L. Eshraghi

Suppression of the auxin response pathway enhances susceptibility to Phytophthora cinnamomi while phosphite-mediated resistance stimulates the auxin signalling pathway

BackgroundPhytophthora cinnamomi is a devastating pathogen worldwide and phosphite (Phi), an analogue of phosphate (Pi) is highly effective in the control of this pathogen. Phi also interferes with Pi starvation responses (PSR), of which auxin signalling is an integral component. In the current study, the involvement of Pi and the auxin signalling pathways in host and Phi-mediated resistance to P. cinnamomi was investigated by screening the Arabidopsis thaliana ecotype Col-0 and several mutants defective in PSR and the auxin response pathway for their susceptibility to this pathogen. The response to Phi treatment was also studied by monitoring its effect on Pi- and the auxin response pathways.ResultsHere we demonstrate that phr1-1 (phosphate starvation response 1), a mutant defective in response to Pi starvation was highly susceptible to P. cinnamomi compared to the parental background Col-0. Furthermore, the analysis of the Arabidopsis tir1-1 (transport inhibitor response 1) mutant, deficient in the auxin-stimulated SCF (Skp1 − Cullin − F-Box) ubiquitination pathway was also highly susceptible to P. cinnamomi and the susceptibility of the mutants rpn10 and pbe1 further supported a role for the 26S proteasome in resistance to P. cinnamomi. The role of auxin was also supported by a significant (P < 0.001) increase in susceptibility of blue lupin (Lupinus angustifolius) to P. cinnamomi following treatment with the inhibitor of auxin transport, TIBA (2,3,5-triiodobenzoic acid). 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. Phi (especially at high concentrations) attenuates the response of some Pi starvation inducible genes such as AT4, AtACP5 and AtPT2 in Pi starved plants. However, Phi enhanced the transcript levels of PHR1 and the auxin responsive genes (AUX1, AXR1and AXR2), suppressed the primary root elongation, and increased root hair formation in plants with sufficient Pi.ConclusionsThe auxin response pathway, particularly auxin sensitivity and transport, plays an important role in resistance to P. cinnamomi in Arabidopsis, and phosphite-mediated resistance may in some part be through its effect on the stimulation of the PSR and auxin response pathways.

Defence Signalling Pathways Involved in Plant Resistance and Phosphite-Mediated Control of Phytophthora Cinnamomi

Phytophthora cinnamomi is one of the most devastating plant pathogens worldwide. Current control of P. cinnamomi in natural ecosystems primarily relies on chemical phosphite (Phi). To investigate host- and Phi-mediated resistance, A. thaliana ecotypes and mutants defective in salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) signalling pathways were screened for susceptibility to P. cinnamomi. In contrast to Col-0, the aba2-4 mutant, deficient in the synthesis of ABA, was susceptible suggesting a role for ABA in resistance to P. cinnamomi. Phi treatment increased resistance in aba2-4, but not to the level of Col-0, suggesting that Phi may act through both ABA-dependent and ABA-independent pathways. Phi treatment or P. cinnamomi inoculation of Col-0 down-regulated AtMYC2, a positive regulator of ABA signalling, which negatively regulates JA/ET-related pathogenesis-related genes, such as PDF1.2, whilst positively regulating JA-mediated herbivore responses such as VSP and PI. Consistent with this, P. cinnamomi or Phi treatment caused up-regulation of PDF1.2 and THI2.1 and down-regulation of VSP2 and the ABA-responsive gene RD22. Despite the up-regulation of JA/ET-dependent defence genes, the JA-defective mutant, jar1-1 and ET-defective mutants, ein2-1 and etr1-3, showed wild-type levels of resistance to P. cinnamomi, suggesting that these JA/ET defences are not required for resistance to P. cinnamomi. These results suggest that the resistance response of Col-0 act, at least in part, through a mechanism dependent on ABA synthesis, which appears independent of the interaction between ABA and elements of the JA/ET pathway, whilst Phi-mediated resistance, although inducing a response resembling the resistance response of Col-0, is independent of ABA signalling.

The pentatricopeptide repeat protein EMB2654 is essential for trans-splicing of a chloroplast small ribosomal subunit transcript

Binding of the pentatricopeptide repeat protein EMB2654 to one-half of the plastid rps12 intron is essential for trans-splicing, production of plastid ribosomes, and embryogenesis in Arabidopsis. We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.

A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.