PCR-based Suppression Subtractive Hybridization Research Articles

Differentially Expressed Long-Term Salinity Responsive Sequences in Halophyte Suaeda maritima (L.) Dumort

Salinity is a major abiotic stress that affects plant growth and productivity. To cope with salt stress, plants express large number of salt responsive genes and proteins that are involved in a wide range of cellular functions. In the present study, halophytic plant Suaeda maritima (L.) Dumort. were hydroponically exposed to NaCl for understanding the molecular mechanisms behind salinity tolerance using PCR-based Suppressive Subtractive Hybridization (SSH). Two cDNA subtraction libraries were constructed between Suaeda maritima X Sesuvium portulacastrum and Suaeda maritima X Salicornia brachiata to identify differentially expressed genes from leaves exposed to 200mM NaCl treatment for 14 days. A total of 224 clones from both libraries were assembled into 109 unique-ESTs grouped into different functional categories. Based on GO functional annotation, the expressed sequences like Oxygen-evolving enhancer protein1, AARF domain-containing kinase protein, V-type proton ATPase subunitd2, RMD5 homologA, and ABC transporter G35 that are involved in photosynthesis, cellular transport, cell rescue and defense, polyubiquitination and secondary metabolism played a significant role implying a complex response to salt in S. maritima. This is the first report that SSH could facilitate screening across species and family specific identification of salt responsive genes provides insight into biological mechanisms underlying salinity response.

A feasible method for detecting unknown GMOs via a combined strategy of PCR-based suppression subtractive hybridization and next-generation sequencing

The rapid development of genetically modified (GM) technologies, increasing GM plant varieties and differentiated policies towards GM organisms (GMOs), make it more challenging for GMO testing nowadays. PCR-based approaches, as current routine methods, require the detailed information of transgenic events before assay. Recently reported probe-based capture strategies coupled with next-generation sequencing (NGS) offer a practical but costly way for detecting unauthorized or unknown GMOs (UGMOs) that still depend on the known GM elements as targets or start-points. Herein, we present a novel method, ‘SSH-seq’, for GMO testing based on the combination of suppressive subtractive hybridization (SSH) and NGS strategies, which has potentials for detecting UGMOs without the prior knowledge of GM cassettes. As a result, single SSH-seq experiment yielded approximately 33.8 times of reads derived from the MON87769 transgene compared to endogenous gene Lectin. For multiple SSH-seq started with eight GM soybean reference materials, part of targets were successfully enriched. Although our method might be influenced by several factors, such as the choice of degrading enzymes, GMO insertion size, sequence specificity of targets, dosage of driver DNA, NGS production, and experimental parameters, we have reasons to believe that SSH-seq will act as a powerful tool in the field of GMO testing after further optimizations, especially for UGMOs containing novel elements.

Identification and characterization of sex related genes in Actinidia arguta by suppression subtractive hybridization

Abstract Actinidia arguta, with sweet flavour, high nutritional value and health promoting role, is a typical dioecious plant. But dioecy represents an important constraint in kiwifruit breeding programmes and also requires identification of male and female genotypes before planting an orchard. Therefore, understanding molecular basis of sex determination in A. arguta is important for molecular identification of seedling gender at early stage. PCR-based suppressive subtractive hybridization (SSH) was performed to screen genes related to sex determination. A total of 533 and 508 positive clones, from forward and reverse libraries respectively, were selected for sequencing. The clean ESTs were assembled into 309 (forwad) and 369 (reverse) unigenes, and 228 and 299 of them hit known homologous sequences of the NCBI nr database. Based on Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, four unigenes that might be involved in sex determination were selected for validation by qRT-PCR. The expression of PME was very strong in male flower at late development stage, with 72 times higher than that of female flower. Reversely, male flower exhibited extremely low expression level of ACO, MAN1 and MYC2 at late development stage as compared with female flower. The variable expression patterns of these genes between male and female flowers suggested their importance in controlling sex expression. These results advanced our understanding of the molecular mechanism of sex determination in A. arguta.

Plant endophytic Pseudomonas putida BP25 induces expression of defense genes in black pepper roots: Deciphering through suppression subtractive hybridization analysis

Black pepper associated endophytic Pseudomonas putida BP25 displayed broad spectrum antagonistic activity against a wide range of plant pathogens. The molecular responses of black pepper upon endophytic colonization by P. putida BP25 was studied using a PCR based suppression subtractive hybridization (SSH) method. The transcripts induced by endophytic P. putida BP25 in black pepper included pathogenesis related proteins such as PR-4 and PR-1; reactive oxygen species (ROS) scavenging catalase and metallothionein; stress induced glutathione S-transferase; 5-enolpyruvylshikimate-3-phosphate synthase involved in phenylpropanoid metabolism. Differential expression of transcripts was validated by quantitative real time PCR (qPCR) for selected ESTs. This information helps in understanding the ability of an endophytic strain P. putida BP25 in inducing a wide array of defence responses in black pepper roots.

Identification of heavy metal pollutant tolerance-associated genes in Avicennia marina (Forsk.) by suppression subtractive hybridization

The halophytic Avicennia marina (Forsk.) is one of the pioneer mangroves along the south coast of China. It is an appropriate material for understanding molecular mechanisms of heavy metal tolerance in mangrove plants. A forward and a reverse cDNA library was constructed by PCR-based suppressive subtractive hybridization (SSH) to isolate these tolerance-associated genes from A. marina leaves. A total of 99 ESTs obtained from the forward and reverse libraries showed significant differential expressions. Twenty-nine genes selected by SSH were studied by real-time PCR in order to analyze their expression level. Most of these genes' expression increased in leaves under Cd stress, which suggests that these genes contribute to the heavy metal tolerance in A. marina. The diversity of these genes indicated that heavy metal stress resulted in a complex response in mangrove plants. This could prove a useful approach for further exploring the molecular mechanisms behind such heavy metal tolerance.

Identification of drought response genes in Zygophyllum xanthoxylum by suppression subtractive hybridization

Drought is an important abiotic stress that seriously limits plant growth and crop productivity in some regions of the world. Zygophyllum xanthoxylum, a succulent xerophyte species with excellent adaptability to adverse arid environments, is an excellent model system for the study of the molecular mechanisms behind drought tolerance in xerophytic species. To better understand the molecular basis of Z. xanthoxylum responses to drought stress and to isolate drought tolerance-related genes from Z. xanthoxylum, we constructed a cDNA library from Z. xanthoxylum and screened the library for stress-related genes by PCR-based suppression subtractive hybridization (SSH). A total of 637 expressed sequence tags were generated by sequencing 672 randomly selected clones, resulting in the identification of 338 unigenes comprising 77 contigs and 261 singlets. The unigenes were assigned to 13 functional categories, such as metabolism, disease- or defense-related genes and energyrelated gene. In addition, we analyzed the expression patterns of 24 genes associated with drought stress-responsive pathways by real-time RT-PCR. The results indicated that osmolyte biosynthesis genes, reactive oxygen scavengers, transporters, signaling components and transcription factors play important roles in drought stress responses. This study provides a valuable resource for studying the drought tolerance mechanism of this xerophytic plant species.

Transcript dynamics at early stages of molecular interactions of MYMIV with resistant and susceptible genotypes of the leguminous host, Vigna mungo.

Initial phases of the MYMIV- Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.

Analysis of differentially expressed genes in Curcuma amada and Zingiber officinale upon infection with Ralstonia solanacearum by suppression subtractive hybridization

Bacterial wilt, one of the major diseases in ginger is caused by Ralstonia solanacearum and lack of resistant genotype adds constraints to the crop improvement programs and for the management of bacterial wilt. Curcuma amada is a potential donor for bacterial wilt resistance to Zingiber officinale, if the exact mechanism of resistance is understood. In this study, a PCR-based suppression subtractive hybridization (SSH) was used to identify C. amada genes that are differentially and early expressed in response to the R. solanacearum infection compared to Z. officinale. A forward subtracted library of differentially expressed genes was synthesized by cloning DNA fragments (300–1,200 bp) from subtracted sample and sequenced. Upon computational analysis of the 150 SSH clones sequenced, 119 produced suitable sequences of high homology with the genes of known functions. These were classified into three potential functional categories: proteins involved in biological processes (31 %), cellular component (43 %) and molecular function (26 %). Among these differentially expressed genes, sequences coding for putative proteins related to defense response (high homology between 85 and 100 % identity) were, cell wall-associated hydrolase, xyloglucan transglycosylase (XTG), cytochrome P 450, metallocarboxypeptidase inhibitor, peroxiredoxin, nitrilase-associated protein, leucine-rich protein (LRR) and glutathione-S-transferase (GST). The three transcripts were discriminative: expression of LRR, GST and XTG was much higher in resistant species (C. amada) than in susceptible species (Z. officinale) at every time point. The isolated ESTs reported here contribute to improve our understanding about this plant–pathogen interaction.

Differential gene expression between summer and winter diapause pupae of the onion maggot Delia antiqua, detected by suppressive subtractive hybridization

To gain a better understanding of the molecular mechanisms regulating pupal diapause of the onion maggot Delia antiqua, PCR-based suppressive subtractive hybridization was performed to identify genes involved in summer and/or winter diapause. A total of 209 unique sequences were obtained including 89 in forward library for winter diapausing pupae and 120 in the reverse library for summer diapausing pupae. 76.4% (68/89) and 68.3% (82/120) unique sequences had significant hits to non-redundant proteins database. Gene functional annotation showed these non-redundant sequences are involved in stress response and innate immunity, metabolism and energy, information processing and regulation, binding, food storage, morphogenesis and development, cell skeleton and cycle, protein synthesis and folding. Approximately 28.2% (59/209) transcripts showed no significant similarity to any other sequence in the public databases, probably representing unique genes of the onion maggot. Semi-quantitative RT-PCR revealed that the relative expression levels of 18 genes were comparable between summer and winter diapause. This study elucidates the temporal expression of diapause-related genes in onion maggot, also provides new insights into the differences in the physiological changes in summer and winter pupae. Functional characterization of some candidate genes will further enhance the understanding of the generating, maintaining, and breaking mechanism of diapause.

Differential gene expression in Ulva prolifera under low light and low temperature conditions

The past several years witnessed the increasing global interest in the marine green macroalga Ulva prolifera as it is a key causative species of the massive green tides successively occurring in the Yellow Sea. Accurate localization of the 'seed' source is one of the principal scientific concerns to be solved before it is possible to manage these algal blooms. It has been suggested that somatic cells of Ulva prolifera which settled in cold benthic sediments might serve as one of the major propagule banks. To identify the molecular mechanisms underlying this hypothesis, PCR-based suppression subtractive hybridization was employed to analyze the differential gene expression of Ulva prolifera under low light and low temperature conditions (matching the cold benthic sediments conditions, 6 °C, 30 μmol photons m(-2) s(-1)). 137 ESTs representing 88 unigenes (80 singletons and 8 contigs) were detected as being over-expressed, whereas 109 unigenes (96 singletons and 13 contigs) in 130 ESTs were found to be down-regulated in this study. BLASTX analysis revealed that 65 % of the over-expressed and 59 % of the down-regulated genes did not belong to any documented functionally annotated or hypothetical proteins in the public database. However, analysis of the functional defined sequences displayed (1) an obvious sign of senescence, (2) enhancements of the photosynthesis system and the pentose phosphate pathway, (3) slow-down of activities in a wide range of processes including the DNA replication, the transcription, the translation, the glycolysis, the citrate cycle and the pyruvate metabolism in Ulva prolifera cells under low light and low temperature conditions. This work disclosed some basic information of the molecular mechanisms of Ulva prolifera cells under low light and low temperature conditions and provides useful clues for future studies on the "seed" source of the massive green tides.

Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

BackgroundTaxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control.ResultsSix separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line.ConclusionsThis study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

English

One concern for garlic (Allium sativum Linnaeus) producers is the damage caused by the pathogen Sclerotium cepivorum Berk. Unfortunately, the genetic and molecular mechanisms that participate during S. cepivorum Berk pathogenesis are currently unknown. In order to identify and isolate genes that are differentially expressed by the fungus during garlic white rot pathogenesis, PCR-based suppression subtractive hybridization (SSH) was used. Combining SSH and cDNA arrays hybridization techniques, 120 ESTs whose expression is restricted to the pathogenic stage were identified and isolated. Fourteen ESTs showing higher expression in cDNA arrays were sequenced, these included homologues to oxaloacetate acetylhydrolase (Oah), cysteine desulfurase (Nfs 1p), regulator of drug sensitivity (Rds 1p), outer membrane protein (Omp 1) and cell wall adhesion (Fig2p). One selected EST with high homology to Oah gene, a putative virulence factor, was analyzed by RT-PCR. The possible role of Oah gene in the pathogenesis of this fungus toward garlic is discussed. The combined applications of SSH and cDNA arrays permitted global analysis of gene expression patterns in S. cepivorum Berk, as an initial stage to improve the knowledge at molecular level from fungal pathogenesis.   Key words: Pathogenesis, differential gene expression, suppression subtractive hybridization, cDNA arrays, Oah gene.

Differential transcript accumulation in chickpea during early phases of compatible interaction with a necrotrophic fungus Ascochyta rabiei

The initial phases of the disease establishment are very crucial for the compatible interactions. Pathogens must overcome the responses generated by the host for the onset of disease invasion. The compatible interaction is inadequately represented in plant-pathogen interaction studies. To gain broader insight into the early responses elicited by chickpea blight fungus Ascochyta rabiei during compatible interaction; we isolated early responsive genes of chickpea using PCR based suppression subtractive hybridization (SSH) strategy. We obtained ~250 unique genes after homology search and redundancy elimination. Based on their potential cellular functions, these genes were broadly classified into eleven different categories viz. stress, signaling, gene regulation, cellular metabolism and genes of unknown functions. Present study revealed few unexpected genes which have a possible role in induced immunity and disease progression. We employed macroarray, northern blot, real-time PCR and cluster analysis to develop transcript profiles. Most of the genes analyzed were early induced and were transcriptionally upregulated upon 24h post inoculation. Our approach has rendered the isolation of early responsive genes involved in signaling and regulation of metabolic changes upon fungal infection. The information obtained will help to dissect the molecular mechanisms during compatible chickpea-Ascochyta interactions.

Differential expression patterns of capping protein, protein phosphatase 1, and casein kinase 1 may serve as diagnostic markers for malignant melanoma

Early and accurate diagnosis of malignant melanoma is critical for patient survival. However, currently used diagnostic markers are insufficiently specific, which limits their utility. We aimed to identify molecular markers that are more specific to malignant melanoma, thereby aiding in melanoma diagnosis and treatment. A PCR-based suppression subtractive hybridization was used to identify capping protein Z-line α1, protein phosphatase 1 catalytic subunit β isoform (PP1CB), and casein kinase 1 α1 (CSNK1A1) as being differentially expressed between melanoma cells and normal melanocytes. Quantitative reverse transcription-PCR and western blot analysis confirmed that these genes were overexpressed in melanoma cells. In addition, immunohistochemical assays revealed that the expression of PP1CB and CSNK1A1 was significantly greater in human melanoma specimens than nevi (P<0.0001). Combined application of PP1CB and CSNK1A showed high sensitivity and specificity for melanoma. Thus, our data suggest that PP1CB and CSNK1A1 are potential biomarkers for distinguishing malignant melanoma from other melanocytic lesions. In addition, because capping protein Z-line α1, PP1CB, and CSNK1A1 are involved in cell motility, which underlies invasion and metastasis of human cancer; they may be novel targets for antimetastatic therapies as well.

Isolation, identification and expression analysis of salt-responsive genes in Oryza sativa var. MR219 using PCR-based suppression subtractive hybridization

Isolation, identification and expression analysis of salt-responsive genes in Oryza sativa var. MR219 using PCR-based suppression subtractive hybridization

Identification of genes involved in bamboo fiber development

Recently bamboo has gained reputation as a major resource of non-wood fiber. The present study was undertaken to generate information about fiber development process in bamboo ( Bambusa balcooa) using PCR-based suppressive subtractive hybridization (PCR-SSH) technique, as molecular mechanism of its fiber development is yet to be explored. SSH was performed between cDNA isolated from leaf (as driver) and internodes (as tester) of B. balcooa which indicated up-regulation of 521 ESTs. Among these 41 were contigs and 65 ESTs were singletons. On the basis of BLASTX search 307 ESTs with known functions were classified into several functional categories including transport, metabolism, information, perception and response to stimuli while others were either non-significant (120) or hypothetical proteins (94). A total of 51 out of 307 functional ESTs were found fiber specific and their global distribution among different plant species like maize, rice, cotton and Arabidopsis ESTs were determined. Net distributions and differential expression patterns of 13 important B. balcooa fiber specific cDNAs among different internodes during bamboo development were studied using RNA slot–blot, semi-quantitative RT-PCR and real time PCR. In-situ localization of mRNA transcript for few selected bamboo fiber ESTs namely, V1Bb147 (protein kinase-like protein) and V1Bb88 (myb domain-containing protein) were detected using Confocal Laser Scanning Microscope. Transcript levels of these genes exhibited an orchestral turn-over during bamboo development, suggesting their close association with fiber development, an event associated with several metabolic and physiological changes. The results clearly suggest that these genes are involved in several concerted mechanisms involving Ca + signaling pathway, cell wall synthesis, hormone regulation, system maintaining cell turgor pressure and cytoskeleton synthesis pathway accountable for bamboo fiber development signifying fiber development as a complex but ordered metabolic process involving differential expression of large scale fiber associated genes. This is the first report on systematic analysis of genes involved in bamboo fiber development.

Isolation of Salt Stress Tolerance Genes from Roots of Mangrove Plant, Rhizophora stylosa Griff., Using PCR-Based Suppression Subtractive Hybridization

Salinity is a major abiotic stress that seriously limits plant growth and crop productivity. The halophytic Rhizophora stylosa is useful for the study of the molecular mechanisms behind salinity tolerance in mangrove trees. To isolate anti-salt stress genes from mangrove plants, a cDNA library of R. stylosa roots was constructed and screened for stress-related genes by polymerase chain reaction (PCR)-based suppressive subtractive hybridization (SSH). The mangrove seedlings were grown for 8 months under two conditions: exposure to water with 3% salt and exposure to fresh water. cDNA of seedlings exposed to water with 3% salt was used as a tester and cDNA for freshwater germination was used as a driver. We isolated and sequenced 240 up-regulated expressed sequence tags (ESTs) from the SSH library. Among these up-regulated ESTs, 48 unique clones were putatively identified and classified into ten functional categories, such as cell rescue and defense, secondary metabolism, protein synthesis, and metabolism. Fifteen genes from different categories were selected and their expression was studied by real-time RT-PCR. Significantly increased expression levels were confirmed for 13 of these 15 transcripts, which suggest that these genes contribute to the salt tolerance of this plant. Among them, two transcription factors and several genes involved in isoprenoid biosynthesis were identified from mangrove trees as salt tolerance genes for the first time. The physiological significance of the increased expression of these genes in the long-term adaptation of mangrove trees to salt stress is discussed.

PCR-based suppression subtractive hybridization analyses of enantioselective gene expression in root tips of wheat treated with optically active urea compounds

The optically active urea compounds R- and S-1-α-methylbenzyl-3- p-tolylurea ( R- and S-MBTU) have qualitatively different effects on plant physiology, especially in Gramineae plants. To elucidate the mechanisms by which S-MBTU inhibits root growth of wheat ( Triticum aestivum L. var. Chinese Spring), we used PCR-based suppression subtractive hybridization (SSH) and quantitative real-time RT-PCR (qPCR) analyses. Genes related to amino acid biosynthesis, cell cycle and skeleton, protein synthesis, cell wall biosynthesis, glycolysis, signaling, DNA modification, and detoxification were enantioselectively regulated. These expression profiles suggested that the putative mode of action of S-MBTU is disruption of primary amino acid biosynthesis, especially the glutamine synthetase (GS)- and acetohydroxyacid synthase (AHAS)-enzyme systems, and disruption of cell membrane skeleton homeostasis related to a putative α-tubulin 2 (α-TUB2), a putative β-actin (ACT) and β-tubulin 1 (β-TUB1). Other genes specifically affected by S-MBTU may result in disruption of metabolic processes, leading to inhibition of root growth.

Involvement of a novel p38 mitogen‐activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell)

Hydroides elegans is a common marine fouling organism in most tropical and subtropical waters. The life cycle of H. elegans includes a planktonic larval stage in which swimming larvae normally take 5 days to attain competency to settle. Larval metamorphosis marks the beginning of its benthic life; however, the endogenous molecular mechanisms that regulate metamorphosis remain largely unknown. In this study, a PCR-based suppressive subtractive hybridization (SSH) library was constructed to screen the genes expressed in competent larvae but not in precompetent larvae. Among the transcripts isolated from the library, 21 significantly matched sequences in the GenBank. Many of these isolated transcripts have putative roles in the reactive oxygen species (ROS) signal transduction pathway or in response to ROS stress. A putative novel p38 mitogen-activated protein kinase (MAPK), which was also isolated with SSH screen, was then cloned and characterized. The MAPK inhibitors assay showed that both p38 MAPK inhibitors SB202190 and SB203580 effectively inhibited the biofilm-induced metamorphosis of H. elegans. A cell stressors assay showed that H(2)O(2) effectively induced larval metamorphosis of H. elegans, but the inductivity of H(2)O(2) was also inhibited by both SB inhibitors. The catalase assay showed that the catalase could effetely inhibit H. elegans larvae from responding to inductive biofilm. These results showed that the p38 MAPK-dependent pathway plays critical role in controlling larval metamorphosis of the marine polychaete H. elegans, and the reactive oxygen radicals produced by biofilm could be the cue inducing larval metamorphosis.

Identification and analysis of in planta expressed genes of Magnaporthe oryzae

BackgroundInfection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.ResultsA cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.ConclusionThis study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.