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
