Spatial IMA1 regulation restricts root iron acquisition on MAMP perception.

Iron is critical during host-microorganism interactions1-4. Restriction of available iron by the host during infection is an important defence strategy, described as nutritional immunity5. However, this poses a conundrum for externally facing, absorptive tissues such as the gut epithelium or the plant root epidermis that generate environments that favour iron bioavailability. For example, plant roots acquire iron mostly from the soil and, when iron deficient, increase iron availability through mechanisms that include rhizosphere acidification and secretion of iron chelators6-9. Yet, the elevated iron bioavailability would also be beneficial for the growth of bacteria that threaten plant health. Here we report that microorganism-associated molecular patterns such as flagellin lead to suppression of root iron acquisition through a localized degradation of the systemic iron-deficiency signalling peptide Iron Man 1 (IMA1) in Arabidopsis thaliana. This response is also elicited when bacteria enter root tissues, but not when they dwell on the outer root surface. IMA1 itself has a role in modulating immunity in root and shoot, affecting the levels of root colonization and the resistance to a bacterial foliar pathogen. Our findings reveal an adaptive molecular mechanism of nutritional immunity that affects iron bioavailability and uptake, as well as immune responses.

Xylem K+ loading modulates K+ and Cs+ absorption and distribution in Arabidopsis under K+-limited conditions.

Potassium (K+) is an essential macronutrient for plant growth. The transcriptional regulation of K+ transporter genes is one of the key mechanisms by which plants respond to K+ deficiency. Among the HAK/KUP/KT transporter family, HAK5, a high-affinity K+ transporter, is essential for root K+ uptake under low external K+ conditions. HAK5 expression in the root is highly induced by low external K+ concentration. While the molecular mechanisms of HAK5 regulation have been extensively studied, it remains unclear how plants sense and coordinates K+ uptake and translocation in response to changing environmental conditions. Using skor mutants, which have a defect in root-to-shoot K+ translocation, we have been able to determine how the internal K+ status affects the expression of HAK5. In skor mutant roots, under K+ deficiency, HAK5 expression was lower than in wild-type although the K+ concentration in roots was not significantly different. These results reveal that HAK5 is not only regulated by external K+ conditions but it is also regulated by internal K+ levels, which is in agreement with recent findings. Additionally, HAK5 plays a major role in the uptake of Cs+ in roots. Therefore, studying Cs+ in roots and having more detailed information about its uptake and translocation in the plant would be valuable. Radioactive tracing experiments revealed not only a reduction in the uptake of 137Cs+ and 42K+in skor mutants compared to wild-type but also a different distribution of 137Cs+ and 42K+ in tissues. In order to gain insight into the translocation, accumulation, and repartitioning of both K+ and Cs+ in plants, long-term treatment and split root experiments were conducted with the stable isotopes 133Cs+ and 85Rb+. Finally, our findings show that the K+ distribution in plant tissues regulates root uptake of K+ and Cs+ similarly, depending on HAK5; however, the translocation and accumulation of the two elements are different.

Yet uninfected? Resolving cell states of plants under pathogen attack.

Although we have made significant strides in unraveling plant responses to pathogen attacks at the tissue or major cell type scale, a comprehensive understanding of individual cell responses still needs to be achieved. Addressing this gap, Zhu et al. employed single-cell transcriptome analysis to unveil the heterogeneous responses of plant cells when confronted with bacterial pathogens.

Multiplexed single-cell 3D spatial gene expression analysis in plant tissue using PHYTOMap.

Retrieving the complex responses of individual cells in the native three-dimensional tissue context is crucial for a complete understanding of tissue functions. Here, we present PHYTOMap (plant hybridization-based targeted observation of gene expression map), a multiplexed fluorescence in situ hybridization method that enables single-cell and spatial analysis of gene expression in whole-mount plant tissue in a transgene-free manner and at low cost. We applied PHYTOMap to simultaneously analyse 28 cell-type marker genes in Arabidopsis roots and successfully identified major cell types, demonstrating that our method can substantially accelerate the spatial mapping of marker genes defined in single-cell RNA-sequencing datasets in complex plant tissue.

Dissecting the cotranscriptome landscape of plants and their microbiota.

Interactions between plants and neighboring microbial species are fundamental elements that collectively determine the structure and function of the plant microbiota. However, the molecular basis of such interactions is poorly characterized. Here, we colonize Arabidopsis leaves with nine plant-associated bacteria from all major phyla of the plant microbiota and profile cotranscriptomes of plants and bacteria six hours after inoculation. We detect both common and distinct cotranscriptome signatures among plant-commensal pairs. In planta responses of commensals are similar to those of a disarmed pathogen characterized by the suppression of genes involved in general metabolism in contrast to a virulent pathogen. We identify genes that are enriched in the genome of plant-associated bacteria and induced in planta, which may be instrumental for bacterial adaptation to the host environment and niche separation. This study provides insights into how plants discriminate among bacterial strains and lays the foundation for in-depth mechanistic dissection of plant-microbiota interactions.

PhcQ mainly contributes to the regulation of quorum sensing-dependent genes, in which PhcR is partially involved, in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal via the methyltransferase PhcB and senses the chemical through the sensor histidine kinase PhcS. This leads to functionalization of the LysR family transcriptional regulator PhcA, regulating QS-dependent genes responsible for the QS-dependent phenotypes including virulence. The phc operon consists of phcB, phcS, phcR, and phcQ, with the latter two encoding regulator proteins with a receiver domain and a histidine kinase domain and with a receiver domain, respectively. To elucidate the function of PhcR and PhcQ in the regulation of QS-dependent genes, we generated phcR-deletion and phcQ-deletion mutants. Though the QS-dependent phenotypes of the phcR-deletion mutant were largely unchanged, deletion of phcQ led to a significant change in the QS-dependent phenotypes. Transcriptome analysis coupled with quantitative reverse transcription-PCR and RNA-sequencing revealed that phcB, phcK, and phcA in the phcR-deletion and phcQ-deletion mutants were expressed at similar levels as in strain OE1-1. Compared with strain OE1-1, expression of 22.9% and 26.4% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcR-deletion mutant. However, expression of 96.8% and 66.9% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcQ-deletion mutant. Furthermore, a strong positive correlation of expression of these QS-dependent genes was observed between the phcQ-deletion and phcA-deletion mutants. Our results indicate that PhcQ mainly contributes to the regulation of QS-dependent genes, in which PhcR is partially involved.

Plant single-cell solutions for energy and the environment.

Progress in sequencing, microfluidics, and analysis strategies has revolutionized the granularity at which multicellular organisms can be studied. In particular, single-cell transcriptomics has led to fundamental new insights into animal biology, such as the discovery of new cell types and cell type-specific disease processes. However, the application of single-cell approaches to plants, fungi, algae, or bacteria (environmental organisms) has been far more limited, largely due to the challenges posed by polysaccharide walls surrounding these species' cells. In this perspective, we discuss opportunities afforded by single-cell technologies for energy and environmental science and grand challenges that must be tackled to apply these approaches to plants, fungi and algae. We highlight the need to develop better and more comprehensive single-cell technologies, analysis and visualization tools, and tissue preparation methods. We advocate for the creation of a centralized, open-access database to house plant single-cell data. Finally, we consider how such efforts should balance the need for deep characterization of select model species while still capturing the diversity in the plant kingdom. Investments into the development of methods, their application to relevant species, and the creation of resources to support data dissemination will enable groundbreaking insights to propel energy and environmental science forward.

Author Correction: Multidimensional gene regulatory landscape of a bacterial pathogen in plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multidimensional gene regulatory landscape of a bacterial pathogen in plants.

Understanding the gene regulation of plant pathogens is crucial for pest control and thus global food security. An integrated understanding of bacterial gene regulation in the host is dependent on multi-omic datasets, but these are largely lacking. Here, we simultaneously characterized the transcriptome and proteome of a bacterial pathogen in plants. We found a number of bacterial processes affected by plant immunity at the transcriptome and proteome levels. For instance, salicylic acid-mediated plant immunity suppressed the accumulation of proteins comprising the tip component of the bacterial type III secretion system. Interestingly, there were instances of concordant and discordant regulation of bacterial messenger RNAs and proteins. Gene co-expression analysis uncovered previously unknown gene regulatory modules underlying virulence. This study provides molecular insights into the multiple layers of gene regulation that contribute to bacterial growth in planta, and elucidates the role of plant immunity in affecting pathogen responses.

Site-specific cleavage of bacterial MucD by secreted proteases mediates antibacterial resistance in Arabidopsis.

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucDF106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.

The plant immune system in heterogeneous environments.

The plant immune system inhibits pathogen growth and contributes to shaping a healthy microbial community in the plant body. Plants must appropriately respond to both microbial signals and abiotic factors that are diverse in time and space, and thus, proper integration of these inputs at local and systemic levels is of crucial importance for optimal plant responses and fitness in nature. Here, we review our current knowledge of three properties of the plant immune system, resilience, tunability, and balance, which enable plants to deal with complex cocktails of environmental factors. We also discuss future challenges on the path towards a comprehensive understanding of the interactions between plant immunity and pathogenic, non-pathogenic, and beneficial microbes.

Balancing trade-offs between biotic and abiotic stress responses through leaf age-dependent variation in stress hormone cross-talk.

In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance.

Molecular networks in plant-pathogen holobiont.

Plant immune receptors enable detection of a multitude of microbes including pathogens. The recognition of microbes activates various plant signaling pathways, such as those mediated by phytohormones. Over the course of coevolution with microbes, plants have expanded their repertoire of immune receptors and signaling components, resulting in highly interconnected plant immune networks. These immune networks enable plants to appropriately respond to different types of microbes and to coordinate immune responses with developmental programs and environmental stress responses. However, the interconnectivity in plant immune networks is exploited by microbial pathogens to promote pathogen fitness in plants. Analogous to plant immune networks, virulence-related pathways in bacterial pathogens are also interconnected. Accumulating evidence implies that some plant-derived compounds target bacterial virulence networks. Thus, the plant immune and bacterial virulence networks intimately interact with each other. Here, we highlight recent insights into the structures of the plant immune and bacterial virulence networks and the interactions between them. We propose that small molecules derived from plants and/or bacterial pathogens connect the two molecular networks, forming supernetworks in the plant-bacterial pathogen holobiont.

Transcriptome landscape of a bacterial pathogen under plant immunity.

Plant pathogens can cause serious diseases that impact global agriculture. The plant innate immunity, when fully activated, can halt pathogen growth in plants. Despite extensive studies into the molecular and genetic bases of plant immunity against pathogens, the influence of plant immunity in global pathogen metabolism to restrict pathogen growth is poorly understood. Here, we developed RNA sequencing pipelines for analyzing bacterial transcriptomes in planta and determined high-resolution transcriptome patterns of the foliar bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana with a total of 27 combinations of plant immunity mutants and bacterial strains. Bacterial transcriptomes were analyzed at 6 h post infection to capture early effects of plant immunity on bacterial processes and to avoid secondary effects caused by different bacterial population densities in planta We identified specific "immune-responsive" bacterial genes and processes, including those that are activated in susceptible plants and suppressed by plant immune activation. Expression patterns of immune-responsive bacterial genes at the early time point were tightly linked to later bacterial growth levels in different host genotypes. Moreover, we found that a bacterial iron acquisition pathway is commonly suppressed by multiple plant immune-signaling pathways. Overexpression of a P. syringae sigma factor gene involved in iron regulation and other processes partially countered bacterial growth restriction during the plant immune response triggered by AvrRpt2. Collectively, this study defines the effects of plant immunity on the transcriptome of a bacterial pathogen and sheds light on the enigmatic mechanisms of bacterial growth inhibition during the plant immune response.