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.

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.

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.

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.

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.

Transcriptomic dataset of Malus domestica young leaves in response to acibenzolar-S-methyl (ASM) and/or nitrogen nutrition.

Plant resistance inducers (PRIs) and nitrogen (N) nutrition are both known to affect plant defence but their interaction has not been well described. We addressed this question in apple (Malus domestica) by generating a transcriptomic data set of young leaves from seedlings grown in subirrigation systems allowing variations in nitrate supply as the sole nitrogen source. Plants under three contrasting N status (high; limited for 10 days; or just resupplied after a 12 days limitation) received foliar applications of the chemical elicitor acibenzolar-S-methyl (ASM), a functional analog of salicylic acid, or water. Two days later, the youngest developed leaves were sampled for total RNA extraction and sequencing analysis (RNAseq). The current dataset includes 1) a detailed protocol of plant sample production and 2) transcriptomic profile description of young leaves as normalized counts obtained from sequence mapping against the Malus domestica GDDH13v1.1 reference transcriptome. The raw data files and processed data are available at the Gene Expression Omnibus (GEO) repository under the accession number GSE264541. This dataset is a valuable resource to investigate further the molecular mechanisms underlying the role of nitrogen and/or ASM treatment in Malus domestica.

Phenotyping data coupled with RNA sequencing of apple genotypes exhibiting contrasted quantitative trait loci architecture for apple scab (Venturia inaequalis) resistance.

Previous studies have highlighted the role of three quantitative trait loci (QTL, i.e. 'qT1', 'qF11' and 'qF17') in partial resistance to apple scab. Underlying molecular mechanisms of these loci are yet unknown. Exploring differential gene expression between apple genotypes carrying contrasting combinations of these QTLs could depict original candidate genes and pathways implicated. We therefore carried out RNA sequencing just before and five days after inoculation of the pathogenic fungi Venturia inaequalis, in sixteen genotypes from a pseudo-F1 progeny segregating for resistant or susceptible alleles of the three QTLs. The current dataset includes i) transcriptomic profile description, ii) analysis of differentially expressed genes related to none or combined QTLs, infected or not with Venturia inaequalis and iii) disease phenotyping of the same genetic materials. The raw data files have been deposited in the Gene Expression Omnibus (GEO) repository with the accession number GSE250309. These outputs represent the first step towards elucidating the genetic basis of quantitative apple scab resistance. In the long term, this data set will improve apple breeding strategies on how to combine qualitative (used so far) and quantitative resistances to apple scab, with the aim of diversifying selective pressures on the pathogen.

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.

Transmission of plant-pathogenic bacteria by nonhost seeds without induction of an associated defense reaction at emergence.

An understanding of the mechanisms involved in the different steps of bacterial disease epidemiology is essential to develop new control strategies. Seeds are the passive carriers of a diversified microbial cohort likely to affect seedling physiology. Among seed-borne plant-pathogenic bacteria, seed carriage in compatible situations is well evidenced. The aims of our work are to determine the efficiency of pathogen transmission to seeds of a nonhost plant and to evaluate bacterial and plant behaviors at emergence. Bacterial transmission from flowers to seeds and from seeds to seedlings was measured for Xanthomonas campestris pv. campestris in incompatible interactions with bean. Transmissions from seeds to seedlings were compared for X. campestris pv. campestris, for Xanthomonas citri pv. phaseoli var. fuscans in compatible interactions with bean, and for Escherichia coli, a human pathogen, in null interactions with bean. The induction of defense responses was monitored by using reverse transcription and quantitative PCR (RT-qPCR) of genes representing the main signaling pathways and assaying defense-related enzymatic activities. Flower inoculations resulted in a high level of bean seed contamination by X. campestris pv. campestris, which transmitted efficiently to seedlings. Whatever the type of interaction tested, dynamics of bacterial population sizes were similar on seedlings, and no defense responses were induced evidencing bacterial colonization of seedlings without any associated defense response induction. Bacteria associated with the spermosphere multiply in this rich environment, suggesting that the colonization of seedlings relies mostly on commensalism. The transmission of plant-pathogenic bacteria to and by nonhost seeds suggests a probable role of seeds of nonhost plants as an inoculum source.

Biotic Stress-Induced Priming and De-Priming of Transcriptional Memory in Arabidopsis and Apple.

Under natural growth conditions, plants experience various and repetitive biotic and abiotic stresses. Salicylic acid (SA) is a key phytohormone involved in the response to biotic challenges. Application of synthetic SA analogues can efficiently prime defense responses, and leads to improved pathogen resistance. Because SA analogues can result in long-term priming and memory, we identified genes for which expression was affected by the SA analogue and explored the role of DNA methylation in this memorization process. We show that treatments with an SA analogue can lead to long-term transcriptional memory of particular genes in Arabidopsis. We found that subsequent challenging of such plants with a bacterial elicitor reverted this transcriptional memory, bringing their expression back to the original pre-treatment level. We also made very similar observations in apple (Malus domestica), suggesting that this expression pattern is highly conserved in plants. Finally, we found a potential role for DNA methylation in the observed transcriptional memory behavior. We show that plants defective in DNA methylation pathways displayed a different memory behavior. Our work improves our understanding of the role of transcriptional memory in priming, and has important implication concerning the application of SA analogues in agricultural settings.

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.

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system.

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora, which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple (Malus x domestica) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E. amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.

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.

DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants.

Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (TTSS) to induce disease on hosts and hypersensitive response (HR) on nonhost plants. The DspA/E protein is essential for E. amylovora pathogenicity and is secreted via the TTSS in vitro. DspA/E belongs to a type III effector family that is conserved in several phytopathogenic bacteria. In E. amylovora, DspA/E has been implicated in the generation of an oxidative stress during disease and the suppression of callose deposition. We investigated the fate of DspA/E in planta. DspA/E delivered artificially to apple or tobacco cells by agroinfection induced necrotic symptoms, indicating that DspA/E was probably injected via the TTSS. We confirmed that DspA/E acts as a major cell-death inducer during disease and HR, because the dspA/E mutant is severely impaired in its ability to induce electrolyte leakage in apple and tobacco leaves. Expression of the defense marker gene PR1 was delayed when dspA/E was transiently expressed in tobacco, suggesting that DspA/E-mediated necrosis may be associated with an alteration of defense responses.

The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions.

In this work we present evidence of an opposite regulation in the phytopathogenic bacteria Erwinia amylovora between the virulence-associated Type III secretion system (TTSS) and the flagellar system. Using loss-of-function mutants we show that motility enhanced the virulence of wild-type bacteria relative to a nonmotile mutant when sprayed on apple seedlings with unwounded leaves. Then we demonstrated through analyses of motility, flagellin export and visualization of flagellar filament that HrpL, the positive key regulator of the TTSS, also down-regulates the flagellar system. Such a dual regulation mediated by an alternative sigma factor of the TTSS appears to be a level of regulation between virulence and motility not yet described among Proteobacteria.

Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora.

Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae. In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E. amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv. tabaci. Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course. This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E. amylovora, because an hrp secretion mutant did not induce such responses. However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E. amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant. This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.

Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear.

Erwinia amylovora, the causal agent of fire blight of Maloideae, induces in its susceptible host plants an oxidative burst as does an incompatible pathogen. In this paper we present evidence that the elicitation of this phenomenon is the result of the combined action of two Hrp effectors of the bacteria, HrpN and DspA. We also confirmed that desferrioxamine, the siderophore of E. amylovora, is necessary for the bacteria to tolerate high levels of hydrogen peroxide. Two other pathogenicity factors of the bacteria, the HrpW effector and the capsule, do not seem to play any role in the elicitation of the oxidative burst nor in the protection of the bacteria.