Occurrence of different Cacopsylla species in apple orchards in South Tyrol (Italy) and detection of apple proliferation phytoplasma in Cacopsylla melanoneura and Cacopsylla picta: (Hemiptera: Psylloidea)

Preventing the diffusion of phytoplasma associated diseases until now is based mainly on indirect control measurements against the transmitting insect vectors. Apple proliferation, one of the economically most important pests in European apple cultivation is caused by the apple proliferation (AP) phytoplasma (‘Candidatus Phytoplasma mali’), which is spread by the psyllids Cacopsylla (C.) picta (Foerster, 1848) and C. melanoneura (Foerster, 1848). Current control measures primarily comprise treatments against these AP phytoplasma transmitting vectors. The surveillance of C. picta and C. melanoneura population dynamics, as well as the determination of their infection rate in the field are crucial prerequisites to develop suitable and appropriate strategies to limit further spread of AP phytoplasma. Furthermore, the analysis of the species composition of the genus Cacopsylla present in apple orchards provides important information about the presence of other insect vectors potentially involved in spreading AP or other diseases. During an intensive monitoring program realized in the valleys of Val Venosta and Burggraviato (South Tyrol, Italy), the hotspots of apple proliferation epidemics, over 13,000 Cacopsylla individuals were captured and the occurrence of 16 species of the genus Cacopsylla was confirmed. The presence of C. picta was recorded in more than 50% of the investigated apple orchards and the natural infection rate of this vector was about 21% in a three-year average. Conversely, C. melanoneura was confirmed in more than 90% of the investigated sites but its low infection rate of about 1 % further supports that it plays a rather secondary role in spreading AP phytoplasma in South Tyrol

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

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

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

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

Abstract 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 SAP11 CaPm . For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11 CaPm 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 SAP11 CaPm 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

Linear regression analysis of TCP expression depending on phytoplasma quantity or <i>SAP11</i>_<i>CaPm</i> expression.

95% confidence interval (CI), goodness of fit and significance level (alpha = 0.05) of linear regression, calculated with GraphPad Prism 7.05 (GraphPad Software Inc.), between normalized phytoplasma quantity and SAP11CaPm, MdTCP16, MdTCP25 and MdTCP24 expression (left panel) and between normalized SAP11CaPm expression and MdTCPs expression in leaves from greenhouse (middle panel) and from foil-tunnel, respectively (right panel).</p

SAP11_CaPm interacts with MdTCP16 <i>in planta</i>.

Nicotiana benthamiana mesophyll protoplasts were co-transformed with a BiFC expression vector encoding SAP11CaPm and MdTCP16. SAP11CaPm and MdTCP16 interact in the nucleus and in the cytoplasm as indicated by the occurrence of a YFP signal in these two cellular compartments. The co-expression of SAP11CaPm and MdNYC1 did not show any YFP signal. The RFP signal (depicted in magenta) indicates a successful transformation of protoplasts with the BiFC vector. Chl (depicted in blue) shows the autofluorescence of chlorophyll within the chloroplasts. Microscopic analysis was performed with a Zeiss LSM 800 confocal microscope. Bars represent 20 μm for all micrographs.</p

SAP11_CaPm interacts with MdTCP16 but not with MdNYC1 in yeast.

Interaction between the bait SAP11CaPm fused to a DNA binding domain (BD) and the prey protein from Malus × domestica fused to an activation domain (AD) is indicated by growth of the Saccharomyces cerevisiae reporter strain NMY51 on SD minimal medium lacking the amino acids histidine (his) and adenine (ade). The white color of BD-SAP11CaPm + AD-MdTCP16 grown on full medium is an additional indication for a strong interaction, since the ADE2 reporter gene is activated upon interaction, while in absence of a protein-protein interaction and thus no ADE2 activation, a red colored intermediate accumulates in the adenine metabolic pathway.</p

<i>MdTCP16</i> expression increases from spring to autumn, is slightly higher in infected samples and strongly correlates to phytoplasma levels in leaves from infected <i>Malus × domestica</i>.

MdTCP expression in spring and autumn of non-infected (green) and naturally infected (black) apple leaves (A) or graft inoculated and grown in greenhouse plants (B). Naturally infected samples (A) comprise one leaf pool from non-infected trees, and three leaf pools from infected trees. Graft inoculated greenhouse plants (B) comprise leaf samples from 5 non-infected and 5 infected trees, each represented by a data point. Correlation of MdTCP and SAP11CaPm expression (C, D, E) in infected Malus × domestica leaf-samples from spring (grey, n = 8) and autumn (black, n = 8). Lines in graphs C, D, E and F show linear regression of the respective samples either from the greenhouse (dashed line) or from the foil tunnel (solid line). Samples in C, D and E were grouped regarding their growing conditions and plotted to different y-axis due to the differences in the concentration ranges of the different sample subsets. Correlation of MdTCP, SAP11CaPm expression and phytoplasma levels (F) in graft-inoculated Malus × domestica leaf-samples from spring (grey, n = 3) and autumn (black, n = 4). Statistical analysis was performed with multiple t-test. Statistical differences were determined using the Holm-Sidak method, with alpha = 0.05 and linear regression analysis, using GraphPad Prism 7.05 (GraphPad Software Inc.). For pools comprising only one biological replicate, technical replicates accounted to the statistical analysis. Significant differences between groups are indicated with asterisks (* P≤0.05, ** P≤0.01, *** P≤0.001). Data points with very similar values might overlap in the graph and can thus misleadingly appear as a single data point. In only seven out of the ten samples from the greenhouse phytoplasma concentration could be determined (F). This was due to a lack of sample material for the DNA preparation which is necessary for phytoplasma detection.</p

Primers used in this study.

Lowercase letters indicate bases for Gateway-attB site overhangs or SfiI restriction site overhangs. (PDF)</p