Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Adjustment of Photosynthetic and Antioxidant Activities to Water Deficit Is Crucial in the Drought Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms.

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.

Genius Architect or Clever Thief-How Plasmodiophora brassicae Reprograms Host Development to Establish a Pathogen-Oriented Physiological Sink.

When plants are infected by Plasmodiophora brassicae, their developmental programs are subjected to extensive changes and the resultant clubroot disease is associated with formation of large galls on underground tissue. The pathogen's need to build an efficient feeding site as the disease progresses drives these changes, ensuring successful production of resting spores. This developmental reprogramming is an outcome of interactions between the pathogen and the infected host. During disease progression, we can observe alteration of growth regulator dynamics, patterns of cell proliferation and differentiation, increased cell expansion, and eventual cell wall degradation as well as the redirection of nutrients toward the pathogen. Recently, detailed studies of anatomical changes occurring during infection and studies profiling transcriptional responses have come together to provide a clearer understanding of the sequence of events and processes underlying clubroot disease. Additionally, genome sequencing projects have revealed P. brassicae's potential for the production of signaling molecules and effectors as well as its requirements and capacities with respect to taking up host nutrients. Integration of these new findings together with physiological studies can significantly advance our understanding of how P. brassicae brings about reprogramming of host development. This article summarizes the current state of knowledge on cellular changes induced by P. brassicae infection and aims to explain their impact and importance for both the host and the pathogen.