The transcription factor VviNAC60 regulates senescence- and ripening-related processes in grapevine.

Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the upregulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being upregulated through a VviNAC60-VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologs to the NAC transcription factor AtNAP, VviNAC60 complemented the nonripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase.

NAC61 regulates late- and post-ripening osmotic, oxidative, and biotic stress responses in grapevine.

During late- and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes involved in berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves resulted in low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61 and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14, the osmotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene WRKY52, and NAC61 itself was validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulatory network that governs stilbenoid metabolism and osmotic, oxidative, and biotic stress responses that are the hallmark of late- and post-ripening grape stages.

Red and Blue Light Differently Influence Actinidia chinensis Performance and Its Interaction with Pseudomonas syringae pv. Actinidiae.

Light composition modulates plant growth and defenses, thus influencing plant-pathogen interactions. We investigated the effects of different light-emitting diode (LED) red (R) (665 nm) and blue (B) (470 nm) light combinations on Actinidia chinensis performance by evaluating biometric parameters, chlorophyll a fluorescence, gas exchange and photosynthesis-related gene expression. Moreover, the influence of light on the infection by Pseudomonas syringae pv. actinidiae (Psa), the etiological agent of bacterial canker of kiwifruit, was investigated. Our study shows that 50%R-50%B (50R) and 25%R-75%B (25R) lead to the highest PSII efficiency and photosynthetic rate, but are the least effective in controlling the endophytic colonization of the host by Psa. Monochromatic red light severely reduced ΦPSII, ETR, Pn, TSS and photosynthesis-related genes expression, and both monochromatic lights lead to a reduction of DW and pigments content. Monochromatic blue light was the only treatment significantly reducing disease symptoms but did not reduce bacterial endophytic population. Our results suggest that monochromatic blue light reduces infection primarily by modulating Psa virulence more than host plant defenses.

Transcriptional Profiling of Three Pseudomonas syringae pv. actinidiae Biovars Reveals Different Responses to Apoplast-Like Conditions Related to Strain Virulence on the Host.

Pseudomonas syringae pv. actinidiae is a phytopathogen that causes devastating bacterial canker in kiwifruit. Among five biovars defined by genetic, biochemical, and virulence traits, P. syringae pv. actinidiae biovar 3 (Psa3) is the most aggressive and is responsible for the most recent reported outbreaks; however, the molecular basis of its heightened virulence is unclear. Therefore, we designed the first P. syringae multistrain whole-genome microarray, encompassing biovars Psa1, Psa2, and Psa3 and the well-established model P. syringae pv. tomato, and analyzed early bacterial responses to an apoplast-like minimal medium. Transcriptomic profiling revealed i) the strong activation in Psa3 of all hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) cluster genes, encoding components of the type III secretion system required for bacterial pathogenicity and involved in responses to environmental signals; ii) potential repression of the hrp/hrc cluster in Psa2; and iii) activation of flagellum-dependent cell motility and chemotaxis genes in Psa1. The detailed investigation of three gene families encoding upstream regulatory proteins (histidine kinases, their cognate response regulators, and proteins with diguanylate cyclase or phosphodiesterase domains) indicated that cyclic di-GMP may be a key regulator of virulence in P. syringae pv. actinidiae biovars. The gene expression data were supported by the quantification of biofilm formation. Our findings suggest that diverse early responses to the host apoplast, even among bacteria belonging to the same pathovar, can lead to different virulence strategies and may explain the differing outcomes of infections. Based on our detailed structural analysis of hrp operons, we also propose a revision of hrp cluster organization and operon regulation in P. syringae.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Grapevine E3 Ubiquitin Ligase VriATL156 Confers Resistance against the Downy Mildew Pathogen Plasmopara viticola.

Downy mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine (Vitis vinifera L.). Genetic resistance is an effective and sustainable control strategy, but major resistance genes (encoding receptors for specific pathogen effectors) introgressed from wild Vitis species, although effective, may be non-durable because the pathogen can evolve to avoid specific recognition. Previous transcriptomic studies in the resistant species Vitis riparia highlighted the activation of signal transduction components during infection. The transfer of such components to V. vinifera might confer less specific and therefore more durable resistance. Here, we describe the generation of transgenic V. vinifera lines constitutively expressing the V. riparia E3 ubiquitin ligase gene VriATL156. Phenotypic and molecular analysis revealed that the transgenic plants were less susceptible to P. viticola than vector-only controls, confirming the role of this E3 ubiquitin ligase in the innate immune response. Two independent transgenic lines were selected for detailed analysis of the resistance phenotype by RNA-Seq and microscopy, revealing the profound reprogramming of transcription to achieve resistance that operates from the earliest stages of pathogen infection. The introduction of VriATL156 into elite grapevine cultivars could therefore provide an effective and sustainable control measure against downy mildew.

Genetic buffering of cyclic AMP in Arabidopsis thaliana compromises the plant immune response triggered by an avirulent strain of Pseudomonas syringae pv. tomato.

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non-specific pharmacological approaches. We therefore developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the 'cAMP-sponge' (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with wild-type (WT) plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24 h post-infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium-calmodulin-binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis-regulation of AtSR1 activity, affected the speed and strength of the immune response.

Host-Mediated S-Nitrosylation Disarms the Bacterial Effector HopAI1 to Reestablish Immunity.

Pathogens deliver effectors into plant cells to suppress immunity-related signaling. However, effector recognition by the host elicits a hypersensitive response (HR) that overcomes the inhibition of host signaling networks, restoring disease resistance. Signaling components are shared between the pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity, and it is unclear how plants inactivate these effectors to execute the HR. Here, we report that, in Arabidopsis thaliana, during the onset of the HR, the bacterial effector HopAI1 is S-nitrosylated and that this modification inhibits its phosphothreonine lyase activity. HopAI1 targets and suppresses mitogen-activated protein kinases (MAPKs). The S-nitrosylation of HopAI1 restores MAPK signaling and is required during the HR for activation of the associated cell death. S-nitrosylation is therefore revealed here as a nitric oxide-dependent host strategy involved in plant immunity that works by directly disarming effector proteins.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Detection and function of nitric oxide during the hypersensitive response in Arabidopsis thaliana: where there's a will there's a way.

Nitric oxide (NO) was identified as a key player in plant defence responses approximately 20 years ago and a large body of evidence has accumulated since then supporting its role as a signalling molecule. However, there are many discrepancies in current NO detection assays and the enzymatic pathways responsible for its synthesis have yet to be determined. This has provoked strong debates concerning the function of NO in plants, even questioning its existence in planta. Here we gather data obtained using the model pathosystem Arabidopsis/Pseudomonas, which confirms the production of NO during the hypersensitive response and supports is role as a trigger of hypersensitive cell death and a mediator of defence gene expression. Finally, we discuss potential sources of NO synthesis, focusing on the role of nitrite as major substrate for NO production during incompatible interactions.

Host-specific signal perception by PsaR2 LuxR solo induces Pseudomonas syringae pv. actinidiae virulence traits.

Plant-associated bacteria, including pathogens, recognise host-derived signals to activate specific responses. The genome of Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of bacterial canker of kiwifruit, encodes for three putative LuxR-like receptors. Proteins of this family are usually involved in the quorum sensing system, through the perception of autoinducers (AHLs) produced by a cognate LuxI. However, Psa does not produce AHLs according to the lack of LuxI-encoding gene. It has been proposed that the so-called LuxR solos may be involved in the perception of environmental stimuli. We thus hypothesised that Psa LuxR-like receptors could be involved in host-derived signal sensing. Psa virulence traits, i.e., biofilm formation, motility and endophytic colonisation, were stimulated by growing the pathogen in host plant extracts, but not in non-host plant extracts or rich medium. Moreover, the phenotypic analyses of Psa mutant strains lacking the LuxR solo-encoding genes, demonstrated that PsaR2 plays a major role in host recognition and induction of virulence responses. The heterologous expression of PsaR2, followed by affinity chromatography and fraction activity assessment, confirmed the specific recognition of plant-derived components by this sensor. Overall, these data provide a deeper understanding of the regulation of Psa virulence through interkingdom communication, which represents a interesting target for the development of tolerant/resistant genotypes or innovative control strategies.

Plant Signals Anticipate the Induction of the Type III Secretion System in Pseudomonas syringae pv. actinidiae, Facilitating Efficient Temperature-Dependent Effector Translocation.

Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipated T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the nonhost plant Arabidopsis thaliana Columbia-0 (Col-0). Our results show that in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. IMPORTANCE Plant diseases-their occurrence and severity-result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to what extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization make the difference in determining the outcome of an interaction and thus the occurrence of plant disease.

Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response.

Nitric oxide (NO) is synthesized in plants in response to stress, and its role in signaling is well-documented. In contrast, very little is known about the physiological role of its derivate peroxynitrite (ONOO(-)), which forms when NO reacts with O(2)(-) and induces protein modification by tyrosine nitration. Infection with an avirulent pathogen triggers the simultaneous production of NO and reactive oxygen species, as well as an increase in tyrosine nitration, so peroxynitrite could be physiologically relevant during this process. To gain insight into the role of peroxynitrite in plants, we measured its accumulation during the hypersensitive response in Arabidopsis thaliana using the specific peroxynitrite-sensitive fluorescent dye HKGreen-2 in a leaf disc assay. The avirulent pathogen Pseudomonas syringae pv. tomato, carrying the AvrB gene (Pst AvrB), induced a strong increase in fluorescence 3-4 h post-infiltration (hpi) which peaked 7-8 hpi. The increase in HKGreen-2 fluorescence was inhibited by co-injecting the peroxynitrite-scavenger urate together with the pathogen, and was almost completely eliminated by co-infiltrating urate with HKGreen-2, confirming that HKGreen-2 fluorescence in planta is induced specifically by peroxynitrite. This establishes a link between peroxynitrite synthesis and tyrosine nitration, and we therefore propose that peroxynitrite transduces the NO signal by modifying protein functions.

Activity and Mechanism of Action of the Bioceramic Silicon Nitride as an Environmentally Friendly Alternative for the Control of the Grapevine Downy Mildew Pathogen Plasmopara viticola.

Downy mildew of grapevine, caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, is one of the most devastating diseases of grapevine, severely affecting grape and wine production and quality worldwide. Infections are usually controlled by the intensive application of synthetic fungicides or by copper-based products in organic farming, rising problems for soil contamination and adverse impacts on environment and human health. While strict regulations attempt to minimize their harmful consequences, the situation calls for the development of alternative fungicidal strategies. This study presents the unprecedented case of a bioceramic, silicon nitride, with antimicrobial properties against P. viticola, but without adverse effects on human cells and environment, opening the way to the possible extension of silicon nitride applications in agriculture. Raman spectroscopic assessments of treated sporangia in conjunction with microscopic observations mechanistically showed that the nitrogen-chemistry of the bioceramic surface affects pathogen's biochemical components and cell viability, thus presenting a high potential for host protection from P. viticola infections.

Protein nitration during defense response in Arabidopsis thaliana.

Nitric oxide and reactive oxygen species play a key role in the plant hypersensitive disease resistance response, and protein tyrosine nitration is emerging as an important mechanism of their co-operative interaction. Up to now, the proteins targeted by this post-translational modification in plants are still totally unknown. In this study, we analyzed for the first time proteins undergoing nitration during the hypersensitive response by analyzing via 1D- and 2D-western blot the protein extracts from Arabidopsis thaliana plants challenged with an avirulent bacterial pathogen (Pseudomonas syringae pv. Tomato). We show that the plant disease resistance response is correlated with a modulation of nitration of proteins involved in important cellular process, such as photosynthesis, glycolysis and nitrate assimilation. These findings shed new light on the signaling functions of nitric oxide and reactive oxygen species, paving the way on studies on the role of this post-translational modification in plants.

Inhibition of Virulence-Related Traits in Pseudomonas syringae pv. actinidiae by Gunpowder Green Tea Extracts.

Green tea is a widely-consumed healthy drink produced from the leaves of Camellia sinensis. It is renowned for its antioxidant and anticarcinogenic properties, but also displays significant antimicrobial activity against numerous human pathogens. Here we analyzed the antimicrobial activity of Gunpowder green tea against Pseudomonas syringae pv. actinidiae (Psa), the agent that causes kiwifruit bacterial canker. At the phenotypic level, tea extracts strongly inhibited Psa growth and swimming motility, suggesting it could reduce Psa epiphytic survival during plant colonization. The loss of bacterial virulence-related traits following treatment with tea extracts was also investigated by large-scale transcriptome analysis, which confirmed the in vitro phenotypes and revealed the induction of adaptive responses in the treated bacteria allowing them to cope with iron deficiency and oxidative stress. Such molecular changes may account for the ability of Gunpowder green tea to protect kiwifruit against Psa infection.

Plant and fungus transcriptomic data from grapevine berries undergoing artificially-induced noble rot caused by Botrytis cinerea.

Noble rot is a latent infection of grape berries caused by the necrotrophic fungus Botrytis cinerea, which develops under specific climatic conditions. The infected berries undergo biochemical and metabolic changes, associated with a rapid withering, which altogether offer interesting organoleptic features to sweet white wines. In this paper, we provide RNAseq datasets (raw and normalized counts as well as differentially expressed genes lists) of the transcriptome profiles of both grapevine berries (Vitis vinifera cv. Garganega) and B. cinerea during the establishment of noble rot, artificially induced in controlled conditions. The sequencing data are available in the NCBI GEO database under accession number GSE116741. These data were exploited in a comprehensive meta-analysis of gene expression during noble rot infection, gray mold and post-harvest withering. This highlighted an important common transcriptional reprogramming in different botrytized grape berry varieties and led to the identification of key genes specifically modulated during noble rot infection, which are described in the article entitled "Specific molecular interactions between Vitis vinifera and Botrytis cinerea are required for noble rot development in grape berries" Lovato et al., 2019.

Recognition of Elicitors in Grapevine: From MAMP and DAMP Perception to Induced Resistance.

In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.

Measurement of Hypersensitive Cell Death Triggered by Avirulent Bacterial Pathogens in Arabidopsis.

The hypersensitive response is one of the most powerful and complex defense reactions to survive to pathogen attacks during an incompatible plant-pathogen interaction. Local programmed cell death accompanies the hypersensitive response at the site of infection to prevent pathogen growth and spread. A precise quantitative assessment of this form of programmed cell death is essential to unravel the genetic and molecular mechanisms underlying the process. Here, we first describe the optimization of a Trypan Blue staining protocol for quantitatively measuring the HR-cell death in Arabidopsis. Furthermore, we provide an electrolyte leakage protocol based on pathogen vacuum infiltration, which allows its simultaneous application to a large number of plants as well as to Arabidopsis mutants affected by small size phenotype.

Measurement of Cyclic GMP During Plant Hypersensitive Disease Resistance Response.

Cyclic guanosine-3',5'-monophosphate (cGMP) is recognized as an important second messenger in plants, mediating intracellular signal in important physiological processes, including the hypersensitive disease resistance response induced by avirulent pathogens. In this context, the analysis of cGMP levels in infected plants requires an accurate and specific detection method allowing its quantification. Here, we describe an assay based on the Alphascreen technology, developed for animal cells and further adapted and optimized for the detection of cGMP in plants. The method is applied for the measurement of cGMP in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the extraction of cGMP, the assay procedure and the calculation of cGMP concentration.

Co-expression network analysis and cis-regulatory element enrichment determine putative functions and regulatory mechanisms of grapevine ATL E3 ubiquitin ligases.

Arabidopsis thaliana Toxicos en Levadura (ATL) proteins are a subclass of the RING-H2 zinc finger binding E3 ubiquitin ligases. The grapevine (Vitis vinifera) ATL family was recently characterized, revealing 96 members that are likely to be involved in several physiological processes through protein ubiquitination. However, the final targets and biological functions of most ATL E3 ligases are still unknown. We analyzed the co-expression networks among grapevine ATL genes across a set of transcriptomic data related to defense and abiotic stress, combined with a condition-independent dataset. This revealed strong correlations between ATL proteins and diverse signal transduction components and transcriptional regulators, in particular those involved in immunity. An enrichment analysis of cis-regulatory elements in ATL gene promoters and related co-expressed genes highlighted the importance of hormones in the regulation of ATL gene expression. Our work identified several ATL proteins as candidates for further studies aiming to decipher specific grapevine resistance mechanisms activated in response to pathogens.