大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.     

Strategies of attack and defense in plant-oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan)

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant-pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

A Phytophthora sojae G-protein alpha subunit is involved in chemotaxis to soybean isoflavones

For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein alpha subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.     

Expressed sequence tags from phytophthora sojae reveal genes specific to development and infection

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象,该文通过建立拟南芥．大豆疫霉菌（Arabidopsis thaliana—Phytophthora sojae）非寄主互作系统,筛选对大豆疫霉菌感病的拟南芥突变体,为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T—DNA插入突变体离体叶片,从代表12000个独立转化株系的40000株T3代T。DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定,离体叶片接菌后3天内出现明显的水渍状病斑,4—5天后产生大量卵孢子和／或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明,581—51突变体含有4个T-DNA插入事件,其感病性状可能由隐性单基因控制。     

Genomewide analysis of phospholipid signaling genes in Phytophthora spp.: novelties and a missing link

Phospholipids are cellular membrane components in eukaryotic cells that execute many important roles in signaling. Genes encoding enzymes required for phospholipid signaling and metabolism have been characterized in several organisms, but only a few have been described for oomycetes. In this study, the genome sequences of Phytophthora sojae and P. ramorum were explored to construct a comprehensive genomewide inventory of genes involved in the most universal phospholipid signaling pathways. Several genes and gene families were annotated, including those encoding phosphatidylinositol synthase (PIS), phosphatidylinositol (phosphate) kinase (PI[P]K), diacylglycerol kinase (DAG), and phospholipase D (PLD). The most obvious missing link is a gene encoding phospholipase C (PLC). In all eukaryotic genomes sequenced to date, PLC genes are annotated based on certain conserved features; however, these genes seem to be absent in Phytophthora spp. Analysis of the structural and regulatory domains and domain organization of the predicted isoforms of PIS, PIK, PIPK, DAG, and PLD revealed many novel features compared with characterized representatives in other eukaryotes. Examples are transmembrane proteins with a C-terminal catalytic PLD domain, secreted PLD-like proteins, and PIPKs that have an N-terminal G-protein-coupled receptor-transmembrane signature. Compared with other sequenced eukaryotes, the genus Phytophthora clearly has several exceptional features in its phospholipid-modifying enzymes.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

Comparative analysis of Phytophthora genes encoding secreted proteins reveals conserved synteny and lineage-specific gene duplications and deletions

Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of P. ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements also have occurred. Deletions and duplications often were found in association with genes encoding secreted proteins, including effectors that are important for interaction with host plants. Among secreted protein genes, different evolutionary patterns were found. Elicitin genes that code for a complex family of highly conserved Phytophthora-specific elicitors show conservation in gene number and order, and often are clustered. In contrast, the race-specific elicitor gene Avrlb-1 appeared to be missing from the region with conserved synteny, as were its five homologs that are scattered over the four genomic regions. Some gene families encoding secreted proteins were found to be expanded in one species compared with the other. This could be the result of either repeated gene duplications in one species or specific deletions in the other. These different evolutionary patterns may shed light on the functions of these secreted proteins in the biology and pathology of the two Phytophthora spp.     

Gene duplication event in family 12 glycosyl hydrolase from Phytophthora spp

A total of 18 paralogs of xyloglucan-specific endoglucanases (EGLs) from the glycosyl hydrolase family 12 were identified and characterized in Phytophthora sojae and Phytophthora ramorum. These genes encode predicted extracellular enzymes, with sizes ranging from 189 to 435 amino acid residues, that would be capable of hydrolyzing the xyloglucan component of the host cell wall. In two cases, four and six functional copies of these genes were found in tight succession within a region of 5 and 18 kb, respectively. The overall gene copy number and relative organization appeared well conserved between P. sojae and P. ramorum, with apparent synteny in this region of their respective genomes. Phylogenetic analyses of Phytophthora endoglucanases of family 12 and other known members of EGL 12, revealed a close relatedness with a fairly conserved gene sub-family containing, among others, sequences from the fungi Emericella desertorum and Aspergillus aculeatus. This is the first report of family 12 EGLs present in plant pathogenic eukaryotes.     

Specific molecular detection of Phytophthora sojae using conventional and real-time PCR

Phytophthora rot, caused by Phytophthora sojae, is one of the most damaging diseases of soybean (Glycine max) worldwide. This disease can be difficult to diagnose and other Phytophthora species can infect soybean. Accurate diagnosis is important for management of Phytophthora rot. The objective of this study was to evaluate polymerase chain reaction (PCR) methods for rapid and specific detection of P. sojae and diagnosis of Phytophthora rot. PCR assays using two sets of primers (PS and PSOJ) that target the ITS region were evaluated for specificity and sensitivity to P. sojae. Genomic DNA extracted from 11 species of Phytophthora and 19 other species of fungal and oomycete pathogens were used to test the specificity of each primer set. The previously published PS primers amplified DNA from P.?sojae and from four other Phytophthora species using conventional PCR, indicating they are not specific for P. sojae. The new PSOJ primers amplified DNA only from P. sojae using conventional and real-time PCR and not from Phytophthora sansomeana, which has been found in soybean production areas, indicating that they are specific for P. sojae. The PSOJ primers were also used to detect P. sojae in diseased soybean tissue and infested soil. The PCR assays based on the PSOJ primers are specific, rapid, and sensitive tools for the detection of P. sojae.     

Extensive variation in nuclear mitochondrial DNA content between the genomes of Phytophthora sojae and Phytophthora ramorum

Fragments of mitochondrial DNA (mtDNA) transferred to the nuclear genome are called nuclear mitochondrial DNAs (NUMTs). We report here a comparison of NUMT content between genomes from two species of the same genus. Analysis of the genomes of Phytophthora sojae and P. ramorum revealed large differences in the NUMT content of the two genomes: 16.27 x 10(-3) and 2.28 x 10(-3)% of each genome, respectively. Substantial differences also exist between the two species in the sizes of the NUMTs found in each genome, with ranges of 20 to 405 bp for P. sojae and 19 to 137 bp for P. ramorum. Furthermore, in P. sojae, fragments from the mitochondrial genes rns, rnl, coxl, and nad (various subunits) are found most frequently, whereas P. ramorum NUMTs most often originate from the cox3, rpsl4, nad4, and nad5 genes. The large differences in the presumptive mtDNA insertions suggest that the insertions occurred subsequent to the divergence of the two species, and this is supported by sequence comparisons among the NUMTs and the mtDNA sequences of the two species. P. sojae mtDNA sequences inserted in the nuclear genome appear to have been altered as a result of insertions, deletions, inversions, and translocations and provide insights into active mechanisms of sequence divergence in this plant pathogen. No clear examples were found of NUMTs forming functional nuclear genes or of NUMTs inserted into exons or introns of any nuclear gene.     

The repertoire of transfer RNA genes is tuned to codon usage bias in the genomes of Phytophthora sojae and Phytophthora ramorum

In all, 238 and 155 transfer (t)RNA genes were predicted from the genomes of Phytophthora sojae and P. ramorum, respectively. After omitting pseudogenes and undetermined types of tRNA genes, there remained 208 P. sojae tRNA genes and 140 P. ramorum tRNA genes. There were 45 types of tRNA genes, with distinct anticodons, in each species. Fourteen common anticodon types of tRNAs are missing altogether from the genome in the two species; however, these appear to be compensated by wobbling of other tRNA anticodons in a manner which is tied to the codon bias in Phytophthora genes. The most abundant tRNA class was arginine in both P. sojae and P. ramorum. A codon usage table was generated for these two organisms from a total of 9,803,525 codons in P. sojae and 7,496,598 codons in P. ramorum. The most abundant codon type detected from the codon usage tables was GAG (encoding glutamic acid), whereas the most numerous tRNA gene had a methionine anticodon (CAT). The correlation between the frequencies of tRNA genes and the codon frequencies in protein-coding genes was very low (0.12 in P. sojae and 0.19 in P. ramorum); however, the correlation between amino acid tRNA gene frequency and the corresponding amino acid codon frequency in P. sojae and P. ramorum was substantially higher (0.53 in P. sojae and 0.77 in P. ramorum). The codon usage frequencies of P. sojae and P ramorum were very strongly correlated (0.99), as were tRNA gene frequencies (0.77). Approximately 60% of orthologous tRNA gene pairs in P sojae and P. ramorum are located in regions that have conserved synteny in the two species.     

Characterization of intronic structures and alternative splicing in Phytophthora sojae by comparative analysis of expressed sequence tags and genomic sequences

The oomycetes, a distinct phylogenetic lineage of fungus-like microorganisms, are heterokonts (stramenopiles) belonging to the supergroup Chromalveolata. Although the complete genomic sequences of a number of oomycetes have been reported, little information regarding the introns therein is available. Here, we investigated the introns of Phytophthora sojae, a pathogen that causes soybean root and stem rot, by a comparative analysis of genomic sequences and expressed sequence tags. A total of 4013 introns were identified, of which 96.6% contained canonical splice sites. The P. sojae genome possessed features distinct from other organisms at 5' splice sites, polypyrimidine tracts, branch sites, and 3' splice sites. Diverse repeating sequences, ranging from 2 to 10 nucleotides in length, were found at more than half of the intron-exon boundaries. Furthermore, 122 genes underwent alternative splicing. These data indicate that P. sojae has unique splicing mechanisms, and recognition of those mechanisms may lead to more accurate predictions of the location of introns in P. sojae and even other oomycete species.     

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences.

Phytophthora species are devastating plant pathogens in both agricultural and natural environments. Due to their significant economic and environmental impact, there has been increasing interest in Phytophthora genetics and genomics, culminating in the recent release of three complete genome sequences (P. ramorum, P. sojae, and P. infestans). In this study, genome and other large sequence databases were used to identify over 225 potential genetic markers for phylogenetic analyses. Here, we present a genus-wide phylogeny for 82 Phytophthora species using seven of the most informative loci (approximately 8700 nucleotide sites). Our results support the division of the genus into 10 well-supported clades. The relationships among these clades were rigorously evaluated using a number of phylogenetic methods. This is the most comprehensive study of Phytophthora relationships to date, and many newly discovered species have been included. A more resolved phylogeny of Phytophthora species will allow for better interpretations of the overall evolutionary history of the genus.     

Computational and comparative analyses of 150 full-length cDNA sequences from the oomycete plant pathogen Phytophthora infestans

Phytophthora infestans is a devastating phytopathogenic oomycete that causes late blight on tomato and potato. Recent genome sequencing efforts of P. infestans and other Phytophthora species are generating vast amounts of sequence data providing opportunities to unlock the complex nature of pathogenesis. However, accurate annotation of Phytophthora genomes will be a significant challenge. Most of the information about gene structure in these species was gathered from a handful of genes resulting in significant limitations for development of ab initio gene-calling programs. In this study, we collected a total of 150 bioinformatically determined near full-length cDNA (FLcDNA) sequences of P. infestans that were predicted to contain full open reading frame sequences. We performed detailed computational analyses of these FLcDNA sequences to obtain a snapshot of P. infestans gene structure, gauge the degree of sequence conservation between P. infestans genes and those of Phytophthora sojae and Phytophthora ramorum, and identify patterns of gene conservation between P. infestans and various eukaryotes, particularly fungi, for which genome-wide translated protein sequences are available. These analyses helped us to define the structural characteristics of P. infestans genes using a validated data set. We also determined the degree of sequence conservation within the genus Phytophthora and identified a set of fast evolving genes. Finally, we identified a set of genes that are shared between Phytophthora and fungal phytopathogens but absent in animal fungal pathogens. These results confirm that plant pathogenic oomycetes and fungi share virulence components, and suggest that eukaryotic microbial pathogens that share similar lifestyles also share a similar set of genes independently of their phylogenetic relatedness.