Peasy t1

PmCesA2 was cloned from a cDNA library constructed of RNA isolated from the needle tissue of P. massoniana using the Prime Script 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). A pair of primers was designed according to the full-length coding region of the CesA2 gene sequence of P. taeda (GenBank: AY789651.1). The PCR products were cloned into the pEASY-T1 (Transgen, Beijing, China) vector and transformed into E. coli DH5α, and then sequenced. All of the primers used in these assays are listed in Supplementary Table S2.

Sequence information of tea Clp complex subunits used in this study was obtained from our previous transcriptomic data (Liu et al., 2017 ) and tea genomic sequence information (Xia et al., 2019 ). Sequences of tobacco NtClpP3a and NtClpP3b were obtained according to the previously published literature (Moreno et al., 2017 (link)). Full-length cDNA was cloned with specific primers (Supplementary Table 1) and inserted into pEASY-T1 (TransGen, Beijing, China) for sequencing. COBALT protein sequence alignment analyses were conducted using the BLATA tool at the National Center for Biotechnology Information.¹ Intron retention of CsClpP3 was found in one of the two sequenced samples and confirmed with re-sequencing data. Active site prediction of the deduced CsClpP3 and CsClpP3m proteins was conducted using Hmmer (version 3.2.1).²

The 3D gene of FMDV (GenBank accession no. DQ533483.2), the N gene of VSV (GenBank accession no. M31846.1) and VP7 of BTV (GenBank accession no. AY776331.1) were previously cloned into pEASY-T1, an in vitro transcription vector (Transgen Biotech, China) containing the T7 promoter priming site, generating the recombinant plasmids pEASY-FDMV-3D, pEASY-VSV-N, and pEASY-BTV-VP7. These plasmids were linearized by endonuclease enzymatic digestion using EcoRV. RNA transcription was performed using the In vitro Transcription T7 Kit (Takara, Dalian, China). Then, the RNA was purified using the EasyPure RNA Purification Kit (Transgen Biotech, China). The purified RNA transcripts obtained were then quantified by absorbance at 260 nm using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, USA). Each concentration of the purified RNA transcripts was adjusted to 3.0 ×10⁹ copies/μL and mixed equally to prepare the RNA standards by serial 10-fold dilution with RNase-free H₂O. The standards contained the three in vitro‐transcribed RNAs with specific gene sequences of the three target viruses, and the concentrations of each RNA ranged from 1×10⁸ to 1 copies/μL. Two microliters of standard RNA transcript was used as a template in the mLAMP assay for analysis of the sensitivity of the mLAMP assay.

DNA was isolated from H460 cells by using the Wizard SV Genomic DNA Purification System (Promega, Southampton, UK). Bisulphite conversion was performed with the EZ DNA Methylation kit (Zymo Research, Orange, CA, USA) according to the manufacturer's recommendations. Bisulphite converted DNA was amplified with the primers listed in Table S1. The PCR products were then cloned into the vector pEASY-T1 (Transgene, Beijing, China), and at least 10 clones from each sample were subjected to DNA sequencing.

In this study, SYBR Green method was used for qPCR. The primer sequences for CTV and for the three endosymbionts detection were shown in Table 1. qPCR was carried out in 20 μL reaction mixtures containing 10 μL of SYBR Green Mix (TransGen Biotech, Beijing, China), 8 μL of ddH₂O, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM) and 1 μL of template DNA or cDNA (~50 ng). The qPCR cycling parameters were 95°C for 2 min, 40 cycles of denaturation at 95°C for 15 s, and 20 s extension at 60°C. All qPCR reactions were performed in triplicate. Samples with Ct values lower than 33 were judged as CTV positive. At the same time, in order to relatively quantify the CTV or endosymbiont titers, standard curves were drawn using pEASY-T1 (TransGenBiotech, Beijing, China) recombinant cloning plasmids containing the target fragments and the relative primers, with 8 gradients (10(7)-fold). CTV titers was assessed by the copy number of CTV in per ng cDNA using a formula referencing to the study of Ruiz-Ruiz et al. (2009) (link). β-actin was selected as endogenous internal control. The relative microbial titers was calculated by the method of 2^-△△CT (Livak and Schmittgen, 2001 (link)).

Bisulfite conversion was performed using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Bisulfite-treated DNA was then used to amplify. The amplified regions were cloned into pEASYT1 (TransGen Biotech) and sequenced. The primers for PCR amplification were listed in Table S1.