Julie D. Scholes

Infection of Arabidopsis thaliana leaves with Albugo candida (white blister rust) causes a reprogramming of host metabolism

Abstract Albugo candida (Pers.) (O.) Kunze is a biotrophic pathogen which infects the crucifer Arabidopsis thaliana (L.) Heynh forming discrete areas of infection. Eight days after inoculation of leaves, white blisters became visible on the under surface of the leaf although no symptoms were apparent on the upper surface. By day 14, the region of leaf invaded by fungal mycelium had become chlorotic. Recently it has been hypothesized that an accumulation of soluble carbohydrates, following an increase in invertase activity, may trigger sugar signal transduction pathways leading to the repression of photosynthetic gene expression and to the induction of defence proteins. This hypothesis was investigated by quantifying localized changes in carbohydrate and photosynthetic metabolism and the expression of genes encoding photosynthetic and defence proteins. Quantitative imaging of chlorophyll fluorescence revealed that the rate of photosynthesis declined progressively in the invaded regions of the leaf. However, in uninfected regions of the infected leaf the rate of photosynthesis was similar to that measured in the control leaf until late on during the infection cycle when it declined. Images of nonphotochemical fluorescence quenching (NPQ) suggested that the capacity of the Calvin cycle had been reduced in infected regions and that there was a complex metabolic heterogeneity within the infected leaf. A. candida also caused localized changes in the carbohydrate metabolism of the leaf; soluble carbohydrates accumulated in the infected region whereas the amount of starch declined. The reverse was seen in uninfected regions of the infected leaf; carbohydrates did not accumulate until late on during infection and the amount of starch increased as the infection progressed. There was an increase in the activity of invertases which was confined to regions of the leaf invaded by the fungal mycelium. The increase in apoplastic invertase activity was of host origin, as mRNA levels of the ATbetaFRUCT1 gene (measured by semiquantitative RT-PCR) increased 40-fold in the infected region. The increase in soluble invertase activity resulted from the appearance of a new isoform in the invaded region of the leaf. Current evidence suggests that this was of fungal origin. Northern blot analysis of cab and rbcS showed that photosynthetic gene expression was repressed in the infected leaf from 6 days after inoculation (DAI) when compared to control leaves. In contrast, there was no detectable induction of defence proteins in the infected leaf. These data are discussed in the context of the sugar-sensing hypothesis presented above.

Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata) : quantitative imaging of chlorophyll fluorescence

Localised changes in photosynthesis in oat leaves infected with the biotrophic rust fungus Puccinia coronata Corda were examined at different stages of disease development by quantitative imaging of chlorophyll fluorescence. Following inoculation of oat leaves with crown rust the rate of whole-leaf gas exchange declined. However, crown rust formed discrete areas of infection which expanded as the disease progressed and these localised regions of infection gave rise to heterogeneous changes in photosynthesis. To quantify these changes, images of chlorophyll fluorescence were taken 5, 8 and 11 d after inoculation and used to calculate images representing two parameters; ΦII, a measure of PSII photochemical efficiency and ΔFm/Fm′, a measure of non-photochemical energy dissipation (qN). Five days after inoculation, disease symptoms appeared as yellow flecks which were correlated with the extent of the fungal mycelium within the leaf. At this stage, ΔII was slightly reduced in the infected regions but, in uninfected regions of the leaf, values of ΦII were similar to those of healthy leaves. In contrast, qN (ΔFm/Fm′) was greatly reduced throughout the infected leaf in comparison to healthy leaves. We suggest that the low value of qN in an infected leaf reflects a high demand for ATP within these leaves. At sporulation, 8 d after inoculation, ΦII was reduced throughout the infected leaf although the reduction was most marked in areas invaded by fungal mycelium. In the infected leaf the pattern of non-photochemical quenching was complex; qN was low within invaded regions, perhaps reflecting high metabolic activity, but was now much higher in uninfected regions of the infected leaf, in comparison to healthy leaves. Eleven days after inoculation “green islands” formed in regions of the leaf associated with the fungal mycelium. At this stage, photosynthesis was severely inhibited over the entire leaf; however, heterogeneity was still apparent. In the region not invaded by the fungal mycelium, ΦII and qN were very low and these regions of the leaf were highly fluorescent, indicating that the photosynthetic apparatus was severely damaged. In the greenisland tissue, ΦII was low but detectable, indicating that some photosynthetic processes were still occurring. Moreover, qN was high and fluorescence low, indicating that the cells in this region were not dead and were capable of significant quenching of chlorophyll fluorescence.

Host plant resistance to parasitic weeds; recent progress and bottlenecks.

Parasitic witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) directly invade the roots of crop plants connecting to the vascular system and abstracting nutrients and water. As a consequence they cause devastating losses in crop yield. Genetic resistance to parasitic weeds is a highly desirable component of any control strategy. Resistance to parasitic plants can occur at different stages of the parasite lifecycle: before attachment to the host, during penetration of the root or after establishment of vascular connections. New studies are beginning to shed light on the molecular mechanisms and signaling pathways involved in plant-plant resistance. The first resistance gene to Striga, encoding a CC-NBS-LRR Resistance protein (R) has been identified and cloned suggesting that host plants resist attack from parasitic plants using similar surveillance mechanisms as those used against fungal and bacterial pathogens. It is becoming clear that the salicylic acid (SA) signaling pathway plays an important role in resistance to parasitic plants and genes encoding pathogenesis-related (PR) proteins are upregulated in a number of the resistant interactions. New strategies for engineering resistance to parasitic plants are also being explored, including the expression of parasite-specific toxins in host roots and RNAi to silence parasite genes crucial for development.

Localization of photosynthetic metabolism in the parasitic angiosperm Cuscuta reflexa

Abstract. Cells capable of photosynthesis in the parasitic angiosperm Cuscuta reflexa Roxb. (dodder) are highly localized. Immunolocalization of ribulose-1,5 bisphosphate carboxylase-oxygenase (Rubisco) and autofluorescence of chlorophyll in transverse sections of stems showed that they were largely restricted to a band of cells adjacent to the vascular bundles, consequently, the concentrations of Rubisco and chlorophyll were low per unit area or fresh weight. When 14CO2 was supplied to stem segments of C. reflexa it preferentially accumulated in these cells adjacent to the vasculature. Although the conductance for CO2 movement to the cells containing chlorophyll and Rubisco was very low, both the light reactions and dark reactions of photosynthesis appeared to be functional. De-epoxidation of the xanthophyll-cycle pigments after exposure to high light, and the chlorophyll fluorescence parameters, photochemical quenching (qP), non-photochemical quenching (NPQ) and the quantum efficiency of photosystem II (φPSII) responded normally to changes in photon flux density, indicating functional light-driven electron transport. The response of CO2 exchange to photon flux density followed a typical hyperbolic curve, and positive rates of CO2 fixation occurred when external CO2 was increased to 5%. We propose that CO2 for carbon assimilation is derived from internally respired CO2 and that this layer of photosynthetic cells makes a positive contribution to the carbon budget of C. reflexa.

Chlorophyll fluorescence imaging of plant-pathogen interactions.

Chlorophyll fluorescence imaging provides a noninvasive, non-destructive method with which to measure heterogenous changes in photosynthetic metabolism in plants infected by pathogens. The availability of commercial imaging fluorimeters has helped make this technique available to the wider scientific community, but considerable care is needed, both in experimental design and in the interpretation of results, to make the most of this powerful analytical tool. The origins of changes in chlorophyll fluorescence yield are discussed and the use of conventional and novel combinatorial imaging approaches explored, together with complementary techniques such as thermal imaging. This review examines the use of chlorophyll fluorescence imaging as a method for the early detection of viral, bacterial and fungal infection, before symptoms are visible by eye, and also as a means with which to probe underlying pathogen-induced changes in host physiology in both compatible and incompatible interactions. The use of chlorophyll fluorescence imaging to study host physiology is greatly enhanced when the atmosphere around the leaf is manipulated and simultaneous measurements of gas exchange made: The cost to the host plant of different resistance mechanisms can be calculated, the fate of the products of photosynthetic electron transport determined and localised alterations in the source–sink status of host tissue visualised.

Metabolism and Plant Hormone Action During Clubroot Disease

Infection of Brassicaceae with the obligate biotrophic pathogen Plasmodiophora brassicae results in the development of root galls (clubroots). During the transformation of a healthy root to a root gall a plethora of changes in primary and secondary metabolism occur. The upper part of an infected plant is retarded in growth due to redirection of assimilates from the shoot to the root. In addition, changes in the levels of plant growth regulators, especially auxins and cytokinins, contribute to the hypertrophy of infected roots. Also, defense reactions are manipulated after inoculation of suitable host plants with P. brassicae. This review summarizes our current knowledge on the changes in these parameters. A model is presented for how primary metabolism and secondary metabolism, including plant hormones, interact to induce clubroot formation.

Chlorophyll fluorescence imaging as tool for understanding the impact of fungal diseases on plant performance: a phenomics perspective

Chlorophyll fluorescence imaging is a non-invasive, non-destructive means with which to examine the impact of fungal pathogens on the photosynthetic metabolism of host plants. As such, it has great potential for screening purposes in high-throughput phenomics environments. However, there is great diversity in the responses of plants to different plant-fungal pathogens and the choice of suitable experimental conditions and protocols and interpretation of the results requires both preliminary laboratory experiments and an understanding of the biology of the specific plant-pathogen interaction. In this review, we examine the interaction between biotrophic, hemi-biotrophic and necrotrophic fungal pathogens and their hosts to illustrate the extent to which chlorophyll fluorescence imaging can be used to detect the presence of disease before the appearance of visible symptoms, distinguish between compatible and incompatible fungal interactions, identify heterogeneity in photosynthetic performance within the infected leaf and provide insights into the underlying mechanisms. The limitations and challenges of using chlorophyll fluorescence imaging in high throughput screens is discussed.

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of β-fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a Km for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.

Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species

The formation of biofilms by diverse bacteria isolated from contaminated soil and groundwater on model substrata with different surface properties was assessed in a multifactorial screen. Diverse attachment phenotypes were observed as measured by crystal violet dye retention and confocal laser scanning microscopy (CLSM). Bulk measurements of cell hydrophobicity had little predictive ability in determining whether biofilms would develop on hydrophobic or hydrophilic substrata. Therefore selected pairs of bacteria from the genera Rhodococcus, Pseudomonas and Sphingomonas that exhibited different attachment phenotypes were examined in more detail using CLSM and the lipophilic dye, Nile Red. The association of Rhodococcus sp. cell membranes with lipids was shown to influence the attachment properties of these cells, but this approach was not informative for Pseudomonas and Sphingomonas sp. Confocal Raman Microspectroscopy of Rhodococcus biofilms confirmed the importance of lipids in their formation and indicated that in Pseudomonas and Sphingomonas biofilms, nucleic acids and proteins, respectively, were important in identifying the differences in attachment phenotypes of the selected strains. Treatment of biofilms with DNase I confirmed a determining role for nucleic acids as predicted for Pseudomonas. This work demonstrates that the attachment phenotypes of microbes from environmental samples to different substrata varies markedly, a diverse range of macromolecules may be involved and that these differ significantly between genera. A combination of CLSM and Raman spectroscopy distinguished between phenotypes and could be used to identify the key macromolecules involved in cell attachment to surfaces for the specific cases studied.