Functional characterization of a gene family encoding Polygalacturonases in Phytophthora parasitica.

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of plant species. In our previous study, we discovered a multigene family encoding endopolygalacturonases (endoPG) in Phytophthora parasitica. Here, we screened the genomic library of Phytophthora parasitica for the genes encoding endoPG named pppg2 through pppg10, and analyzed their functions. Results obtained by real-time quantitative reverse transcriptase-polymerase chain reaction demonstrated that some of these genes are highly induced during plant infection, which suggests their important roles in the pathogenesis of Phytophthora parasitica. Analysis by in-gel activity assay of recombinant proteins obtained from Pichia pastoris indicated that each of these genes encodes a functional endoPG. Investigation of the function of pppg genes in planta by a Potato virus X agroinfection system in tobacco revealed that each pppg caused specific effects, varying from no symptoms to dwarfism, necrosis, leaf curl, silvery leaf, and cracks in leaf stalks. Appearance of these effects depends on the expression of a pppg protein with a normal active site in the apoplast. These results indicated that each pppg plays a distinct role in the decomposition of plant cell wall.

A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance.

The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

Selection of internal control genes for real-time quantitative RT-PCR assays in the oomycete plant pathogen Phytophthora parasitica.

Real-time quantitative reverse transcription-PCR (qRT-PCR) has become one of the most commonly used methods for RNA quantification in recent years. To obtain reliable results with biological significance, it is important that qRT-PCR data are normalized with a proper internal control. In this study, 18 housekeeping genes were selected and evaluated for their potential as a suitable internal control for study of gene expression in the oomycete plant pathogen Phytophthora parasitica. Analysis of qRT-PCR data using the geNorm software indicated that, although commonly used as internal controls, beta-actin (ACT) and translation elongation factor 1alpha (eEF1A) might not be the best choice due to variable expression across different life stages of P. parasitica. Instead, other genes would serve as better controls, including ubiquitin-conjugating enzyme (Ubc), WS21, and beta-tubulin (Tub-b) for 'asexual stage,' Ubc and Tub-b for 'sexual reproduction,' while Ubc and WS21 for the stage of pathogenesis, because of their constant expression levels in each given subset of RNA samples. Although normalization with more than one gene would generate more reliable results, use of a single stably expressed gene as an internal control would suffice for accurate data normalization in some experiments.

Cloning and analysis of pppg1, an inducible endopolygalacturonase gene from the oomycete plant pathogen Phytophthora parasitica.

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of crops. Here, we report the isolation of a gene, named pppg1, which encodes an extracellular endopolygalacturonase in P. parasitica. Both cDNA and a genomic clone were isolated and sequenced. The pppg1 gene showed standard characteristics with respect to core promoter and intron sequences of Phytophthora. The predicted protein of pppg1 has a calculated molecular mass of 39.7 kDa and a pI value of 5.2, and contains a putative signal peptide of 20 amino acid residues on the N-terminus. The deduced amino acid sequence is highly conserved with those of other Phytophthora and fungal endopolygalacturonases. Analysis by reverse transcription followed by real-time quantitative polymerase chain reaction showed that transcription of pppg1 was repressed by glucose, but induced by pectin in the culture. Moreover, pppg1 is highly expressed during interaction of P. parasitica with the host plant, suggesting its involvement in the process of host infection. Heterologous expression of pppg1 in Pichia pastoris produced proteins with molecular mass ranging from 75 to 200 kDa, very likely due to differential glycosylation by the yeast. Deglycosylation of the recombinant protein resulted in a complete loss of the endopolygalacturonase activity.