Generation and characterization of single and multigene Arabidopsis thaliana mutants in LSU1-4 (RESPONSE TO LOW SULFUR) genes.

In Arabidopsis thaliana, there are four members of the LSU (RESPONSE TO LOW SULFUR) gene family which are tandemly located on chromosomes 3 (LSU1 and LSU3) and 5 (LSU2 and LSU4). The LSU proteins are small, with coiled-coil structures, and they are able to form homo- and heterodimers. LSUs are involved in plant responses to environmental challenges, such as sulfur deficiency, and plant immune responses. Assessment of the role and function of these proteins was challenging due to the absence of deletion mutants. Our work fulfills this gap through the construction of a set of LSU deletion mutants (single, double, triple, and quadruple) by CRISPR/Cas9 technology. The genomic deletion regions in the obtained lines were mapped and the level of expression of each LSUs was assayed in each mutant. All lines were viable and capable of seed production. Their growth and development were compared at several different stages with the wild-type. No significant and consistent differences in seedlings' growth and plant development were observed in the optimal conditions. In sulfur deficiency, the roots of 12-day-old wild-type seedlings exhibited increased length compared to optimal conditions; however, this difference in root length was not observed in the majority of lsu-KO mutants.

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.

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.

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities.

Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species-with the most prominent being Centaurea rhenana and Cirsium canum-have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

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.

Clubroot Disease Stimulates Early Steps of Phloem Differentiation and Recruits SWEET Sucrose Transporters within Developing Galls.

Successful biotrophic plant pathogens can divert host nutrition toward infection sites. Here we describe how the protist Plasmodiophora brassicae establishes a long-term feeding relationship with its host by stimulating phloem differentiation and phloem-specific expression of sugar transporters within developing galls. Development of galls in infected Arabidopsis (Arabidopsis thaliana) plants is accompanied by stimulation of host BREVIS RADIX, COTYLEDON VASCULAR PATTERN, and OCTOPUS gene expression leading to an increase in phloem complexity. We characterized how the arrest of this developmental reprogramming influences both the host and the invading pathogen. Furthermore, we found that infection leads to phloem-specific accumulation of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS11 and 12 facilitating local distribution of sugars toward the pathogen. Utilizing Fourier-transform infrared microspectroscopy to monitor spatial distribution of carbohydrates, we found that infection leads to the formation of a strong physiological sink at the site of infection. High resolution metabolic and structural imaging of sucrose distributions revealed that sweet11 sweet12 double mutants are impaired in sugar transport toward the pathogen, delaying disease progression. This work highlights the importance of precise regulation of sugar partitioning for plant-pathogen interactions and the dependence of P brassicae's performance on its capacity to induce a phloem sink at the feeding site.