Bp clonase 2 enzyme

Total RNA was isolated from fresh Arabidopsis leaf tissue using IRIS Kit (Bangalore, Genei) according to manufacturer’s instructions (S1 Fig). The RNA was reverse transcribed using RevertAid H Minus first stand cDNA synthesis Kit (Fermentas Life Sciences, USA). The first strand of cDNA was used to amplify AtGLYI2 (AT1G08110.1), AtGLYI3 (AT1G11840.1) and AtGLYI6 (AT1G67280.1) genes with Q5 polymerase (NEB) using gene specific forward and reverse primer containing attB1 and attB2 sequences for subsequent cloning into donor vector (S1 Table & S1 Fig). Full-length AtGLYI genes were recombined into pDONOR221 vector (Addgene) using BP clonase II enzyme (Invitrogen, instructions for cloning followed as per manufacturer’s protocol). Sequence of all the three AtGLYI genes recombined into pDONOR vector were confirmed by Sanger sequencing, only the correct sequences were further recombined into pEARLEY100 [14 (link)], a plant expression vector in presence of LR clonase II enzyme (Invitrogen, instructions for cloning followed as per manufacture’s protocol).

For infiltration into the leaf epidermal cells of N. benthamiana, the full-length open reading frames of PpDUC1, and parts of PpPHY (1–3621 bp in DUC1) and PpCRY (3115–5070 bp in DUC1 without stop codon) were amplified from P. provasolii genomic DNA by PCR with each primer set (Supplementary Table 2) and cloned into the pDONR207 ENTRY vector by BP recombination using with BP Clonase II enzyme (Invitrogen Corp.). These open reading frames were transformed into the pEarleyGate 103 destination vector^{50 (link)} to fuse in frame with GFP and 6× His by LR reaction. A synthetic gene of Arabidopsis thaliana HY5 (At5g11260) fused to mCherry (Sequence ID: MH976504.1) was synthesized by Eurofins Genomics. The DNA fragment was amplified by PCR with primers (Supplementary Table 2) using the synthetic gene as a template and cloned into the pSK1 plasmid vector^{51 (link)}.

The cDNA library was constructed using Gateway recombination technology. Human universal reference total RNA (Catalog No. 636538, Clontech, Mountain View, CA) was used as a template to synthesize cDNA by reverse transcription. The cDNA was ligated to adaptors with the attB1 site. To construct the Gateway Entry vectors, pDONR222 (Catalog No. 54648, Invitrogen) was mixed with the purified cDNA fragments and the BP Clonase II enzyme (Catalog No. 11789100, Invitrogen). The reactions were incubated at 25 °C overnight and then treated with Proteinase K at 37 °C for 10 min for termination. The reaction products were transformed into E. coli DH10B competent cells, colonies were grown in LB medium with kanamycin selection, and plasmids were extracted with a Plasmid Mini Kit (Catalog No. 12125, Qiagen, Hilden, Germany). To construct the cDNA library, pDONR222 entry vectors were mixed with the pPC86-YN157-CCDB vectors and the Gateway LR Clonase II enzyme (Catalog No. 11791100, Invitrogen). The reactions were incubated at 25 °C overnight and then treated with Proteinase K at 37 °C for 10 min for termination. The reaction products were transformed into E. coli DH10B competent cells, colonies were grown in LB medium with ampicillin selection, and plasmids were extracted and stored at −80 °C.

The ORF of TvAP33 was cloned and inserted into the bait vector pDHB1 to form pDHB1-TvAP33. The cDNA library of VK2/E6E7 cells was constructed according to the instructions of the SMART cDNA Library Construction Kit (TaKaRa, Clontech Laboratories, CA, USA). In brief, RNase-free DNase I (TaKaRa, Clontech Laboratories) was used to remove the genomic DNA contamination from the prepared RNA samples. The mRNA was then isolated using the Oligotex mRNA Midi Kit (Qiagen, Hilden, Germany). The purified mRNA was used to synthesize double-stranded cDNA, and both ends of the cDNAs were equipped with connectors for recombination into the prey vector. The cDNAs were purified and cloned into the pray plasmid pPR3-N with the BP Clonase^® II enzyme (Thermo Fisher Scientific). These recombinant plasmids were electroporated into E. coli DH10B, according to the following electrotransfer procedure: voltage 1500 V, resistance 200 Ω and capacity 25 μF. These bacterial solutions were diluted and coated onto solid medium, the number of bacterial clones was counted and the capacity of the library was calculated. The plasmid DNA was randomly extracted from 24 clones and restriction digested by SfiI to confirm the size of the inserts in the clones.

To knock down the tomato gene BiP, the gene fragments were amplified from tomato plant DNA using the specific primer pair TRV-Sl-BiP-F/TRV-Sl-BiP-R (Supplementary Table S1), respectively. The amplified gene fragment was then cloned into the pDONR221 entry vector (Thermo Fisher Scientific, Waltham, MA, USA) using BP Clonase II enzyme (Thermo Fisher Scientific, Waltham, MA, USA) to create a pDONR221-BiP plasmid clone. Next, the BiP fragment was cleaved from the pDONR221 vector using HpaI and EcoRV restriction enzymes (NEB, Ipswich, MA, USA) and cloned into the vector YL279 using LR Clonase II enzyme (Thermo Fisher Scientific, Waltham, MA, USA). YL279 is a pTRV2 vector that is commonly used for virus-induced gene silencing in plants and was obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH, USA). The resulting plasmid constructs, pTRV2-BiP and pTRV2-GFP (as a control), were transformed into agrobacterium strain C58C1, which was given by Dr. Rose Hammond. Two-week-old tomato plants were then agroinfiltrated with the TRV constructs. Ten days after the TRV inoculation, the tomato plants were infected with PPT phytoplasma via graft inoculation. Newly grown leaves were harvested one month after the PPT phytoplasma infection, and both DNAs and RNAs were extracted for further analysis.