Submitted to: Genesis
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/15/2009
Publication Date: 7/1/2009
Publication URL: hdl.handle.net/10113/36493
Citation: Sun, W.X., Jia, Y.J., Feng, B.Z., Zhu, X.P., Xie, B., Oneill, N.R., Zhang, X.G. 2009. Functional analysis of Pcipg2 from the straminopilous plant Pathogen Phytophthora capsici. Genesis. 47(8):535-544. Available: http://dx.doi.org/10.1002/dvg.20530. Interpretive Summary: Phytophthora blight caused by the fungus Phytophthora capsici is a common, serious disease of numerous crops in the U.S., China, and many parts of the world. The fungus is able to attack plants by producing specific cell-wall degrading enzymes involved in the infection process. We conducted molecular studies to identify DNA sequences and compare the activities of different enzymes produced by P. capsici. Results showed that the activity of specific enzymes was correlated with the destruction of cell wall components. A molecular DNA analysis showed that the enzymes from Phytophthora were different from those from insects, plants, bacteria, and other fungi. The activity of specific enzymes suggested that they are responsible for differences in ability to cause disease. This information improves our understanding of the infection process and why some fungi are able to cause disease. Results will lead to the rapid detection of virulent fungi in soils and crops. The research will be useful to scientists, breeders, and seed companies concerned with understanding and controlling diseases caused by fungal pathogens.
Technical Abstract: Phytophthora capsici is an oomycete plant pathogen that causes severe diseases in a wide variety of crops. Polygalacturonases (PGs) play a major role in the degradation pectin in plant cell walls. A genomic library was made from a highly virulent strain of P. capsici with high PGs activity. Seven pg genes of P. capsici were isolated using degenerate primers based on conserved regions in P. cinnamomi, P. parasitica, and P. infestans, including three complete sequences for pg genes (pcipg2, pcipg3, and pcipg5) and four incomplete sequences for pg genes (pcipg1, pcipg4, pcipg6, and pcipg7). Three complete sequence pg genes encoded a polypeptide of 362 amino acid residues with a predicted molecular mass of 37 kDa. Pcipg2 has a signal peptide with 19-amino acid and 3 N-glycosylation sites, whereas both pcipg3 and pcipg5 have signal peptides with 20-amino acid. There was only one N-glycosylation site in pcipg3, and none in pcipg5. The position of Phytophthora PGs in the phylogenetic tree was clearly separate from fungi, insects, bacteria, and plant PGs, but closer to fungi and insect PGs than to plant and bacterial PGs. Three P. capsici pg genes and seven pg genes from other Phytophthora spp. fell into subgroups most of which exhibited no N-glycosylation sites. In this study, we explored the functions of pcipg2 based on its number of N-glycosylation sites. Heterologous expression of pcipg2 in Pichia pastoris produced a protein of 42 kDa (PGC) that did not correspond to the mass of this protein. Western blot of the purified protein confirmed that PGC was specifically expressed in Pichia pastoris from 1d to 7d during culture. The PGC exhibited high activity toward PCA, citrus pectin and pepper pectin that was derived from the cell walls of pepper leaves, and PGs activity variation in PGC treated pepper leaves was consistent with symptom development in pepper leaves. These results suggested that PGC might also contribute to cell wall death and variation in pathogen virulence on the host. RT-PCR and northern blot analysis of pcipg2 expression in the host showed that pcipg2 was highly expressed during interaction of P. capsici with the host, so pcipg2 might be involved in the infection process. All of these results were confirmed by the presence of disease symptoms in pepper leaves inoculated with PGC compared with plants inoculated with a zoospore suspension.