Complete Genome Sequences of the Obligate Symbionts “ Candidatus Sulcia muelleri” and “ Ca. Nasuia deltocephalinicola” from the Pestiferous Leafhopper Macrosteles quadripunctulatus (Hemiptera: Cicadellidae)

Two bacterial symbionts of the European pest leafhopper, Macrosteles quadripunctulatus (Hemiptera: Cicadellidae), were fully sequenced. “ Candidatus Sulcia muelleri” and “ Ca . Nasuia deltocephalinicola” represent two of the smallest known bacterial genomes at 190 kb and 112 kb, respectively. Genome sequences are nearly identical to strains reported from the closely related host species, M. quadrilineatus .

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

ABSTRACTTo highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of “CandidatusPhytoplasma asteris,” chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatusandEuscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection ofA. thalianaandM. quadripunctulatusbut were more constant inE. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant.

Temperature-dependent transmission of Candidatus phytoplasma asteris by the vector leafhopper Macrosteles quadripunctulatus Kirschbaum

A set of experiments was carried out to characterize how temperature affects the spread of chrysanthemum yellows phytoplasma (CYP), a strain of Candidatus Phytoplasma asteris, in Chrysanthemum carinatum plants transmitted by the Macrosteles quadripunctulatus leafhopper. Experiments provided data on CYP latency period in insect and plant host, M. quadripunctulatus adult mortality rate, and epidemics progression in plants under controlled conditions inside climatic chambers. Experiments were conducted at temperatures ranging between 15 and 30°C. Empirical laws for temperature-dependent epidemiological parameters were next derived and used in a dynamical model of the epidemics progression. Experiments showed that CYP epidemics was faster at higher temperatures and the model could replicate these observations with relatively high accuracy (correlation >98.03% and residuals <14.5%). The epidemics spreading rate increased linearly from 0.2 plants infected per day at 15°C to about 0.7 plants per day at 30°C, possibly due to: i) faster CYP multiplication in the host plants and ii) higher frequency of feeding bouts of M. quadripunctulatus at higher temperatures.

Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.