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Crop improvement can be facilitated through efficient gene transfer, leading to pRAP plasmid development. Comparative hairy root transformation results from 24 previously published articles examining 29,756 roots show a 70% transformation efficiency. Average gene overexpression was 11.24-fold and −3.84-fold in RNAi roots. New studies show Glycine max BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) overexpression leads to a 67% decrease in Heterodera glycines parasitism while BAK1-1 RNAi led to a 4.8-fold increase in parasitism. The results show pathogen associated molecular pattern triggered immunity (PTI) functions in the G. max-H. glycines pathosystem during defense. Consequently, the pRAP vectors have applicability for studying basic biology and defense in other agricultural plants including Manihot esculenta (cassava), Zea mays (maize), Oryza sativa (rice), Triticum aestivum (wheat), Sorghum bicolor (sorghum), Brassica rapa (rape seed), Solanum tuberosum (potato), Solanum lycopersicum (tomato), Elaes guineensis (oil palm), Saccharum officinalis (sugarcane) and Beta vulgaris (sugar beet) since each have BAK1 homologs.

Vesicle and target membrane fusion involves tethering, docking and fusion. The GTPase SECRETORY4 (SEC4) positions the exocyst complex during vesicle membrane tethering, facilitating docking and fusion. Glycine max (soybean) Sec4 functions in the root during its defense against the parasitic nematode Heterodera glycines as it attempts to develop a multinucleate nurse cell (syncytium) serving to nourish the nematode over its 30-day life cycle. Results indicate that other tethering proteins are also important for defense. The G. max exocyst is encoded by 61 genes: 5 EXOC1 (Sec3), 2 EXOC2 (Sec5), 5 EXOC3 (Sec6), 2 EXOC4 (Sec8), 2 EXOC5 (Sec10) 6 EXOC6 (Sec15), 31 EXOC7 (Exo70) and 8 EXOC8 (Exo84) genes. At least one member of each gene family is expressed within the syncytium during the defense response. Syncytium-expressed exocyst genes function in defense while some are under transcriptional regulation by mitogen-activated protein kinases (MAPKs). The exocyst component EXOC7-H4-1 is not expressed within the syncytium but functions in defense and is under MAPK regulation. The tethering stage of vesicle transport has been demonstrated to play an important role in defense in the G. max-H. glycines pathosystem, with some of the spatially and temporally regulated exocyst components under transcriptional control by MAPKs.

Expression of the central circadian oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), TIMING OF CAB1 (TOC1), GIGANTEA (GI), and CONSTANS (CO) occurs in Glycine max root cells (syncytia) parasitized by the nematode Heterodera glycines while undergoing resistance, indicating a defense role. GmCCA1-1 relative transcript abundance (RTA) in roots experiencing overexpression (OE) or RNA interference (RNAi) is characterized by rhythmic oscillations, compared to a ribosomal protein gene (GmRPS21) control. A GmCCA1-1 RTA change, advancing by 12 h, exists in H. glycines-infected as compared to uninfected controls in wild-type, H. glycines-resistant, G. max

Glycine max has 32 mitogen activated protein kinases (MAPKs), nine of them exhibiting defense functions (defense MAPKs) to the plant parasitic nematode Heterodera glycines. RNA seq analyses of transgenic G. max lines overexpressing (OE) each defense MAPK has led to the identification of 309 genes that are increased in their relative transcript abundance by all 9 defense MAPKs. Here, 71 of those genes are shown to also have measurable amounts of transcript in H. glycines-induced nurse cells (syncytia) produced in the root that are undergoing a defense response. The 71 genes have been grouped into 7 types, based on their expression profile. Among the 71 genes are 8 putatively-secreted proteins that include a galactose mutarotase-like protein, pollen Ole e 1 allergen and extensin protein, endomembrane protein 70 protein, O-glycosyl hydrolase 17 protein, glycosyl hydrolase 32 protein, FASCICLIN-like arabinogalactan protein 17 precursor, secreted peroxidase and a pathogenesis-related thaumatin protein. Functional transgenic analyses of all 8 of these candidate defense genes that employ their overexpression and RNA interference (RNAi) demonstrate they have a role in defense. Overexpression experiments that increase the relative transcript abundance of the candidate defense gene reduces the ability that the plant parasitic nematode Heterodera glycines has in completing its life cycle while, in contrast, RNAi of these genes leads to an increase in parasitism. The results provide a genomic analysis of the importance of MAPK signaling in relation to the secretion apparatus during the defense process defense in the G. max-H. glycines pathosystem and identify additional targets for future studies.

Anthropogenic sound is increasingly considered a major environmental issue, but its effects are relatively unstudied. Organisms may be directly affected by anthropogenic sound in many ways, including interference with their ability to detect mates, predators, or food, and disturbances that directly affect one organism may in turn have indirect effects on others. Thus, to fully appreciate the net effect of anthropogenic sound, it may be important to consider both direct and indirect effects. We report here on a series of experiments to test the hypothesis that anthropogenic sound can generate cascading indirect effects within a community. We used a study system of lady beetles, soybean aphids, and soybean plants, which are a useful model for studying the direct and indirect effects of global change on food webs. For sound treatments, we used several types of music, as well as a mix of urban sounds (e.g., sirens, vehicles, and construction equipment), each at volumes comparable to a busy city street or farm tractor. In 18‐hr feeding trials, rock music and urban sounds caused lady beetles to consume fewer aphids, but other types of music had no effect even at the same volume. We then tested the effect of rock music on the strength of trophic cascades in a 2‐week experiment in plant growth chambers. When exposed to music by AC/DC, who articulated the null hypothesis that “rock and roll ain't noise pollution” in a song of the same name, lady beetles were less effective predators, resulting in higher aphid density and reduced final plant biomass relative to control (no music) treatments. While it is unclear what characteristics of sound generate these effects, our results reject the AC/DC hypothesis and demonstrate that altered interspecific interactions can transmit the indirect effects of anthropogenic noise through a community.

The conserved oligomeric Golgi (COG) complex maintains correct Golgi structure and function during retrograde trafficking. Glycine max has 2 paralogs of each COG gene, with one paralog of each gene family having a defense function to the parasitic nematode Heterodera glycines. Experiments presented here show G. max COG paralogs functioning in defense are expressed specifically in the root cells (syncytia) undergoing the defense response. The expressed defense COG gene COG7-2-b is an alternate splice variant, indicating specific COG variants are important to defense. Transcriptomic experiments examining RNA isolated from COG overexpressing and RNAi roots show some COG genes co-regulate the expression of other COG complex genes. Examining signaling events responsible for COG expression, transcriptomic experiments probing MAPK overexpressing roots show their expression influences the relative transcript abundance of COG genes as compared to controls. COG complex paralogs are shown to be found in plants that are agriculturally relevant on a world-wide scale including Manihot esculenta, Zea mays, Oryza sativa, Triticum aestivum, Hordeum vulgare, Sorghum bicolor, Brassica rapa, Elaes guineensis and Saccharum officinalis and in additional crops significant to U.S. agriculture including Beta vulgaris, Solanum tuberosum, Solanum lycopersicum and Gossypium hirsutum. The analyses provide basic information on COG complex biology, including the coregulation of some COG genes and that MAPKs functioning in defense influence their expression. Furthermore, it appears in G. max and likely other crops that some level of neofunctionalization of the duplicated genes is occurring. The analysis has identified important avenues for future research broadly in plants.

A Glycine max homolog of the Arabidopsis thaliana NON-RACE SPECIFIC DISEASE RESISTANCE 1 (NDR1) coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene (Gm-NDR1-1) is expressed in root cells undergoing a defense response to the root pathogenic nematode, Heterodera glycines. Gm-NDR1-1 overexpression in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] impairs parasitism. In contrast, Gm-NDR1-1 RNA interference (RNAi) in the H. glycines-resistant genotype G. max[Peking/PI 548402] facilitates parasitism. The broad effectiveness of Gm-NDR1-1 in impairing parasitism has then been examined by engineering its heterologous expression in Gossypium hirsutum which is susceptible to the root pathogenic nematode Meloidogyne incognita. The heterologous expression of Gm-NDR1-1 in G. hirsutum effectively impairs M. incognita parasitism, reducing gall, egg mass, egg and juvenile numbers. In contrast to our prior experiments examining the effectiveness of the heterologous expression of a G. max homolog of the A. thaliana salicyclic acid signaling (SA) gene NONEXPRESSOR OF PR1 (Gm-NPR1-2), no cumulative negative effect on M. incognita parasitism has been observed in G. hirsutum expressing Gm-NDR1-1. The results indicate a common genetic basis exists for plant resistance to parasitic nematodes that involves Gm-NDR1. However, the Gm-NDR1-1 functions in ways that are measurably dissimilar to Gm-NPR1-2. Notably, Gm-NDR1-1 overexpression leads to increased relative transcript levels of its homologs of A. thaliana genes functioning in SA signaling, including NPR1-2, TGA2-1 and LESION SIMULATING DISEASE1 (LSD1-2) that is lost in Gm-NDR1-1 RNAi lines. Similar observations have been made regarding the expression of other defense genes.

Genes functioning in membrane fusion were originally identified genetically in Saccharomyces cerevisiae and are found in all eukaryotes. Components of the unit, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE), function in the plant genetic model Arabidopsis thaliana during its defense to shoot pathogens. Regarding defense, little is understood about SNARE in roots or its regulation. Experiments in Glycine max (soybean) have provided an opportunity to perform such studies, revealing that SNARE genes are expressed under natural conditions in root cells undergoing defense to parasitism by the nematode Heterodera glycines. Presented here, the G. max homolog of S. cerevisiae suppressor of sec1 (SSO1), identified genetically in A. thaliana as PENETRATION1 (PEN1) and named in its genomic annotation as syntaxin 121 (SYP121) functions in the resistance of G. max to H. glycines. Genetic experiments demonstrate Gm-SYP121 is co-expressed with homologs of other SNARE genes...

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 pla...

Mitogen activated protein kinases (MAPKs) play important signal transduction roles. However, little is known regarding how they influence the gene expression of other family members and the relationship to a biological process, including the Glycine max defense response to Heterodera glycines. Transcriptomics have identified MAPK gene expression occurring within root cells undergoing a defense response to a pathogenic event initiated by H. glycines in the allotetraploid Glycine max. Functional analyses are presented for its 32 MAPKs revealing 9 have a defense role, including homologs of Arabidopsis thaliana MAPK (MPK) MPK2, MPK3, MPK4, MPK5, MPK6, MPK13, MPK16 and MPK20. Defense signaling occurring through pathogen activated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) have been determined in relation to these MAPKs. Five different types of gene expression relate to MAPK expression, influencing PTI and ETI gene expression and proven defense genes including an ABC-G transporter, 20S membrane fusion particle components, glycoside biosynthesis, carbon metabolism, hemicellulose modification, transcription and secretion. The experiments show MAPKs broadly influence defense MAPK gene expression, including the co-regulation of parologous MAPKs and reveal its relationship to proven defense genes. The experiments reveal each defense MAPK induces the expression of a G. max homolog of a PATHOGENESIS RELATED1 (PR1), itself shown to function in defense in the studied pathosystem.

Mitogen activated protein kinases (MAPKs) play important signal transduction roles. However, little is known regarding whether MAPKs influence the gene expression of other family members and the relationship that expression has to a biological process. Transcriptomic studies have identified MAPK gene expression occurring within root cells undergoing a defense response to a pathogenic event in the allotetraploidGlycine max. Furthermore, functional analyses are presented for its 32 MAPKs revealing 9 of the 32 MAPKs have a defense role, including homologs ofArabidopsis thalianaMAPK (MPK) MPK2, MPK3, MPK4, MPK5, MPK6, MPK13, MPK16 and MPK20. Defense signal transduction processes occurring through pathogen activated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) have been determined in relation to these MAPKs. PTI has been analyzed by examiningBOTRYTIS INDUCED KINASE1(BIK1),ENHANCED DISEASE SUSCEPTIBILITY1(EDS1) andLESION SIMULATING DISEASE1(LSD1). ETI has been analyzed by examining the role of the bacterial effector protein harpin and the downstream cell membrane receptorNON-RACE SPECIFIC DISEASE RESISTANCE1(NDR1). Experiments have identified 5 different types of gene expression relating to MAPK expression. The MAPKs are shown to influence PTI and ETI gene expression and a panel of proven defense genes including an ABC-G type transporter, 20S membrane fusion particle components, glycoside biosynthesis, carbon metabolism, hemicellulose modification, transcription andPATHOGENESIS RELATED 1(PR1). The experiments show MAPKs broadly influence the expression of other defense MAPKs, including the co-regulation of parologous MAPKs and reveal its relationship to proven defense genes.

Soybean (Glycine max) infection by the charcoal rot (CR) ascomycete Macrophomina phaseolina is enhanced by the soybean cyst nematode (SCN) Heterodera glycines. We hypothesized that G. max genetic lines impairing infection by M. phaseolina would also limit H. glycines parasitism, leading to resistance. As a part of this M. phaseolina resistance process, the genetic line would express defense genes already proven to impair nematode parasitism. Using G. max[DT97-4290/PI 642055], exhibiting partial resistance to M. phaseolina, experiments show the genetic line also impairs H. glycines parasitism. Furthermore, comparative studies show G. max[DT97-4290/PI 642055] exhibits induced expression of the effector triggered immunity (ETI) gene NON-RACE SPECIFIC DISEASE RESISTANCE 1/HARPIN INDUCED1 (NDR1/HIN1) that functions in defense to H. glycines as compared to the H. glycines and M. phaseolina susceptible line G. max[Williams 82/PI 518671]. Other defense genes that are induced in G. max[DT97-4290/PI 642055] include the pathogen associated molecular pattern (PAMP) triggered immunity (PTI) genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), NONEXPRESSOR OF PR1 (NPR1) and TGA2. These observations link G. max defense processes that impede H. glycines parasitism to also potentially function toward impairing M. phaseolina pathogenicity. Testing this hypothesis, G. max[Williams 82/PI 518671] genetically engineered to experimentally induce GmNDR1-1, EDS1-2, NPR1-2 and TGA2-1 expression leads to impaired M. phaseolina pathogenicity. In contrast, G. max[DT97-4290/PI 642055] engineered to experimentally suppress the expression of GmNDR1-1, EDS1-2, NPR1-2 and TGA2-1 by RNA interference (RNAi) enhances M. phaseolina pathogenicity. The results show components of PTI and ETI impair both nematode and M. phaseolina pathogenicity.

The study of biological processes has been aided greatly by the development of procedures to identify the large numbers of associated genes. However, the ability to study the identified genes experimentally is often impeded by the absence of technologies to perform such functional analyses. Here, a nonaxenic plant transformation system has been developed in Gossypium hirsutum (cotton) for the study of genes associated with root functions and root–organism interactions. The plant transformation system is compatible with modern high throughput plant transformation goals and the processing of large numbers of genes intended for the study of root function in G. hirsutum.

The ability to analyze the gene activity occurring within a single cell has ushered in a new understanding of complex biological processes. Furthermore, this capability has established the prerequisite technologies for the analysis of cells involved in complex pathogenic and/or symbiotic interactions. Collectively, the identification of biological models permitting the analysis of individual cells and improvements in histological technology are allowing for analyses of cells positioned within tissues and involved in complex cellular interactions at unprecedented resolution. Here, a plastic embedding procedure is used for laser microdissection of plant tissues infected with a pathogen. This technique enabled the acquisition of nucleic acids from semi-thin sections that can be used for downstream biological studies of host–pathogen interaction at the single-cell resolution.

The endosporic male gametophyte of the water fern, Marsilea vestita, provides a unique opportunity to study the mechanisms that control cell fate determination during a burst of rapid development. In this review, we show how the spatial and temporal control of development in this simple gametophyte involves several distinct modes of RNA processing that allow the translation of specific mRNAs at distinct stages during gametogenesis. During the early part of development, nine successive cell division cycles occur in precise planes within a closed volume to produce seven sterile cells and 32 spermatids. There is no cell movement in the gametophyte; so, cell position and size within the spore wall define cell fate. After the division cycles have been completed, the spermatids become sites for the de novo formation of basal bodies, for the assembly of a complex cytoskeleton, for nuclear and cell elongation, and for ciliogenesis. In contrast, the adjacent sterile cells exhibit none of these changes. The spermatids differentiate into multiciliated, corkscrew-shaped gametes that resemble no other cells in the entire plant. Development is controlled post-transcriptionally. The transcripts stored in the microspore are released (unmasked) in the gametophyte at different times during development. At the start of these studies, we identified several key mRNAs that undergo translation at specific stages of gametophyte development. We developed RNA silencing protocols that enabled us to block the translation of these proteins and thereby establish their necessity and sufficiency for the completion of specific stages of gametogenesis. In addition, RNAi enabled us to identify additional proteins that are essential for other phases of development. Since the distributions of mRNAs and the proteins they encode are not identical in the gametophyte, transcript processing is apparently important in allowing translation to occur under strict temporal and spatial control. Transcript polyadenylation occurs in the spermatogenous cells in ways that match the translation of specific mRNAs. We have found that the exon junction complex plays key roles in transcript regulation and modifications that underlie cell specification in the gametophyte. We have recently become interested in the mechanisms that control the unmasking of the stored transcripts and have linked the synthesis and redistribution of spermidine in the gametophyte to the control of mRNA release from storage during early development and later to basal body formation, cytoskeletal assembly, and nuclear and cell elongation in the differentiating spermatids.

Glycine max L. Merr. (soybean) resistance to Heterodera glycines Ichinohe occurs at the site of infection, a nurse cell known as the syncytium. Resistance is classified into two cytologically-defined responses, the G. max ([Peking])- and G. max ([PI 88788])-types. Each type represents a cohort of G. max genotypes. Resistance in G. max ([Peking]) occurs by a potent and rapid localized response, affecting parasitic second stage juveniles (p-J2). In contrast, resistance occurs by a potent but more prolonged reaction in the genotype G. max ([PI 88788]) that affects nematode development at the J3 and J4 stages. Microarray analyses comparing these cytologically and developmentally distinct resistant reactions reveal differences in gene expression in pericycle and surrounding cells even before infection. The differences include higher relative levels of the differentially expressed in response to arachidonic acid 1 gene (DEA1 [Gm-DEA1]) (+224.19-fold) and a protease inhibitor (+68.28-fold) in G. max ([Peking/PI 548402]) as compared to G. max ([PI 88788]). Gene pathway analyses compare the two genotypes (1) before, (2) at various times during, (3) constitutively throughout the resistant reaction and (4) at all time points prior to and during the resistant reaction. The amplified levels of transcriptional activity of defense genes may explain the rapid and potent reaction in G. max ([Peking/PI 548402]) as compared to G. max ([PI 88788]). In contrast, the shared differential expression levels of genes in G. max ([Peking/PI 548402]) and G. max ([PI 88788]) may indicate a conserved genomic program underlying the G. max resistance on which the genotype-specific gene expression programs are built off.

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 major pest of soybean ( Glycine max ) is the soybean cyst nematode (SCN), Heterodera glycines . One population of SCN can evoke a resistant response while a second population can evoke a susceptible response from the same soybean cultivar. Recently, interactions between SCN and soybean roots

Syncytial cells in soybean (Glycine max cultivar [cv.] Peking) roots infected by incompatible and compatible populations of soybean cyst nematode (SCN [Heterodera glycines]) were collected using laser capture microdissection (LCM). Gene transcript abundance was assayed using Affymetrix® soybean GeneChips®, each containing 37,744 probe sets. Our analyses identified differentially expressed genes in syncytial cells that are not differentially expressed in the whole root analyses. Therefore, our results show that the mass of transcriptional activity occurring in the whole root is obscuring identification of transcriptional events occurring within syncytial cells. In syncytial cells from incompatible roots at three dpi, genes encoding lipoxygenase (LOX), heat shock protein (HSP) 70, superoxidase dismutase (SOD) were elevated almost tenfold or more, while genes encoding several transcription factors and DNA binding proteins were also elevated, albeit at lower levels. In syncytial cells formed during the compatible interaction at three dpi, genes encoding prohibitin, the epsilon chain of ATP synthase, allene oxide cyclase and annexin were more abundant. By 8 days, several genes of unknown function and genes encoding a germin-like protein, peroxidase, LOX, GAPDH, 3-deoxy-D-arabino-heptolosonate 7-phosphate synthase, ATP synthase and a thioesterase were abundantly expressed. These observations suggest that gene expression is different in syncytial cells as compared to whole roots infected with nematodes. Our observations also show that gene expression is different between syncytial cells that were isolated from incompatible and compatible roots and that gene expression is changing over the course of syncytial cell development as it matures into a functional feeding site.

Experiments show the membrane fusion genes α soluble NSF attachment protein (α-SNAP) and syntaxin 31 (Gm-SYP38) contribute to the ability of Glycine max to defend itself from infection by the plant parasitic nematode Heterodera glycines. Accompanying their expression is the transcriptional activation of the defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1) that function in salicylic acid (SA) signaling. These results implicate the added involvement of the antiapoptotic, environmental response gene LESION SIMULATING DISEASE1 (LSD1) in defense. Roots engineered to overexpress the G. max defense genes Gm-α-SNAP, SYP38, EDS1, NPR1, BOTRYTIS INDUCED KINASE1 (BIK1) and xyloglucan endotransglycosylase/hydrolase (XTH) in the susceptible genotype G. max[Williams 82/PI 518671] have induced Gm-LSD1 (Gm-LSD1-2) transcriptional activity. In reciprocal experiments, roots engineered to overexpress Gm-LSD1-2 in the susceptible genotype G. max[Williams 82/PI 518671] have induced levels of SYP38, EDS1, NPR1, BIK1 and XTH, but not α-SNAP prior to infection. In tests examining the role of Gm-LSD1-2 in defense, its overexpression results in ∼52 to 68% reduction in nematode parasitism. In contrast, RNA interference (RNAi) of Gm-LSD1-2 in the resistant genotype G. max[Peking/PI 548402] results in an 3.24-10.42 fold increased ability of H. glycines to parasitize. The results identify that Gm-LSD1-2 functions in the defense response of G. max to H. glycines parasitism. It is proposed that LSD1, as an antiapoptotic protein, may establish an environment whereby the protected, living plant cell could secrete materials in the vicinity of the parasitizing nematode to disarm it. After the targeted incapacitation of the nematode the parasitized cell succumbs to its targeted demise as the infected root region is becoming fortified.

Spermiogenesis in the male gametophytes of the water fern Marsilea vestita is a precise and rapid process resulting in the production of ciliated gametes. Development begins from a single cell within the microspore wall that undergoes nine rapid cell division cycles in distinct planes to produce 32 spermatids that are surrounded by 7 sterile cells. Thereafter, the de novo formation of basal bodies occurs in a discrete cytoplasmic particle known as a blepharoplast, with the subsequent formation of a complex ciliary apparatus in elongating spermatids. The rate and extent of development appear to be controlled at a post-transcriptional level, where the sudden translation of specific stored mRNAs (e.g., centrin) results in the formation of particular structures in the cells (e.g., blepharoplasts). We show here that additional centrosomal and cytoskeletal antigens known as SF assemblin, p95 kDa protein, δ tubulin, γ tubulin, Xgrip109, Aik, CTR453, RanBPM, BX63, RSP6, and α tubulin each exhibit specific localization patterns both on immunoblots of gametophyte protein isolates and in fixed cells. BAp90, PP4, and RLC exhibit specific localization patterns in fixed cells. We show that the antigens exhibit complex patterns of abundance during spermiogenesis. In an attempt to identify regulatory agents involved in spermiogenesis, we employed a RNAi-based screen of 41 randomly selected gametophyte cDNAs on developing populations of synchronously growing gametophytes. The gametophytes treated with each of the RNAi probes exhibited arrest at a specific stage of development. A consequence of anomalous development was the block to assembly of the ciliary apparatus, an effect highlighted by altered staining with anti-centrin, anti-β-tubulin, and anti-RSP6 antibodies. Our results show that complex, integrated processes of translation and protein partitioning apparently underlie the assembly of the ciliary apparatus during spermiogenesis in male gametophytes of M. vestita. Cell Motil. Cytoskeleton 56:57–73, 2003. © 2003 Wiley-Liss, Inc.

The term regulon has been coined in the genetic model plant Arabidopsis thaliana, denoting a structural and physiological defense apparatus defined genetically through the identification of the penetration (pen) mutants. The regulon is composed partially by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) syntaxin PEN1. PEN1 has homology to a Saccharomyces cerevisae gene that regulates a Secretion (Sec) protein, Suppressor of Sec 1 (Sso1p). The regulon is also composed of the β-glucosidase (PEN2) and an ATP binding cassette (ABC) transporter (PEN3). While important in inhibiting pathogen infection, limited observations have been made regarding the transcriptional regulation of regulon genes until now. Experiments made using the model agricultural Glycine max (soybean) have identified co-regulated gene expression of regulon components. The results explain the observation of hundreds of genes expressed specifically in the root cells undergoing the natural process of defense. Data regarding additional G. max genes functioning within the context of the regulon are presented here, including Sec 14, Sec 4 and Sec 23. Other examined G. max homologs of membrane fusion genes include an endosomal bromo domain-containing protein1 (Bro1), syntaxin6 (SYP6), SYP131, SYP71, SYP8, Bet1, coatomer epsilon (ε-COP), a coatomer zeta (ζ-COP) paralog and an ER to Golgi component (ERGIC) protein. Furthermore, the effectiveness of biochemical pathways that would function within the context of the regulon ave been examined, including xyloglucan xylosyltransferase (XXT), reticuline oxidase (RO) and galactinol synthase (GS). The experiments have unveiled the importance of the regulon during defense in the root and show how the deposition of callose relates to the process.

A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil.

The carpel is the female reproductive organ of flowering plants. In Arabidopsis, congenital fusion of two carpels leads to the formation of an enclosed gynoecium. The margins of the two fused carpels are meristematic in nature and give rise to placentas, ovules, septa, abaxial repla, and the majority of the stylar and stigmatic tissues. Thus, understanding how the marginal tissues are specified and identifying genes that direct their development may provide important insight into higher plant reproductive development. In this study, we show that LEUNIG and AINTEGUMENTA are two critical regulators of marginal tissue development. Double mutants of leunig aintegumenta fail to develop placentas, ovules, septa, stigma, and style. This effect is specific to the leunig aintegumenta double mutant and is not found in other double mutant combinations such as leunig apetala2 or aintegumenta apetala2 . Additional analyses indicate that the absence of marginal tissues in leunig aintegumenta double mutants is not mediated by ectopic AGAMOUS . We propose that LEUNIG and AINTEGUMENTA act together to control the expression of common target genes that regulate cell proliferation associated with marginal tissue development.

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.

A Glycine max syntaxin 31 homolog (Gm-SYP38) was identified as being expressed in nematode-induced feeding structures known as syncytia undergoing an incompatible interaction with the plant parasitic nematode Heterodera glycines. The observed Gm-SYP38 expression was consistent with prior gene expression analyses that identified the alpha soluble NSF attachment protein (Gm-α-SNAP) resistance gene because homologs of these genes physically interact and function together in other genetic systems. Syntaxin 31 is a protein that resides on the cis face of the Golgi apparatus and binds α-SNAP-like proteins, but has no known role in resistance. Experiments presented here show Gm-α-SNAP overexpression induces Gm-SYP38 transcription. Overexpression of Gm-SYP38 rescues G. max [Williams 82/PI 518671], genetically rhg1 −/−, by suppressing H. glycines parasitism. In contrast, Gm-SYP38 RNAi in the rhg1 +/+ genotype G. max [Peking/PI 548402] increases susceptibility. Gm-α-SNAP and Gm-SYP38 overexpression induce the transcriptional activity of the cytoplasmic receptor-like kinase BOTRYTIS INDUCED KINASE 1 (Gm-BIK1-6) which is a family of defense proteins known to anchor to membranes through a 5′ MGXXXS/T(R) N-myristoylation sequence. Gm-BIK1-6 had been identified previously by RNA-seq experiments as expressed in syncytia undergoing an incompatible reaction. Gm-BIK1-6 overexpression rescues the resistant phenotype. In contrast, Gm-BIK1-6 RNAi increases parasitism. The analysis demonstrates a role for syntaxin 31-like genes in resistance that until now was not known.

The ability to isolate and perform nucleic acid analyses of individual cells is critical to studying the development of various cell types and structures. We present a novel biological sample preparation method developed for laser capture microdissection-assisted nucleic acid analysis of ultrathin cell/tissue sections. We used cells of the mitotic bed of the tadpole teeth of Lithobates sphenocephalus (Southern Leopard Frog). Cells from the mitotic beds at the base of the developing teeth series were isolated and embedded in the methacrylate resin, Technovit® 9100®. Intact cells of the mitotic beds were thin sectioned and examined by bright-field and transmission electron microscopy. The cytological and ultrastructural anatomy of the immature and progressively more mature tooth primordia appeared well preserved and intact. A developmental series of tooth primordia were isolated by laser capture microdissection (LCM). Processing of these cells for RNA showed that intact RNA could be isolated. The study demonstrates that Technovit® 9100® can be used as an embedding medium for extremely small tissues and from individual cells, a prerequisite step to LCM and nucleic acid analyses. A relatively small amount of sample material was needed for the analysis, which makes this technique ideal for cell-specific analyses when the desired cells are limited in quantity.

Living organisms conduct biological processes by transducing biotic and abiotic stimuli through gene regulation into well-orchestrated growth and development. Analyzing RNA from a transcribed genome (the transcriptome) is fairly easy due to the availability of various nucleotide sequencing technologies. The translation of a transcriptome provides a blueprint of tens of thousands of different proteins that is known as the proteome (Wasinger et al., 1995). The analysis of proteins from whole tissues, organs or organisms has been made easier thanks to various technologies, including the Edman degradation technique, which is used in sequencing polypeptides (Edman, 1950), and 2 dimensional gel electrophoresis (2DE), which could resolve up to 10,000 polypeptides (Barrett & Gould, 1973; O'Farrell, 1975; O'Farrell et al., 1977). Thus, the problems of assaying a transcriptome and a proteome from samples isolated from tissues, organs or whole organisms have largely been overcome. However, although it is fairly easy to assay a transcriptome and a proteome at these higher levels of biological organization, assaying a proteome at a cellular resolution level involves a set of problems that in primarily centered on the ability to collect sufficient cells for meaningful studies. The central focus of this chapter is a discussion on the technologies that have allowed the proteomic analyses of cells, isolated from complex samples thanks to a procedure that was first called laser microdissection (Isenberg et al., 1976).

1.1 Plant nematodes Plant parasitic nematodes cause severe damage to plants and are responsible for billions of dollars of losses worldwide (Koenning et al. 2007). Soybean cyst nematode (SCN; Hederodera glycines; Fig. 1a ) and root-knot nematode (RKN; Meloidogyne spp.; Fig. 1b) are sedentary obligate parasites of plants. SCN is the major pest of soybean and causes an estimated one billion dollars in losses annually in the US (Wrather & Koenning 2006). RKN is a major pest of vegetables and can become a serious problem on soybean, especially on edible soybean planted in areas used to grow vegetables (Adegbite & Adesiyan 2005). The genera Meloidogyne is widespread and is considered, economically and agriculturally, as a very important group of plant pathogens. The host range of Meloidogyne is very wide as it attacks almost all plant species (Sasser 1980). Both SCN and RKN are sedentary endoparasites and they cause dramatic morphological and physiological changes in plant cells while inflicting severe decreases on yield. Chemical methods of nematode control are costly and can damage the environment, especially with contamination of ground water. Therefore, the preferred method of nematode control is the use of resistant or tolerant varieties, when available. Unfortunately, a plant with resistance to one population of nematode is often susceptible to a different population due to the wide genetic variation of nematode populations. When a plant parasitic nematode infects a plant root, the nematode and the plant enters an intricate interactive relationship with the host that is attempting to inhibit nematode development, while the nematode’s goal is to develop and reproduce. The life cycle of SCN and cellular responses of soybean to SCN infection have been documented and reviewed extensively (Bird & Koltai 2000; Endo 1964; Endo, 1965; Endo, 1992; Goverse et al. 2000; Lilley et al. 2005; Mitchum & Baum 2008; Niblack et al. 2006; Williamson & Gleason 2003; Abad & Williamson 2010; Klink et al. 2011a). The SCN egg can be found in soil and within the mature female. The second stage juvenile (J2) hatches from the egg, searches for a root of a plant host, penetrates the root epidermis, and migrates intracellularly, using its stylet and enzyme secretions to disrupt cells and force its way toward the vascular tissue.

The soybean defense response to the soybean cyst nematode was used as a model to map at cellular resolution its genotype-defined cell fate decisions occurring during its resistant reactions. The defense responses occur at the site of infection, a nurse cell known as the syncytium. Two major genotype-defined defense responses exist, the G. max [Peking]- and G. max [PI 88788]-types. Resistance in G. max [Peking] is potent and rapid, accompanied by the formation of cell wall appositions (CWAs), structures known to perform important defense roles. In contrast, defense occurs by a potent but more prolonged reaction in G. max [PI 88788], lacking CWAs. Comparative transcriptomic analyses with confirmation by Illumina® deep sequencing were organized through a custom-developed application, Pathway Analysis and Integrated Coloring of Experiments (PAICE) that presents gene expression of these cytologically and developmentally distinct defense responses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) framework. The analyses resulted in the generation of 1,643 PAICE pathways, allowing better understanding of gene activity across all chromosomes. Analyses of the rhg1 resistance locus, defined within a 67 kb region of DNA demonstrate expression of an amino acid transporter and an α soluble NSF attachment protein gene specifically in syncytia undergoing their defense responses.

Soybeans are hosts to a number of pests and pathogens. Of those numerous pathogens, the parasitic nematodes represent the major, ubiquitous, dominant and persistent problem for its cultivation, worldwide. Remarkably, soybeans are hosts to over 100 species of nematodes (reviewed in Sinclair and Backman, 1989). However, the most devastating pathogen of soybean is the soybean cyst nematode, Heterodera glycines. Heterodera glycines were first observed on soybean in Japan in 1881 (reviewed in Schmitt and Noel, 1984). The first scientific description of H. glycines was published in 1952 (Ichinohe, 1952). Up until this time there were no records of H. glycines in the New World. The combination of the growing agronomic status of soybean, the lack of agricultural practices to prevent the spread of any parasitic nematode and the biology of H. glycines set the stage for their rapid dispersal. Heterodera glycines were first identified in the U.S. in 1954 in North Carolina (Winstead et al. 1955). By 1957, H. glycines had been identified as far west as the state of Mississippi (reviewed in Riggs, 2004). Now H. glycines are nearly ubiquitous, found worldwide in 93.5% of the total acreage where soybean is cultivated (reviewed in Riggs 2004). In the U.S., reports rank H. glycines infection as causing more agronomic loss of soybean production than the rest of its pathogens combined (Wrather et al. 2006). H. glycines infection, itself, causes more damage to soybeans in the U.S. (~1.5 billion $) than the entire value of some agricultural crops. These facts demonstrate the significant drain that H. glycines have on soybean production.

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells.Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types.Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

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 6dpi) and is completed by 9dpi. 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 9dpi. 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 3dpi (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 9dpi to 6dpi (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]).

Laser Microdissection (LM) is a technology that allows the rapid procurement of selected cell populations from a section of heterogeneous tissues in a manner conducive to the extraction of DNA, RNA, proteins and even metabolites. In the past few years, it has also been applied to plant biology in order to study gene expression in plant-nematode and plant-microbe interactions. LM represents a powerful tool since cells associated with a particular infection stage can be visualized under the microscope and harvested. Therefore, verification of the response of the plant during the progression of the colonization can be performed in different cell types. Applications of LM to study the interaction between the plant and both pathogenic and symbiotic organisms (i.e. nematode and fungi, respectively) are explored in this review.

Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway®-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix® soybean GeneChip® allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81–93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.