Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

ABSTRACT: Understanding protein and gene function requires identifying interaction partners usingbiochemical, molecular or genetic tools. In plants, searching for novel protein-proteininteractions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel proteinpartners in living plant cells. We demonstrate that it is possible to screen for novel proteinproteininteractions from a random library in protoplasted Arabidopsis plant cells and recoversome of the interacting partners. Our screen is based on capturing the bi-molecularcomplementation of mYFP between an YN-bait fusion partner and a completely random preyYC-cDNA library with FACS. The candidate interactions were confirmed using in plantaBiFC assays and in planta FRET-FLIM assays. From this work, we show that the wellcharacterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3,HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This isone of the first random in planta screens to be successfully employed.     

Caribbean Plant Quarantine Officers’ Self-Perceived Competencies and Training Needs for Regional Food Security

Plant quarantine is a key strategy in international plant protection efforts. This study identified the areas for knowledge and skills improvement among 108 Caribbean plant protection personnel from 20 countries. The Borich (1980) model of needs assessment was used. Results showed that respondents perceived all areas assessed as very important to their job functions. However, they perceived their proficiencies as low to average. Areas with highest training needs, as well as lowest need areas, were identified. The results can be used to revise the curricula for the regional training of plant quarantine personnel.     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Differential alterations in ultrastructural morphology of chicken heterophils and lymphocytes induced by corticosterone and lipopolysaccharide

Birds are continuously confronted by a large number of stressors, including pathogens. Despite their variety, all stressors induce an elevation in plasma corticosterone concentration, and consequently increase heterophil to leukocyte (H/L) ratio. In order to evaluate and differentiate effects of endocrine (non-bacterial) and bacterial stress on the proportions and ultrastructural characteristics of chicken leukocytes, a series of experiments were conducted with seven-week old chickens exposed either to dietary corticosterone or to intravenous (i.v.)-injected lipopolysaccharide (LPS). Samples were taken for haematological, endocrine, and electron microscopy examination. Administration of corticosterone and LPS significantly elevated plasma corticosterone concentrations and increased H/L ratios. Electron microscopy observations indicated changes in heterophil size, shape, and granulation, and lymphocyte cytoplasmic characteristics. Immature heterophils were observed in the peripheral blood, suggesting that corticosterone and LPS both stimulate an earlier release of heterophils from bone marrow and enhance their influx into blood circulation. The LPS induced a degenerative morphology and the destruction of lymphocytes, whereas corticosterone appeared to stimulate their redistribution rather than destruction. The results indicate that exposure to corticosterone or LPS similarly increase H/L ratios, but differentially alter the ultrastructure of heterophils and lymphocytes. Elucidation of the mechanisms that cause such changes may play an important role in distinguishing between a nonimmune and immune stress challenge at the molecular level.     

Calf-level risk factors for neonatal diarrhea and shedding of Cryptosporidium parvum in Ontario dairy calves

This work was conducted to investigate calf-level factors that influence the risk of neonatal diarrhea and shedding of Cryptosporidium parvum oocysts in calves, on dairy farms in Ontario with histories of calf diarrhea or cryptosporidiosis. Fecal samples were collected weekly for 4 weeks from each of 1045 calves under 30 days of age on 11 dairy farms in south-western Ontario during the summer of 2003 and the winter of 2004. A questionnaire designed to gather information on calf-level management factors was administered on farm for each calf in the study. Samples were examined for C. parvum oocysts by microscopy, and a subset of specimens was also tested for enterotoxigenic Escherichia coli, Salmonella, bovine rotavirus and bovine coronavirus. The consistency of each sample was scored and recorded at the time of collection in order to assess the presence or absence of diarrhea. In addition, a blood sample was taken from each calf upon enrollment in the study, for assessment of maternal antibody transfer and for polymerase chain reaction testing for persistent bovine viral diarrhea virus infection. Using the GLLAMM function in Stata 9.0, multilevel regression techniques were employed to investigate associations between management practices and the risk of C. parvum shedding or diarrhea. C. parvum oocysts were detected in the feces of 78% of the 919 calves from which all four fecal samples had been collected. Furthermore, 73% of the 846 calves for which all four fecal consistency scores had been recorded were diarrheic at the time of collection of at least one sample. Significant predictors of the calf-level risk of C. parvum shedding included the use of calf diarrhea prophylaxis in pregnant cows, and the type of maternity facilities in which the calves were born. Factors associated with an increased risk of diarrhea were leaving the calf with the dam for more than an hour after birth, and the birth of a calf in the summer as opposed to winter. Calves shedding C. parvum oocysts had 5.3 (95% CI 4.4, 6.4) times the odds of diarrhea than non-shedding calves, controlling for other factors included in the final multivariable model. Furthermore, infected calves shedding more than 2.2 x 10(5) oocysts per gram of feces were more likely to scour than infected calves shedding lower numbers of oocysts (OR= 6.1, 95% CI 4.8, 7.8). The odds of diarrhea in calves shedding oocysts that had been allowed to remain with their dams for more than an hour were higher than the odds of diarrhea in shedding calves that had been separated from their dams within an hour after birth.