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.

Plasmodiophora brassicae-Triggered Cell Enlargement and Loss of Cellular Integrity in Root Systems Are Mediated by Pectin Demethylation.

Gall formation on the belowground parts of plants infected with Plasmodiophora brassicae is the result of extensive host cellular reprogramming. The development of these structures is a consequence of increased cell proliferation followed by massive enlargement of cells colonized with the pathogen. Drastic changes in cellular growth patterns create local deformities in the roots and hypocotyl giving rise to mechanical tensions within the tissue of these organs. Host cell wall extensibility and recomposition accompany the growth of the gall and influence pathogen spread and also pathogen life cycle progression. Demethylation of pectin within the extracellular matrix may play an important role in P. brassicae-driven hypertrophy of host underground organs. Through proteomic analysis of the cell wall, we identified proteins accumulating in the galls developing on the underground parts of Arabidopsis thaliana plants infected with P. brassicae. One of the key proteins identified was the pectin methylesterase (PME18); we further characterized its expression and conducted functional and anatomic studies in the knockout mutant and used Raman spectroscopy to study the status of pectin in P. brassicae-infected galls. We found that late stages of gall formation are accompanied with increased levels of PME18. We have also shown that the massive enlargement of cells colonized with P. brassicae coincides with decreases in pectin methylation. In pme18-2 knockout mutants, P. brassicae could still induce demethylation; however, the galls in this line were smaller and cellular expansion was less pronounced. Alteration in pectin demethylation in the host resulted in changes in pathogen distribution and slowed down disease progression. To conclude, P. brassicae-driven host organ hypertrophy observed during clubroot disease is accompanied by pectin demethylation in the extracellular matrix. The pathogen hijacks endogenous host mechanisms involved in cell wall loosening to create an optimal cellular environment for completion of its life cycle and eventual release of resting spores facilitated by degradation of demethylated pectin polymers.

Transcriptional profiling identifies critical steps of cell cycle reprogramming necessary for Plasmodiophora brassicae-driven gall formation in Arabidopsis.

Plasmodiophora brassicae is a soil-borne biotroph whose life cycle involves reprogramming host developmental processes leading to the formation of galls on its underground parts. Formation of such structures involves modification of the host cell cycle leading initially to hyperplasia, increasing the number of cells to be invaded, followed by overgrowth of cells colonised by the pathogen. Here we show that P. brassicae infection stimulates formation of the E2Fa/RBR1 complex and upregulation of MYB3R1, MYB3R4 and A- and B-type cyclin expression. These factors were previously described as important regulators of the G2-M cell cycle checkpoint. As a consequence of this manipulation, a large population of host hypocotyl cells are delayed in cell cycle exit and maintained in the proliferative state. We also report that, during further maturation of galls, enlargement of host cells invaded by the pathogen involves endoreduplication leading to increased ploidy levels. This study characterises two aspects of the cell cycle reprogramming efforts of P. brassicae: systemic, related to the disturbance of host hypocotyl developmental programs by preventing cell cycle exit; and local, related to the stimulation of cell enlargement via increased endocycle activity.

Nictaba Homologs from Arabidopsis thaliana Are Involved in Plant Stress Responses.

Plants are constantly exposed to a wide range of environmental stresses, but evolved complicated adaptive and defense mechanisms which allow them to survive in unfavorable conditions. These mechanisms protect and defend plants by using different immune receptors located either at the cell surface or in the cytoplasmic compartment. Lectins or carbohydrate-binding proteins are widespread in the plant kingdom and constitute an important part of these immune receptors. In the past years, lectin research has focused on the stress-inducible lectins. The Nicotiana tabacum agglutinin, abbreviated as Nictaba, served as a model for one family of stress-related lectins. Here we focus on three non-chimeric Nictaba homologs from Arabidopsis thaliana, referred to as AN3, AN4, and AN5. Confocal microscopy of ArathNictaba enhanced green fluorescent protein (EGFP) fusion constructs transiently expressed in N. benthamiana or stably expressed in A. thaliana yielded fluorescence for AN4 and AN5 in the nucleus and the cytoplasm of the plant cell, while fluorescence for AN3 was only detected in the cytoplasm. RT-qPCR analysis revealed low expression for all three ArathNictabas in different tissues throughout plant development. Stress application altered the expression levels, but all three ArathNictabas showed a different expression pattern. Pseudomonas syringae infection experiments with AN4 and AN5 overexpression lines demonstrated a significantly higher tolerance of several transgenic lines to P. syringae compared to wild type plants. Finally, AN4 was shown to interact with two enzymes involved in plant defense, namely TGG1 and BGLU23. Taken together, our data suggest that the ArathNictabas represent stress-regulated proteins with a possible role in plant stress responses. On the long term this research can contribute to the development of more stress-resistant plants.

Glycan-binding F-box protein from Arabidopsis thaliana protects plants from Pseudomonas syringae infection.

BACKGROUND: A small group of F-box proteins consisting of a conserved F-box domain linked to a domain homologous to the glycan-binding protein has been identified within the genome of Arabidopsis thaliana. Previously, the so-called F-box-Nictaba protein, encoded by the gene At2g02360, was shown to be a functional lectin which binds N-acetyllactosamine structures. Here, we present a detailed qRT-PCR expression analysis of F-box-Nictaba in Arabidopsis plants upon different stresses and hormone treatments. RESULTS: Expression of the F-box-Nictaba gene was enhanced after plant treatment with salicylic acid and after plant infection with the virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). ß-glucuronidase histochemical staining of transgenic Arabidopsis plants displayed preferential activity of the At2g02360 promoter in trichomes present on young rosette leaves. qRT-PCR analyses confirmed high expression of F-box-Nictaba in leaf trichomes. A. thaliana plants overexpressing the gene showed less disease symptoms after Pst DC3000 infection with reduced bacterial colonization compared to infected wild type and F-box-Nictaba knock-out plants. CONCLUSIONS: Our data show that the Arabidopsis F-box-Nictaba gene is a stress-inducible gene responsive to SA, bacterial infection and heat stress, and is involved in salicylic acid related plant defense responses. This knowledge enriched our understanding of the physiological importance of F-box-Nictaba, and can be used to create plants with better performance in changing environmental conditions.

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galß1-3GlcNAc and Galß1-4GlcNAc) and poly-N-acetyllactosamine ([Galß1-4GlcNAc]n) as well as Lewis A (Galß1-3(Fucα1-4)GlcNAc), Lewis X (Galß1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galß1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galß1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.