They might also pave the way to identify genes that can be targeted to elevate plant resistance or inhibit the growth and reproduction of the pathogen. However, further research is required to elucidate the roles of these genes in the susceptibility/resistance of Mexican

lime tress to “” Ca. Phytoplasma aurantifolia”", and to determine how strategies might be developed to TGF-beta pathway incorporate these genes into molecular breeding programmes. Methods Plant material and inoculation Ten healthy 1-year-old Mexican lime trees grown in the greenhouse were used Captisol mouse in this experiment. Specimens from Mexican lime trees infected with witches’ broom were grafted to healthy trees, and specimens from healthy Mexican lime trees were grafted to other healthy trees. The grafted plants were covered for 1 month with plastic bags to increase humidity and were arranged randomly on the greenhouse bench. They were kept under natural light conditions at a temperature of 25-28°C. The branches infected with witches’ broom were sampled 20 weeks after inoculation and used for RNA extraction. As a control, RNA was extracted from non-grafted healthy plant leaves that has been grown under similar conditions.

Detection of Phytoplasma infection by nested PCR Total RXDX-101 DNA was extracted from leaf samples (vascular tissues from leaf veins and petioles) using the method described originally by Daire et al [28] with some modifications [29]. Samples of tissue (1 g) were homogenised at room temperature in 7 ml of cetyl trimethyl ammonium DNA ligase bromide (CTAB) buffer (3% CTAB, 1 M Tris-HCl pH 8.2, mM EDTA, 1.4 M NaCl), with addition of 0.2% 2-mercaptoethanol, in disposable plastic bags

using a ball-bearing device. Aliquots of 1 ml of homogenate were transferred to Eppendorf tubes and incubated in a water bath at 65°C for 20 min. After extraction with 1 ml of chloroform, nucleic acids were precipitated from the aqueous phase with an equal volume of isopropanol, collected by centrifugation, washed with 70% ethanol, dried, dissolved in 150 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) and stored at -20°C until use. The region of the phytoplasma 16 S rRNA gene was amplified by PCR in a total reaction volume of 25 μl in an Applied Biosystems thermal cycler. The first set of PCR primers was P1 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′) [30] and P7 (5′-CGTCCTTCATCGGCTCTT -3′) [31]. The resulting P1-P7 amplicons were then used as template DNA in a nested-PCR amplification with the universal primer pair for phytoplasmas r16r2/r16F2n [32]. The purified PCR products were cloned into the pGEM-T Easy vector (Promega), and sequenced at the fluorescent automated sequencing facility at Fazabiotech (Tehran, Iran). The phytoplasma strains were classified using iPhyClassifier, as described by Zhao et al [33].