Workflow of analysis.

<p>Transcriptomes of sorghum and <i>Bipolaris sorghicola</i> were analyzed simultaneously by using different approaches. The mixed transcriptome obtained from <i>B. sorghicola</i>-infected sorghum leaves was sequenced by using Illumina mRNA-Seq technology. The sequenced reads contain the reads derived from sorghum (green bars), <i>B. sorghicola</i> (purple bars), and other organisms such as normal inhabitants of plant tissue (red bars). The reads were aligned to the sorghum reference genome (black bars), and the aligned and unaligned reads were used to analyze gene expression in sorghum (left) and <i>B. sorghicola</i> (right), respectively. Gene expression in sorghum was analyzed for each transcript (green arrows), including transcripts annotated with Phytozome and unannotated transcripts identified on the basis of the piling-up of aligned reads by using the Cufflinks program. For <i>B. sorghicola</i>, unaligned reads were assembled by using the Oases program to retrieve the pathogen transcripts expressed during growth of the fungus in the plant. Expression of the assembled transcripts was analyzed by aligning the reads back to the transcripts. The assembled transcripts contained not only the transcripts of <i>B. sorghicola</i> (purple arrows) but also those of other organisms (red arrows). The transcripts of other organisms were removed as contaminants, and those of <i>B. sorghicola</i> was validated experimentally.</p

Pathogen (<i>Bipolaris sorghicola</i>)-induced genes encoding receptors in sorghum.

<p>(A) Fold changes of pathogen-induced transcripts for each family. RPKM fold changes at 24 h were calculated for infected samples compared with mock-infected samples. Genes with a fold change value of ≥3 were shown. To avoid division by 0, 1 was added to the RPKM values of mock-infected samples. Families of the receptors, shown on the top, were defined on the basis of the Pfam domain structures (See Materials and Methods). (B) Domain structures of the 2 receptors for which the genes were highly induced. Predicted structures included SP, signal peptide; LRRNT_2, Leucine rich repeat N-terminal domain (PF08263); LRR_4, Leucine Rich repeats (2 copies) (PF12799); and LRR_8, Leucine rich repeat (PF13855). (C) Phylogenetic tree of the 2 receptors for which the genes were particularly strongly induced, along with their homologous proteins. The amino acid sequences of LRRNT_2 domain (PF08263) of each protein were aligned by using ClustalW and the tree was created by using MEGA5. Red underlines show the 2 receptors for which the genes were particularly strongly induced. Abbreviations are as follows: Sb, <i>Sorghum bicolor</i>; Os, <i>Oryza sativa</i> (rice); At, <i>Arabidopsis thaliana</i>; Nt, <i>Nicotiana tabacum</i> (tobacco); Ca, <i>Capsicum annuum</i> (pepper); Sp, <i>Solanum pimpinellifolium</i> (currant tomato).</p

Surveillance of norovirus among children with diarrhea in four major hospitals in Bhutan: Replacement of GII.21 by GII.3 as a dominant genotype

<div><p>Background</p><p>Diarrhea is a major cause of morbidity and mortality among Bhutanese children. The etiology of diarrhea is not well known due to the challenges of conducting routine surveillance with Bhutan’s modest research facilities. Establishing an etiology is crucial toward generating evidence that will contribute to policy discussions on a diarrheal disease control program. Our previous study, during 2010–2012, revealed that norovirus (NoV) is an important cause of diarrhea among Bhutanese children, and that GII.21 was the major genotype circulating at that time. In other countries, GII.4 is the major genotype responsible for NoV infections. In this update report, we provide new prevalence data to describe the progression of the transformation and distribution of the NoV genotype among Bhutanese children.</p><p>Methods</p><p>From June 2013 through May 2014, diarrheal stool samples were collected at one national referral hospital in Thimphu, two regional referral hospitals in the eastern and central regions, and one general hospital in the western region of Bhutan. NoV was detected by reverse transcription–polymerase chain reaction (RT–PCR), by amplifying the capsid gene. The RT–PCR results were confirmed by nucleotide sequencing of the amplicons.</p><p>Results</p><p>The proportion of NoV-positive stool samples was 23.6% (147/623), of which 76.9% were NoV GII and the remainders were NoV GI. The median age of infected children was 15.5 months, with a fairly balanced female: male ratio. NoV GII was most prevalent in the colder months (late November–mid April) and NoV GI had the highest prevalence in the summer (mid April–late September). Nucleotide sequencing was successful in 99 samples of GII strains. The most common genotypes were GII.3 (42.6%), GII.4 Sydney 2012 (15.8%), and GII.4 unassigned (11.9%). No GII.21 was found in any child in the present study. Phylogenetic analysis showed that GII.3 strains in the present study belonged to an independent cluster in lineage B. These strains shared an ancestor with those from different countries and Bhutanese strains circulating during 2010.</p><p>Conclusion</p><p>NoV remains an important cause of diarrhea among Bhutanese children. Genotype GII.3 from a single ancestor strain has spread, replacing the previously circulating GII.21. Current NoV genotypes are similar to the strains circulating worldwide but are primarily related to those in neighboring countries. NoV GII is prevalent during the cold season, while GI is prevalent during the summer. To develop a NoV infection control policy, further studies are needed.</p></div

Pathogen (<i>Bipolaris sorghicola</i>)-induced genes encoding peroxidases in sorghum.

<p>(A) Pathogen-induced fold changes in expression. RPKM fold changes at 24 h were calculated for infected samples compared with those of mock-infected samples. To avoid division by 0, 1 was added to the RPKM values of mock-infected samples. (B) Phylogenetic tree of peroxidases for which the encoding genes were strongly induced. The amino acid sequences of peroxidase domain (PF00141) of Sb02g042860 (SbPrx18) (red underline) and its best 10 BLAST hits in the Phytozome sorghum protein database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062460#pone.0062460-Pruitt1" target="_blank">[16]</a> and the Rice Annotation Project (<a href="http://rapdb.dna.affrc.go.jp" target="_blank">http://rapdb.dna.affrc.go.jp</a>) protein database, as well as the sequences of wheat TaPrx103 and barley HvPrx08, were aligned by using ClustalW. Phylogenetic tree was constructed by using MEGA5. Abbreviations are as follows: Sb, <i>Sorghum bicolor</i>; Os, <i>Oryza sativa</i> (rice); Hv, <i>Hordeum vulgare</i>; Ta, <i>Triticum aestivum</i>.</p

Pathogen (<i>Bipolaris sorghicola</i>)-induced genes encoding transcriptional factors in sorghum.

<p>(A) Number of pathogen-induced transcripts for each family. Families of the transcriptional factors (TFs), shown at left, were defined on the basis of a homology search in the Plant Transcription Factor Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062460#pone.0062460-PrezRodrguez1" target="_blank">[22]</a>. (B) Expression of the 10 induced WRKY TFs. RPKMs of each transcript were compared in mock- and pathogen-infected leaves by using the disease-resistant cultivar SIL-05 in the early stages of infection (24 h) (this study) and a related cultivar, BTx623, at a relatively late infection stage (7 days) (our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062460#pone.0062460-Mizuno1" target="_blank">[13]</a>) (C) Phylogenetic tree of the 2 WRKY TFs for which the genes were expressed at relatively high levels. The amino acid sequences of WRKY domain (PF03106) of Sb08g005080 (red underline in right panel) and Sb04g005520 (red underline in left panel), and their best 10 BLAST hits in the Phytozome sorghum protein database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062460#pone.0062460-Pruitt1" target="_blank">[16]</a> and the Rice Annotation Project (RAP) protein database (<a href="http://rapdb.dna.affrc.go.jp" target="_blank">http://rapdb.dna.affrc.go.jp</a>), were aligned by using ClustalW. Phylogenetic trees were constructed by using MEGA5. For Sb04g005520, HvWRKY1/2 and ATWRKY40/60 were also aligned. Abbreviations are as follows: Sb, <i>Sorghum bicolor</i>; Os, <i>Oryza sativa</i> (rice); Hv, <i>Hordeum vulgare</i>; At, <i>Arabidopsis thaliana</i>.</p

Monthly distribution of norovirus GII genotypes.

<p>Graph showing the relationship of different GII genotypes with the months of occurrence. The Y-axis represents the number of norovirus GII genotypes and the X- axis represents the months of occurrence within each year. On the lower part of the graph, each GII genotype is represented by a specific color.</p

Phylogenetic tree of norovirus GII cases.

<p>Phylogenetic tree constructed with the nucleotide sequences of the capsid gene at the C region of norovirus GII strains. Each strain begins with the species name, followed by strain name, year of detection, and country of origin. Bhutanese strains from this and the previous study are preceded by blue- and black-filled circles, respectively. The strain detected in water in the previous study is preceded by a black-filled diamond. Bovine GIII norovirus strain Aba-Z5 was used as an outgroup. The number adjacent to the node represents the bootstrap value. The scale bar shows genetic distance expressed as nucleotide substitutions per site. The DNA Data Bank of Japan/European molecular Biology Laboratory/GenBank accession numbers for the Bhutanese strains of the present study are LC209682–LC209785.</p

Pathogen (<i>Bipolaris sorghicola</i>)-induced genes encoding proteins for signaling cascade in <i>Sorghum bicolor.</i>

1<p>data from our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062460#pone.0062460-Mizuno1" target="_blank">[13]</a>.</p

Simultaneous RNA-Seq Analysis of a Mixed Transcriptome of Rice and Blast Fungus Interaction

<div><p>A filamentous fungus, <em>Magnaporthe oryzae</em>, is a causal agent of rice blast disease, which is one of the most serious diseases affecting cultivated rice, <em>Oryza sativa</em>. However, the molecular mechanisms underlying both rice defense and fungal attack are not yet fully understood. Extensive past studies have characterized many infection-responsive genes in the pathogen and host plant, separately. To understand the plant-pathogen interaction comprehensively, it is valuable to monitor the gene expression profiles of both interacting organisms simultaneously in the same infected plant tissue. Although the host-pathogen interaction during the initial infection stage is important for the establishment of infection, the detection of fungal gene expression in infected leaves at the stage has been difficult because very few numbers of fungal cells are present. Using the emerging RNA-Seq technique, which has a wide dynamic range for expression analyses, we analyzed the mixed transcriptome of rice and blast fungus in infected leaves at 24 hours post-inoculation, which is the point when the primary infection hyphae penetrate leaf epidermal cells. We demonstrated that our method detected the gene expression of both the host plant and pathogen simultaneously in the same infected leaf blades in natural infection conditions without any artificial treatments. The upregulation of 240 fungal transcripts encoding putative secreted proteins was observed, suggesting that these candidates of fungal effector genes may play important roles in initial infection processes. The upregulation of transcripts encoding glycosyl hydrolases, cutinases and LysM domain-containing proteins were observed in the blast fungus, whereas pathogenesis-related and phytoalexin biosynthetic genes were upregulated in rice. Furthermore, more drastic changes in expression were observed in the incompatible interactions compared with the compatible ones in both rice and blast fungus at this stage. Our mixed transcriptome analysis is useful for the simultaneous elucidation of the tactics of host plant defense and pathogen attack.</p> </div

Highly Enantioselective Monofluoromethylation of C2-Arylindoles Using FBSM under Chiral Phase-Transfer Catalysis

The highly enantioselective addition of 1-fluoro-1,1-bis(phenylsulfonyl)methane (FBSM) to vinylogous imines generated in situ from 2-aryl-3-(1-arylsulfonylmethyl)indoles was achieved using chiral ammonium salts derived from cinchona alkaloids. One-pot conversion from 2-arylindoles with FBSM was also adaptable under the same reaction conditions. The key for this transformation is the effective use of the arylsulfonyl group