In 2016, the QH sample was mixed from five early HLB-infected green orange leaves with asymptomatic in Qionghai County, Hainan Province, and the QZ sample was mixed from another five early HLB-infected green orange leaves with asymptomatic in Qiongzhong County, Hainan Province. Both pathogens of QH and QZ were identical with the Candidatus Liberibacter asiaticus psy62 isolate (GenBank accession number: CP001677.5) [22] . Wild-type Nicotiana benthamiana (N. benthamiana) (Ferox genus) was kept in the Laboratory of Molecular Virology, Institute of Tropical Bioscience and Biotechnology (ITBB), Chinese Academy of Tropical Agricultural Sciences (CATAS). The GV1300 plasmid (pCAMBIA1300: GFP) was provided by Professor Ming Peng of ITBB, CATAS.

In order to identify one or two high-expression SPGs, six SPGs were chosen from the different protein secretory systems according to the complete genome sequence of Candidatus Liberibacter asiaticus [22] (Table 1). Based on the nucleotide sequences of these SPGs, 6 primer-pairs of the 408, 24A, fATP, pap, msp, and 377 genes were designed for qPCR or RT-qPCR using the online website (https://www.idtdna.com/Scitools/Applications/RealTimePCR/) (Table 2). 18S rRNA of citrus was used as an internal reference gene [23] (Table 2). All primers were synthesized by the Beijing Genomics Institute (BGI).

Total DNA extraction of the QH and QZ samples was performed according to the manufacturer’s instructions with a Plant Genomic DNA Kit (TIANGEN BIOTECH, Beijing, China) and total RNA extraction was performed according to the Trizol Universal Regent (TIANGEN BIOTECH, Beijing, China). First-strand cDNA was synthesized from 2.0 μL of total RNA using 0.5 μL of random hexamer primer (10 μM) and the Fast Quant RT Kit (with gDNase) (TIANGEN BIOTECH, Beijing, China).

In order to measure the efficiency and correlation coefficients of six SPG primer pairs in qPCR, the initial amplified DNA template was further diluted to 1:10⁻¹, 1:10⁻², 1:10⁻³, 1:10⁻⁴, and 1:10⁻⁵ by ddH₂O, and these samples were used to establish the standard curves by each pair primer in the Stratagene Mx3005 machine. The results showed that six qPCR reactions by the specific primer pairs amplified SPGs highly efficiently (efficiencies of 86.8% to 92.2%) with correlation coefficients between 0.981 and 0.999.

In order to measure the relative DNA amounts and their RNA expression levels of six SPGs, qPCR and RT-qPCR analyses were performed on an Agilent Stratagen Mx3005P instrument using the Hieff^TM qPCR SYBR Green Master Mix with Low Rox Plus (Yeasan, Shanghai, China) according to the instructions, and

The database sources: NCBI Reference Sequence database (http://www.ncbi.nlm.nih.gov).

Note: The bold sequences represent the restriction enzymes.

each gene was measured three times independently. The total DNA from healthy green orange leaves was used as a negative control. The qPCR or RT-qPCR mixture was 10 μL of Hieff^TM qPCR SYBR Green Master Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1 μL of template DNA or cDNA, and 8.2 μL of ddH₂O. The qPCR and RT-qPCR programs involved pre-denaturing at 95˚C for 5 min, followed by 40 cycles of denaturing at 95˚C for 10 s, annealing at 55˚C for 30 s, extending at 72˚C for 20 s, and a dissolution curves program using the Agilent Strata Mx3005P instrument.

The 408 gene was amplified with the 408-gF/408-gR primers, while the pap gene was amplified by using the pap-gF/pap-gR primers (Table 2). The PCR reaction was conducted using PrimeSTAR HS DNA Polymerase kit (Takara, Dalian, China): 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U/μL), 10 μL of 5 × PrimeSTAR Buffer (Mg²⁺ plus), 4 μL of dNTP Mixture (2.5 mM each), 2 μL of F/R (5 μM) primer, and 2 μL of total DNA, and ddH₂O was added up to 50 μL. The PCR program involved pre-denaturing at 98˚C for 3 min, followed by 35 cycles of denaturing at 98˚C for 30 s, annealing at 55˚C for 30 s, extending at 72˚C for 50 s; and finally, the reaction was terminated by post-extending at 72˚C for 10 min. The amplified target fragments of 408 and pap were gel-extracted by using the DNA Gel Extraction Kit (Omega Bio-Tek, Doraville, GA, USA) and subsequently, cloned into the plant expression vector GV1300 using T4 DNA ligase (Takara, Dalian, China). The recombinant plasmid was further transformed into Escherichia coli (E. coli) Trans 5a competent cells (TransGen, Beijing, China), and three positive clones were selected for bidirectional sequencing by 1300-F and 1300-R primers at Thermo Fisher (Guangzhou, China).

The recombinant plasmids of GV1300, GV1300-408, and GV1300-pap were transformed into Agrobacterium tumefaciens GV3101 competent cells by the freeze-thaw method, as described in Sparkes and Al [24] . The transfected tobacco leaves were cut into pieces of 1 cm × 1 cm, and fluorescence images were visualized on a microscope (FluoView FV1000D IX81; Olympus, Tokyo, Japan) to observe the subcellular localization of the fusion protein under wavelengths of 488 nm and 546 nm.

Total DNA extracted from two mixed samples were visualized on a 1% agarose gel, and the specific DNA bands of more than 10 kbp in lengths were consistent with the predicted sizes (data not shown). Total RNA samples were also extracted from these two samples and were visualized on a 1% agarose gel (data not shown). The results indicated that the RNA bands of 28S, 18S, and 5S were abundant which suggests that a high quality of total RNA was obtained. The total RNA was further used to synthesize the first strand cDNA (1^st cDNA) which subsequently could be used for RT-qPCR.

Analysis of the real-time qPCR showed that the amplification plot of six SPGs and the internal reference gene shown in the dissociation curve of QH sample (Figure 1(a)), similar result was observed in QZ sample. Further analysis indicated that the DNA contents of the 408 and pap genes were 26.48 and 9.36 times that of the 377 gene, while the other genes were 2.17 - 4.11 times that of the

377 gene in the QH sample (Figure 1(b)). In the QZ sample, the DNA contents of the 408 and pap genes were 15.20 and 8.35 times that of the 377 gene, while the DNA content of the 24A gene was also relatively high, about 9.22 times that of the 377 gene (Figure 1(b)). In summary, the relative DNA contents of the 408, pap, and 24A genes were relatively high in QH and QZ samples.

The results from the RT-qPCR quantification did not match the DNA amount shown in the qPCR reactions. Of these six SPGs, only the 408 and pap genes had amplification curves, and the Ct value was between 15 and 35. Other genes did not have an obvious amplification curve, or their Ct value was more than 35 which should be insignificant (Very low amount or unspecific amplification). Further analysis revealed that the relative transcription level of the 408 gene was 2.43 higher than the transcription level of the pap gene in QH sample. In the QZ sample, the relative transcription level of the 408 gene was 9.45 higher than the transcription level of pap gene, and the relative transcript RNA level was much higher than that of the 408 gene in the QH sample (Figure 2). In this study, two relatively high transcription levels of SPGs were screened from the ten candidate SPGs.

In order to further clarify the distribution of the 408 and pap proteins in the host cells, the recombinant plasmids of GV1300-408 and GV1300-pap were transformed into Agrobacterium tumefaciens GV1301 competent cells. Then, the positive clones were identified by colony PCR (Single colony was used as template), as described above. After injection of GV1300-408/GV1301 and GV1300-pap/GV1301 into the tobacco leaves, fluorescence images were visualized

by microscopy at 72 hours post inoculation (h.p.i.). The green fluorescence signal from the 408-GFP fusion protein was observed in the nuclei and cytoplasm of tobacco leaf cells, while the green fluorescence signal of the pap-GFP fusion protein was observed in the cytoplasm of tobacco leaf cells. These results indicate that the 408 protein localizes in the nucleus and cytoplasm tobacco mesophyll cells, but the pap protein localizes in the cytoplasm of tobacco leaf cells. In addition, the GFP protein is localized in the cytoplasm and the nuclei of tobacco mesophyll cells (Figure 3).