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Genetic analysis of host and phosphite mediated resistance to Phytophthora cinnamomi in Arabidopsis thaliana

Eshraghi, Leila (2012) Genetic analysis of host and phosphite mediated resistance to Phytophthora cinnamomi in Arabidopsis thaliana. PhD thesis, Murdoch University.

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Phosphite (Phi), an analogue of phosphate (Pi) is highly effective for the control of Phytophthora cinnamomi, a devastating necrotrophic pathogen worldwide. This study describes the effect of phosphite (Phi) on the induction of defence responses in Phytophthora cinnamomi-infected Arabidopsis thaliana accessions Ler and Col-0, and mutants defective in salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), phosphate starvation response (PSR) and auxin response signalling pathways. The inoculation of the resistant Col-0 with P. cinnamomi induced a rapid increase in callose deposition (by 6 h after inoculation) and hydrogen peroxide (H2O2) production (by 24 h after inoculation) whereas inoculation of susceptible Ler showed a delayed and reduced response. Treatment of Ler with Phi produced a response to P. cinnamomi inoculation similar to that observed in Col-0 in terms of timing and magnitude suggesting Phi primes the plant for a rapid and intense response to infection involving heightened activation of a range of defence responses.

A reliable method for measuring disease progression is important when evaluating susceptibility in host–pathogen interactions. A sensitive quantitative polymerase chain reaction (QPCR) assay was developed for the quantitative measurement of P. cinnamomi DNA (biomass) in planta that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and the pathogen’s biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. It was demonstrated that normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to an overestimation of the pathogen’s biomass.

Inoculation of mutants in the SA, JA, and ET defence signalling pathways did not affect the resistance of Col-0 suggesting alternative pathways are involved. A high level susceptibility was observed in the aba2-4 mutant suggesting a role for ABA signalling in the induction of resistance to P. cinnamomi. Phi treatment of aba2-4 increased resistance but not to the wild type levels indicating a possible role for ABA-dependent and ABA independent signalling in Phi mediated resistance. Application of Phi to non-inoculated A. thaliana seedlings elevated transcription of defence genes in the SA (PR1 and PR5) and JA/ET (THI2.1 and PDF1.2) pathways. Furthermore, analysis of gene expression in Col-0 revealed that either Phi or P. cinnamomi caused the down-regulation of the transcriptional level of AtMYC2 (a positive regulator of ABA signalling which also negatively regulates JA-related genes) and increased the transcriptional abundance of PDF1.2. Together these results suggest that the resistance response of Col-0 and Phi treatment both act partially through an ABA dependent mechanism which is independent of the antagonism between ABA and elements of the JA/ET pathway such as PDF1.2.

Phosphite has been suggested to interfere with various plant processes including Pi homeostasis therefore the potential involvement of the Pi and auxin signalling pathways in resistance to P. cinnamomi was investigated using several PSR and auxin response pathway mutants. The mutants tir1-1, an auxin response mutant deficient in the auxin-stimulated SCF (Skp1−Cullin−F-Box) ubiquitination pathway and phr1-1, a mutant defective in response to Pi starvation were highly susceptible to P. cinnamomi compared to their parental background Col-0. Complementation restored resistance to the level observed in Col-0. Moreover, inhibition of auxin transporters by TIBA (2,3,5-triiodobenzoic acid) led to a significant increase in susceptibility of Lupinus angustifolius seedlings to P. cinnamomi supporting the importance of the auxin signalling pathway in P. cinnamomi resistance. The 26S proteasome subunits mutants; rpn10-1 (Defective in ubiquitin/26S proteasome-mediated proteolysis) and pbe1-1 (proteasome subunit beta type-5-A) were also susceptible to P. cinnamomi. The rpn10-1 mutant has also been associated with the auxin signalling pathway and the susceptibility of rpn10-1 and pbe1-1 indicates that the 26S proteasome and auxin signalling could play a role in resistance to P. cinnamomi. Given the apparent involvement of auxin and PSR signalling in the resistance to P. cinnamomi, the possible involvement of these pathways in Phi mediated resistance was also investigated. Application of Phi at both low and high concentrations attenuated some of the Pi starvation inducible genes such as At4, AtACP5 and AtPT2. However, in phosphate sufficient plants, Phi treatment mimicked Pi starvation responses in terms of enhanced expression of PHR1, AUX1, AXR1, AXR2 and SGT1B; suppression of primary root elongation, and increased root hair formation. Together, these results suggest that the auxin response pathway, particularly auxin sensitivity and transport, plays a role in the plant’s resistance to P. cinnamomi and suggest that phosphite-mediated resistance may in some part be through its effect on stimulation of the auxin response pathway.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Hardy, Giles, McComb, Jen, Shearer, Bryan and Anderson, Johnathan
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