Fungal wood-degrading enzymes in esca-diseased grapevine and effects of carbohydrate environment on fungal development.

In esca disease affecting grapevines, Phaeomoniella chlamydospora and Phaeoacremonium minimum colonize the woody parts of the trunks and arms, where they obtain nutrition from xylem sap and, potentially, from residues resulting from the enzymatic breakdown of lignified cell walls, particularly osidic residues. We quantified the secretion of lignin peroxidase, manganese peroxidase and laccase by these fungi in woody tissues of selectively infected cuttings using immunolabeling and transmission electron microscopy. Our results indicated that the detection of these enzymes was generally higher in tissues infected with Phaeoacremonium minimum. These data were confirmed through immunodetection of enzymes secreted by hyphae of fungi grown in vitro. Additionally, we observed that the supply of various carbohydrates (mono, di, tri and tetrasaccharides and polymers) differentially influenced fungal growth and polypeptide secretion. Since some secreted polypeptides display detrimental effects on grapevine cells, these results raise the question of whether the carbohydrate environment could be a factor affecting the aggressiveness of these pathogens.

Expression of Arabidopsis sugar transport protein STP13 differentially affects glucose transport activity and basal resistance to Botrytis cinerea.

Botrytis cinerea is the causing agent of the grey mold disease in more than 200 crop species. While signaling pathways leading to the basal resistance against this fungus are well described, the role of the import of sugars into host cells remains to be investigated. In Arabidopsis thaliana, apoplastic hexose retrieval is mediated by the activity of sugar transport proteins (STPs). Expression analysis of the 14 STP genes revealed that only STP13 was induced in leaves challenged with B. cinerea. STP13-modified plants were produced and assayed for their resistance to B. cinerea and glucose transport activity. We report that STP13-deficient plants exhibited an enhanced susceptibility and a reduced rate of glucose uptake. Conversely, plants with a high constitutive level of STP13 protein displayed an improved capacity to absorb glucose and an enhanced resistance phenotype. The correlation between STP13 transcripts, protein accumulation, glucose uptake rate and resistance level indicates that STP13 contributes to the basal resistance to B. cinerea by limiting symptom development and points out the importance of the host intracellular sugar uptake in this process. We postulate that STP13 would participate in the active resorption of hexoses to support the increased energy demand to trigger plant defense reactions and to deprive the fungus by changing sugar fluxes toward host cells.

The glutaredoxin ATGRXS13 is required to facilitate Botrytis cinerea infection of Arabidopsis thaliana plants.

Botrytis cinerea is a major pre- and post-harvest necrotrophic pathogen with a broad host range that causes substantial crop losses. The plant hormone jasmonic acid (JA) is involved in the basal resistance against this fungus. Despite basal resistance, virulent strains of B. cinerea can cause disease on Arabidopsis thaliana and virulent pathogens can interfere with the metabolism of the host in a way to facilitate infection of the plant. However, plant genes that are required by the pathogen for infection remain poorly described. To find such genes, we have compared the changes in gene expression induced in A. thaliana by JA with those induced after B. cinerea using genome-wide microarrays. We have identified genes that are repressed by JA but that are induced by B. cinerea. In this study, we describe one candidate gene, ATGRXS13, that encodes for a putative glutaredoxin and that exhibits such a crossed expression. In plants that are infected by this necrotrophic fungus, ATGRXS13 expression was negatively controlled by JA and TGA transcription factors but also through a JA-salicylic acid (SA) cross-talk mechanism as B. cinerea induced SA production that positively controlled ATGRXS13 expression. Furthermore, plants impaired in ATGRXS13 exhibited resistance to B. cinerea. Finally, we present a model whereby B. cinerea takes advantage of defence signalling pathways of the plant to help the colonization of its host.

Synthesis, phloem mobility and induced plant resistance of synthetic salicylic acid amino acid or glucose conjugates.

BACKGROUND: The growing demand for food, combined with a strong social expectation for a diet produced with fewer conventional agrochemical inputs, has led to the development of new alternatives in plant protection worldwide. Among different possibilities, the stimulation of the plant innate immune system by chemicals represents a novel and promising way. The vectorization strategy of an active ingredient that we previously developed with fungicides can potentially extend to salicylic acid (SA) or its halogenated analogues. RESULTS: Using the click chemistry method, six new conjugates combining SA or two mono- or di-halogenated analogues with L-glutamic acid or ß-D-glucose via a 1,2,3-triazole nucleus have been synthesized. Conjugate 8a, which is derived from SA and glutamic acid, showed high phloem mobility in the Ricinus model, similar to that of SA alone despite a much higher steric hindrance. In vivo bioassays of the six conjugates against two maize pathogenic fungi Bipolaris maydis and Fusarium graminearum revealed that, unlike SA, the amino acid conjugate 8a with good phloem mobility exerted a protective effect not only locally at the application site, but also in distant stem tissues after foliar application. Moreover, compounds 8a and 8b induced up-regulation of both defense-related genes ZmNPR1 and ZmPR1 similar to their parent compounds upon challenge inoculation with B. maydis. CONCLUSION: The vectorization of salicylic acid or its halogenated derivatives by coupling them with an α-amino acid can be a promising strategy to stimulate SA-mediated plant defenses responses against pathogens outside the application site. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses.

The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans-activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1) or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses.

The Arabidopsis patatin-like protein 2 (PLP2) plays an essential role in cell death execution and differentially affects biosynthesis of oxylipins and resistance to pathogens.

We previously reported that patatin-like protein 2 (PLP2), a pathogen-induced patatin-like lipid acyl hydrolase, promotes cell death and negatively affects Arabidopsis resistance to the fungus Botrytis cinerea and to the bacteria Pseudomonas syringae. We show here that, on the contrary, PLP2 contributes to resistance to Cucumber mosaic virus, an obligate parasite inducing the hypersensitive response. These contrasted impacts on different pathosystems were also reflected by differential effects on defense gene induction. To examine a possible link between PLP2 lipolytic activity and oxylipin metabolism, gene expression profiling was performed and identified B. cinerea among these pathogens as the strongest inducer of most oxylipin biosynthetic genes. Quantitative oxylipin profiling in wild-type and PLP2-modified, Botrytis-challenged plants established the massive accumulation of oxidized fatty acid derivatives in infected leaves. Several compounds previously described as modulating plant tissue damage and issued from the alpha-dioxygenase pathway were found to accumulate in a PLP2-dependent manner. Finally, the contribution of PLP2 to genetically controlled cell death was evaluated using PLP2-silenced or -overexpressing plants crossed with the lesion mimic mutant vascular-associated death 1 (vad1). Phenotypic analysis of double-mutant progeny showed that PLP2 expression strongly promotes necrotic symptoms in vad1 leaves. Collectively, our data indicate that PLP2 is an integral component of the plant cell death execution machinery, possibly providing fatty acid precursors for the biosynthesis of specific oxylipins and differentially affecting resistance to pathogens with distinct lifestyles.

Overexpression of the VvSWEET4 Transporter in Grapevine Hairy Roots Increases Sugar Transport and Contents and Enhances Resistance to Pythium irregulare, a Soilborne Pathogen.

Sugar transport and partitioning play key roles in the regulation of plant development and responses to biotic and abiotic factors. During plant/pathogen interactions, there is a competition for sugar that is controlled by membrane transporters and their regulation is decisive for the outcome of the interaction. SWEET sugar transporters are the targets of extracellular pathogens, which modify their expression to acquire the sugars necessary to their growth (Chen et al., 2010). The regulation of carbon allocation and sugar partitioning in the interaction between grapevine (Vitis vinifera) and its pathogens is poorly understood. We previously characterized the SWEET family in V. vinifera and showed that SWEET4 could be involved in resistance to the necrotrophic fungus Botrytis cinerea in Arabidopsis (Chong et al., 2014). To study the role of VvSWEET4 in grapevine, we produced V. vinifera cv. Syrah hairy roots overexpressing VvSWEET4 under the control of the CaMV 35S promoter (VvSWEET4 OX). High levels of VvSWEET4 expression in hairy roots resulted in enhanced growth on media containing glucose or sucrose and increased contents in glucose and fructose. Sugar uptake assays further showed an improved glucose absorption in VvSWEET4 overexpressors. In parallel, we observed that VvSWEET4 expression was significantly induced after infection of wild type grapevine hairy roots with Pythium irregulare, a soilborne necrotrophic pathogen. Importantly, grapevine hairy roots overexpressing VvSWEET4 exhibited an improved resistance level to P. irregulare infection. This resistance phenotype was associated with higher glucose pools in roots after infection, higher constitutive expression of several genes involved in flavonoid biosynthesis, and higher flavanol contents. We propose that high sugar levels in VvSWEET4 OX hairy roots provides a better support to the increased energy demand during pathogen infection. In addition, high sugar levels promote biosynthesis of flavonoids with antifungal properties. Overall, this work highlights the key role of sugar transport mediated by SWEET transporters for secondary metabolism regulation and pathogen resistance in grapevine.

Comparison of the Molecular Responses of Tolerant, Susceptible and Highly Susceptible Grapevine Cultivars During Interaction With the Pathogenic Fungus Eutypa lata.

Eutypa lata is the causal agent of eutypa dieback, one of the most destructive grapevine trunk disease that causes severe economic losses in vineyards worldwide. This fungus causes brown sectorial necrosis in wood which affect the vegetative growth. Despite intense research efforts made in the past years, no cure currently exists for this disease. Host responses to eutypa dieback are difficult to address because E. lata is a wood pathogen that causes foliar symptoms several years after infection. With the aim to classify the level of susceptibility of grapevine cultivars to the foliar symptoms caused by E. lata, artificial inoculations of Merlot, Cabernet Sauvignon, and Ugni Blanc were conducted over 3 years. Merlot was the most tolerant cultivar, whereas Ugni Blanc and Cabernet Sauvignon exhibited higher and differential levels of susceptibility. We took advantage of their contrasting phenotypes to explore their defense responses, including the activation of pathogenesis-related (PR) genes, oxylipin and phenylpropanoid pathways and the accumulation of stilbenes. These analyses were carried out using the millicell system that enables the molecular dialogue between E. lata mycelium and grapevine leaves to take place without physical contact. Merlot responded to E. lata by inducing the expression of a large number of defense-related genes. On the contrary, Ugni Blanc failed to activate such defense responses despite being able to perceive the fungus. To gain insight into the role of carbon partitioning in E. lata infected grapevine, we monitored the expression of plant genes involved in sugar transport and cleavage, and measured invertase activities. Our results evidence a coordinated up-regulation of VvHT5 and VvcwINV genes, and a stimulation of the cell wall invertase activity in leaves of Merlot elicited by E. lata, but not in Ugni Blanc. Altogether, this study indicates that the degree of cultivar susceptibility is associated with the activation of host defense responses, including extracellular sucrolytic machinery and hexose uptake during the grapevine/E. lata interaction. Given the role of these activities in governing carbon allocation through the plant, we postulate that the availability of sugar resources for either the host or the fungus is crucial for the outcome of the interaction.

The molecular dialogue between Arabidopsis thaliana and the necrotrophic fungus Botrytis cinerea leads to major changes in host carbon metabolism.

Photoassimilates play crucial roles during plant-pathogen interactions, as colonizing pathogens rely on the supply of sugars from hosts. The competition for sugar acquisition at the plant-pathogen interface involves different strategies from both partners which are critical for the outcome of the interaction. Here, we dissect individual mechanisms of sugar uptake during the interaction of Arabidopsis thaliana with the necrotrophic fungus Botrytis cinerea using millicell culture insert, that enables molecular communication without physical contact. We demonstrate that B. cinerea is able to actively absorb glucose and fructose with equal capacities. Challenged Arabidopsis cells compete for extracellular monosaccharides through transcriptional reprogramming of host sugar transporter genes and activation of a complex sugar uptake system which displays differential specificity and affinity for hexoses. We provide evidence that the molecular dialogue between Arabidopsis cells and B. cinerea triggers major changes in host metabolism, including apoplastic sucrose degradation and consumption of carbohydrates and oxygen, suggesting an enhanced activity of the glycolysis and the cellular respiration. We conclude that beside a role in sugar deprivation of the pathogen by competing for sugar availability in the apoplast, the enhanced uptake of hexoses also contributes to sustain the increased activity of respiratory metabolism to fuel plant defences.

Targeting the AtCWIN1 Gene to Explore the Role of Invertases in Sucrose Transport in Roots and during Botrytis cinerea Infection.

Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink, or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H+ symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e., STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and AtVIN genes accompanied with an enhanced vacuolar invertase activity, suggesting that the AtCWIN1-independent pathway is efficient upon infection. In absence of AtCWIN1, we postulate that intracellular sucrose hydrolysis is sufficient to provide intracellular hexoses to maintain sugar homeostasis in host cells and to fuel plant defenses. Finally, we demonstrated that Botrytis cinerea possesses its own functional sucrolytic machinery and hexose uptake system, and does not rely on the host apoplastic invertases.

Armadillidin H, a Glycine-Rich Peptide from the Terrestrial Crustacean Armadillidium vulgare, Displays an Unexpected Wide Antimicrobial Spectrum with Membranolytic Activity.

Antimicrobial peptides (AMPs) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, there are currently 15 distinct AMP families published so far in the literature, mainly isolated from members of the Decapoda order. Up to now, armadillidin is the sole non-decapod AMP isolated from the haemocytes of Armadillidium vulgare, a crustacean isopod. Its first description demonstrated that armadillidin is a linear glycine-rich (47%) cationic peptide with an antimicrobial activity directed toward Bacillus megaterium. In the present work, we report identification of armadillidin Q, a variant of armadillidin H (earlier known as armadillidin), from crude haemocyte extracts of A. vulgare using LC-MS approach. We demonstrated that both armadillidins displayed broad spectrum antimicrobial activity against several Gram-positive and Gram-negative bacteria, fungi, but were totally inactive against yeasts. Membrane permeabilization assays, only performed with armadillidin H, showed that the peptide is membrane active against bacterial and fungal strains leading to deep changes in cell morphology. This damaging activity visualized by electronic microscopy correlates with a rapid decrease of cell viability leading to highly blebbed cells. In contrast, armadillidin H does not reveal cytotoxicity toward human erythrocytes. Furthermore, no secondary structure could be defined in this study [by circular dichroism (CD) and nuclear magnetic resonance (NMR)] even in a membrane mimicking environment. Therefore, armadillidins represent interesting candidates to gain insight into the biology of glycine-rich AMPs.

Source-to-sink transport of sugar and regulation by environmental factors.

Source-to-sink transport of sugar is one of the major determinants of plant growth and relies on the efficient and controlled distribution of sucrose (and some other sugars such as raffinose and polyols) across plant organs through the phloem. However, sugar transport through the phloem can be affected by many environmental factors that alter source/sink relationships. In this paper, we summarize current knowledge about the phloem transport mechanisms and review the effects of several abiotic (water and salt stress, mineral deficiency, CO2, light, temperature, air, and soil pollutants) and biotic (mutualistic and pathogenic microbes, viruses, aphids, and parasitic plants) factors. Concerning abiotic constraints, alteration of the distribution of sugar among sinks is often reported, with some sinks as roots favored in case of mineral deficiency. Many of these constraints impair the transport function of the phloem but the exact mechanisms are far from being completely known. Phloem integrity can be disrupted (e.g., by callose deposition) and under certain conditions, phloem transport is affected, earlier than photosynthesis. Photosynthesis inhibition could result from the increase in sugar concentration due to phloem transport decrease. Biotic interactions (aphids, fungi, viruses…) also affect crop plant productivity. Recent breakthroughs have identified some of the sugar transporters involved in these interactions on the host and pathogen sides. The different data are discussed in relation to the phloem transport pathways. When possible, the link with current knowledge on the pathways at the molecular level will be highlighted.

A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis.

Genes and proteins related to patatin, the major storage protein of potato tubers, have been identified in many plant species and shown to be induced by a variety of environmental stresses. The Arabidopsis patatin-like gene family (PLPs) comprises nine members, two of which (PLP2 and PLP7) are strongly induced in leaves challenged with fungal and bacterial pathogens. Here we show that accumulation of PLP2 protein in response to Botrytis cinerea or Pseudomonas syringae pv. tomato (avrRpt2) is dependent on jasmonic acid and ethylene signaling, but is not dependent on salicylic acid. Expression of a PLP2-green fluorescent protein (GFP) fusion protein and analysis of recombinant PLP2 indicates that PLP2 encodes a cytoplasmic lipid acyl hydrolase with wide substrate specificity. Transgenic plants with altered levels of PLP2 protein were generated and assayed for pathogen resistance. Plants silenced for PLP2 expression displayed enhanced resistance to B. cinerea, whereas plants overexpressing PLP2 were much more sensitive to this necrotrophic fungus. We also established a positive correlation between the level of PLP2 expression in transgenic plants and cell death or damage in response to paraquat treatment or infection by avirulent P. syringae. Interestingly, repression of PLP2 expression increased resistance to avirulent bacteria, while PLP2-overexpressing plants multiplied avirulent bacteria close to the titers reached by virulent bacteria. Collectively, the data indicate that PLP2-encoded lipolytic activity can be exploited by pathogens with different lifestyles to facilitate host colonization. In particular PLP2 potentiates plant cell death inflicted by Botrytis and reduces the efficiency of the hypersensitive response in restricting the multiplication of avirulent bacteria. Both effects are possibly mediated by providing fatty acid precursors of bioactive oxylipins.

Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways.

In their environment, plants interact with a multitude of living organisms and have to cope with a large variety of aggressions of biotic or abiotic origin. To survive, plants have acquired, during evolution, complex mechanisms to detect their aggressors and defend themselves. Receptors and signaling pathways that are involved in such interactions with the environment are just beginning to be uncovered. What has been known for several decades is the extraordinary variety of chemical compounds the plants are capable to synthesize, and many of these products are implicated in defense responses. The number of natural products occurring in plants may be estimated in the range of hundreds of thousands, but only a fraction have been fully characterized. Despite the great importance of these metabolites for plant and also for human health, our knowledge about their biosynthetic pathways and functions is still fragmentary. Recent progress has been made particularly for phenylpropanoid and oxylipin metabolism, which are emphasized in this review. Both pathways are involved in plant resistance at several levels: by providing building units of physical barriers against pathogen invasion, by synthesizing an array of antibiotic compounds, and by producing signals implicated in the mounting of plant resistance.