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The plant pathogen Phytophthora cinnamon the causal agent of disease in numerous species, is a major threat to natural vegetation and has economic impacts in agriculture. The pathogen principally invades the root system, which, in susceptible species, is rapidly colonised and functionally destroyed. Few species are resistant, however, where resistance is expressed the pathogen is restricted to small, localised lesions. The molecular mechanisms that underpin this response in resistant species are not well understood. Lomandra longifolia, an Australian native species, is highly resistant to P. cinnamomi. In an earlier study, we showed induction of resistance-related components such as callose, lignin and hydrogen peroxide (H2 O2 ) in L. longifolia roots that had been inoculated with P. cinnamomi. Here, in order to further identify, during the very early stages of infection, the molecular components and regulatory networks that may trigger resistance, a comprehensive root transcriptome analysis was performed using next generation sequencing. Overall, 18 cDNA libraries were produced generating 52.8 GB 126 base pair reads, which were de novo assembled into contigs. Differentially expressed genes (DEGs) were identified allowing the identification of infection-responsive candidate genes that were putatively related to resistance, and from this set ten were selected for qRT-PCR to validate the RNA-Seq expression value. Further analysis of individual candidates revealed that many were involved in PAMP-triggered immunity (PTI; pattern recognition receptors, glutathione S-transferase, callose synthases, pathogenesis-related protein-1, mitogen activated protein kinases) and effector-triggered immunity (ETI) (NBS-LRR, signalling genes, transcription factors and anti-pathogenic compound synthase genes). As these candidate genes or mediated components activate different defence signalling systems, they may have potential for investigation of novel approaches to disease control and in transgenic approaches for improvement, in susceptible species, of resistance to P. cinnamomi.

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Exposure of plants to UV-C irradiation induces gene expression and cellular responses that are commonly associated with wounding and pathogen defence, and in some cases can lead to increased resistance against pathogen infection. We examined, at a physiological, molecular and biochemical level, the effects of and responses to, sub-lethal UV-C exposure on Arabidopsis plants when irradiated with increasing dosages of UV-C radiation. Following UV-C exposure plants had reduced leaf areas over time, with the severity of reduction increasing with dosage. Severe morphological changes that included leaf glazing, bronzing and curling were found to occur in plants treated with the 1000 J·m(-2) dosage. Extensive damage to the mesophyll was observed, and cell death occurred in both a dosage- and time-dependent manner. Analysis of H2O2 activity and the pathogen defence marker genes PR1 and PDF1.2 demonstrated induction of these defence-related responses at each UV-C dosage tested. Interestingly, in response to UV-C irradiation the production of callose (ß-1,3-glucan) was identified at all dosages examined. Together, these results show plant responses to UV-C irradiation at much lower doses than have previously been reported, and that there is potential for the use of UV-C as an inducer of plant defence.

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Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

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Etiolated hypocotyls of the resistant soybean (Glycine max [L.] Merr.) cultivar Harosoy 63 became susceptible to Phytophthora megasperma (Drechs.) f.sp. glycinea (Hildeb.) Kuan and Erwin race 1 after treatment with abscisic acid. Susceptibility was expressed by increases in lesion size and a major decrease in accumulation of the isoflavonoid phytoalexin, glyceollin. In untreated hypocotyls, activity of phenylalanine ammonia-lyase and accumulation of mRNA for this enzyme increased rapidly after infection, but these increases were suppressed in abscisic acid-treated hypocotyls. The results suggest the possibility that biosynthesis of glyceollin in the resistance response of soybeans may be controlled at the transcriptional level by changes in abscisic acid concentrations caused by infection.

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The concentrations of zeatin-type and isopentenyladenine-type cytokinins were reduced in the xylem extrudate collected from seedlings of Eucalyptus species following infection by Phytophthora cinnamomi Rands. The use of an enzyme-linked immunosorbent assay (ELISA) allowed the detection of these cytokinins over the range of 0.3 to 7 picomoles for the isopentenyladenine-type and 1 to 1000 picomoles for the zeatin-type. Isopentenyladenine-type cytokinins occurred in concentrations less than 10% of the zeatin-type, but they could be readily detected and measured. This is the first report of their presence in xylem. The sensitivity of the assay allowed a short collection period (30 minutes) reducing any confusion with trauma-induced changes. Infection of the susceptible species Eucalyptus marginata Donn. ex Sm. resulted in significant reduction of zeatin-type cytokinins within 3 days of infection, and at 14 days postinfection the concentration of both cytokinin types was reduced to 26% of uninoculated controls. No reduction in cytokinins occurred with the field resistant Eucalyptus calophylla R. Br. It is suggested that failure of cytokinin transport from the root system may be responsible for the failure in water transport and symptoms of P. cinnamomi infection observed in infected susceptible eucalypts.

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Freeze fracture and lanthanum tracer experiments have shown that gap junctions exist throughout folliculogenesis between granulosa cells and growing mouse oocytes (Anderson and Albertini, J. Cell Biol. 71, 680-686, 1976). The following lines of experimentation in the present study suggest that metabolic cooperativity exists between granulosa cells and their enclosed oocytes, i.e., gap junctions are functional, and that in most cases examined, greater than 85% of the metabolites present in follicle-enclosed oocytes were originally taken up by the granulosa cells and transferred to the oocyte via gap junctions: (1) When incubated with various radiolabeled compounds, follicle-enclosed oocytes contained more intracellular radioactivity than did oocytes with no attached granulosa cells (denuded oocytes); (2) for two radiolabeled ribonucleosides examined, the distribution of phosphorylated metabolites in follicle-enclosed oocytes resembled that of granulosa cells and differed significantly from that in denuded oocytes; (3) pulse-chase experiments with radiolabeled ribonucleosides revealed that during the chase period more radioactivity became associated with the follicle-enclosed oocyte; (4) treatments known to disrupt gap junctions in other cell types were effective in reversibly uncoupling metabolic cooperativity between granulosa cells and oocytes; and (5) a series of control experiments using (a) medium conditioned by granulosa cells and (b) cocultures of denuded oocytes and granulosa cells in which physical contact between the two cell types was not permitted demonstrated that contact between follicle cells and oocytes was necessary for observing metabolic cooperativity. Metabolic cooperativity was also found between follicle cells and oocytes in the two culture systems which support growth of mouse oocytes in vitro. The fact that oocytes do not grow well, if at all, in the absence of follicle cells and the large contribution of nutrients apparently furnished to the oocyte by the granulosa cells is consistent with the concept that gap junction mediated metabolic cooperativity between follicle cells and their enclosed oocytes is vital for mammalian oocyte growth.

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