematodes.     

Caenorhabditis elegans: A Genetic Guide to Parasitic Nematode Biology

The advent of parasite genome sequencing projects, as well as an increase in biology-directed gene discovery, promises to reveal genes encoding many of the key molecules required for nematode-host interactions. However, distinguishing parasitism genes from those merely required for nematode viability remains a substantial challenge. Although this will ultimately require a functional test in the host or parasite, the free-living nematode Caenorhabditis elegans can be exploited as a heterologous system to determine function of candidate parasitism genes. Studies of C. elegans also have revealed genetic networks, such as the dauer pathway, that may also be important adaptations for parasitism. As a more directed means of identifying parasitism traits, we developed classical genetics for Heterodera glycines and have used this approach to map genes conferring host resistance-breaking phenotypes. It is likely that the C. elegans and H. glycines genomes will be at least partially syntenic, thus permitting predictive physical mapping of H. glycines genes of interest.     

NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD⁺ level. NAMPT is a rate-limiting enzyme for NAD⁺ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD⁺ production to maintain mitochondrial NAD⁺ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD⁺ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD⁺ content to promote cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression rescued the mitochondrial membrane potential and content, suggesting that maintained mitochondrial content and integrity by NAMPT overexpression might be one of the key mechanisms to maintain mitochondrial NAD⁺ level and subsequently dictate cell survival under oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and membrane potential.     

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.