NBR1-mediated selective autophagy of ARF7 modulates root branching.

Auxin dictates root architecture via the Auxin Response Factor (ARF) family of transcription factors, which control lateral root (LR) formation. In Arabidopsis, ARF7 regulates the specification of prebranch sites (PBS) generating LRs through gene expression oscillations and plays a pivotal role during LR initiation. Despite the importance of ARF7 in this process, there is a surprising lack of knowledge about how ARF7 turnover is regulated and how this impacts root architecture. Here, we show that ARF7 accumulates in autophagy mutants and is degraded through NBR1-dependent selective autophagy. We demonstrate that the previously reported rhythmic changes to ARF7 abundance in roots are modulated via autophagy and might occur in other tissues. In addition, we show that the level of co-localization between ARF7 and autophagy markers oscillates and can be modulated by auxin to trigger ARF7 turnover. Furthermore, we observe that autophagy impairment prevents ARF7 oscillation and reduces both PBS establishment and LR formation. In conclusion, we report a novel role for autophagy during development, namely by enacting auxin-induced selective degradation of ARF7 to optimize periodic root branching.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

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.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Determination of De Novo Suberin-Lignin Ferulate Deposition in Xylem Tissue Upon Vascular Pathogen Attack.

Plant vascular pathogens use different ways to reach the xylem vessels and cause devastating diseases in plants. Resistant and tolerant plants have evolved various defense mechanisms against vascular pathogens. Inducible physico-chemical structures, such as the formation of tyloses and wall reinforcements with phenolic polymers, are very effective barriers that confine the pathogen and prevent colonization. Here, we use a combination of classical histochemistry along with bright-field and fluorescence microscopy and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy to visualize and characterize wall reinforcements containing phenolic wall polymers, namely, lignin, ferulates, and suberin, which occur in different xylem vasculature in response to pathogen attack.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

The Tomato Feruloyl Transferase FHT Promoter Is an Accurate Identifier of Early Development and Stress-Induced Suberization.

As a wall polymer, suberin has a multifaceted role in plant development and stress responses. It is deposited between the plasma membrane and the primary cell wall in specialized tissues such as root exodermis, endodermis, phellem, and seed coats. It is formed de novo in response to stresses such as wounding, salt injury, drought, and pathogen attack and is a complex polyester mainly consisting of fatty acids, glycerol, and minor amounts of ferulic acid that are associated to a lignin-like polymer predominantly composed of ferulates. Metabolomic and transcriptomic studies have revealed that cell wall lignification precedes suberin deposition. The ferulic acid esterified to ω-hydroxy fatty acids, synthetized by the feruloyl transferase FHT (or ASFT), presumably plays a role in coupling both polymers, although the precise mechanism is not understood. Here, we use the promoter of tomato suberin feruloyl transferase (FHT/ASFT) fused to GUS (ß-glucuronidase) to demonstrate that ferulate deposition agrees with the site of promoter FHT activation by using a combination of histochemical staining and UV microscopy. Hence, FHT promoter activation and alkali UV microscopy can be used to identify the precise localization of early suberizing cells rich in ferulic acid and can additionally be used as an efficient marker of early suberization events during plant development and stress responses. This line can be used in the future as a tool to identify emerging suberization sites via ferulate deposition in tomato plants, which may contribute to germplasm screening in varietal improvement programs.

Cell death as a defense strategy against pathogens in plants and animals.

Eukaryotes are endowed with sophisticated innate immune systems to recognize non-self and halt pathogen proliferation. Activation of cell death at the site of attempted pathogen ingress is a common strategy used by plants and animals to restrict pathogen proliferation and trigger immune responses in the surrounding tissues. As such, immunogenic cell death shares several features in both plants and animals that will be discussed in this article, namely: (i) it is triggered by activation of NLR immune receptors-often through oligomerization; (ii) it results in disruption of the plasma membrane (PM)/endomembrane integrity driving an imbalance in ion fluxes; and (iii) it results in the release of signaling molecules from dying cells.

Detection and Quantification of Protein Aggregates in Plants.

Protein quality control is an important aspect of stress recovery. It maintains protein homeostasis through a machinery of regulatory proteins such as chaperones and proteases. When the system recognizes accumulation of misfolded or aggregated proteins, the cell recruits a set of regulatory proteins to initiate protein quality control. To understand the dynamics of stress-mediated aggregate protein formation and recovery in plants, robust methods aimed at detecting and measuring such protein aggregates are needed. This will help us to deepen our understanding of protein quality control mechanisms in plants.

Dying in self-defence: a comparative overview of immunogenic cell death signalling in animals and plants.

Host organisms utilise a range of genetically encoded cell death programmes in response to pathogen challenge. Host cell death can restrict pathogen proliferation by depleting their replicative niche and at the same time dying cells can alert neighbouring cells to prepare environmental conditions favouring future pathogen attacks. As expected, many pathogenic microbes have strategies to subvert host cell death to promote their virulence. The structural and lifestyle differences between animals and plants have been anticipated to shape very different host defence mechanisms. However, an emerging body of evidence indicates that several components of the host-pathogen interaction machinery are shared between the two major branches of eukaryotic life. Many proteins involved in cell death execution or cell death-associated immunity in plants and animals exert direct effects on endomembrane and loss of membrane integrity has been proposed to explain the potential immunogenicity of dying cells. In this review we aim to provide a comparative view on how cell death processes are linked to anti-microbial defence mechanisms in plants and animals and how pathogens interfere with these cell death programmes. In comparison to the several well-defined cell death programmes in animals, immunogenic cell death in plant defence is broadly defined as the hypersensitive response. Our comparative overview may help discerning whether specific types of immunogenic cell death exist in plants, and correspondingly, it may provide new hints for previously undiscovered cell death mechanism in animals.

Robust transcriptional indicators of immune cell death revealed by spatiotemporal transcriptome analyses.

Recognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response (HR). This type of cell death occurs precisely at the site of pathogen recognition, and it is restricted to a few cells. Extensive research has shed light on how plant immune receptors are mechanistically activated. However, two central key questions remain largely unresolved: how does cell death zonation take place, and what are the mechanisms that underpin this phenomenon? Consequently, bona fide transcriptional indicators of HR are lacking, which prevents deeper insight into its mechanisms before cell death becomes macroscopic and precludes early or live observation. In this study, to identify the transcriptional indicators of HR we used the paradigmatic Arabidopsis thaliana-Pseudomonas syringae pathosystem and performed a spatiotemporally resolved gene expression analysis that compared infected cells that will undergo HR upon pathogen recognition with bystander cells that will stay alive and activate immunity. Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes, expression profiles, and biological processes derived from tissue undergoing HR and that of its surroundings. Furthermore, we generated a pipeline based on concatenated pairwise comparisons between time, zone, and treatment that enabled us to define 13 robust transcriptional HR markers. Among these genes, the promoter of an uncharacterized AAA-ATPase was used to obtain a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death. This valuable set of genes can be used to define cells that are destined to die upon infection with HR-triggering bacteria, opening new avenues for specific and/or high-throughput techniques to study HR processes at a single-cell level.

Detection and Quantification of the Hypersensitive Response Cell Death in Arabidopsis thaliana.

In plants, the hypersensitive response (HR) is a programmed cell death modality that occurs upon recognition of harmful non-self. It occurs at the site of pathogen infection, thus preventing pathogens to live off plant tissue and proliferate. Shedding light on the molecular constituents underlying this process requires robust and quantitative methods that can determine whether plants lacking functional genes are defective in HR execution compared to wild-type controls. In this chapter, we provide two quantitative protocols in which we measure cell death from Arabidopsis thaliana leaves infected with avirulent HR-causing bacterial strains. Firstly, we use trypan blue staining to quantify the stained area of leaves upon bacterial infection using a personalized macro in the Image J (Fiji) software. Alternately, we incorporate an electrolyte leakage protocol in order to measure HR caused by different avirulent bacterial strains at different bacterial titers. We encourage users to perform a combination of both methods when assessing HR in different plant genotypes.

Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato.

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses.

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1-107 ) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant-pathogen co-evolution.

The Bacterial Wilt Reservoir Host Solanum dulcamara Shows Resistance to Ralstonia solanacearum Infection.

Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum.

Molecular Detection of Ralstonia solanacearum to Facilitate Breeding for Resistance to Bacterial Wilt in Potato.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.