Draft Genome Sequence of Aspergillus oryzae Strain 3.042

Aspergillus oryzae is the most important fungus for the traditional fermentation in China and is particularly important in soy sauce fermentation. We report the 36,547,279-bp draft genome sequence of A. oryzae 3.042 and compared it to the published genome sequence of A. oryzae RIB40.     

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.     

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。     

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.     

Global Metabolic Reconstruction and Metabolic Gene Evolution in the Cattle Genome

The sequence of cattle genome provided a valuable opportunity to systematically link genetic and metabolic traits of cattle. The objectives of this study were 1) to reconstruct genome-scale cattle-specific metabolic pathways based on the most recent and updated cattle genome build and 2) to identify duplicated metabolic genes in the cattle genome for better understanding of metabolic adaptations in cattle. A bioinformatic pipeline of an organism for amalgamating genomic annotations from multiple sources was updated. Using this, an amalgamated cattle genome database based on UMD_3.1, was created. The amalgamated cattle genome database is composed of a total of 33,292 genes: 19,123 consensus genes between NCBI and Ensembl databases, 8,410 and 5,493 genes only found in NCBI or Ensembl, respectively, and 266 genes from NCBI scaffolds. A metabolic reconstruction of the cattle genome and cattle pathway genome database (PGDB) was also developed using Pathway Tools, followed by an intensive manual curation. The manual curation filled or revised 68 pathway holes, deleted 36 metabolic pathways, and added 23 metabolic pathways. Consequently, the curated cattle PGDB contains 304 metabolic pathways, 2,460 reactions including 2,371 enzymatic reactions, and 4,012 enzymes. Furthermore, this study identified eight duplicated genes in 12 metabolic pathways in the cattle genome compared to human and mouse. Some of these duplicated genes are related with specific hormone biosynthesis and detoxifications. The updated genome-scale metabolic reconstruction is a useful tool for understanding biology and metabolic characteristics in cattle. There has been significant improvements in the quality of cattle genome annotations and the MetaCyc database. The duplicated metabolic genes in the cattle genome compared to human and mouse implies evolutionary changes in the cattle genome and provides a useful information for further research on understanding metabolic adaptations of cattle.     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

第二代测序技术用于水稻和稻瘟菌互作早期转录组的分析

稻瘟菌为了实现对水稻的有效侵染,在侵染水稻时可能通过表达和转运一定数量的效应蛋白进入到水稻细胞,抑制和干扰水稻的先天免疫机制。文章利用Solexa第二代测序技术,通过开展水稻和稻瘟菌互作早期转录组的测定和分析,克隆和鉴定在互作早期表达的稻瘟菌效应蛋白基因。利用序列同源比对,我们从总计约12.5 M条序列标签中,分离和鉴定了338 942条来源于稻瘟菌的序列,并最终定位到779个稻瘟菌预测基因。其中108个基因很可能参与了水稻和稻瘟菌互作过程,42个基因为预测的分泌蛋白基因。通过RT-PCR分析,最终确认了42个预测分泌蛋白基因中有12个基因在侵染水稻早期有显著的表达,而其中有4个基因表现为侵染早期特异表达。文章尝试利用第二代测序技术实现稻瘟菌侵染早期特异表达基因,尤其是分泌蛋白基因的快速克隆和鉴定,为稻瘟菌效应蛋白基因的克隆和功能鉴定提供了较为有意义的探索。     

Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.     

Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99-880 polygalacturonase gene family

A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with twelve genes encoding for endo-polygalacturonase enzymes and three genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygalacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times.     

Comparative genomics of two closely related unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae

Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria.     

Automated interpretation of metabolic capacity from genome and metagenome sequences

The KEGG pathway maps are widely used as a reference data set for inferring high-level functions of the organism or the ecosystem from its genome or metagenome sequence data. The KEGG modules, which are tighter functional units often corresponding to subpathways in the KEGG pathway maps, are designed for better automation of genome interpretation. Each KEGG module is represented by a simple Boolean expression of KEGG Orthology (KO) identifiers (K numbers), enabling automatic evaluation of the completeness of genes in the genome. Here we focus on metabolic functions and introduce reaction modules for improving annotation and signature modules for inferring metabolic capacity. We also describe how genome annotation is performed in KEGG using the manually created KO database and the computationally generated SSDB database. The resulting KEGG GENES database with KO (K number) annotation is a reference sequence database to be compared for automated annotation and interpretation of newly determined genomes.     

Genetic organization of the lexA,recA and recX genes in Xanthomonas campestris

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.     

Three new loci of insertion element IS1112 in Chinese strains of Xanthomonas oryzae pv. oryzae

Insertion sequence IS1112 is a repetitive element with a relatively high number of copies in Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice (Oryza sativa L.). Three new loci of IS1112 were identified in seven Chinese strains of Xoo using a single oligonucleotide primer J3; 5'-GCTCAGGTCAGGTGGCCTGG-3' by insertion-sequence-based polymerase chain reaction (IS-PCR). Among the three new loci of IS1112, two were located in the open-reading frame region of genes fhuA and cirA, which encode TonB-dependent receptors, and the third in ISXo2, another type of insertion sequence in Xoo genome. Three variants of IS1112 were identified in those three loci based on their sequence similarities: two were identical to IS1112a and IS1112b, reported in strain PXO86 from the Philippines, while the third was a new member of IS1112, defined as IS1112d. Inserting IS1112 in gene fhuA caused three bases, GGT, to be duplicated at the target site, but inserting it in gene cirA did not cause any duplication in the target site. The diversity of IS1112 sequence and insertion loci in Xoo genome and their potential effects are discussed.     

Functional analysis of <Emphasis Type="Italic">pilQ</Emphasis> gene in <Emphasis Type="Italic">Xanthomanas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>, bacterial blight pathogen of rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.     

Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is the most important fungal disease of rice. To understand the molecular basis of interaction between the fungus and rice, we constructed a cDNA library from a rice-resistant line inoculated with M. oryzae. One hundred and fifty-three cDNA clones were sequence analyzed, of which 129 exhibited significant nucleotide sequence homology to known genes, 21 were homologous to unknown genes, while three clones did not match to any database. However, these three unmatched clones showed sequence homology at protein level in the protein databases and one of them encoded a disease resistance-related protein kinase and was abundant in the EST collection. Northern analysis showed that this disease resistance-related protein kinase gene was induced by inoculation and only expressed in the rice-resistant, but not susceptible, lines. Southern analysis showed that this gene was present in a single copy in the rice genome and co-segregated with the M. oryzae resistance in the cross of the resistant and susceptible lines. This study illustrates that sequencing of ESTs from inoculated resistant plants can reveal genes responsive to pathogen infection, which could help understand plant defense mechanisms.     

A substitution mutation in OsPELOTA confers bacterial blight resistance by activating the salicylic acid pathway

We previously reported a spotted-leaf mutant pelota(originally termed HM_(47)) in rice displaying arrested growth and enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae. Here, we report the mapbased cloning of the causal gene OsPELOTA(originally termed spl~(HM47)). We identified a single base substitution from T to A at position 556 in the coding sequence of OsPELOTA, effectively mutating phenylalanine to isoleucine at position 186 in the translated protein sequence. Both functional complementation and over-expression could rescue the spotted-leaf phenotype. OsPELOTA, a paralogue to eukaryotic release factor 1(eRF_1), shows high sequence similarity to Drosophila Pelota and also localizes to the endoplasmic reticulum and plasma membrane.OsPELOTA is constitutively expressed in roots, leaves,sheaths, stems, and panicles. Elevated levels of salicylic acid and decreased level of jasmonate were detected in the pelota mutant. RNA-seq analysis confirmed that genes responding to salicylic acid were upregulated in the mutant. Our results indicate that the rice PELOTA protein is involved in bacterial leaf blight resistance by activating the salicylic acid metabolic pathway.     

A Survey of the Brassica rapa genome by BAC-end sequence analysis and comparison with Arabidopsis thaliana

Brassica rapa ssp. pekinensis (Chinese cabbage) is an economically important crop and a model plant for studies on polyploidization and phenotypic evolution. To gain an insight into the structure of the B. rapa genome we analyzed 12,017 BAC-end sequences for the presence of transposable elements (TEs), SSRs, centromeric satellite repeats and genes, and similarity to the closely related genome of Arabidopsis thaliana. TEs were estimated to occupy 14% of the genome, with 12.3% of the genome represented by retrotransposons. It was estimated that the B. rapa genome contains 43,000 genes, 1.6 times greater than the genome of A. thaliana. A number of centromeric satellite sequences, representing variations of a 176-bp consensus sequence, were identified. This sequence has undergone rapid evolution within the B. rapa genome and has diverged among the related species of Brassicaceae. A study of SSRs demonstrated a non-random distribution with a greater abundance within predicted intergenic regions. Our results provide an initial characterization of the genome of B. rapa and provide the basis for detailed analysis through whole-genome sequencing.