Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of anindica rice

To decipher the genes associated with drought stress response and to identify novel genes in rice, we utilized 1540 high-quality expressed sequence tags (ESTs) for functional annotation and mapping to rice genomic sequences. These ESTs were generated earlier by 3'-end single-pass sequencing of 2000 cDNA clones from normalized cDNA libraries constructed from drought-stressed seedlings of anindica rice. A rice UniGene set of 1025 transcripts was constructed from this collection through the BLASTN algorithm. Putative functions of 559 nonredundant ESTs were identified by BLAST similarity search against public databases. Putative functions were assigned at a stringency E value of 10-6 in BLASTN and BLASTX algorithms. To understand the gene structure and function further, we have utilized the publicly available finished and unfinished rice BAC/PAC (BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome) sequences for similarity search using the BLASTN algorithm. Further, 603 nonredundant ESTs have been mapped to BAC/PAC clones. BAC clones were assigned by a homology of above 95% identity along 90% of EST sequence length in the aligned region. In all, 700 ESTs showed rice EST hits in GenBank. Of the 325 novel ESTs, 128 were localized to BAC clones. In addition, 127 ESTs with identified putative functions but with no homology in IRGSP (International Rice Genome Sequencing Program) BAC/PAC sequences were mapped to the Chinese WGS (whole genome shotgun contigs) draft sequence of the rice genome. Functional annotation uncovered about a hundred candidate ESTs associated with abiotic stress in rice andArabidopsis that were previously reported based on microarray analysis and other studies. This study is a major effort in identifying genes associated with drought stress response and will serve as a resource to rice geneticists and molecular biologists

Characterizing the role of Glycine max NHL gene family members in plant-nematode interactions [abstract]

Abstract only availableSoybean cyst nematode (SCN; Heterodera glycines) is a microscopic parasitic roundworm of soybean that causes nearly $1 billion dollars in annual yield loss in the United States. SCN damages the plant by attaching itself to the soybean root system, where it forms a complex feeding site and drains vital nutrients from the plant. Naturally resistant soybean lines have been used as the primary strategy to manage SCN, because they have evolved a natural mechanism for resisting SCN infection. However, soybean resistance against SCN is derived from a small genetic base and repeated annual plantings of these same resistant lines has selected for populations of SCN that can reproduce on the resistant lines. Therefore, understanding the molecular mechanisms of how some soybean plants have the ability to naturally resist infection by SCN is critical for designing new strategies to improve crop plant resistance to SCN. My project focuses on soybean NDR1/HIN1-like (NHL) genes found to be expressed at higher levels specifically within SCN-induced feeding cells of resistant soybean as compared to susceptible soybean. To gain insight into the potential role of these genes in soybeans ability to resist SCN, full-length gene and cDNA sequences have been isolated using techniques known as genome walking and RACE PCR. RNAi and overexpression constructs have been generated to directly test the function of these genes in SCN resistance. To gain insight into the nematode-responsive regulation of each gene, the endogenous promoter sequences have been isolated and fused to the _-glucuronidase reporter gene for expression studies. This project will give insight into the mechanisms the soybean plant uses to defend itself against SCN infection and hopefully reveal crucial results which aid in the goal of developing SCN resistant soybean.Life Sciences Undergraduate Research Opportunity Progra

Parallel Genome-Wide Expression Profiling of Host and Pathogen During Soybean Cyst Nematode Infection of Soybean

Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root

Rice flavonoid pathway genes, OsDfr and OsAns, are induced by dehydration, high salt and ABA, and contain stress responsive promoter elements that interact with the transcription activator, OsC1-MYB

Molecular basis of regulation of abiotic stress responses and the flavonoid biosynthesis in rice was investigated. The role of the regulatory gene OsC1-Myb, encoding a MYB class of transcription activators in the stress-induced expression of the structural genes, OsDfr and OsAns, was analyzed. Northern analysis of shoot tissues of rice, Nagina 22, (Oryza sativa L. sub sp. indica) seedlings under dehydration stress or high salt or abscisic acid (ABA) showed a significant enhancement of transcript level and/or transcript stability of OsDfr and OsAns. Enhanced levels of the OsC1-myb transcript were also detected. The expression pattern of these three genes indicates that the stress responsive accumulation of OsDfr and OsAns transcripts is mediated by the transcription factor, OsC1-MYB. The 5' upstream region of the OsDfr and OsAns genes carry several regulatory domains, which share homology with some of the known stress responsive genes in plants. In addition, several putative myb and myc responsive domains were identified in the promoter region of the genes, OsDfr and OsAns. The recombinant OsC1-MYB protein binds in vitro to the myb responsive elements (MREs) in the OsDfr and OsAns promoters, suggesting that it is a potential transcription activator of stress-induced expression of structural genes of the flavonoid pathway

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance. Molecular Breeding 7: 73 – 83

Abstract The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R 0 C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R 0 generation, suggesting gene silencing. Preliminary screening of these R 1 plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance

Developmental Transcript Profiling of Cyst Nematode Feeding Cells in Soybean Roots

Cyst nematodes of the genus Heterodera are obligate, sedentary endoparasites that have developed highly evolved relationships with specific host plant species. Successful parasitism involves significant physiological and morphological changes to plant root cells for the formation of specialized feeding cells called syncytia. To better understand the molecular mechanisms that lead to the development of nematode feeding cells, transcript profiling was conducted on developing syncytia induced by the soybean cyst nematode Heterodera glycines in soybean roots by coupling laser capture microdissection with high-density oligonucleotide microarray analysis. This approach has identified pathways that may play intrinsic roles in syncytium induction, formation, and function. Our data suggest interplay among phytohormones that likely regulates synchronized changes in the expression of genes encoding cell-wall-modifying proteins. This process appears to be tightly controlled and coordinately regulated with cell wall rigidification processes that may involve lignification of feeding cell walls. Our data also show local downregulation of jasmonic acid biosynthesis and responses in developing syncytia, which suggest a local suppression of plant defense mechanisms. Moreover, we identified genes encoding putative transcription factors and components of signal transduction pathways that may be important in the regulatory processes governing syncytium formation and function. Our analysis provides a broad mechanistic picture that forms the basis for future hypothesis-driven research to understand cyst nematode parasitism and to develop effective management tools against these pathogens.This article is published as Ithal, Nagabhushana, Justin Recknor, Dan Nettleton, Tom Maier, Thomas J. Baum, and Melissa G. Mitchum. "Developmental transcript profiling of cyst nematode feeding cells in soybean roots." Molecular Plant-Microbe Interactions 20, no. 5 (2007): 510-525, doi: 10.1094/MPMI-20-5-0510. Posted with permission.</p

Characterization of soybean genes involved in soybean cyst nematode (SCN) resistance [abstract]

Abstract only availableThe expansion of the soybean cyst nematode (SCN; Heterodera glycines) across soybean producing regions of the United States shows no signs of slowing. Resistant soybean germplasm has been used as the primary strategy to manage this pest. However, soybean resistance against SCN is derived from a narrow genetic base and repeated plantings have selected for populations of SCN that can break the resistance. Therefore, understanding the molecular mechanisms of soybean resistance is critical for designing novel strategies to improve crop plant resistance to SCN. To identify genes potentially involved in SCN resistance, we previously coupled laser capture microdissection with microarray profiling to compare gene expression profiles of nematode feeding cells induced in resistant and susceptible near-isogenic lines (NILs) of soybean. We identified 390 soybean genes that were differentially expressed between the resistant and susceptible NILs. Bacterial artificial chromosome (BAC) pools of soybean DNA were then screened for the presence of a subset of these genes. Three genes, believed to be involved in soybean stress and defense responses, were selected for further functional studies. Full length gene and cDNA sequences are being isolated using genome walking and RACE PCR approaches. RNAi and overexpression studies will be used to test the function of these genes in resistance to SCN.MU Monsanto Undergraduate Research Fellowshi

Parallel Genome-Wide Expression Profiling of Host and Pathogen During Soybean Cyst Nematode Infection of Soybean

Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential expression across different stages of nematode parasitism and development. Differentially expressed SCN genes were grouped into nine different clusters based on their expression profiles during parasitism of soybean roots. The patterns of gene expression we observed in SCN suggest coordinated regulation of genes involved in parasitism. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of our microarray analysis. The simultaneous genome-wide analysis of gene expression changes in the host and pathogen during a compatible interaction provides new insights into soybean responses to nematode infection and the first profile of transcript abundance changes occurring in the nematode as it infects and establishes a permanent feeding site within a host plant root.This article is published as Ithal, Nagabhushana, Justin Recknor, Dan Nettleton, Leonard Hearne, Tom Maier, Thomas J. Baum, and Melissa G. Mitchum. "Parallel genome-wide expression profiling of host and pathogen during soybean cyst nematode infection of soybean." Molecular Plant-Microbe Interactions 20, no. 3 (2007): 293-305, doi: 10.1094/MPMI-20-3-0293. Posted with permission.</p

The Soybean Rhg1 Locus for Resistance to the Soybean Cyst Nematode Heterodera glycines Regulates the Expression of a Large Number of Stress- and Defense-Related Genes in Degenerating Feeding Cells1[C][W][OA]

To gain new insights into the mechanism of soybean (Glycine max) resistance to the soybean cyst nematode (Heterodera glycines), we compared gene expression profiles of developing syncytia in soybean near-isogenic lines differing at Rhg1 (for resistance to Heterodera glycines), a major quantitative trait locus for resistance, by coupling laser capture microdissection with microarray analysis. Gene expression profiling revealed that 1,447 genes were differentially expressed between the two lines. Of these, 241 (16.8%) were stress- and defense-related genes. Several stress-related genes were up-regulated in the resistant line, including those encoding homologs of enzymes that lead to increased levels of reactive oxygen species and proteins associated with the unfolded protein response. These results indicate that syncytia induced in the resistant line are undergoing severe oxidative stress and imbalanced endoplasmic reticulum homeostasis, both of which likely contribute to the resistance reaction. Defense-related genes up-regulated within syncytia of the resistant line included those predominantly involved in apoptotic cell death, the plant hypersensitive response, and salicylic acid-mediated defense signaling; many of these genes were either partially suppressed or not induced to the same level by a virulent soybean cyst nematode population for successful nematode reproduction and development on the resistant line. Our study demonstrates that a network of molecular events take place during Rhg1-mediated resistance, leading to a highly complex defense response against a root pathogen