CDC48 regulates immunity pathway in tobacco plants.

The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.

New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i.

BACKGROUND: For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. 'Chardonnay'). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy. RESULTS: By comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. 'Chardonnay'. These results are directly correlated with Cas9 expression level. CONCLUSIONS: Taken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.

Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.

The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Biostimulation can prime elicitor induced resistance of grapevine leaves to downy mildew.

Using plant defense elicitors to protect crops against diseases is an attractive strategy to reduce chemical pesticide use. However, development of elicitors remains limited because of variable effectiveness in the field. In contrast to fungicides that directly target pathogens, elicitors activate plant immunity, which depends on plant physiological status. Other products, the biostimulants, can improve certain functions of plants. In this study, the objective was to determine whether a biostimulant via effects on grapevine physiology could increase effectiveness of a defense elicitor. A new methodology was developed to study biostimulant activity under controlled conditions using in vitro plantlets. Both biostimulant and defense elicitor used in the study were plant extracts. When added to the culture medium, the biostimulant accelerated the beginning of plantlet growth and affected the shoot and root development. It also modified metabolomes and phytohormone contents of leaves, stems, and roots. When applied on shoots, the defense elicitor changed metabolite and phytohormone contents, but effects were different depending on whether plantlets were biostimulated or controls. Defense responses and protection against Plasmopara viticola (downy mildew agent) were induced only for plantlets previously treated with the biostimulant, Therefore, the biostimulant may act by priming the defense elicitor action. In this study, a new method to screen biostimulants active on grapevine vegetative growth was used to demonstrate that a biostimulant can optimize the efficiency of a plant defense elicitor.

In situ Phenotyping of Grapevine Root System Architecture by 2D or 3D Imaging: Advantages and Limits of Three Cultivation Methods.

The root system plays an essential role in the development and physiology of the plant, as well as in its response to various stresses. However, it is often insufficiently studied, mainly because it is difficult to visualize. For grapevine, a plant of major economic interest, there is a growing need to study the root system, in particular to assess its resistance to biotic and abiotic stresses, understand the decline that may affect it, and identify new ecofriendly production systems. In this context, we have evaluated and compared three distinct growing methods (hydroponics, plane, and cylindric rhizotrons) in order to describe relevant architectural root traits of grapevine cuttings (mode of grapevine propagation), and also two 2D- (hydroponics and rhizotron) and one 3D- (neutron tomography) imaging techniques for visualization and quantification of roots. We observed that hydroponics tubes are a system easy to implement but do not allow the direct quantification of root traits over time, conversely to 2D imaging in rhizotron. We demonstrated that neutron tomography is relevant to quantify the root volume. We have also produced a new automated analysis method of digital photographs, adapted for identifying adventitious roots as a feature of root architecture in rhizotrons. This method integrates image segmentation, skeletonization, detection of adventitious root skeleton, and adventitious root reconstruction. Although this study was targeted to grapevine, most of the results obtained could be extended to other plants propagated by cuttings. Image analysis methods could also be adapted to characterization of the root system from seedlings.

Cross Kingdom Immunity: The Role of Immune Receptors and Downstream Signaling in Animal and Plant Cell Death.

Both plants and animals are endowed with sophisticated innate immune systems to combat microbial attack. In these multicellular eukaryotes, innate immunity implies the presence of cell surface receptors and intracellular receptors able to detect danger signal referred as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Membrane-associated pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are employed by these organisms for sensing different invasion patterns before triggering antimicrobial defenses that can be associated with a form of regulated cell death. Intracellularly, animals nucleotide-binding and oligomerization domain (NOD)-like receptors or plants nucleotide-binding domain (NBD)-containing leucine rich repeats (NLRs) immune receptors likely detect effectors injected into the host cell by the pathogen to hijack the immune signaling cascade. Interestingly, during the co-evolution between the hosts and their invaders, key cross-kingdom cell death-signaling macromolecular NLR-complexes have been selected, such as the inflammasome in mammals and the recently discovered resistosome in plants. In both cases, a regulated cell death located at the site of infection constitutes a very effective mean for blocking the pathogen spread and protecting the whole organism from invasion. This review aims to describe the immune mechanisms in animals and plants, mainly focusing on cell death signaling pathways, in order to highlight recent advances that could be used on one side or the other to identify the missing signaling elements between the perception of the invasion pattern by immune receptors, the induction of defenses or the transmission of danger signals to other cells. Although knowledge of plant immunity is less advanced, these organisms have certain advantages allowing easier identification of signaling events, regulators and executors of cell death, which could then be exploited directly for crop protection purposes or by analogy for medical research.

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.

Hydrophobized laminarans as new biocompatible anti-oomycete compounds for grapevine protection.

Laminaran, a ß-(1→3)-glucan extracted from Laminaria digitata, is a known elicitor of plant defenses, but provides only low level of disease control in vineyard trials. In this context, laminaran was partly hydrophobized by grafting from 1.6 to 7.6 lauryl chains to the native saccharidic chain and the impact of sulfation of the hydrophobized glucans was studied. The activity of the different synthetized laminaran derivatives as antimicrobial agents against Plasmopara viticola, the causal agent of grape downy mildew, and as elicitors of defense reactions in planta, was evaluated. Our results showed that acylation imparts an antimicrobial activity to laminaran which is related to the degree of acylation, AL3, with 7.6 lauryl chains, being the most effective derivative. Sulfation of the acylated laminarans did not further increase the antimicrobial activity. Our results also demonstrated that the efficacy of AL3 against Plasmopara viticola was most likely due to the direct antimicrobial activity of the lauryl chains rather than to an elicitation of plant defenses.

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation.

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.

Dual Mode of Action of Grape Cane Extracts against Botrytis cinerea.

Crude extracts of Vitis vinifera canes represent a natural source of stilbene compounds with well characterized antifungals properties. In our trials, exogenous application of a stilbene extract (SE) obtained from grape canes on grapevine leaves reduces the necrotic lesions caused by Botrytis cinerea. The SE showed to possess a direct antifungal activity by inhibiting the mycelium growth. The activation of some grapevine defense mechanism was also investigated. H2O2 production and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascades as well as accumulation of stilbenoid phytoalexins were explored on grapevine cell suspension. Moreover, the transcription of genes encoding for proteins affecting defense responses was analyzed on grapevine plants. The SE induced some grapevine defense mechanisms including MAPK activation, and the expression of pathogenesis-related (PR) genes and of a gene encoding the glutathione-S-transferase 1 ( GST1) . By contrast, treatment of grapevine leaves with SE negatively regulates de novo stilbene production.

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity.

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator.

The Cell Wall-Derived Xyloglucan Is a New DAMP Triggering Plant Immunity in Vitis vinifera and Arabidopsis thaliana.

Damage-associated molecular patterns (DAMPs) are endogenous molecules that can activate the plant innate immunity. DAMPs can derive from the plant cell wall, which is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides. Fragments of pectin, called oligogalacturonides (OG), can be released after wounding or by pathogen-encoded cell wall degrading enzymes (CWDEs) such as polygalacturonases (PGs). OG are known to induce innate immune responses, including the activation of mitogen-activated protein kinases (MAPKs), production of H2O2, defense gene activation, and callose deposition. Thus, we hypothesized that xyloglucans (Xh), derived from the plant cell wall hemicellulose, could also act as an endogenous elicitor and trigger a signaling cascade similar to OG. Our results indicate that purified Xh elicit MAPK activation and immune gene expression in grapevine (Vitis vinifera) and Arabidopsis (Arabidopsis thaliana) to trigger induced resistance against necrotrophic (Botrytis cinerea) or biotrophic (Hyaloperonospora arabidopsidis) pathogens. Xh also induce resveratrol production in grapevine cell suspension and callose deposition in Arabidopsis which depends on the callose synthase PMR4. In addition, we characterized some signaling components of Xh-induced immunity using Arabidopsis mutants. Our data suggest that Xh-induced resistance against B. cinerea is dependent on the phytoalexin, salicylate, jasmonate, and ethylene pathways.

Assessment of the impact of PS3-induced resistance to downy mildew on grapevine physiology.

Elicitor-induced resistance against diseases is an attractive strategy that could contribute to reduce the use of fungicides for plant protection. However, activation of defenses has an energetic cost that plants have to fuel by a mobilization of their primary metabolism with possible adverse effect on their physiology. In this context, this study was performed to determine whether elicitor-induced resistance of grapevine leaves against downy mildew impacted its development and metabolism. The elicitor PS3 (sulfated ß-glucan laminarin) was sprayed on grapevine herbaceous cuttings grown in greenhouses once or three times, and its impact was studied on young and older grapevine leaves, prior to, and after Plasmopara viticola inoculation. PS3 did not affect grapevine development during the time course of the experiment. A metabolomic analysis, mainly focused on primary metabolites, highlighted a leaf age dependent effect of PS3 treatment. Nitrogen compounds, and sugars to a lesser extent, were impacted. The results obtained complete the current knowledge of the impact of elicitor-induced resistance on plant physiology. They will be helpful to guide further experiments required to better determine the costs and benefits of elicitor-induced resistance in plants.

A Plant Extract Acts Both as a Resistance Inducer and an Oomycide Against Grapevine Downy Mildew.

Protecting vineyards from cryptogamic diseases such as downy mildew, caused by Plasmopara viticola, generally requires a massive use of phytochemicals. However, the issues on unintentional secondary effects on environment and human health, and the occurrence of P. viticola resistant strains, are leading to the development of alternative strategies, such as the use of biocontrol products. In this paper, we evidenced the ability of a plant extract to protect grapevine from P. viticola. Further experiments carried out both on cell suspensions and on plants revealed that plant extract activates typical defense-related responses such as the production of H2O2, the up-regulation of genes encoding pathogenesis-related proteins and stilbene synthase, as well as the accumulation of resveratrol or its derivative piceid. We also brought to light a strong direct effect of PE on the release and motility of P. viticola zoospores. Furthermore, we found out that PE application left dried residues on leaf surface, impairing zoospores to reach stomata. Altogether, our results highlight the different modes of action of a new biocontrol product able to protect grapevine against downy mildew.

Combined enzymatic and metabolic analysis of grapevine cell responses to elicitors.

Elicitors trigger plant defense responses, including phytoalexin production and cell-wall reinforcement. Primary metabolism plays an important role in these responses as it fuels the associated energetic costs and provides precursors for the synthesis of the numerous secondary metabolites involved in defenses against pathogens. In this context, we aimed to determine whether oligosaccharidic elicitors differing in their capacity to activate defense-associated secondary metabolism in grapevine would differently impact primary metabolism. To answer this question, cell suspensions were treated with two elicitors: an oligogalacturonide, and the ß-glucan laminarin. Enzymatic activity assays together with targeted (HPLC) and global (GC-MS) analyses of metabolites were next performed to compare their impact on plant primary or secondary metabolism. The results showed that the oligogalacturonide, which induced the highest level of the phytoalexin resveratrol and the highest activity of stilbene synthase, also induced the highest activity of shikimate hydroxycinnamoyltransferase, a key enzyme involved in the synthesis of lignin. The oligogalacturonide-induced defenses had a significant impact on primary metabolism 24 h following elicitor treatment, with a reduced abundance of pyruvate and 2-oxoglutarate, together with an increase of a set of metabolites including carbohydrates and amino acids. Interestingly, an accumulation of galacturonate and gentiobiose was observed in the oligogalacturonide- and laminarin-treated cells, respectively, suggesting that both elicitors are rapidly hydrolyzed in grapevine cell suspension cultures.

Metabolic Fingerprint of PS3-Induced Resistance of Grapevine Leaves against Plasmopara viticola Revealed Differences in Elicitor-Triggered Defenses.

Induction of plant resistance against pathogens by defense elicitors constitutes an attractive strategy to reduce the use of fungicides in crop protection. However, all elicitors do not systematically confer protection against pathogens. Elicitor-induced resistance (IR) thus merits to be further characterized in order to understand what makes an elicitor efficient. In this study, the oligosaccharidic defense elicitors H13 and PS3, respectively, ineffective and effective to trigger resistance of grapevine leaves against downy mildew, were used to compare their effect on the global leaf metabolism. Ultra high resolution mass spectrometry (FT-ICR-MS) analysis allowed us to obtain and compare the specific metabolic fingerprint induced by each elicitor and to characterize the associated metabolic pathways. Moreover, erythritol phosphate was identified as a putative marker of elicitor-IR.

Proteomics towards the understanding of elicitor induced resistance of grapevine against downy mildew.

Elicitors are known to trigger plant defenses in response to biotic stress, but do not systematically lead to effective resistance to pathogens. The reasons explaining such differences remain misunderstood. Therefore, elicitation and induced resistance (IR) were investigated through the comparison of two modified ß-1,3 glucans applied on grapevine (Vitis vinifera) leaves before and after inoculation with Plasmopara viticola, the causal agent of downy mildew. The sulfated (PS3) and the shortened (H13) forms of laminarin are both known to elicit defense responses whereas only PS3 induces resistance against downy mildew. The analysis of the 2-DE gel electrophoresis revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Our results point out that the PS3-induced resistance is associated with the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could be considered as useful markers of induced resistance. SIGNIFICANCE: One strategy to reduce the application of fungicides is the use of elicitors which induce plant defense responses. Nonetheless, the elicitors do not systematically lead to resistance against pathogens. The lack of correlation between plant defense activation and induced resistance (IR) requires the investigation of what makes the specificity of elicitor-IR. In this study, the two ß-glucans elicitors, sulfated (PS3) and short (H13) laminarins, were used in the grapevine/Plasmopara viticola interaction since only the first one leads to resistance against downy mildew. To disclose IR specificity, proteomic approach has been employed to compare the two treatments before and after P. viticola inoculation. The analysis of the 2-DE revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Significant increase of the number of proteins regulated by PS3, relative to both H13 and time-points, is correlated with the resistance process establishment. Our results point that the PS3-induced resistance requires the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could constitute useful markers of PS3 induced resistance.