Transcription profiling and identification of infection-related genes in Phytophthora cactorum.

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8 million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.

SCR96, a small cysteine-rich secretory protein of Phytophthora cactorum, can trigger cell death in the Solanaceae and is important for pathogenicity and oxidative stress tolerance.

Peptides and small molecules produced by both the plant pathogen Phytophthora and host plants in the apoplastic space mediate the relationship between the interplaying organisms. Various Phytophthora apoplastic effectors, including small cysteine-rich (SCR) secretory proteins, have been identified, but their roles during interaction remain to be determined. Here, we identified an SCR effector encoded by scr96, one of three novel genes encoding SCR proteins in P. cactorum with similarity to the P. cactorum phytotoxic protein PcF. Together with the other two genes, scr96 was transcriptionally induced throughout the developmental and infection stages of the pathogen. These genes triggered plant cell death (PCD) in the Solanaceae, including Nicotiana benthamiana and tomato. The scr96 gene did not show single nucleotide polymorphisms in a collection of P. cactorum isolates from different countries and host plants, suggesting that its role is essential and non-redundant during infection. Homologues of SCR96 were identified only in oomycetes, but not in fungi and other organisms. A stable protoplast transformation protocol was adapted for P. cactorum using green fluorescent protein as a marker. The silencing of scr96 in P. cactorum caused gene-silenced transformants to lose their pathogenicity on host plants and these transformants were significantly more sensitive to oxidative stress. Transient expression of scr96 partially recovered the virulence of gene-silenced transformants on plants. Overall, our results indicate that the P. cactorum scr96 gene encodes an important virulence factor that not only causes PCD in host plants, but is also important for pathogenicity and oxidative stress tolerance.

Effect of retS gene on antibiotics production in Pseudomonas fluorescens FD6.

A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6.

Transcriptomic analysis of the phytopathogenic oomycete Phytophthora cactorum provides insights into infection-related effectors.

BACKGROUND: Phytophthora cactorum, a hemibiotrophic oomycete pathogen, can cause destructive diseases on numerous crops worldwide, leading to essential economic losses every year. However, little has been known about its molecular pathogenicity mechanisms. To gain insight into its repertoire of effectors, the P. cactorum transcriptome was investigated using Illumina RNA-seq. RESULTS: We first demonstrated an in vitro inoculation method that can be used to mimic natural cyst germination on host plants. Over 28 million cDNA reads were obtained for five life cycle stages (mycelium, sporangium, zoospore, cyst and germinating cyst) and de novo assembled into 21,662 unique genes. By comparisons with 11 public databases, 88.99% of the unique genes were annotated, including 15,845 mapped to the gene models of the annotated relative Phytophthora infestans. Using TribeMCL, 5,538 gene families conserved across P. cactorum and other three completely sequenced Phytophthora pathogen species were determined. In silico analyses revealed that 620 P. cactorum effector homologues including 94 RXLR effector candidates matched known or putative virulence genes in other oomycetes. About half of the RXLR effector candidates were predicted to share a conserved structure unit, termed the WY-domain fold. A subset of the effector genes were checked and validated by PCR amplification. Transcriptional experiments indicated that effector genes were differentially expressed during the life cycle and host infection stages of P. cactorum. Ectopic expression in Nicotiana benthamiana revealed that RXLR, elicitin and NLP effectors can trigger plant cell death. These effectors are highly conserved across oomycete species. Single nucleotide polymorphisms for RXLR effectors were detected in a collection of P. cactorum isolates from different countries and hosts. CONCLUSIONS: This study demonstrates the comprehensive sequencing, de novo assembly, and analyses of the transcriptome of P. cactorum life cycle stages. In the absence of genome sequence, transcriptome data is important for infection-related gene discovery in P. cactorum, as demonstrated here for the effector genes. The first look at the transcriptome and effector arsenal of P. cactorum provides valuable data to elucidate the pathogenicity basis of this broad-host-range pathogen.

[Cloning, prokaryotic expression and bioinformatics of Rspg1 gene of Rhizoctonia solani].

OBJECTIVE: The study was aimed at understanding the roles of polygalacturonases in the pathogenicity and the interaction between Rhizoctonia solani and rice. METHODS: According to the sequences of Rspg1 of R. solani deposited in GenBank, a pair of specific primers was designed. The gene Rspg1 was cloned and expressed using prokaryotic expression tool to elucidate its biological characteristics. The structures of the protein RsPG1 were predicted using bioinformatics tools. RESULTS: A 1395-bp fragment including an open reading frame (OFR) of Rspg1 was amplified from the genomic DNA of the pathogen. Compared with RT-PCR results, it was found that this sequence fragment contains five introns (positions 278-334, 545-601, 657-715, 1090-1155 and 1244-1304) and one 1095 bp ORF. The ORF was predicted to encode 364 amino acids. Bioinformatics analysis showed that RsPG1 contains an 18-amino acid signal peptide and 4 conserved sequence segments (180NTD, 202DD, 223GHG and 255RIK) characteristic of all the polygalacturonases. The main structural elements of the secondary structure are alpha-helix, beta-sheet and random coil. Six cysteines form three disulfide bonds (Cys24-Cys40, Cys204-Cys220 and Cys329-Cys333). Transmembrane prediction analysis suggested that RsPG1 could be secreted outside the cell. Tertiary structure is a right-handed helix which consisted of ten repeated beta-sheet, forming an opening activity cleft. CONCLUSION: RsPG1 is tentatively a 40 kDa protein with polygalacturonase enzyme activity at 277.78 U/mg. It is probably a secreted protein and has characteristics of all the polygalacturonases. The results can help to further understand the roles that R. solani polygalacturonases play during the pathogenicity and how the pathogen interacts with the host.

RNA-Seq reveals infection-related gene expression changes in Phytophthora capsici.

Phytophthora capsici is a soilborne plant pathogen capable of infecting a wide range of plants, including many solanaceous crops. However, genetic resistance and fungicides often fail to manage P. capsici due to limited knowledge on the molecular biology and basis of P. capsici pathogenicity. To begin to rectify this situation, Illumina RNA-Seq was used to perform massively parallel sequencing of three cDNA samples derived from P. capsici mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). Over 11 million reads were generated for each cDNA library analyzed. After read mapping to the gene models of P. capsici reference genome, 13,901, 14,633 and 14,695 putative genes were identified from the reads of the MY, ZO and GC libraries, respectively. Comparative analysis between two of samples showed major differences between the expressed gene content of MY, ZO and GC stages. A large number of genes associated with specific stages and pathogenicity were identified, including 98 predicted effector genes. The transcriptional levels of 19 effector genes during the developmental and host infection stages of P. capsici were validated by RT-PCR. Ectopic expression in Nicotiana benthamiana showed that P. capsici RXLR and Crinkler effectors can suppress host cell death triggered by diverse elicitors including P. capsici elicitin and NLP effectors. This study provides a first look at the transcriptome and effector arsenal of P. capsici during the important pre-infection stages.

[Isolation, purification and characterization of antifungal protein from rice endophytic bacterim Bacillus subtilis G87].

OBJECTIVE: In order to obtain the antagonistic protein of Bacillus subtilis G87 and definitude its characterization. METHODS: Methods of ammonium sulfate precipitating and column chromatography analyzing were used to isolate and purify the protein. RESULTS: A purified protein (peak 6-2-1) was obtained which molecular weight was 50.8 kD by SDS-PAGE and isoelectric point was 5.90 by IEF-PAGE. The antifungal protein contained 0.62% saccharide and some proline or hydroxyproline, but no lipid and aromatic amino acid. The inhibitory activity of the antifungal protein would decreased distinctly at the higher temperature (> or = 60 degrees C) and in the condition of alkalinity (pH > 8), but tolerant to ultraviolet radiation, chloroform, trypsin, proteinase K and pepsin. CONCLUSION: Antifungal protein of Bacillus subtilis G87 was a kind of glycoprotein without aromatic hydrocarbon. It was sensitive to higher temperature and tight alkalinity but not to proteinase analog and ultraviolet radiation et al.