Phytophthora parasitica and lupin (Lupinus angustifolius) interactions: changes in gene expression during infection and after phosphite treatment

Phytophthora species are Oomycete pathogens that cause highly destructive diseases in a variety of agricultural and horticultural crops, and natural ecosystems. An understanding of the key biological processes that occur during development and infection of hosts is important for the development of effective Phytophthora control mechanisms. An infection assay model system was developed for P. parasitica based on lupin (Lupinus angustifolius) seedlings. The progress of lesion development and colonisation of P. parasitica in inoculated root tissues was assessed macroscopically and using light microscopy of sectioned material. At 24 hours post inoculation (hpi), a few hyphae were observed in the epidermal and outer cortical cells in the region of the root that had been at the surface of the zoospore suspension during the inoculation period. As root infection progressed, the hyphae grew both towards the vascular tissue at the centre of the root and longitudinally along the root. At 42 hpi, P. parasitica hyphae developed haustoria within root cortical cells. No evidence of callose deposition, a typical plant defence response, by the lupin root cells was observed after infected roots stained with aniline blue. Development of the model lupin-P. parasitica infection assay system facilitated ensuing studies of this plant-pathogen interaction, including the cellular and molecular basis of plant infection. The model assay system was used to examine levels of resistance of different lupin cultivars following inoculation with P. parasitica and to analyse temporal patterns of P. parasitica gene expression using quantitative real-time PCR (qPCR) during lupin root infection. One crucial component of Phytophthora pathogenicity is the digestion of the plant cell wall to allow penetration of the plant surface and colonisation within the plant tissues. Plant cell walls are complicated structures that are composed of a wide range of complex polysaccharides (i.e. cellulose, hemicelluloses and pectins) and proteins and they constitute an effective barrier that impedes the entry of many potential pathogens. In order to penetrate the plant cell wall, pathogens secrete a diverse array of cell wall degrading enzymes (CWDEs). The identity and timing of the expression of genes encoding P. parasitica CWDEs was analysed using qPCR. It is believed that pathogens secrete cascades of CWDEs during the infection process and evidence supporting this hypothesis was obtained from the lupin-P. parasitica data. One management strategy used in the control of Phytophthora diseases is the application of the chemical phosphite. Our understanding of the mechanism(s) underlying phosphite inhibition of Phytophthora diseases in plants is limited. Phosphite is known to have effects on both host plants and Phytophthora pathogens. In the present study, RNA-Seq was used to investigate the effects of phosphite on P. parasitica gene expression in vitro and in planta. Phosphite treatment was found to induce extensive changes in the expression of many pathogen genes both in vitro and in planta. One of the exciting results was the discovery that there was a general tendency for phosphite to up-regulate the expression of genes that are normally expressed early in lupin infection (30-36 hpi) and to down-regulate the expression of genes that are normally expressed during late infection (54-60 hpi). This was exemplified in particular by P. parasitica genes encoding pectinase and cellulase CWDEs and RxLR effectors. In conclusion, the research described in this thesis has developed a new and robust model infection assay for use in studies of plant infection by P. parasitica and, potentially, by other Phytophthora species. The research also presents the results of using this assay in transcriptomic studies of pathogen gene expression during plant infection. The results that have been obtained provide a better understanding of Phytophthora pathogenicity mechanisms and should aid the future development of improved methods of controlling Phytophthora diseases

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response

Phytophthora cinnamomi in Australia

Members of the genus Phytophthora cause serious damage to a huge array of plants. From the nineteenth century Irish potato famine to current widespread threats to forests and ecosystems in North and South America, Europe and Australia, the genus lives up to its reputation as the plant destroyer. This book provides an overview of Phytophthora species impacting crops, forests, nurseries, greenhouses and natural areas worldwide. Chapters cover major hosts, identification, epidemiology, management, current research, future perspectives and the impacts of globalization on Phytophthora. Phytophthora: A Global Perspective is an essential resource for researchers and extension workers in plant pathology and crop protection