PDA Genes Research Articles

Lentivirus Mediated GOLPH3 shRNA Inhibits Growth and Metastasis of Esophageal Squamous Cancer

To investigate the role of Golgi phosphoprotein 3 (GOLPH3) in tumour growth and metastasis of esophageal squamous cancer. A lentiviral shRNA-vector was utilized to stably knockdown GOLPH3 in Eca-109 esophageal squamous cancer cells. mRNA transcription and protein expression of GOLPH3 were examined by real-time quantitative PCR and Western blotting, respectively. Cell proliferation activity was assessed by MTT assay and invasion and migration potentials by matrigel invasion and transwell motility assays. Stable knockdown in the GOLPH3 cell line was established. PD-A gene expression was significantly suppressed by lentivirus-mediated RNAi, which resulted in reducing the capacity for cell proliferation, migration, invasion and adhesion in vitro. In vivo, GOLPH3 depletion resulted in inhibition of tumour growth, with stable decrease in the expression of GOLPH3 in tumor xenografts. Our findings suggest that lentivirus mediated silencing of the GOLPH3 gene has a significant anti-tumour effect on esophageal squamous cancer in vitro and in vivo. In addition, the results indicate that GOLPH3 might be an effective molecular target for gene therapy in esophageal squamous cancer.

Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum.

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda(?) F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.

Introduction of plant and fungal genes into pea (Pisum sativum L.) hairy roots reduces their ability to produce pisatin and affects their response to a fungal pathogen.

Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less pisatin. To evaluate the role of pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.

Identification of new pisatin demethylase genes ( PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda + isolate (34-18) was used as the recurrent parent in backcrosses to Pda − isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 ( PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/ PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.

Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca.

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda- isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda-progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.

Pisatin demethylation by fungal pathogens and nonpathogens of pea: association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts' phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates ofPythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts' phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.

Biochemical properties of the products of cytochrome P450 genes (PDA) encoding pisatin demethylase activity in nectria haematococca

Pea plants produce the antibiotic (+)pisatin in response to infection by the fungus Nectria haematococca, which can detoxify pisatin utilizing a cytochrome P450 monooxygenase called pisatin demethylase. Genes (PDA) have been identified that encode different whole-cell Pda phenotypes that can be distinguished by the length of the lag period and the resulting amount of enzyme activity produced: PdaSH = short lag, high activity; PdaSM = short lag, moderate activity; and PdaLL = long lag, low activity. Only the PdaSH and PdaSM phenotypes have been correlated with pathogenicity on pea. In this study, we utilize heterologous expression of the PDALL gene PDA6-1 in Aspergillus nidulans to compare the biochemical properties of the product of this gene with the products of the PDASH gene PDA1 expressed in N. haematococca. Preliminary measurements were also done on the PDASM gene PDA5 expressed in N. haematococca. The PDA gene products differed somewhat in their substrate specificity and in their sensitivity to a few cytochrome P450 inhibitors. However, the enzymes produced by PDA6-1 and PDA1 both had low apparent Km values toward (+)pisatin (< 0.25 &mgr;M) and a common high degree of insensitivity to most P450 inhibitors, suggesting similar shared biochemical traits as would be expected of products of a highly homologous gene family. Our results indicate that the different whole-cell phenotypes of N. haematococca are not due to significant differences in the biochemical properties of the gene products and are consistent with recent results that indicate that the phenotypic differences are due to different degrees of expression of the genes.

Expression of the pisatin detoxifying genes (PDA) of Nectria haematococca in vitro and in planta.

The phytopathogenic fungus Nectria haematococca detoxifies pisatin, a phytoalexin produced by pea. Pisatin demethylating ability (a phenotype called Pda) is due to pisatin demethylase (pdm) and the genes encoding this enzyme are called PDA. Some isolates rapidly acquire a high to moderate rate of pisatin demethylating activity culture in response to pisatin (phenotypes PdaSH and PdaSM), while other isolates only slowly demethylate pisatin (phenotype PdaLL). Here we report that PDA-specific RNA levels increased more quickly in response to pisatin in isolates with PDA genes confering a PdaSH or PdaSM phenotype than in isolates with gene conferring a PdaLL phenotype. In addition, the pdm activity of transformants of N. haematococca containing chimeric constructs of PDASH and PDALL genes in which the 5' regulatory regions of these genes had been switched supports the conclusion that differential expression of PDA genes is responsible for the different Pda phenotypes detected in vitro. Northern analysis of pea tissue infected with isolates carrying PDASH or PDALL genes indicated that differential induction of these genes also occurred in planta. Only PDASH-specific RNA is readily detected in tissue infected with isolates containing PDASH and PDALL genes. Recently a pisatin biosynthetic gene, isoflavone reductase (IFR), has been identified. Using the polymerase chain reaction, qualitative detection of IFR and PDASH transcripts in infected tissue were made to assess the relative timing of these genes' expression. No transcripts were detected 6 h after inoculation, but transcripts of both genes were detected a 12 h, suggesting an interplay between the regulatory systems controlling the plants's defense response and the pathogen's counter response.

Characterization of the PDA1 promoter of Nectria haematococca and identification of a region that binds a pisatin-responsive DNA binding factor.

Isolates of Nectria haematococca (anamorph: Fusarium solani) are able to detoxify the pea phytoalexin pisatin through expression of pisatin demethylase (pda). This enzyme is a substrate-inducible cytochrome P450 monooxyenase that is encoded by the PDA gene family. In the current study, PDA1, a highly inducible PDA gene, was cloned and the 5' untranslated region was sequenced. The PDA mRNA levels were measured in pisatin-treated mycelium and found to increase by 20-fold over untreated control. Gel shift assays identified a 35-bp region, -514 to -480 bp relative to the first mRNA start site, that binds a factor found in extracts of pisatin-treated mycelium and absent in untreated mycelium. The function of the binding site in pisatin regulation of the PDA1 gene was tested in an in vivo competition assay by introduction of multiple ectopic copies of the binding site into N. haematococca through transformation. In such transformants, induction of pda activity by pisatin was delayed and reduced, consistent with the titration of a trans-acting factor which responds to pisatin. These results suggest the 35-bp region is functioning as a pisatin-responsive activator binding site for PDA1. Additional controls were characterized that act on PDA1 expression. Induction of pda by pisatin was suppressed by the addition of 0.8% Casamino Acids or 5% glucose to the suspended mycelium. A unique DNA binding factor was detected only in extracts from mycelia treated with the Casamino Acids that bind to the same 35-bp region of the PDA1 gene as the pisatin-responsive factor.

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectrla haematococca

The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda+ isolates) hybridized to PdaT9, while only one Pda- isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda- phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6-2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.

Genetic evidence for a single functional deficiency in isolates of Nectria haematococca unable to demethylate pisatin

Previous evidence suggests that pisatin-demethylating ability (PDA+) is necessary for tolerance of pisatin and for high virulence of Nectria haematococca on pea and that positive alleles at any one of at least three loci can give a PDA+ phenotype. A number of naturally occurring isolates of this fungus lack the ability to demethylate pisatin, but it is not known whether the PDA− phenotype is always due to the presence of negative alleles at all three of the previously studied genes. We have attempted to identify other functional classes of pda genes by searching for PDA+ recombinants among the ascospore progeny of crosses between PDA− isolates. A method was developed to detect rare PDA+ recombinants in a mixed culture containing a large number of PDA− progeny. No PDA+ recombinants were detected from 99 crosses of PDA− isolates when analysed by this method, nor were any detected among 171 ascospore progeny assayed individually. Complementary heterokaryons for the PDA+ phenotype were not formed from mixtures of 37 PDA− isolates when they were grown together on a pisatin-amended medium that was inhibitory to the PDA− isolates. No evidence was found to suggest that the PDA− phenotype can be caused by genes with different functions in N. haematococca.

Regulation of Pisatin Demethylation in Nectria haematococca and its Influence on Pisatin Tolerance and Virulence

Nectria haematococca, a fungal pathogen of pea, demethylates the pea isoflavonoid phytoalexin pisatin to yield the less inhibitory product, 3,6a-dihydroxy-8,9-methylenedioxypterocarpan. Among naturally occurring isolates that demethylate pisatin (PDA+ isolates), some can be induced to do so at a high rate by pretreatment with the substrate (PDA1 isolates), while others demethylate pisatin only slowly, regardless of pretreatment (PDAn isolates). Genetic analysis of these pisatin demethylation phenotypes has indicated that one gene confers the PDA1 phenotype and that two distinct genes at different loci each confer the PDAn phenotype. We report here the relationship of these PDA phenotypes to pisatin tolerance and to virulence toward pea. The contribution of each PDA gene to these traits was also evaluated. In initial crosses between tolerant, PDA+ parents, recombinant progeny lacking demethylating activity (PDA- isolates) occurred. These PDA1 progeny were uniformly more sensitive to pisatin than the PDA* parents and all PDA+ progeny. Progeny having PDA1 phenotypes were more tolerant to pisatin than PDAn progeny, which in turn were more tolerant than PDA1 phenotype. Further genetic analyses confirmed that a characteristic pattern of pisatin tolerance was associated with each PDA gene. The gene conferring the PDA1 phenotype was associated with the highest level of tolerance, while the two genes conferring PDAn phenotypes were each associated with an intermediate level of pisatin tolerance. Thus relative tolerance to pisatin appears to depend upon both the presence and amount of pisatin demethylase activity. Virulence toward pea may also be determined by the ability to demethylate pisatin rapidly. In numerous crosses between vV. haematococca isolates with different pisatin demethylation phenotypes, high virulence in progeny was always correlated to the PDA1 phenotype. AH PDAn and PDA1 progeny were low in virulence. Thus the gene that confers high pisatin demethylase activity in N. haematococca is, or is closely linked to, a gene for virulence to pea.