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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 caused by ‘CandidatusPhytoplasma mali’ which is transmitted by two psyllid species. While Cacopsylla picta is a confirmed vector in different regions, the vector efficiency of Cacopsylla 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, we set-up a phytoplasma acquisition experiment using single mating couples including 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 family. The phytoplasma concentration in the psyllids was again influenced by the phytoplasma subtype, the psyllid family 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 shedding new light on the importance of C. melanoneura as AP vector.

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.

The 52 members of the Teosinte-Branched 1/Cycloidea/Proliferating Cell Factors (TCP) Transcription Factor gene family in

Mountain agriculture is a vital social-economic activity in Europe, including the alpine Province of South Tyrol, Italy. Here, apple orchards and vineyards are extensively cultivated. Besides the difficulty to cultivate in mountain terrain (steep slopes, difficult accessibility, extreme weather conditions), the plants are exposed to a combination of biotic and abiotic stresses that can result in diseases caused by pathogens. It results in the loss of the yield and quality of products, economic losses, reducing food security with severe ecological impacts, and affects many ecosystem services (such as agrotourism).This work presents a proximal sensing technique based on an unmanned aerial platform with a payload consisting of multi and hyperspectral optical cameras. Such platforms are suitable to access rugged terrains in a short time to map the presence of diseases and pests, as well they provide imagery for the optimal management of farms. We study three different experiments: apple orchard, vineyard, and forestry, observing Apple proliferation, Flavescence dorée, and Pine processionary, respectively. We aim at a non-invasive and non-destructive method to monitor plant diseases in the direction of high-precision mapping agriculture applications by exploring supervised classification methods based on ground data to distinguish healthy and unhealthy trees.

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.

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 &rsquo, Candidatus Phytoplasma mali&rsquo, 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.

Apple proliferation is an economically important disease and a threat for commercial apple cultivation. The causative pathogen, the bacterium &lsquo, Candidatus Phytoplasma mali&rsquo, 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.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus &times, domestica) and transmit the bacterium &lsquo, Candidatus Phytoplasma mali&rsquo, 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 &lsquo, 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.

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.

Phytoplasmas, microorganisms associated with severe plant diseases, are obligate parasites transmitted by phloem-feeding insects. Cacopsylla melanoneura and Cacopsylla picta are involved in the transmission of ‘Candidatus Phytoplasma mali,’ the etiological agent of apple proliferation (AP) disease. Research conducted in different geographic regions showed different transmission efficiencies for the two psyllids. In this study, acquisition and transmission trials were carried out to investigate the role of different life stages of these vectors in the epidemiology of AP after a sudden outbreak in northeastern Italy. Both species resulted able to acquire ‘Ca. P. mali,’ with higher infection rates recorded in C. picta. F1 generations showed a higher acquisition ability compared to adults in both species. ‘Ca. P. mali’ transmission was successful: Up to 1.5% of test plants were infected in trials with C. melanoneura and up to 10.2% in trials with C. picta. Overwintered adults of C. melanoneura showed a lower vectoring ability compared to C. picta. F1 nymphs and F1 adults, developed on infected plants, resulted in being competent vectors. Data on phytoplasma acquisition suggest a different relationship of ‘Ca. P. mali’ with the two species, evidencing a stronger affinity with C. picta. Moreover, taking into account the different factors influencing AP transmission, the probability of infection is mainly influenced by the presence of F1 nymphs and the phytoplasma load. In conclusion, this study evidences that C. picta is the most effective AP vector in the studied area, even if C. melanoneura can represent a potential risk in the presence of high inoculum sources.

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.

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...

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 &times, domestica), which can be infected by &lsquo, Candidatus Phytoplasma mali&rsquo, (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 &ldquo, Protein in Malus Expressed 2&rdquo, (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.

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.

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.

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.

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.

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 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.

The plant pathogen ‘Candidatus Phytoplasma mali’ infects apple trees and is associated with apple proliferation, an important disease in European apple growing regions. ‘Ca. P. mali’ manipulates its plant host by secreting so called effector proteins. The characterization of these effector proteins and the determination of their in planta targets is important to unravel the molecular mechanisms underlying disease and symptom development. Preliminary results show that a ‘Ca. P. mali’ effector targets the ubiquitination system of the plant, transcription factors of different classes and other host proteins that are involved in immune response regulation. Furthermore a regional genetic variant of another potential ‘Ca. P. mali’ effector that functionally differs from the variant of the ‘Ca. P. mali’ strain AT was found.

SAP11CaPM is the only bacterial effector known to date to be identified as secreted by the apple proliferation phytoplasma. In Malus spp. this effector binds and deactivates two transcription factors of the TCP family, subgroup CIN-class 2. To elucidate the role of SAP11CaPM and its targets in the context of resistance against apple proliferation phytoplasma (AP), the sequences of the target genes from susceptible and resistant Malus have been compared. Interestingly, some mutations exclusively present in AP resistant Malus x domestica TCP 25 (MdTCP25) could be observed. The MdTCP25 sequences of many other AP-resistant plants were sequenced and a good degree of correlation was found between resistance and presence of the previously detected resistant exclusive mutations. Using a yeast two hybrid-based reporter gene assay, the interaction strength between SAP11CaPM and MdTCP25 from resistant plants was evaluated. However, no visible differences in interaction strengths between SAP11CaPM and MdTCP25 from susceptible or resistant Malus could be observed.

Apple proliferation is associated with the presence of the bacterium ‘Candidatus Phytoplasma mali’. An infection with this pathogen leads to changes of the physiological state of the apple tree. In this study, it was applied near-infrared spectroscopy to measure these changes. In preliminary analysis based on a quadratic discriminant analysis the first indications that it might be possible to discriminate diseased from uninfected sample material were achieved.

Background Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. Objective Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. Methods The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. Results We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. Conclusion H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes . Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes , as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1 −/− mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480 + Gr1 + inflammatory monocytes and lower numbers of F480 − Gr1 + neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1 −/− mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1 −/− BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1 −/− BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.

Suppressor of cytokine signaling 1 (SOCS1) plays a major role in the inhibition of STAT1-mediated responses. STAT1-dependent responses are critical for resistance against infection with Chlamydia pneumoniae. We studied the regulation of expression of SOCS1 and SOCS3, and the role of SOCS1 during infection with C. pneumoniae in mice. Bone marrow-derived macrophages (BMM) and dendritic cells in vitro or lungs in vivo all showed enhanced STAT1-dependent SOCS1 mRNA accumulation after infection with C. pneumoniae. Infection-increased SOCS1 mRNA levels were dependent on IFN-αβ but not on IFN-γ. T or B cells were not required for SOCS1 mRNA accumulation in vivo. Infection-induced STAT1-phosphorylation occurred more rapidly in SOCS1−/− BMM. In agreement, expression of IFN-γ responsive genes, but not IL-1β, IL-6, or TNF-α were relatively increased in C. pneumoniae-infected SOCS1−/− BMM. Surprisingly, C. pneumoniae infection-induced IFN-α, IFN-β, and IFN-γ expression in BMM were attenuated by SOCS1. C. pneumoniae infection of RAG1−/−/SOCS1−/− mice induced a rapid lethal inflammation, accompanied by diminished pulmonary bacterial load and increased levels of iNOS and IDO but not IL-1β, IL-6, or TNF-α mRNA. In summary, C. pneumoniae infection induces a STAT1, IFN-αβ-dependent and IFN-γ independent SOCS1 mRNA accumulation. Presence of SOCS1 controls the infection-induced lethal inflammatory disease but impairs the bacterial control.

Apple proliferation is one of the economically most important apple diseases in Europe and infection rates are increasing over the last years in many apple growing areas. ‘Candidatus Phytoplasma mali’, the agent associated with the disease, is transmitted by two psyllid species, Cacopsylla melanoneura and C. picta. A multilocus sequence typing analysis was performed to determine genetic variability of ‘Ca. P. mali’ in infected trees and insect vectors. Two major genetic clusters were identified and occurrence of sequence variants is different in the geographic areas of South Tyrol (North Italy).

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3(-/-) mice succumbing to infection after day 9. At a low infective dose, C3(-/-) mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3(-/-) mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.