A dual-flow RootChip enables quantification of bi-directional calcium signalling in primary roots

One sentence summary: Bi-directional-dual-flow-RootChip to track calcium signatures in Arabidopsis primary roots responding to osmotic stress. Plant growth and survival is fundamentally linked with the ability to detect and respond to abiotic and biotic factors. Cytosolic free calcium (Ca2+) is a key messenger in signal transduction pathways associated with a variety of stresses, including mechanical, osmotic stress and the plants’ innate immune system. These stresses trigger an increase in cytosolic Ca2+ and thus initiate a signal transduction cascade, contributing to plant stress adaptation. Here we combine fluorescent G-CaMP3 Arabidopsis thaliana sensor lines to visualise Ca2+ signals in the primary root of 9-day old plants with an optimised dual-flow RootChip (dfRC). The enhanced polydimethylsiloxane (PDMS) bi-directionaldual-flow-RootChip (bi-dfRC) reported here adds two adjacent inlet channels at the base of the observation chamber, allowing independent or asymmetric chemical stimulation at either the root differentiation zone or tip. Observations confirm distinct early spatio-temporal patterns of salinity (sodium chloride, NaCl) and drought (polyethylene glycol, PEG)-induced Ca2+ signals throughout different cell types dependent on the first contact site. Furthermore, we show that the primary signal always dissociates away from initially stimulated cells. The observed early signaling events induced by NaCl and PEG are surprisingly complex and differ from long-term changes in cytosolic Ca2+ reported in roots. Bi-dfRC microfluidic devices will provide a novel approach to challenge plant roots with different conditions simultaneously, while observing bi-directionality of signals. Future applications include combining the bi-dfRC with H2O2 and redox sensor lines to test root systemic signaling responses to biotic and abiotic factors

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula G.) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed

Alterations in Soluble Class III Peroxidases of Maize Shoots by Flooding Stress

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed

Proteomic Profiling of the Microsomal Root Fraction: Discrimination of Pisum sativum L. Cultivars and Identification of Putative Root Growth Markers

Legumes are a large and economically important family, containing a variety of crop plants. Alongside different cereals, some fruits, and tropical roots, a number of leguminosae evolved for millennia as crops with human society. One of these legumes is Pisum sativum L., the common garden pea. In the past, breeding has been largely selective on improved above-ground organs. However, parameters, such as root-growth, which determines acquisition of nutrients and water, have largely been underestimated. Although the genome of P. sativum is still not fully sequenced, multiple proteomic studies have been published on a variety of physiological aspects in the last years. The presented work focused on the connection between root length and the influence of the microsomal root proteome of four different pea cultivars after five days of germination (cultivar Vroege, Girl from the Rhineland, Kelvedon Wonder, and Blauwschokker). In total, 60 proteins were identified to have significantly differential abundances in the four cultivars. Root growth of five-days old seedlings and their microsomal proteome revealed a similar separation pattern, suggesting that cultivar-specific root growth performance is explained by differential membrane and ribosomal protein levels. Hence, we reveal and discuss several putative root growth protein markers possibly playing a key role for improved primary root growth breeding strategies

Alterations in Soluble Class III Peroxidases of Maize Shoots by Flooding Stress

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed

Exploiting Structural Modelling Tools to Explore Host-Translocated Effector Proteins

Oomycete and fungal interactions with plants can be neutral, symbiotic or pathogenic with different impact on plant health and fitness. Both fungi and oomycetes can generate so-called effector proteins in order to successfully colonize the host plant. These proteins modify stress pathways, developmental processes and the innate immune system to the microbes’ benefit, with a very different outcome for the plant. Investigating the biological and functional roles of effectors during plant–microbe interactions are accessible through bioinformatics and experimental approaches. The next generation protein modeling software RoseTTafold and AlphaFold2 have made significant progress in defining the 3D-structure of proteins by utilizing novel machine-learning algorithms using amino acid sequences as their only input. As these two methods rely on super computers, Google Colabfold alternatives have received significant attention, making the approaches more accessible to users. Here, we focus on current structural biology, sequence motif and domain knowledge of effector proteins from filamentous microbes and discuss the broader use of novel modelling strategies, namely AlphaFold2 and RoseTTafold, in the field of effector biology. Finally, we compare the original programs and their Colab versions to assess current strengths, ease of access, limitations and future applications

Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta

Multiple downy mildew effectors target the stress‐related NAC transcription factor LsNAC 069 in lettuce

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector‐target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast‐two‐hybrid (Y2H) screening. Interaction of the lettuce tail‐anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N‐terminal NAC domain but depends on the C‐terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail‐anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC‐domain (LsNAC069ΔNAC). Relocalization was significantly reduced in the presence of the Ser/Cys‐protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co‐expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses

Multiple downy mildew effectors target the stress‐related NAC transcription factor LsNAC 069 in lettuce

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector‐target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast‐two‐hybrid (Y2H) screening. Interaction of the lettuce tail‐anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N‐terminal NAC domain but depends on the C‐terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail‐anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC‐domain (LsNAC069ΔNAC). Relocalization was significantly reduced in the presence of the Ser/Cys‐protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co‐expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses