Multiple factors driving the acquisition efficiency of apple proliferation phytoplasma in Cacopsylla melanoneura.

Phytoplasmas are bacterial pathogens located in the plant's phloem that are responsible for several plant diseases and are mainly transmitted by phloem-sucking insects. Apple proliferation (AP) is an economically important disease associated with the presence of 'Candidatus Phytoplasma mali' which is transmitted by two psyllid species. While Cacopsylla picta is a vector in different regions, the vector efficiency of C. melanoneura varies between different populations. This species is considered the main AP vector in Northwestern Italy but plays a minor role in Northeastern Italy and other European regions. To investigate whether the psyllid and/or the phytoplasma subtype drive the phytoplasma acquisition in C. melanoneura, a phytoplasma acquisition experiment was set up using single mating couples of overwintered individuals from different psyllid populations and phytoplasma subtypes. All analyzed insect populations acquired phytoplasma, but with different efficiencies and concentrations. The main factors driving the acquisition were the phytoplasma subtype and its concentration in the leaves of the infected trees together with the psyllid lineage. The phytoplasma concentration in the psyllids was again influenced by the phytoplasma subtype, the psyllid lineage and the region of origin, whereas the phytoplasma concentration in the leaves and the psyllid haplotype defined with the cytochrome oxidase I gene had only a minor impact on the phytoplasma concentration. This is the first study evaluating the roles of both the psyllid haplotype and the phytoplasma subtype on the acquisition process and highlights the importance of C. melanoneura as an additional AP vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-023-01699-1.

Definition of the effector landscape across 13 phytoplasma proteomes with LEAPH and EffectorComb.

'Candidatus Phytoplasma' genus, a group of fastidious phloem-restricted bacteria, can infect a wide variety of both ornamental and agro-economically important plants. Phytoplasmas secrete effector proteins responsible for the symptoms associated with the disease. Identifying and characterizing these proteins is of prime importance for expanding our knowledge of the molecular bases of the disease. We faced the challenge of identifying phytoplasma's effectors by developing LEAPH, a machine learning ensemble predictor composed of four models. LEAPH was trained on 479 proteins from 53 phytoplasma species, described by 30 features. LEAPH achieved 97.49% accuracy, 95.26% precision and 98.37% recall, ensuring a low false-positive rate and outperforming available state-of-the-art methods. The application of LEAPH to 13 phytoplasma proteomes yields a comprehensive landscape of 2089 putative pathogenicity proteins. We identified three classes according to different secretion models: 'classical', 'classical-like' and 'non-classical'. Importantly, LEAPH identified 15 out of 17 known experimentally validated effectors belonging to the three classes. Furthermore, to help the selection of novel candidates for biological validation, we applied the Self-Organizing Maps algorithm and developed a Shiny app called EffectorComb. LEAPH and the EffectorComb app can be used to boost the characterization of putative effectors at both computational and experimental levels, and can be employed in other phytopathological models.

'Candidatus Phytoplasma mali' SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves.

BACKGROUND: 'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11CaPm which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11CaPm. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11CaPm and changes in the transcriptome were recorded until 5 days post infiltration. RESULTS: The RNA-seq analysis revealed that presence of SAP11CaPm in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced. CONCLUSIONS: The results indicate that early SAP11CaPm expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.

Identification of spectral ranges that contribute to phytoplasma detection in apple trees - A step towards an on-site method.

'Candidatus Phytoplasma mali' is the bacterial agent associated with Apple Proliferation, a disease that causes high economic losses in affected commercial apple growing regions. The identification of the disease is carried out by visual inspection performed by skilled professionals in the orchards. To confirm an infection, costly molecular laboratory methods must be applied. Furthermore, both methods are very time-consuming. Here, we analysed the potential of a non-destructive method using in-field measurements to differentiate infected from non-infected apple trees (Malus domestica) based on spectral signatures of fresh leaves. By using multivariate statistics, we were able to distinguish infected from non-infected trees and identified the wavelengths relevant for the differentiation. Factors affecting the differentiation performance were the sampling date and bacterial colonization behaviour.

Phylogenetic Triage and Risk Assessment: How to Predict Emerging Phytoplasma Diseases.

Phytoplasma diseases pose a substantial threat to diverse crops of agricultural importance. Management measures are usually implemented only after the disease has already occurred. Early detection of such phytopathogens, prior to disease outbreak, has rarely been attempted, but would be highly beneficial for phytosanitary risk assessment, disease prevention and mitigation. In this study, we present the implementation of a recently proposed proactive disease management protocol (DAMA: Document, Assess, Monitor, Act) for a group of vector-borne phytopathogens. We used insect samples collected during a recent biomonitoring program in southern Germany to screen for the presence of phytoplasmas. Insects were collected with malaise traps in different agricultural settings. DNA was extracted from these mass trap samples and subjected to PCR-based phytoplasma detection and mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding. Phytoplasma DNA was detected in two out of the 152 insect samples analyzed. Phytoplasma identification was performed using iPhyClassifier based on 16S rRNA gene sequence and the detected phytoplasmas were assigned to 'Candidatus Phytoplasma asteris'-related strains. Insect species in the sample were identified by DNA metabarcoding. By using established databases, checklists, and archives, we documented historical associations and records of phytoplasmas and its hosts in the study region. For the assessment in the DAMA protocol, phylogenetic triage was performed in order to determine the risk for tri-trophic interactions (plant-insect-phytoplasma) and associated disease outbreaks in the study region. A phylogenetic heat map constitutes the basis for risk assessment and was used here to identify a minimum number of seven leafhopper species suggested to be monitored by stakeholders in this region. A proactive stance in monitoring changing patterns of association between hosts and pathogens can be a cornerstone in capabilities to prevent future phytoplasma disease outbreaks. To the best of our knowledge, this is the first time that the DAMA protocol has been applied in the field of phytopathology and vector-borne plant diseases.

The 'Candidatus Phytoplasma mali' effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

'Candidatus Phytoplasma mali', is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from 'Candidatus Phytoplasma asteris'. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.

Chasing Consistency: An Update of the TCP Gene Family of Malus × Domestica.

The 52 members of the Teosinte-Branched 1/Cycloidea/Proliferating Cell Factors (TCP) Transcription Factor gene family in Malus × domestica (M. × domestica) were identified in 2014 on the first genome assembly, which was released in 2010. In 2017, a higher quality genome assembly for apple was released and is now considered to be the reference genome. Moreover, as in several other species, the identified TCP genes were named based on the relative position of the genes on the chromosomes. The present work consists of an update of the TCP gene family based on the latest genome assembly of M. × domestica. Compared to the previous classification, the number of TCP genes decreased from 52 to 40 as a result of the addition of three sequences and the deduction of 15. An analysis of the intragenic identity led to the identification of 15 pairs of orthologs, shedding light on the forces that shaped the evolution of this gene family. Furthermore, a revised nomenclature system is proposed that is based both on the intragenic identity and the homology with Arabidopsis thaliana (A. thaliana) TCPs in an effort to set a common standard for the TCP classification that will facilitate any future interspecific analysis.

Investigating the microbial community of Cacopsylla spp. as potential factor in vector competence of phytoplasma.

Phytoplasmas are obligatory intracellular bacteria that colonize the phloem of many plant species and cause hundreds of plant diseases worldwide. In nature, phytoplasmas are primarily transmitted by hemipteran vectors. While all phloem-feeding insects could in principle transmit phytoplasmas, only a limited number of species have been confirmed as vectors. Knowledge about factors that might determine the vector capacity is currently scarce. Here, we characterized the microbiomes of vector and non-vector species of apple proliferation (AP) phytoplasma 'Candidatus Phytoplasma mali' to investigate their potential role in the vector capacity of the host. We performed high-throughput 16S rRNA metabarcoding of the two principal AP-vectors Cacopsylla picta and Cacopsylla melanoneura and eight Cacopsylla species, which are not AP-vectors but co-occur in apple orchards. The microbiomes of all species are dominated by Carsonella, the primary endosymbiont of psyllids and a second uncharacterized Enterobacteriaceae endosymbiont. Each Cacopsylla species harboured a species-specific phylotype of both symbionts. Moreover, we investigated differences between the microbiomes of AP-vector versus non-vector species and identified the predominant endosymbionts but also Wolbachia and several minor taxa as potential indicator species. Our study highlights the importance of considering the microbiome in future investigations of potential factors influencing host vector competence. We investigated the potential role of symbiotic bacteria in the acquisition and transmission of phytoplasma. By comparing the two main psyillid vector species of Apple proliferation (AP) phytoplasma and eight co-occurring species, which are not able to vector AP-phytoplasma, we found differences in the microbial communities of AP-vector and non-vector species, which appear to be driven by the predominant symbionts in both vector species and Wolbachia and several minor taxa in the non-vector species. In contrast, infection with AP-phytoplasma did not affect microbiome composition in both vector species. Our study provides new insights into the endosymbiont diversity of Cacopsylla spp. and highlights the importance of considering the microbiome when investigating potential factors influencing host vector competence.

Detection of apple proliferation disease in Malus × domestica by near infrared reflectance analysis of leaves.

In this study near infrared spectroscopical analysis of dried and ground leaves was performed and combined with a multivariate data analysis to distinguish 'Candidatus Phytoplasma mali' infected from non-infected apple trees (Malus × domestica). The bacterium is the causative agent of Apple Proliferation, one of the most threatening diseases in commercial apple growing regions. In a two-year study, leaves were sampled from three apple orchards, at different sampling events throughout the vegetation period. The spectral data were analyzed with a principal component analysis and classification models were developed. The model performance for the differentiation of Apple Proliferation diseased from non-infected trees increased throughout the vegetation period and gained best results in autumn. Even with asymptomatic leaves from infected trees a correct classification was possible indicating that the spectral-based method provides reliable results even if samples without visible symptoms are analyzed. The wavelength regions that contributed to the differentiation of infected and non-infected trees could be mainly assigned to a reduction of carbohydrates and N-containing organic compounds. Wet chemical analyses confirmed that N-containing compounds are reduced in leaves from infected trees. The results of our study provide a valuable indication that spectral analysis is a promising technique for Apple Proliferation detection in future smart farming approaches.

Surveying Potential Vectors of Apple Proliferation Phytoplasma: Faunistic Analysis and Infection Status of Selected Auchenorrhyncha Species.

Apple proliferation (AP) is one of the economically most important diseases in European apple cultivation. The disease is caused by the cell-wall-less bacterium ' Candidatus Phytoplasma mali', which is transmitted by Cacopsylla picta (Foerster) and Cacopsylla melanoneura (Foerster) (Hemiptera: Psylloidea). In South Tyrol (Italy), severe outbreaks were documented since the 1990s. Infestation rates of AP do not always correlate with the population densities of the confirmed vectors, implying the presence of other, so far unknown, hemipterian vectors. By elucidating the species community of Auchenorrhyncha (Insecta: Hemiptera) at a regional scale, more than 31,000 specimens were captured in South Tyrolean apple orchards. The occurrence of 95 species was confirmed, whereas fourteen species are new records for this territory. Based on the faunistical data, more than 3600 individuals out of 25 species were analyzed using quantitative PCR to assess the presence of AP phytoplasma. The pathogen was sporadically detected in some individuals of different species, for example in Stictocephala bisonia Kopp and Yonk (Hemiptera: Membracidae). However, the concentration of phytoplasma was much lower than in infected C. picta and C. melanoneura captured in the same region, confirming the role of the latter mentioned psyllids as the main insect vectors of AP- phytoplasma in South Tyrol.

Identification of Plant DNA in Adults of the Phytoplasma Vector Cacopsylla picta Helps Understanding Its Feeding Behavior.

Apple proliferation is an economically important disease and a threat for commercial apple cultivation. The causative pathogen, the bacterium 'Candidatus Phytoplasma mali', is mainly transmitted by Cacopsylla picta, a phloem-feeding insect that develops on the apple tree (Malus spp.). To investigate the feeding behavior of adults of the phytoplasma vector Cacopsylla picta in more detail, we used deep sequencing technology to identify plant-specific DNA ingested by the insect. Adult psyllids were collected in different apple orchards in the Trentino-South Tyrol region of northern Italy. DNA from the whole body of the insect was extracted and analyzed for the presence of plant DNA by performing PCR with two plant-specific primers that target the chloroplast regions trnH-psbA and rbcLa. DNA from 23 plant genera (trnH) and four plant families (rbcLa) of woody and herbaceous plant taxa was detected. Up to six and three plant genera and families, respectively, could be determined in single specimens. The results of this study contribute to a better understanding of the feeding behavior of adult Cacopsylla picta.

Effect of Daytime and Tree Canopy Height on Sampling of Cacopsylla melanoneura, a 'Candidatus Phytoplasma mali' Vector.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium 'Candidatus Phytoplasma mali', the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in 'Candidatus Phytoplasma mali' transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

Development of a universal endogenous qPCR control for eukaryotic DNA samples.

BACKGROUND: Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing. RESULTS: We developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single- or multiplex assays. CONCLUSIONS: Quality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.

A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Hydrogen peroxide stimulates uropathogenic Escherichia coli strains to cellulose production.

Reactive oxygen intermediates, such as hydrogen peroxide, are toxic molecules produced by immune cells in response to bacterial invasion into the host. Bacteria try to protect themselves against the immune system through specific properties such as biofilm formation. This phenomenon occurs also during urinary tract infections. Cellulose is an important factor of Escherichia coli biofilm and contributes to building a protective shield around bacterial cells upon the host immune response. In this study, we aimed to analyze the effect of hydrogen peroxide on the production of this biofilm component. To achieve this goal, 25 clinical E. coli strains isolated from patients with urinary tract infections were used. These bacterial strains were characterized based on their growth characteristics, their ability to form biofilm and their capacity to produce cellulose upon exposure to sub-lethal concentrations of hydrogen peroxide growth, and the biofilm formation of these strains was analyzed. Our results revealed that the analyzed uropathogenic E. coli strains slightly, but significantly, reduced growth and biofilm production upon hydrogen peroxide treatment. However, when separating these strains regarding their ability to produce cellulose, we found that general biofilm production was reduced but cellulose expression was induced upon peroxide treatment. This finding contributes to a better understanding of how bacterial biofilm formation is triggered and provides interesting insights into how uropathogenic E. coli protect themselves in an inhospitable environment.

Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC).

Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.

Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown Found in Degreened Leaves of Phytoplasma-Infected Apple (Malus × domestica Borkh.) and Apricot (Prunus armeniaca L.) Trees Relate to the Pheophorbide a Oxygenase/Phyllobilin Pathway.

Phytoplasmoses such as apple proliferation (AP) and European stone fruit yellows (ESFY) cause severe economic losses in fruit production. A common symptom of both phytoplasma diseases is early yellowing or leaf chlorosis. Even though chlorosis is a well-studied symptom of biotic and abiotic stresses, its biochemical pathways are hardly known. In particular, in this context, a potential role of the senescence-related pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway is elusive, which degrades chlorophyll (Chl) to phyllobilins (PBs), most notably to colorless nonfluorescent Chl catabolites (NCCs). In this work, we identified the Chl catabolites in extracts of healthy senescent apple and apricot leaves. In extracts of apple tree leaves, a total of 12 Chl catabolites were detected, and in extracts of leaves of the apricot tree 16 Chl catabolites were found. The seven major NCC fractions in the leaves of both fruit tree species were identical and displayed known structures. All of the major Chl catabolites were also found in leaf extracts from AP- or ESFY-infected trees, providing the first evidence that the PaO/PB pathway is relevant also for pathogen-induced chlorosis. This work supports the hypothesis that Chl breakdown in senescence and phytoplasma infection proceeds via a common pathway in some members of the Rosaceae family.

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called "effectors," it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant's molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

An effector of apple proliferation phytoplasma targets TCP transcription factors-a generalized virulence strategy of phytoplasma?

The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple-growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11-like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11-like proteins seem to be key players in phytoplasmal infection.

Temperature and host cell-dependent changes in virulence of Chlamydia pneumoniae CWL029 in an optimized mouse infection model.

The obligate intracellular bacterium Chlamydia (C.) pneumoniae causes respiratory infections and is associated with vascular diseases. To elucidate how temperature and host cells used for propagation alter chlamydial virulence, C. pneumoniae CWL0129 (Cpn) was cultured at 35 or 37°C in two different cell lines and then applied to mice. These mice infected with differentially propagated chlamydiae showed differences in clinical score, body weight and inflammatory cytokines in the lung. Our study demonstrates that Cpn cultured at 37°C in hamster fibroblast BHK-21 are able to colonize the mouse lung faster and better, and induce stronger symptoms and cytokine induction than bacteria cultured at 35°C. The temperature-triggered virulence alteration could not be observed for Cpn propagated in HeLa cells and was independent of host cell protein synthesis. Transcriptome analysis did not reveal temperature-induced effects on chlamydial gene expression, suggesting that the observed virulence changes are regulated on a different, so far unknown level. Preculture close to the central body temperature of its warm-blooded human or murine host might 'prepare' Cpn for subsequent in vivo infection. Our identification of culture-dependent virulence alteration helps to establish an optimized mouse lung infection model for Cpn and provides the basis to further unravel the molecular mechanisms underlying chlamydial pathogenicity.