Bacterial Immobilization and Toxicity Induced by a Bean Plant Immune System.

Pseudomonas savastanoi pv. phaseolicola causes halo blight disease in the common bean Phaseolus vulgaris. The bacterium invades the leaf apoplast and uses a type III secretion system to inject effector proteins into a bean cell to interfere with the bean immune system. Beans counter with resistance proteins that can detect effectors and coordinate effector-triggered immunity responses transduced by salicylic acid, the primary defense hormone. Effector-triggered immunity halts bacterial spread, but its direct effect on the bacterium is not known. In this study, mass spectrometry of bacterial infections from immune and susceptible beans revealed that immune beans inhibited the accumulation of bacterial proteins required for virulence, secretion, motility, chemotaxis, quorum sensing, and alginate production. Sets of genes encoding these proteins appeared to function in operons, which implies that immunity altered the coregulated genes in the bacterium. Immunity also reduced amounts of bacterial methylglyoxal detoxification enzymes and their transcripts. Treatment of bacteria with salicylic acid, the plant hormone produced during immunity, reduced bacterial growth, decreased gene expression for methylglyoxal detoxification enzymes, and increased bacterial methylglyoxal concentrations in vitro. Increased methylglyoxal concentrations reduced bacterial reproduction. These findings support the hypothesis that plant immunity involves the chemical induction of adverse changes to the bacterial proteome to reduce pathogenicity and to cause bacterial self-toxicity.

The Proteomics of Resistance to Halo Blight in Common Bean.

Halo blight disease of beans is caused by a gram-negative bacterium, Pseudomonas syringae pv. phaseolicola. The disease is prevalent in South America and Africa and causes crop loss for indigent people who rely on beans as a primary source of daily nutrition. In susceptible beans, P. syringae pv. phaseolicola causes water-soaking at the site of infection and produces phaseolotoxin, an inhibitor of bean arginine biosynthesis. In resistant beans, P. syringae pv. phaseolicola triggers a hypersensitive response that limits the spread of infection. Here, we used high-throughput mass spectrometry to interrogate the responses to two different P. syringae pv. phaseolicola isolates on a single line of common bean, Phaseolus vulgaris PI G19833, with a reference genome sequence. We obtained quantitative information for 4,135 bean proteins. A subset of 160 proteins with similar accumulation changes during both susceptible and resistant reactions included salicylic acid responders EDS1 and NDR1, ethylene and jasmonic acid biosynthesis enzymes, and proteins enabling vesicle secretion. These proteins revealed the activation of a basal defense involving hormonal responses and the mobilization of extracellular proteins. A subset of 29 proteins specific to hypersensitive immunity included SOBIR1, a G-type lectin receptor-like kinase, and enzymes needed for glucoside and phytoalexin production. Virus-induced gene silencing revealed that the G-type lectin receptor-like kinase suppresses bacterial infection. Together, the results define the proteomics of disease resistance to P. syringae pv. phaseolicola in beans and support a model whereby the induction of hypersensitive immunity reinstates defenses targeted by P. syringae pv. phaseolicola.

Benzothiadiazole Conditions the Bean Proteome for Immunity to Bean Rust.

The common bean rust fungus reduces harvests of the dry, edible common bean. Natural resistance genes in the plant can provide protection until a fungal strain that breaks resistance emerges. In this study, we demonstrate that benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) sprayed on susceptible beans induces resistance to common bean rust. Protection occurred as soon as 72 h after treatment and resulted in no signs of disease 10 days after inoculation with rust spores. By contrast, the susceptible control plants sustained heavy infections and died. To understand the effect BTH has on the bean proteome, we measured the changes of accumulation for 3,973 proteins using mass spectrometry. The set of 409 proteins with significantly increased accumulation in BTH-treated leaves included receptor-like kinases SOBIR1, CERK1, and LYK5, which perceive pathogens, and EDS1, a regulator of the salicylic acid defense pathway. Other proteins that likely contributed to resistance included pathogenesis-related proteins, a full complement of enzymes that catalyze phenylpropanoid biosynthesis, and protein receptors, transporters, and enzymes that modulate other defense responses controlled by jasmonic acid, ethylene, brassinosteroid, abscisic acid, and auxin. Increases in the accumulation of proteins required for vesicle-mediated protein secretion and RNA splicing occurred as well. By contrast, more than half of the 168 decreases belonged to chloroplast proteins and proteins involved in cell expansion. These results reveal a set of proteins needed for rust resistance and reaffirm the utility of BTH to control disease by amplifying the natural immune system of the bean plant.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana.

Pre-mRNA alternative splicing is a conserved mechanism for eukaryotic cells to leverage existing genetic resources to create a diverse pool of protein products. It is regulated in coordination with other events in RNA metabolism such as transcription, polyadenylation, RNA transport, and nonsense-mediated decay via protein networks. SERINE/ARGININE-RICH 45 (SR45) is thought to be a neutral splicing regulator. It is orthologous to a component of the apoptosis and splicing-associated protein (ASAP) complex functioning to regulate RNA metabolism at multiple levels. Within this context, we try to understand why the sr45-1 mutant Arabidopsis has malformed flowers, delayed flowering time, and increased disease resistance. Prior studies revealed increased expression for some disease resistance genes and the flowering suppressor Flowering Locus C (FLC) in sr45-1 mutants and a physical association between SR45 and reproductive process-related RNAs. Here, we used Tandem Mass Tag-based quantitative mass spectrometry to compare the protein abundance from inflorescence between Arabidopsis wild-type (Col-0) and sr45-1 mutant plants. A total of 7,206 proteins were quantified, of which 227 proteins exhibited significantly different accumulation. Only a small percentage of these proteins overlapped with the dataset of RNAs with altered expression. The proteomics results revealed that the sr45-1 mutant had increased amounts of enzymes for glucosinolate biosynthesis which are important for disease resistance. Furthermore, the mutant inflorescence had a drastically reduced amount of the Sin3-associated protein 18 (SAP18), a second ASAP complex component, despite no significant reduction in SAP18 RNA. The third ASAP component protein, ACINUS, also had lower abundance without significant RNA changes in the sr45-1 mutant. To test the effect of SR45 on SAP18, a SAP18-GFP fusion protein was overproduced in transgenic Arabidopsis Col-0 and sr45-1 plants. SAP18-GFP has less accumulation in the nucleus, the site of activity for the ASAP complex, without SR45. Furthermore, transgenic sr45-1 mutants overproducing SAP18-GFP expressed even more FLC and had a more severe flowering delay than non-transgenic sr45-1 mutants. These results suggest that SR45 is required to maintain the wild-type level of SAP18 protein accumulation in the nucleus and that FLC-regulated flowering time is regulated by the correct expression and localization of the ASAP complex.

Quantitative Proteomic Analysis of Staphylococcus aureus Treated With Punicalagin, a Natural Antibiotic From Pomegranate That Disrupts Iron Homeostasis and Induces SOS.

Staphylococcus aureus, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. While physical and chemical methods are available to control S. aureus, scientists are searching for inhibitory phytochemicals from plants. One promising compound from pomegranate is punicalagin, a natural antibiotic. To get a broader understanding of the inhibitory effect of punicalagin on S. aureus growth, high-throughput mass spectrometry and quantitative isobaric labeling was used to investigate the proteome of S. aureus after exposure to a sublethal dose of punicalagin. Nearly half of the proteins encoded by the small genome were interrogated, and nearly half of those exhibited significant changes in accumulation. Punicalagin treatment altered the accumulation of proteins and enzymes needed for iron acquisition, and it altered amounts of enzymes for glycolysis, citric acid cycling, protein biosynthesis, and purine and pyrimidine biosynthesis. Punicalagin treatment also induced an SOS cellular response to damaged DNA. Transcriptional comparison of marker genes shows that the punicalagin-induced iron starvation and SOS responses resembles those produced by EDTA and ciprofloxacin. These results show that punicalagin adversely alters bacterial growth by disrupting iron homeostasis and that it induces SOS, possibly through DNA biosynthesis inhibition.

A Proteomic Network for Symbiotic Nitrogen Fixation Efficiency in Bradyrhizobium elkanii.

Rhizobia colonize legumes and reduce N2 to NH3 in root nodules. The current model is that symbiotic rhizobia bacteroids avoid assimilating this NH3. Instead, host legume cells form glutamine from NH3, and the nitrogen is returned to the bacteroid as dicarboxylates, peptides, and amino acids. In soybean cells surrounding bacteroids, glutamine also is converted to ureides. One problem for soybean cultivation is inefficiency in symbiotic N2 fixation, the biochemical basis of which is unknown. Here, the proteomes of bacteroids of Bradyrhizobium elkanii USDA76 isolated from N2 fixation-efficient Peking and -inefficient Williams 82 soybean nodules were analyzed by mass spectrometry. Nearly half of the encoded bacterial proteins were quantified. Efficient bacteroids produced greater amounts of enzymes to form Nod factors and had increased amounts of signaling proteins, transporters, and enzymes needed to generate ATP to power nitrogenase and to acquire resources. Parallel investigation of nodule proteins revealed that Peking had no significantly greater accumulation of enzymes needed to assimilate NH3 than Williams 82. Instead, efficient bacteroids had increased amounts of enzymes to produce amino acids, including glutamine, and to form ureide precursors. These results support a model for efficient symbiotic N2 fixation in soybean where the bacteroid assimilates NH3 for itself.

Putative Rust Fungal Effector Proteins in Infected Bean and Soybean Leaves.

The plant-pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but their effector repertoires are not understood. The discovery of rust fungus effectors may eventually help guide decisions and actions that mitigate crop production loss. Therefore, we used mass spectrometry to identify thousands of proteins in infected beans and soybeans and in germinated fungal spores. The comparative analysis between the two helped differentiate a set of 24 U. appendiculatus proteins targeted for secretion that were specifically found in infected beans and a set of 34 U. appendiculatus proteins targeted for secretion that were found in germinated spores and infected beans. The proteins specific to infected beans included family 26 and family 76 glycoside hydrolases that may contribute to degrading plant cell walls. There were also several types of proteins with structural motifs that may aid in stabilizing the specialized fungal haustorium cell that interfaces the plant cell membrane during infection. There were 16 P. pachyrhizi proteins targeted for secretion that were found in infected soybeans, and many of these proteins resembled the U. appendiculatus proteins found in infected beans, which implies that these proteins are important to rust fungal pathology in general. This data set provides insight to the biochemical mechanisms that rust fungi use to overcome plant immune systems and to parasitize cells.

Manipulation of two α-endo-ß-1,4-glucanase genes, AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots.

Plant endo-ß-1,4-glucanases (EGases) include cell wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the α- and ß-subfamilies contain signal peptides and are secreted, whereas those in the γ-subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis α-EGase At1g48930, designated as AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α-EGase, AtCel2, is up-regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α-EGases are likely to be more useful than membrane-bound γ-EGases in the development of an SCN-resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis-H. schachtii interaction as a model for the soybean-H. glycines pathosystem.

Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots.

BACKGROUND: Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. RESULTS: Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). CONCLUSIONS: Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.

Modification of the expression of two NPR1 suppressors, SNC1 and SNI1, in soybean confers partial resistance to the soybean cyst nematode, Heterodera glycines.

Systemic acquired resistance (SAR) is an enhanced defence response triggered when plants detect a pathogen. The response is extended to uninfected organs to protect against future attack. NPR1 is a nuclear leucine-rich repeat protein with a key role in SAR. It binds specifically to salicylic acid, and acts as a transcriptional coregulator of SAR activators and an inhibitor of transcriptional repressors. The proteins encoded by Suppressor of NPR1, Constitutive (SNC1) and Suppressor of NPR1, Inducible (SNI1) interact with NPR1 to regulate the expression of pathogenesis-related genes. The Arabidopsis thaliana (L.) Heynh. snc1 mutant exhibits a constitutive resistance response, but in the sni1 mutant, the SNI1 protein is rendered incapable of suppressing pathogen resistance genes. To study the influence of SNC1 and SNI1 on resistance to the soybean cyst nematode (Heterodera glycines), soybean (Glycine max (L.) Merr.) roots were separately transformed with four constructs designed to: (i) overexpress GmSNC1, the soybean orthologue of AtSNC1; (ii) overexpress AtSNI1; (iii) silence GmSNC1 and (iv) silence GmSNI1. A significant reduction of the female nematode population was observed in Treatments (i) and (iv). The expression of SAR marker genes was analysed in these treatments. The unusual pattern of expression of pathogen resistance genes shows there are differences in the effect resistance genes have on soybean and A. thaliana. Although NPR1 is involved in the cross-talk between the salicylic acid, jasmonic acid and ethylene pathways, understanding the nematode resistance mechanism in plants is still imprecise. These results provide further insights into the soybean defence response.

Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode.

During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, ß-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.

A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max.

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.

Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions.

Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode).

Changes in gene expression within roots of Glycine max (soybean), cv. Kent, susceptible to infection by Heterodera glycines (the soybean cyst nematode [SCN]), at 6, 12, and 24 h, and 2, 4, 6, and 8 days post-inoculation were monitored using microarrays containing more than 6,000 cDNA inserts. Replicate, independent biological samples were examined at each time point. Gene expression was analyzed statistically using T-tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). These analyses allow the user to query the data in several ways without importing the data into third-party software. RT-PCR confirmed that WRKY6 transcription factor, trehalose phosphate synthase, EIF4a, Skp1, and CLB1 were differentially induced across most time-points. Other genes induced across most timepoints included lipoxygenase, calmodulin, phospholipase C, metallothionein-like protein, and chalcone reductase. RT-PCR demonstrated enhanced expression during the first 12 h of infection for Kunitz trypsin inhibitor and sucrose synthase. The stress-related gene, SAM-22, phospholipase D and 12-oxophytodienoate reductase were also induced at the early time-points. At 6 and 8 dpi there was an abundance of transcripts expressed that encoded genes involved in transcription and protein synthesis. Some of those genes included ribosomal proteins, and initiation and elongation factors. Several genes involved in carbon metabolism and transport were also more abundant. Those genes included glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase and sucrose synthase. These results identified specific changes in gene transcript levels triggered by infection of susceptible soybean roots by SCN.

Ascaris suum: cDNA microarray analysis of 4th stage larvae (L4) during self-cure from the intestine.

There is spontaneous cure of a large portion of Ascaris suum 4th-stage larvae (L4) from the jejunum of infected pigs between 14 and 21 days after inoculation (DAI). Those L4 that remain in the jejunum continue to develop while those that have moved to the ileum are eventually expelled from the intestines. Although increases in intestinal mucosal mast cells and changes in local host immunity are coincidental with spontaneous cure, the population of L4 that continue to develop in the jejunum may counteract host protective mechanisms by the differential production of factors related to parasitism. To this end, a cDNA library was constructed from L4 isolated from pig jejunum at 21 DAI, and 93% of 1920 original clones containing a single amplicon in the range 400-1500 bp were verified by gel electrophoresis and printed onto glass slides for microarray analysis. Fluorescent probes were prepared from total RNA isolated from: (1) 3rd stage-larvae from lung at 7 DAI, (L3); (2) L4 from jejunum at 14 DAI (L4-14-J); (3) L4 from jejunum at 21 DAI (L4-21-J); (4) L4 from ileum at 21 DAI (L4-21-I, and; (5) adults (L5). Cy3-labeled L3, L4-14-J, L4-21-I and L5 cDNA, and Cy5-labeled L4-21-J cDNA were simultaneously used to screen the printed arrays containing the L4-21-J-derived cDNA library. Several clones showed consistent differential gene expression over two separate experiments and were grouped into 3 distinct transcription patterns. The data showed that sequences from muscle actin and myosin, ribosomal protein L11, glyceraldehyde-3-phosphate dehydrogenase and the flavoprotein subunit of succinate dehydrogenase were highly expressed in L4-21-J, but not in L4-21-I; as were a collection of un-annotated genes derived from a worm body wall-hypodermis library, and a testes germinal zone tissue library. These results suggest that only actively developing A. suum L4 are destined to parasitize the host and successfully neutralize host protective responses.