Analysis of Defense-Related Gene Expression and Leaf Metabolome in Wheat During the Early Infection Stages of Blumeria graminis f. sp. tritici.

Blumeria graminis f. sp. tritici (Bgt) is an obligate biotrophic fungal pathogen responsible for powdery mildew in bread wheat (Triticum aestivum). Upon Bgt infection, the wheat plant activates basal defense mechanisms, namely PAMP-triggered immunity, in the leaves during the first few days. Understanding this early stage of quantitative resistance is crucial for developing new breeding tools and evaluating plant resistance inducers for sustainable agricultural practices. In this sense, we used a combination of transcriptomic and metabolomic approaches to analyze the early steps of the interaction between Bgt and the moderately susceptible wheat cultivar Pakito. Bgt infection resulted in an increasing expression of genes encoding pathogenesis-related (PR) proteins (PR1, PR4, PR5, and PR8) known to target the pathogen, during the first 48 h postinoculation. Moreover, RT-qPCR and metabolomic analyses pointed out the importance of the phenylpropanoid pathway in quantitative resistance against Bgt. Among metabolites linked to this pathway, hydroxycinnamic acid amides containing agmatine and putrescine as amine components accumulated from the second to the fourth day after inoculation. This suggests their involvement in quantitative resistance via cross-linking processes in cell walls for reinforcement, which is supported by the up-regulation of PAL (phenylalanine ammonia-lyase), PR15 (oxalate oxidase) and POX (peroxidase) after inoculation. Finally, pipecolic acid, which is considered a signal involved in systemic acquired resistance, accumulated after inoculation. These new insights lead to a better understanding of basal defense in wheat leaves after Bgt infection.

Formulated hydroxy fatty acids from fruit pomaces reduce apple scab development caused by Venturia inaequalis through a dual mode of action.

The outermost hydrophobic layer of plants, i.e. the cuticle, is mainly composed of cutin, a polyester of hydroxy fatty acids with reported eliciting and/or antimicrobial activities for some of them. By-products of the fruit processing industry (fruit pomaces), often strongly enriched in cuticular material, are therefore a potential source of bioactive compounds for crop protection against pathogen attack. We investigated the utilization of tomato and apple pomaces in the development of a cutin-based biocontrol solution against apple scab, a major apple disease caused by Venturia inaequalis. Several cutin monomer extracts obtained through different strategies of depolymerization and purification were first compared for their ability to induce a targeted set of defense genes in apple seedlings after foliar application. After a step of formulation, some extracts were chosen for further investigation in planta and in vitro. Our results show that formulated cutin monomers could trigger a significant transcriptome reprogramming in apple plants and exhibit an antifungal effect on V. inaequalis. Cutin monomers-treated apple seedlings were significantly protected against infection by the apple scab agent. Altogether, our findings suggest that water-dispersed cutin monomers extracted from pomaces are potential new bio-based solutions for the control of apple scab.

The Use of Potassium Phosphonate (KHP) for the Control of Major Apple Pests.

Phosphonate-based products have demonstrated diverse abilities to protect crops against pests, with various modes of action proposed. In this article, we specifically investigated potassium phosphonate (KHP) on apple crops. Its performance to control three major apple bioagressors (Venturia inaequalis, Erwinia amylovora, and Dysaphis plantaginea) was evaluated under semicontrolled conditions. The product was able to confer significant protection rates (40 to 75% for apple scab, 40% for fire blight, and 30% for rosy aphid), which can be explained by its more or less efficient biocidal activity against the three pests, and by its ability to induce apple immunity (pathogenesis-related proteins and secondary metabolites genes). A cumulative effect of treatments as well as the systemic behavior of the product was demonstrated. Fields trials against apple scab and the postharvest disease bull's eyes rot (Neofabraea vagabunda) were performed on different apple varieties by applying KHP combined with light pest management programs either reducing (dessert orchards) or suppressing (cider orchards) fungicide applications. KHP was able to reduce apple scab by 70 to 90% on shoots and young and harvested fruit, and bull's eyes rot by 70 to 90% on harvested fruit. Overall, our results indicate that KHP is useful for the protection of apple trees against its major pests by direct effect and by triggering the host defense system.

An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance.

KEY MESSAGE: pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

Acibenzolar-S-Methyl and Resistance Quantitative Trait Loci Complement Each Other to Control Apple Scab and Fire Blight.

Diversifying disease control methods is a key strategy to sustainably reduce pesticides. Plant genetic resistance has long been used to create resistant varieties. Plant resistance inducers (PRI) are also considered to promote crop health, but their effectiveness is partial and can vary according to the environment and the plant genotype. We investigated the putative interaction between intrinsic (genetic) and PRI-induced resistance in apple when affected by scab and fire blight diseases. A large F1 mapping population was challenged by each disease after a pre-treatment with acibenzolar-S-methyl (ASM) and compared with the water control. Apple scab and fire blight resistance quantitative trait loci (QTLs) were detected in both conditions and compared. ASM exhibited a strong effectiveness in reducing both diseases. When combined, QTL-controlled and ASM-induced resistance acted complementarily to reduce the symptoms from 85 to 100%, depending on the disease. In our conditions, resistance QTLs were only slightly or rarely affected by ASM treatment, despite their probable implication in various stages of the resistance buildup. Implications of these results are discussed considering already known results, the underlying mechanisms, cross protection of both types of resistance against pathogen adaptation, and practical application in orchard conditions.

Evaluation of λ-Carrageenan, CpG-ODN, Glycine Betaine, Spirulina platensis, and Ergosterol as Elicitors for Control of Zymoseptoria tritici in Wheat.

Wheat crops are constantly challenged by the pathogen Zymoseptoria tritici, responsible for Septoria tritici Blotch (STB) disease. The present study reports the evaluation of five elicitor compounds (λ-carrageenan, cytosine-phosphate-guanine oligodesoxynucleotide motifs [CpG ODN], glycine betaine, Spirulina platensis, and ergosterol) for the protection of wheat against STB in order to offer new alternative tools to farmers for sustainable crop protection. Screening of elicitors of wheat defenses was carried out through a succession of experiments: biocidal in vitro tests enabled checking for any fungicidal activities, glasshouse experiments allowed determination of the efficacy of a given compound in protecting wheat against STB, and quantitative reverse-transcription polymerase chain reaction biomolecular tests investigated the relative expression of 23 defense genes in treated versus untreated plants. Therefore, we demonstrated that λ-carrageenan, CpG-ODN, glycine betaine, S. platensis, and ergosterol are potential elicitors of wheat defenses. Foliar treatment with these compounds conferred protection of wheat by up to approximately 70% against Z. tritici under semicontrolled conditions and induced both salicylic acid- and jasmonic acid-dependent signaling pathways in the plant. These findings contribute to extending the narrow list of potential elicitors of wheat defenses against Z. tritici.

Setting-up a fast and reliable cytokinin biosensor based on a plant histidine kinase receptor expressed in Saccharomyces cerevisiae.

Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

Acibenzolar-S-Methyl Reprograms Apple Transcriptome Toward Resistance to Rosy Apple Aphid.

Acibenzolar-S-methyl (ASM) is a chemical compound, which is able to induce resistance in several model and non-model plants, but the end-players of this induced defense remain ill-defined. Here, we test the hypothesis that treatment with ASM can protect apple (Malus × domestica) against the rosy apple aphid (Dysaphis plantaginea) and investigate the defense molecules potentially involved in resistance. We measured aphid life traits and performed behavioral assays to study the effect of ASM on plant resistance against the aphid, and then combined transcriptomic, bioinformatics, metabolic and biochemical analyses to identify the plant compounds involved in resistance. Plants treated with ASM negatively affected several life traits of the aphid and modified its feeding and host seeking behaviors. ASM treatment elicited up-regulation of terpene synthase genes in apple and led to the emission of (E,E)-α-farnesene, a sesquiterpene that was repellent to the aphid. Several genes encoding amaranthin-like lectins were also strongly up-regulated upon treatment and the corresponding proteins accumulated in leaves, petioles and stems. Our results link the production of specific apple proteins and metabolites to the antibiosis and antixenosis effects observed against Dysaphis plantaginea, providing insight into the mechanisms underlying ASM-induced herbivore resistance.

When a Plant Resistance Inducer Leaves the Lab for the Field: Integrating ASM into Routine Apple Protection Practices.

Plant resistance inducers, also called elicitors, could be useful to reduce the use of pesticides. However, their performance in controlling diseases in the field remains unsatisfactory due to lack of specific knowledge of how they can integrate crop protection practices. In this work, we focused on apple crop and acibenzolar-S-methyl (ASM), a well-known SAR (systemic acquired resistance) inducer of numerous plant species. We provide a protocol for orchard-effective control of apple scab due to the ascomycete fungus Venturia inaequalis, by applying ASM in combination with a light integrated pest management program. Besides we pave the way for future optimization levers by demonstrating in controlled conditions (i) the high influence of apple genotypes, (ii) the ability of ASM to prime defenses in newly formed leaves, (iii) the positive effect of repeated elicitor applications, (iv) the additive effect of a thinning fruit agent.

Using molecular tools to decipher the complex world of plant resistance inducers: an apple case study.

Exogenous application of plant resistance inducers (PRIs) able to activate plant defenses is an interesting approach for new integrated pest management practices. The full integration of PRIs into agricultural practices requires methods for the fast and objective upstream screening of efficient PRIs and optimization of their application. To select active PRIs, we used a molecular tool as an alternative to methods involving plant protection assays. The expressions of 28 genes involved in complementary plant defense mechanisms were simultaneously determined by quantitative real-time PCR in PRI-treated tissues. Using a set of 10 commercial preparations and considering the pathosystem apple/Erwinia amylovora, this study shows a strong correlation between defense activation and protection efficiency in controlled conditions, thus enabling the easy identification of promising PRIs in fire blight protection. Hence this work clearly highlights the benefits of using a molecular tool to discriminate nonactive PRI preparations and provides useful molecular markers for the optimization of their use in orchard.

Histolocalization and physico-chemical characterization of dihydrochalcones: Insight into the role of apple major flavonoids.

Flavonoids, like other metabolites synthesized via the phenylpropanoid pathway, possess a wide range of biological activities including functions in plant development and its interaction with the environment. Dihydrochalcones (mainly phloridzin, sieboldin, trilobatin, phloretin) represent the major flavonoid subgroup in apple green tissues. Although this class of phenolic compounds is found in very large amounts in some tissues (≈200mg/g of leaf DW), their physiological significance remains unclear. In the present study, we highlight their tissue-specific localization in young growing shoots suggesting a specific role in important physiological processes, most notably in response to biotic stress. Indeed, dihydrochalcones could constitute a basal defense, in particular phloretin which exhibits a strong broad-range bactericidal and fungicidal activity. Our results also indicate that sieboldin forms complexes with iron with strong affinity, reinforcing its antioxidant properties and conferring to this dihydrochalcone a potential for iron seclusion and/or storage. The importance of localization and biochemical properties of dihydrochalcones are discussed in view of the apple tree defense strategy against both biotic and abiotic stresses.

Same ammo, different weapons: enzymatic extracts from two apple genotypes with contrasted susceptibilities to fire blight (Erwinia amylovora) differentially convert phloridzin and phloretin in vitro.

The necrogenic bacterium Erwinia amylovora responsible for the fire blight disease causes cell death in apple tissues to enrich intercellular spaces with nutrients. Apple leaves contain large amounts of dihydrochalcones (DHCs), including phloridzin and its aglycone phloretin. Previous work showed an important decrease in the constitutive DHCs stock in infected leaves, probably caused by transformation reactions during the infection process. At least two flavonoid transformation pathways have been described so far: deglucosylation and oxidation. The aim of the present study was to determine whether DHCs are differentially converted in two apple genotypes displaying contrasted susceptibilities to the disease. Different analyses were performed: i) enzymatic activity assays in infected leaves, ii) identification/quantification of end-products obtained after in vitro enzymatic reactions with DHCs, iii) evaluation of the bactericidal activity of end-products. The results of the enzymatic assays showed that deglucosylation was dominant over oxidation in the susceptible genotype MM106 while the opposite was observed in the resistant genotype Evereste. These data were confirmed by LC-UV/Vis-MS analysis of in vitro reaction mixtures, especially because higher levels of o-quinoid oxidation products of phloretin were measured by using the enzymatic extracts of Evereste infected leaves. Their presence correlated well with a strong bactericidal activity of the reaction mixtures. Thus, our results suggest that a differential transformation of DHCs occur in apple genotypes with a potential involvement in the establishment of the susceptibility or the resistance to fire blight, through the release of glucose or of highly bactericidal compounds respectively.

T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora.

Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea.

Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction.

Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.