ScAnalyzer: an image processing tool to monitor plant disease symptoms and pathogen spread in Arabidopsis thaliana leaves.

BACKGROUND: Plants are known to be infected by a wide range of pathogenic microbes. To study plant diseases caused by microbes, it is imperative to be able to monitor disease symptoms and microbial colonization in a quantitative and objective manner. In contrast to more traditional measures that use manual assignments of disease categories, image processing provides a more accurate and objective quantification of plant disease symptoms. Besides monitoring disease symptoms, computational image processing provides additional information on the spatial localization of pathogenic microbes in different plant tissues. RESULTS: Here we report on an image analysis tool called ScAnalyzer to monitor disease symptoms and bacterial spread in Arabidopsis thaliana leaves. Thereto, detached leaves are assembled in a grid and scanned, which enables automated separation of individual samples. A pixel color threshold is used to segment healthy (green) from chlorotic (yellow) leaf areas. The spread of luminescence-tagged bacteria is monitored via light-sensitive films, which are processed in a similar manner as the leaf scans. We show that this tool is able to capture previously identified differences in susceptibility of the model plant A. thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris. Moreover, we show that the ScAnalyzer pipeline provides a more detailed assessment of bacterial spread within plant leaves than previously used methods. Finally, by combining the disease symptom values with bacterial spread values from the same leaves, we show that bacterial spread precedes visual disease symptoms. CONCLUSION: Taken together, we present an automated script to monitor plant disease symptoms and microbial spread in A. thaliana leaves. The freely available software ( https://github.com/MolPlantPathology/ScAnalyzer ) has the potential to standardize the analysis of disease assays between different groups.

Specific members of the TOPLESS family are susceptibility genes for Fusarium wilt in tomato and Arabidopsis.

Vascular wilt diseases caused by Fusarium oxysporum are a major threat to many agriculturally important crops. Genetic resistance is rare and inevitably overcome by the emergence of new races. To identify potentially durable and non-race-specific genetic resistance against Fusarium wilt diseases, we set out to identify effector targets in tomato that mediate susceptibility to the fungus. For this purpose, we used the SIX8 effector protein, an important and conserved virulence factor present in many pathogenic F. oxysporum isolates. Using protein pull-downs and yeast two-hybrid assays, SIX8 was found to interact specifically with two members of the tomato TOPLESS family: TPL1 and TPL2. Loss-of-function mutations in TPL1 strongly reduced disease susceptibility to Fusarium wilt and a tpl1;tpl2 double mutant exerted an even higher level of resistance. Similarly, Arabidopsis tpl;tpr1 mutants became significantly less diseased upon F. oxysporum inoculation as compared to wildtype plants. We conclude that TPLs encode susceptibility genes whose mutation can confer resistance to F. oxysporum.

Interaction of Whitefly Effector G4 with Tomato Proteins Impacts Whitefly Performance.

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci. Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome sequence of a novel phlebovirus associated with lettuce big vein disease infecting lettuce (Lactuca spp.).

Lettuce big vein disease is a disease complex involving at least two RNA viruses, both transmitted by the soilborne fungus Olpidium virulentus. Here, we present the genomic sequence of a novel unrelated third negative-stranded RNA virus, belonging to the family Phenuiviridae, recovered from infected lettuce plants.

Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens.

Plants prevent disease by passively and actively protecting potential entry routes against invading microbes. For example, the plant immune system actively guards roots, wounds, and stomata. How plants prevent vascular disease upon bacterial entry via guttation fluids excreted from specialized glands at the leaf margin remains largely unknown. These so-called hydathodes release xylem sap when root pressure is too high. By studying hydathode colonization by both hydathode-adapted (Xanthomonas campestris pv. campestris) and non-adapted pathogenic bacteria (Pseudomonas syringae pv. tomato) in immunocompromised Arabidopsis mutants, we show that the immune hubs BAK1 and EDS1-PAD4-ADR1 restrict bacterial multiplication in hydathodes. Both immune hubs effectively confine bacterial pathogens to hydathodes and lower the number of successful escape events of an hydathode-adapted pathogen toward the xylem. A second layer of defense, which is dependent on the plant hormones' pipecolic acid and to a lesser extent on salicylic acid, reduces the vascular spread of the pathogen. Thus, besides glands, hydathodes represent a potent first line of defense against leaf-invading microbes.

Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana.

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus-induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants' performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative-stranded TSWV and the positive-stranded potato virus X.

An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in Nicotiana benthamiana.

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development.

Synergistic interaction between the type III secretion system of the endophytic bacterium Pantoea agglomerans DAPP-PG 734 and the virulence of the causal agent of olive knot Pseudomonas savastanoi pv. savastanoi DAPP-PG 722.

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722.

The protein modifier SUMO is critical for integrity of the Arabidopsis shoot apex at warm ambient temperatures.

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis - a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype.

Antagonism between SUMO1/2 and SUMO3 regulates SUMO conjugate levels and fine-tunes immunity.

The attachment of SMALL UBIQUITIN-LIKE MODIFIER (SUMO) to target proteins regulates a plethora of cellular processes across eukaryotes. In Arabidopsis thaliana, mutants with abnormal SUMO1/2 conjugate levels display a dwarf stature, autoimmunity, and altered stress responses to adverse environmental conditions. Since the SUMO pathway is known to autoregulate its biochemical activity (via allosteric interactions), we assessed whether the emergence of additional SUMO paralogs in Arabidopsis has introduced the capacity of self-regulation by means of isoform diversification in this model plant. By studying the plant defense responses elicited by the bacterial pathogen Pseudomonas syringae pv. tomato, we provide genetic evidence that SUM3, a divergent paralog, acts downstream of the two main SUMO paralogues, SUM1/2. The expression of SUM3 apparently buffers or suppresses the function of SUM1/2 by controlling the timing and amplitude of the immune response. Moreover, SUM1 and SUM2 work additively to suppress both basal and TNL-specific immunity, a specific branch of the immune network. Finally, our data reveal that SUM3 is required for the global increase in SUMO1/2 conjugates upon exposure to biotic and abiotic stresses, namely heat and pathogen exposure. We cannot exclude that this latter effect is independent of the role of SUM3 in immunity.

A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells.

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.

The impact of green spaces on mental health in urban settings: a scoping review.

BACKGROUND: Our planet is currently experiencing the largest wave of urban growth in history, with 55% of the world's population (4.2 billion people) currently living in urban areas, a figure set to rise to 70% by 2050. Primary care is the first point of treatment for most mental health disorders. Since the urban environment and health are intrinsically linked, it is useful to examine the effectiveness of Nature-Based Solutions in addressing mental health. AIMS: The aim of this scoping review was to examine "How urban green spaces can improve mental health and wellbeing among patients attending general practice and primary care?" METHODS: The scoping review framework comprised a five-stage process developed by Arksey and O'Malley. RESULTS: Twenty-five studies were included in the review, mostly cross-sectional studies. A wide range of mental health outcomes were identified. Out of the 25 included studies, 23 showed positive associations between mental health and green space characteristics. CONCLUSION: Our findings suggest that while green spaces are associated with positive mental health outcomes, most studies were cross-sectional in nature and provided limited evidence for association. Furthermore, the population samples used in most of the studies were taken from national survey data and were not specifically primary care patients. Further studies that assess primary care patients specifically are warranted.

Regulation of Aluminum Resistance in Arabidopsis Involves the SUMOylation of the Zinc Finger Transcription Factor STOP1.

Aluminum (Al) is a primary constraint for crop production on acid soils, which make up more than 30% of the arable land in the world. Al resistance in Arabidopsis (Arabidopsis thaliana) is achieved by malate secretion mediated by the Al-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) transporter. The C2H2-type transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) is essential and required for Al resistance, where it acts by inducing the expression of Al-resistance genes, including AtALMT1 In this study, we report that STOP1 protein function is modified by SUMOylation. The SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protease ESD4, but not other SUMO proteases, specifically interacts with and deSUMOylates STOP1. Mutation of ESD4 increases the level of STOP1 SUMOylation and the expression of the STOP1-regulated gene AtALMT1, which contributes to the increased Al resistance in esd4 The esd4 mutation does not influence STOP1 protein abundance but increases the association of STOP1 with the AtALMT1 promoter, which might explain the elevated expression of AtALMT1 in esd4 We demonstrate that STOP1 is mono-SUMOylated at K40, K212, or K395 sites, and blocking STOP1 SUMOylation reduces STOP1 stability and the expression of STOP1-regulated genes, leading to the reduced Al resistance. Our results thus reveal the involvement of SUMOylation in the regulation of STOP1 and Al resistance in Arabidopsis.

Identification of Tomato Proteins That Interact With Replication Initiator Protein (Rep) of the Geminivirus TYLCV.

Geminiviruses are plant-infecting DNA viruses that reshape the intracellular environment of their host in order to create favorable conditions for viral replication and propagation. Viral manipulation is largely mediated via interactions between viral and host proteins. Identification of this protein network helps us to understand how these viruses manipulate their host and therefore provides us potentially with novel leads for resistance against this class of pathogens, as genetic variation in the corresponding plant genes could subvert viral manipulation. Different studies have already yielded a list of host proteins that interact with one of the geminiviral proteins. Here, we use affinity purification followed by mass spectrometry (AP-MS) to further expand this list of interacting proteins, focusing on an important host (tomato) and the Replication initiator protein (Rep, AL1, C1) from Tomato yellow leaf curl virus (TYLCV). Rep is the only geminiviral protein proven to be essential for geminiviral replication and it forms an integral part of viral replisomes, a protein complex that consists of plant and viral proteins that allows for viral DNA replication. Using AP-MS, fifty-four 'high confidence' tomato proteins were identified that specifically co-purified with Rep. For two of them, an unknown EWS-like RNA-binding protein (called Geminivirus Rep interacting EWS-like protein 1 or GRIEP1) and an isoform of the THO complex subunit 4A (ALY1), we were able to confirm this interaction with Rep in planta using a second method, bimolecular fluorescence complementation (BiFC). The THO subunit 4 is part of the THO/TREX (TRanscription-EXport) complex, which controls RNA splicing and nuclear export of mRNA to the cytoplasm and is also connected to plant disease resistance. This work represents the first step towards characterization of novel host factors with a putative role in the life cycle of TYLCV and possibly other geminiviruses.

Infection Assay for Xanthomonas campestris pv. campestris in Arabidopsis thaliana Mimicking Natural Entry via Hydathodes.

Xanthomonas campestris pv. campestris (Xcc) causes the devastating disease Black rot in Brassicaceae. Typically Xcc enters the plant through specialized organs on the leaf margin, called hydathodes, and spreads from there through the vasculature. In order to mimic natural entry as closely as possible, we here describe a "hydathode guttation"-based entry assay for Xcc in Arabidopsis. This disease assay combines spray inoculation with the induction of guttation and allows reabsorption of guttation droplets by the plant. Moreover, our assay relies on a bioluminescent reporter strain of Xcc to allow direct visualization of both entry and subsequent spreading of Xcc in its host. The assay allows the routine infection from one to two hydathodes per Arabidopsis leaf. Infections are scored 14 days post inoculation, just before the infection goes systemic.

A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein.

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined.IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme.

A Na+ /Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence.

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+ /Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+ /Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+ /Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a 'host signal' that allows and promotes Psav pathogenicity on olive plants.

The SUMO Conjugation Complex Self-Assembles into Nuclear Bodies Independent of SIZ1 and COP1.

Attachment of the small ubiquitin-like modifier (SUMO) to substrate proteins modulates their turnover, activity, or interaction partners. However, how this SUMO conjugation activity concentrates the proteins involved and the substrates into uncharacterized nuclear bodies (NBs) remains poorly understood. Here, we characterized the requirements for SUMO NB formation and for their subsequent colocalization with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a master regulator of plant growth. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by darkness and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity alone was sufficient to target the SUMO machinery into NBs. Colocalization of these bodies with COP1 required, in addition to SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SAP and Miz 1 (SIZ1). We found that SIZ1 docks in the substrate-binding pocket of COP1 via two valine-proline peptide motifs, which represent a known interaction motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRYPTOCHROME 1 and CRYPTOCHROME 2. Our findings thus suggest that sumoylation stimulates COP1 activity within NBs. Moreover, the presence of SIZ1 and SUMO in these NBs explains how both the timing and amplitude of the high-temperature growth response is controlled. The strong colocalization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated.