Abstract
Plant cell walls are pivotal battlegrounds between microbial pathogens and their hosts. To penetrate the cell wall and thereby to facilitate infection, microbial pathogens are equipped with a wide array of cell wall-degrading enzymes to depolymerize the polysaccharides in the cell wall. However, many of these enzymes and their role in the pathogenesis of microbial pathogens are not characterized, especially those from Oomycetes. In this study, we analyzed the function of four putative endo-beta-1,4-xylanase-encoding genes (ppxyn1–ppxyn4) from Phytophthora parasitica, an oomycete plant pathogen known to cause severe disease in a wide variety of plant species. All four genes belong to the glycoside hydrolase family 10 (GH10). Recombinant proteins of ppxyn1, ppxyn2, and ppxyn4 obtained from the yeast Pichia pastoris showed degrading activities toward birch wood xylan, but they behaved differently in terms of the conditions for optimal activity, thermostability, and durability. Quantitative RT-PCR revealed upregulated expression of all four genes, especially ppxyn1 and ppxyn2, during plant infection. In contrast, ppxyn3 was highly expressed in cysts and its close homolog, ppxyn4, in germinating cysts. To uncover the role of ppxyn1 and ppxyn2 in the pathogenesis of P. parasitica, we generated silencing transformants for these two genes by double-stranded RNA-mediated gene silencing. Silencing ppxyn1 and ppxyn2 reduced the virulence of P. parasitica toward tobacco (Nicotiana benthamiana) and tomato plants. These results demonstrate the crucial role of xylanase-encoding ppxyn1 and ppxyn2 in the infection process of P. parasitica.
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Acknowledgements
We are grateful to Dr. Y. C. Wang (Department of Plant Pathology, Nanjing Agricultural University) for providing the plasmid vectors and technical assistance. This work was supported by the Ministry of Science and Technology, Taiwan.
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294_2018_814_MOESM1_ESM.tif
Figure S1. Analysis of the enzymatic activity of ppxyn3 and ppxyn4 recombinant proteins obtained from Pichia pastoris by using different substrates. The reactions were performed with indicated substrate, pH, temperature, and reaction time as detailed in the figure, using different amounts of PPXYN3 (A-C) or PPXYN4 (D). The reducing sugar was detected by a modified DNS method, with the color of DNS reagent turning from bright yellow to red brown in the presence of reducing sugar. The experiments were repeated 3 times with similar results (TIF 1211 KB)
294_2018_814_MOESM2_ESM.tif
Figure S2. The electrophoretic pattern of ppxyn recombinant proteins obtained from E. coli. PPXYN1 and PPXYN3 from E. coli were separated by 12.5% SDS-PAGE and stained with Coomassie brilliant blue. M: molecular weight markers (TIF 125 KB)
294_2018_814_MOESM3_ESM.tif
Figure S3. PCR analysis of putative ppxyn1- and ppxyn2-silenced and empty-vector transformants. Genomic DNA isolated from each transformant was analyzed by PCR with NptII_F.1 and Trans_bb_R.2 used as primers. Genomic DNA of the wild-type strain of Phytophthora parasitica (Wt) and plasmid DNA of the empty silencing vector (pHPdest) were used as negative and positive controls, respectively. M: DNA size markers, with the 1.65-kb band marked with an arrowhead (TIF 194 KB)
294_2018_814_MOESM4_ESM.tif
Figure S4. The morphology of sporangia produced by ppxyn1- and ppxyn2-silenced and empty-vector transformants of Phytophthora parasitica. Mycelial disks of the wild-type strain (Wt), and empty-vector (V27, V49, and V85), ppxyn1-silenced (I39, I66, and I84), and ppxyn2-silenced transformants (II31, II50, and II84) were incubated in 1x Petri’s solution and incubated at 25oC under light to induce sporangia formation. Pictures of sporangia were taken under a microscope at 2 days after induction. Numbers under each picture are mean (± SD) of three biological repeats to show the mean number of sporangia present in a mycelial block (5 x 5 mm2). Scale bar = 5 μm (TIF 3267 KB)
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Lai, MW., Liou, RF. Two genes encoding GH10 xylanases are essential for the virulence of the oomycete plant pathogen Phytophthora parasitica. Curr Genet 64, 931–943 (2018). https://doi.org/10.1007/s00294-018-0814-z
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DOI: https://doi.org/10.1007/s00294-018-0814-z