Overtime expression of selected chrysanthemum yellows phytoplasma genes during infection of plant and leafhopper vectors

Background and objectives Microarrays analysis suggests that phytoplasma genome expression in the plant can be modulated according to the infection stage (Oshima et al., 2011). A real time PCR protocol was set up to study the expression profile of different 'Candidatus Phytoplasma asteris' Chrysanthemum yellows isolate (CYP) genes during infection of Arabidopsis thaliana and the two vector species Macrosteles quadripunctulatus and Euscelidius variegatus. Target genes were selected among secreted proteins, known effectors, general metabolism and unique CYP ORFs, obtained following Illumina sequencing. A detailed study on gene expression of an obligate parasite in the plant and insect hosts is a prerequisite for the identification of molecular mechanisms involved in phytoplasma perception and regulation of host switch. Materials and Methods Expression of 14 CYP genes coding three secreted proteins, four generic transporters (a mechanosensitive channel, the translocation component of SecY translocase, a multidrug and an oligopeptide transporters), two specific transporters (for arginine and Zn++ ion), a protein involved in phospholipid metabolism and the 30S rRNA ribosomal subunits obtained by the Illumina sequencing of CYP genome, one effector protein (Tengu, Hoshi et al., 2009), the major antigenic membrane protein (Amp, Galetto et al., 2008), and the immunodominant membrane protein (Imp, Kakizawa et al., 2009) was analyzed in the plant (A. thaliana) and vector (M. quadripunctulatus, E. variegatus) hosts. For each selected target, specific primers were designed and a qPCR protocol with SYBR Green chemistry was optimized. Amplification efficiencies and melting temperatures of the amplicons were obtained by analyses of qPCR amplification plots using serial dilutions of plasmids containing the specific fragment of each target gene. To study phytoplasma gene expression in plant, CYP-infected M. quadripunctulatus were fed on 10 A. thaliana plants and leaf samples were collected at different days post inoculation (dpi). To study phytoplasma gene expression in the vector, several individuals of the two analyzed species were collected at different days post acquisition (dpa) on the same CYP-infected daisies. Total DNA and RNA were separately extracted from each plant and insect sample. Presence and titre of CYP were determined by PCR (Lee et al., 1994) and qPCR (Marzachì and Bosco, 2005) , respectively, with total DNA as reaction template. Total RNA was DNase treated, reverse transcribed and amplified by qPCR to quantify the transcripts of each selected gene. Results and Discussion Phytoplasma specific symptoms appeared on A. thaliana plants at two weeks post infection. Fifteen and 83% of the plants were infected at 5 and 25 dpi, first and last sampling dates, respectively. At the first two sampling dates (7 and 14 dpa), 91 and 71% of sampled M. quadripunctulatus and E. variegatus were already CYP positive. Later on (up to the last sampling date, 35 dpa) all M. quadripunctulatus and about 90% E. variegatus sampled adults were CYP infected, in line with the acquisition efficiencies already observed for these species (Bosco et al., 2007). CYP titre in A. thaliana increased from 5 to 28 dpi, and the final concentration was similar to that observed for other herbaceous hosts (Saracco et al., 2006). In the two vector species, CYP titre increased up to 21 dpa. . The newly designed primers produced specific amplicons and the standard curves obtained following the amplification of serial dilutions of appropriate plasmids showed R2 values above 0.99 and efficiencies ranging from 74% to 100%. The expression level of each target gene was calculated with respect to the CYP titre measured in 100 mg of each leaf tissue sample or in each entire inse