Abstract: Clones of genes activated in mating cultures of A1 and A2 mating-type strains of Phytophthora infestans were isolated using the cDNA-representational difference analysis subtraction method. Clone cET58 was selected based on its accumulation in mating cultures and then was used as a probe to isolate cDNA clone cET58L2 from a cDNA library that was constructed from mycelia grown under mating conditions. Sequence analysis revealed that cET58L2 was 1043thinspbp long and contained a complete open reading frame of 789thinspbp. The amino acid sequence of the putative protein was similar to a pectate lyase, PLD, of Fusarium solani f. sp. pisi. The central region of the predicted protein was highly similar to the sequence of other pectate lyases. The gene from which the cDNA clones were derived was designated mpl1. A probe corresponding to the protein-coding region of mpl1 was prepared (probe p58L) for Northern and Southern analyses. The maximum rate of oogonia increase and mpl1 transcript accumulation reached a maximum after 5 days in mating culture. More than 13 genes with sequences similar to that of mpl1 were found in the genome, revealing mpl1 to be a multicopy gene. The mpl1 may be a pectate lyase gene that is activated in P. infestans during mating.
Abstract: The deletion of glnA, encoding the glutamine synthetase (GS), had led to the down-regulation of genes involved in secondarymetabolismand up-regulation of cpc1, the cross-pathway control transcription factor. In the present study, a Dcpc1 mutant was created and used for transcriptional profiling bymacroarray analysis. Most of the Cpc1 target genes were amino acid biosynthesis genes besides a homologue of the multi-protein bridging factor MBF1 that binds to the yeast Cpc1 homologue GCN4. We show that Dmbf1 mutants exhibit no Cpc1-related phenotype and that both proteins do not interact with each other in Fusarium fujikuroi. Moreover, results presented here suggest that Cpc1 is not responsible for the GS-dependent down-regulation of secondary metabolism and that its role is focused on the activation of amino acid biosynthesis in response to the amino acid status of the cell. Surprisingly, cross-pathway control is repressed by nitrogen limitation in an AreA-dependent manner.
Abstract: Transcription factors containing two or three imperfect tandem repeats of the Myb DNA-binding domain (named R2R3 and R1R2R3, respectively) regulate important processes in growth and development. This study characterizes the structure, evolution, and expression of these proteins in the potato pathogen Phytophthora infestans and other oomycetes. P. infestans was found to encode five R2R3 and nine R1R2R3 transcription factor-like proteins, plus several with additional configurations of Myb domains. Sets of R2R3 and R1R2R3 orthologs are well-conserved in three Phytophthora species. Analyses of sites that bind DNA in canonical Myb transcription factors, such as mammalian c-Myb, revealed unusual diversification in the DNA recognition helices of the oomycete proteins. While oomycete R2R3 proteins contain c-Myb-like helices, R1R2R3 proteins exhibit either c-Myb-like or novel sequences. This suggests divergence in their DNA-binding specificities, which was confirmed by electrophoretic mobility shift assays. Eight of the P. infestans R2R3 and R1R2R3 genes are up-regulated during sporulation and three during zoospore release, which suggests their involvement in spore development. This is supported by the observation that an oomycete that does not form zoospores, Hyaloperonospora arabidopsidis, contains one-third fewer of these genes than Phytophthora.
Abstract: Really interesting new gene (RING) finger proteins function as ubiquitin ligase and play key roles in biotic and abiotic stresses. A new RING-H2 finger protein gene, StRFP1, was cloned from Phytophthora infestans-inoculated leaves of potato (Solanum tuberosum) clone 386209.10, which is free of R1-R11 genes. The deduced amino acid sequence was characterized by an N-terminal transmembrane domain, a GLD region and a RING-H2 finger signature. StRFP1 is homologous to the tobacco NtACRE132 protein and belongs to the ATL family. The DNA gel blot analysis and mapping revealed that StRFP1, an intron-free gene, had one to two copies in the potato genome and was located on chromosome 3. RT-PCR assays showed that StRFP1 was constitutively expressed in potato plants and significantly induced in detached potato leaves by P. infestans and plant defense-related signal molecules, abscisic acid, salicylic acid and methyl jasmonate. Transient expression studies revealed that StRFP1 fused with GFP localized to the plasma membrane or out of that in onion epidermal cells. The function of StRFP1 in potato resistance against late blight was further investigated by constructing overexpression and RNA interference (RNAi) vectors, which were introduced into potato cv. E-potato 3, respectively. By challenging the detached leaves with mixture races of P. infestans, all of the StRFP1-overexpressing plants displayed slower disease development than non-transformed controls in terms of the lesion growth rate (LGR). In contrast, StRFP1-silencing plants through RNAi were more susceptible to pathogen infection. The present results demonstrate that StRFP1 contributes to broad-spectrum resistance against P. infestans in potato.
Abstract: Background: Oomycete pathogens have attracted significant attention in recent years due to their economic impact. With improving sequencing technologies, large amounts of oomycete transcriptomics data are now available which have great biological utility. A known bottleneck with next generation sequencing data however lies with their analysis, interpretation, organization, storage and visualization. A number of efforts have been made in this respect resulting in development of a myriad of resources. Most of the existing NGS browsers work as standalone applications that need processed data to be uploaded to the browser locally for visualization. At the same time, several oomycete EST databases such as PFGD, ESTAP and SPC, are not available anymore, so there is an immediate need for a database resource that can store and disseminate this legacy information in addition to NGS data. Description Oomycetes Transcriptomics Database is an integrated transcriptome and EST data resource for oomycete pathogens. The database currently stores processed ABI SOLiD transcript sequences from Phytophthora sojae and its host soybean (P. sojae mycelia, healthy soybean and P. sojae-infected soybean) as well as Illumina transcript sequences from five Hyaloperonospora arabidopsidis libraries. In addition to those resources, it has also a complete set of Sanger EST sequences from P. sojae, P. infestans and H. arabidopsidis grown under various conditions. A web-based transcriptome browser was created for visualization of assembled transcripts, their mapping to the reference genome, expression profiling and depth of read coverage for particular locations on the genome. The transcriptome browser merges EST-derived contigs with NGS-derived assembled transcripts on the fly and displays the consensus. OTD possesses strong query features and the database interacts with the VBI Microbial Database as well as the Phytophthora Transcriptomics Database. Conclusion: Oomycete Transcriptomics Database provides access to NGS transcript and EST data for oomycete pathogens and soybean. The OTD browser is a light weight transcriptome browser that displays the raw read alignment as well as the transcript assembly and expression information quantitatively. The query features offer a wide variety of options including querying data from the VBI microbial database and the Phytophthora transcriptomics database. The database is publicly available at http://www.eumicrobedb.org/transcripts/.
Abstract: Black shank, caused by the pathogen Phytophthora nicotianae, is an important disease affecting tobacco (Nicotiana tabacum L.) production in many parts of the world. Host resistance offers an efficient means of reducing economic loss due to this pathogen. Previous observations suggested that a genomic region introgressed from Nicotiana rustica L. may affect resistance to black shank. The effect of this genomic region, designated as Wz, on resistance to multiple races of P. nicotianae had not previously been investigated in a systematic way, however. We used growth chamber and field experiments to evaluate resistance of a doubled haploid (DH) mapping population segregating for the Wz region. We also genotyped the population with amplified fragment length polymorphism (AFLP) markers found to be polymorphic between the parental lines. In addition, we determined resistance and marker genotypes for individuals of a larger BC1F1 population segregating for Wz. A set of 29 AFLP markers determined to be of N. rustica origin were found to cosegregate with each other and were associated with resistance to both race 0 and race 1 of P. nicotianae in the DH population. A selected subset of these markers was also found to cosegregate with resistance in the BC1F1 population. Consistent with other introgressed alien genomic regions in N. tabacum, no recombination was observed between these selected markers. The N. rustica-derived genetic variation and associated DNA markers will be of value for breeding for black shank resistance in tobacco.
Abstract: Black shank, caused by Phytophthora nicotianae, is typically the most important disease affecting tobacco (Nicotiana tabacum L.) production in the United States. Pedigree information suggests that most black shank resistance was derived from the cigar tobacco cultivar Florida 301. This resistance is thought to be polygenic in nature. The objectives of the current experiment were to (i) evaluate lines from a recombinant inbred line population derived from a cross between Florida 301 and the black shank-susceptible cultivar Hicks for partial resistance using replicated field and greenhouse testing, (ii) genotype the population and use quantitative trait loci (QTL) analyses to identify Florida 301 genomic regions associated with resistance, and (iii) compare results with those obtained from a previous QTL analysis of a population derived from a cross involving 'Beinhart 1000'. A total of 11 QTL affecting area under the disease progress curve were identified in both the field and greenhouse experiments. The QTL with the largest effect explained 16.9 and 18.6% of the phenotypic variation in the field and greenhouse experiments, respectively. This QTL was also found to have the largest effect on resistance in a Beinhart 1000 × Hicks doubled haploid mapping population. A major QTL found to affect resistance on linkage group 15 in the latter population, however, was not found to be important in the current population. Quantitative trait loci identification using greenhouse data was comparable to, if not superior to, use of field data.
Abstract: Background: Genes for the production of a broad range of fungal secondary metabolites are frequently colinear. The prevalence of such gene clusters was systematically examined across the genome of the cereal pathogen Fusarium graminearum. The topological structure of transcriptional networks was also examined to investigate control mechanisms for mycotoxin biosynthesis and other processes. Results: The genes associated with transcriptional processes were identified, and the genomic location of transcription-associated proteins (TAPs) analyzed in conjunction with the locations of genes exhibiting similar expression patterns. Highly conserved TAPs reside in regions of chromosomes with very low or no recombination, contrasting with putative regulator genes. Co-expression group profiles were used to define positionally clustered genes and a number of members of these clusters encode proteins participating in secondary metabolism. Gene expression profiles suggest there is an abundance of condition-specific transcriptional regulation. Analysis of the promoter regions of co-expressed genes showed enrichment for conserved DNA-sequence motifs. Potential global transcription factors recognising these motifs contain distinct sets of DNA-binding domains (DBDs) from those present in local regulators. Conclusions: Proteins associated with basal transcriptional functions are encoded by genes enriched in regions of the genome with low recombination. Systematic searches revealed dispersed and compact clusters of co-expressed genes, often containing a transcription factor, and typically containing genes involved in biosynthetic pathways. Transcriptional networks exhibit a layered structure in which the position in the hierarchy of a regulator is closely linked to the DBD structural class.
Abstract: The complete genome of the biocontrol antagonist Bacillus amyloliquefaciens AS 43.3 is reported. B. amyloliquefaciens AS 43.3 has previously been shown to be effective in reducing Fusarium head blight in wheat. The 3.9 Mbp genome was sequenced, assembled, and annotated. Genomic analysis of the strain identified 9 biosynthetic gene clusters encoding secondary metabolites associated with biocontrol activity. The analysis identified five non-ribosomal peptide synthetase clusters encoding three lipopeptides (surfactin, iturin, and fengycin), a siderophore (bacillibactin), and the antibiotic dipeptide bacilysin. In addition, three polyketide synthetase clusters were identified which encoded for the antibacterials: bacillaene, difficidin, and macrolactin. In addition to the non-ribosomal mediated biosynthetic clusters discovered, we identified a ribosomally encoded biosynthetic cluster that produces the antibiotic plantazolicin. To confirm the gene clusters were functional, cell-free culture supernatant was analyzed using LC-MS/MS. The technique confirmed the presence of all nine metabolites or their derivatives. The study suggests the strain is most likely a member of the B. amyloliquefaciens subsp. plantarium clade. Comparative genomics of eight completed genomes of B. amyloliquefaciens identify the core and pan-genomes for the species, including identifying genes unique to the biocontrol strains. This study demonstrates the growing importance of applying genomic-based studies to biocontrol organisms of plant pathogens which can enable the rapid identification of bioactive metabolites produced by a prospective biological control organism. In addition, this work provides a foundation for a mechanistic understanding of the B. amyloliquefaciens AS 43.3/Fusarium head blight biocontrol interaction.
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