Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Identification of candidate genes associated with positive and negative heterosis in rice.

To identify the genes responsible for yield related traits, and heterosis, massively parallel signature sequencing (MPSS) libraries were constructed from leaves, roots and meristem tissues from the two parents, 'Nipponbare' and '93-11', and their F1 hybrid. From the MPSS libraries, 1-3 million signatures were obtained. Using cluster analysis, commonly and specifically expressed genes in the parents and their F1 hybrid were identified. To understand heterosis in the F1 hybrid, the differentially expressed genes in the F1 hybrid were mapped to yield related quantitative trait loci (QTL) regions using a linkage map constructed from 131 polymorphic simple sequence repeat markers with 266 recombinant inbred lines derived from a cross between Nipponbare and 93-11. QTLs were identified for yield related traits including days to heading, plant height, plant type, number of tillers, main panicle length, number of primary branches per main panicle, number of kernels per main panicle, total kernel weight per main panicle, 1000 grain weight and total grain yield per plant. Seventy one QTLs for these traits were mapped, of which 3 QTLs were novel. Many highly expressed chromatin-related genes in the F1 hybrid encoding histone demethylases, histone deacetylases, argonaute-like proteins and polycomb proteins were located in these yield QTL regions. A total of 336 highly expressed transcription factor (TF) genes belonging to 50 TF families were identified in the yield QTL intervals. These findings provide the starting genomic materials to elucidate the molecular basis of yield related traits and heterosis in rice.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors.

BACKGROUND: Rice blast caused by the fungus Magnaporthe oryzae is an important disease in virtually every rice growing region of the world, which leads to significant annual decreases of grain quality and yield. To prevent disease, resistance genes in rice have been cloned and introduced into susceptible cultivars. However, introduced resistance can often be broken within few years of release, often due to mutation of cognate avirulence genes in fungal field populations. RESULTS: To better understand the pattern of mutation of M. oryzae field isolates under natural selection forces, we used a next generation sequencing approach to analyze the genomes of two field isolates FJ81278 and HN19311, as well as the transcriptome of FJ81278. By comparing the de novo genome assemblies of the two isolates against the finished reference strain 70-15, we identified extensive polymorphisms including unique genes, SNPs (single nucleotide polymorphism) and indels, structural variations, copy number variations, and loci under strong positive selection. The 1.75 MB of isolate-specific genome content carrying 118 novel genes from FJ81278, and 0.83 MB from HN19311 were also identified. By analyzing secreted proteins carrying polymorphisms, in total 256 candidate virulence effectors were found and 6 were chosen for functional characterization. CONCLUSIONS: We provide results from genome comparison analysis showing extensive genome variation, and generated a list of M. oryzae candidate virulence effectors for functional characterization.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

Transcriptome dynamics through alternative polyadenylation in developmental and environmental responses in plants revealed by deep sequencing.

Polyadenylation sites mark the ends of mRNA transcripts. Alternative polyadenylation (APA) may alter sequence elements and/or the coding capacity of transcripts, a mechanism that has been demonstrated to regulate gene expression and transcriptome diversity. To study the role of APA in transcriptome dynamics, we analyzed a large-scale data set of RNA "tags" that signify poly(A) sites and expression levels of mRNA. These tags were derived from a wide range of tissues and developmental stages that were mutated or exposed to environmental treatments, and generated using digital gene expression (DGE)-based protocols of the massively parallel signature sequencing (MPSS-DGE) and the Illumina sequencing-by-synthesis (SBS-DGE) sequencing platforms. The data offer a global view of APA and how it contributes to transcriptome dynamics. Upon analysis of these data, we found that ∼60% of Arabidopsis genes have multiple poly(A) sites. Likewise, ∼47% and 82% of rice genes use APA, supported by MPSS-DGE and SBS-DGE tags, respectively. In both species, ∼49%-66% of APA events were mapped upstream of annotated stop codons. Interestingly, 10% of the transcriptomes are made up of APA transcripts that are differentially distributed among developmental stages and in tissues responding to environmental stresses, providing an additional level of transcriptome dynamics. Examples of pollen-specific APA switching and salicylic acid treatment-specific APA clearly demonstrated such dynamics. The significance of these APAs is more evident in the 3034 genes that have conserved APA events between rice and Arabidopsis.

Large scale identification of genes involved in plant-fungal interactions using Illumina's sequencing-by-synthesis technology.

Deep transcriptome profiling of pathogen-infected tissues enhances the understanding of molecular mechanisms underlying host-pathogen interactions. Illumina's next generation sequencing technology sequencing-by-synthesis (SBS) is a powerful tool to rapidly sequence genomes and transcriptomes at an affordable rate. We modified the procedure for SBS library construction to significantly increase the efficiency of library construction. Using our improved method, two Sclerotinia homoeocarpa libraries were constructed from mycelia grown in potato dextrose broth (PDB) or potato dextrose agar (PDA) for 96 h, respectively, and two creeping bentgrass libraries were constructed from leaves 96 h after inoculation with S. homoeocarpa or water sprayed, respectively. About 4-7 million mRNA signatures were sequenced from each library. Sequence analysis using BLAST was performed against sequenced fungal genomes and rice genomic sequence to identify the expressed genes in both S. homoeocarpa mycelia and creeping bentgrass. Bioinformatic analysis identified many expressed genes in the pathogen and host. A public database to access the sequence data was developed at http://www.dstidb.org . Our results demonstrate how SBS technology can unravel transcriptome complexity during the creeping bentgrass-S. homoeocarpa interaction.

Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins.

Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activity-related protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags (ESTs), full-length cDNAs, or massively parallel signature sequencing (MPSS) tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

RL-SAGE and microarray analysis of the rice transcriptome after Rhizoctonia solani infection.

Sheath blight caused by the fungal pathogen Rhizoctonia solani is an emerging problem in rice production worldwide. To elucidate the molecular basis of rice defense to the pathogen, RNA isolated from R. solani-infected leaves of Jasmine 85 was used for both RL-SAGE library construction and microarray hybridization. RL-SAGE sequence analysis identified 20,233 and 24,049 distinct tags from the control and inoculated libraries, respectively. Nearly half of the significant tags (> or =2 copies) from both libraries matched TIGR annotated genes and KOME full-length cDNAs. Among them, 42% represented sense and 7% antisense transcripts, respectively. Interestingly, 60% of the library-specific (> or =10 copies) and differentially expressed (>4.0-fold change) tags were novel transcripts matching genomic sequence but not annotated genes. About 70% of the genes identified in the SAGE libraries showed similar expression patterns (up or down-regulated) in the microarray data obtained from three biological replications. Some candidate RL-SAGE tags and microarray genes were located in known sheath blight QTL regions. The expression of ten differentially expressed RL-SAGE tags was confirmed with RT-PCR. The defense genes associated with resistance to R. solani identified in this study are useful genomic materials for further elucidation of the molecular basis of the defense response to R. solani and fine mapping of target sheath blight QTLs.

Magnaporthe grisea infection triggers RNA variation and antisense transcript expression in rice.

Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages.

An expression atlas of rice mRNAs and small RNAs.

Identification of all expressed transcripts in a sequenced genome is essential both for genome analysis and for realization of the goals of systems biology. We used the transcriptional profiling technology called 'massively parallel signature sequencing' to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). We sequenced 46,971,553 mRNA transcripts from 22 libraries, and 2,953,855 small RNAs from 3 libraries. The data demonstrate widespread transcription throughout the genome, including sense expression of at least 25,500 annotated genes and antisense expression of nearly 9,000 annotated genes. An additional set of approximately 15,000 mRNA signatures mapped to unannotated genomic regions. The majority of the small RNA data represented lower abundance short interfering RNAs that match repetitive sequences, intergenic regions and genes. Among these, numerous clusters of highly regulated small RNAs were readily observed. We developed a genome browser (http://mpss.udel.edu/rice) for public access to the transcriptional profiling data for this important crop.

Use of robust-long serial analysis of gene expression to identify novel fungal and plant genes involved in host-pathogen interactions.

Identification of important transcripts from fungal pathogens and host plants is indispensable for full understanding the molecular events occurring during fungal-plant interactions. Recently, we developed an improved LongSAGE method called robust-long serial analysis of gene expression (RL-SAGE) for deep transcriptome analysis of fungal and plant genomes. Using this method, we made 10 RL-SAGE libraries from two plant species (Oryza sativa and Zea maize) and one fungal pathogen (Magnaporthe grisea). Many of the transcripts identified from these libraries were novel in comparison with their corresponding EST collections. Bioinformatic tools and databases for analyzing the RL-SAGE data were developed. Our results demonstrate that RL-SAGE is an effective approach for large-scale identification of expressed genes in fungal and plant genomes.

Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods.

BACKGROUND: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. RESULTS: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. CONCLUSION: The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.

Genomic and genetic characterization of rice Cen3 reveals extensive transcription and evolutionary implications of a complex centromere.

The centromere is the chromosomal site for assembly of the kinetochore where spindle fibers attach during cell division. In most multicellular eukaryotes, centromeres are composed of long tracts of satellite repeats that are recalcitrant to sequencing and fine-scale genetic mapping. Here, we report the genomic and genetic characterization of the complete centromere of rice (Oryza sativa) chromosome 3. Using a DNA fiber-fluorescence in situ hybridization approach, we demonstrated that the centromere of chromosome 3 (Cen3) contains approximately 441 kb of the centromeric satellite repeat CentO. Cen3 includes an approximately 1,881-kb domain associated with the centromeric histone CENH3. This CENH3-associated chromatin domain is embedded within a 3,113-kb region that lacks genetic recombination. Extensive transcription was detected within the CENH3 binding domain based on comprehensive annotation of protein-coding genes coupled with empirical measurements of mRNA levels using RT-PCR and massively parallel signature sequencing. Genes <10 kb from the CentO satellite array were expressed in several rice tissues and displayed histone modification patterns consistent with euchromatin, suggesting that rice centromeric chromatin accommodates normal gene expression. These results support the hypothesis that centromeres can evolve from gene-containing genomic regions.