Materials and methods

Biological material. Plants of Arabidopsis thaliana and sweet orange (Citrus sinensis) were grown in a mixture of peat moss-perlite substrate (60:40) under greenhouse conditions at 27-30 °C temperature and 2243 masl, in Montecillo, Texcoco, State of Mexico.

Bioinformatic analysis. The consensus sequences in the Arabidopsis (^{Pajerowska M.K. et al., 2013}; ^{Yu et al., 2013}) and C. sinensis database (^{Lu et al., 2013}) were found using the BLAST (Basic Local Alignment Search Tools) program, and the sequence with the highest similarity (Query) was selected. The analysis of alignment of the conserved sequences of the reported genes was conducted using the Mega6 program. The designed primers were analyzed using the Oligo Analyzer Tool and Primer Questo Tool (IDT, Integrated DNA Technologies) programs, and NEBcutter (BioLabs) was used to detect the possible formation of dimers and identify the restriction sites in the primers.

DNA extraction. To extract DNA from the AZI-1 and PR-1 gene isolates, 0.1 gram of mature A. thaliana leaves was used following the method of 2% CTAB (^{Doyle and Doyle, 1987}). To extract DNA from the CsNDR-1 isolate, young leaves of C. sinensis and the QIAGEN DNAeasy kit were used following the manufacturer’s instructions. DNA concentration was measured using 1 µL of the sample in a Nanodrop (Thermo SCIENTIFIC, NANODROP 2000).

Design of primers. The gene search was conducted using BLAST. For the CsNDR-1 gene the following sequence was found: gi|568842565|ref|XM_006475151.1| PREDICTED: Citrus sinensis protein CsNDR-1-like (LOC102630232), mRNA. Based on the sequence found, one ‘Forward’ primer and one ‘Reverse’ primer were designed, both containing a series of 6 A/T followed by the sequence of the restriction enzyme selected for cloning plus the Kozac ACC sequence, and, finally, the primer sequence.

The following record of the AZI-1 gene was found: gi|30682133ǀrefǀNM_117317.4ǀ Arabidopsis thaliana azelaic acid induced 1 (AT4G12470), mRNA, and for PR-1 giǀ 17381133ǀAY064023ǀ Arabidopsis thaliana putative pathogenesis-related PR-1 protein (At2g14610), mRNA. Based on the sequences found, the ‘Forward’ and ‘Reverse’ primers were designed for each gene.

Gene identification and amplification. Gene detection and amplification through end-point PCR was performed using specific primers for each gene, including restriction points for cloning (Table 1).

To design the primers, restriction sites of Apa1 (GGGCCC) and Spe1 (ACTAGT) were incorporated, as well as several adenines following the restriction sites. For this, we used a final volume of 25 µL of a mixture containing 5 µL of DNA, 2.5 µL of 10X PCR Buffer (Invitrogen), 1 µL of MgCl₂ 50 (Invitrogen), 0.5 µL of dNTPs 10 mM (Promega), 1 µL of each primer (10 pmol), RNAsa/DNAsa-free H₂O (Invitrogen) and 0.2 µL of Taq polymerase (Invitrogen). The amplification program consisted of 35 cycles for AZI-1 and PR-1: 95 °C for 30 s, 68 °C for 30 s and 68 °C for 30 s. For CsNDR-1, 35 cycles: 95 °C for 30 s, 66 °C for 30 s and 68 °C for 40 s. The three genes had an initial pre-denaturation stage at 95 °C for 2 min and a final extension stage at 68 °C for 5 min.

The PCR products were analyzed in 1% agarose gel, with 5 µL of the PCR product and 3 µL of load buffer (Promega). The gel was run at 90 volts, 400 amperes for one hour, and was dyed in ethidium bromide for 5 min. The PCR products that amplified at the expected size were purified using the QIAquick^® PCR Purification kit and sequenced to corroborate the isolation of the tested gene before using engineering.

Vector construction. The pUC118-FMV-Poly-2-1 vector (^{Febres et al., 2003}) was grown in three millimeters of 2xYT liquid culture medium (yeast extract 10 g L, tryptone 16 g L, NaCl 5 g L) containing 100 µg mL^-1 of ampicillin (amp) at 37 °C for 12 h and then the plasmid was isolated using the QIAprep^®Spin miniprep kit, following the manufacturer’s instructions. The purified AZI-1, PR-1 and CsNDR-1 genes, as well as the pUC118-FMV-Poly-2-1 isolated vector, were digested with Apa1 and Spe1 restriction enzymes at the temperature and time recommended for each enzyme. The double digestion of the three genes and vector pUC118-FMV-Poly-2-1 produce a single fragment, so they were purified using a QIAquick^® PCR Purification kit, and then ligated for 12 h at 4 °C. The digestion products were cloned in Escherichia coli DH5α (Invitrogen) using 80 to 100 ng µL^-1 of DNA by electroporation under one pulse conditions at 1.8 kV (Bio-Rad, 165-2100). The bacteria that were supposedly transformed were sown in a 2xYT solid culture medium containing 100 µg mL^-1 of amp; three aliquots of transformed cells (10, 20 and 50 µL) were tested using a sterile microinjector to distribute them on the medium surface. After 12 h at 37 °C, several individual colonies formed and were analyzed using PCR with the previously mentioned primers in order to corroborate the presence of the genes. The PCR products were run in 0.8% agarose gel at 100 volts for 30 min.

The positive colonies were sown in three ml of 2xYT liquid culture medium containing 100 µg mL^-1 of amp at 37 °C at 230 rpm for 12 h. Using half of the bacterial culture that had grown (1.5 ml), the plasmid was extracted using the QIAprep Spin Miniprep kit from QIAGEN and sent out for sequencing to verify the sequences of the three genes. With the other half of the bacterial cultures, “stocks” were prepared using glycerol and then stored at -80 °C.

To continue inserting the genes of interest now into pCAMBIA transformation vector plasmids, the following procedure was used: the bacteria with the three genes independently contained in pUC118-FMV-Poly-2-1 were grown in 2xYT solid culture medium with 100 µg mL^-1 of ampicillin, while those containing the pCAMBIA2201 and pCAMBIA 2301 plasmids were grown in solid 2xYT culture medium with 25 µg mL^-1 of chloramphenicol (Cap) or 50 µg mL^-1 of kanamycin (Kan), respectively, for 12 h at 37 °C. The plasmids were extracted with a QIAprep® Spin Miniprep kit and then digested with BamH1 and SphI restriction enzymes at the temperature and time recommended for each enzyme. The double digestion products of pUC118-FMV-Poly-2-1 were purified using the QIAgen gel purification kit, while the double digestion products of the pCAMBIA vectors were purified using the Qiaprep Miniprep kit. Product ligation was carried out by incubating them at 4 °C for 12 h. The digestion products were cloned in Escherichia coli DH5α (Invitrogen) using 80 to 100 ng µL^-1 of DNA by electroporation under one pulse conditions at 1.8 kV (Bio-Rad, 165-2100). The bacteria that were supposedly transformed were sown in 2xYT solid culture medium containing 25 µg mL^-1 of Cap for pCAMBIA2201, or 50 µg mL^-1 of Kan for pCAMBIA 2301; three aliquots of transformed cells (10, 20 and 50 µL) were tested using a sterile microinjector to distribute them on the medium surface. After 12 h at 37 °C, several individual colonies formed and were analyzed through PCR using the specific primers in order to corroborate the presence of each of the three genes.

Once more, the positive colonies were individually grown in a liquid culture medium containing the corresponding antibiotics for each plasmid at 37 °C at 230 rpm for 12 h. Similarly, one aliquot was sent out for sequencing, and the other aliquot of bacteria was used to prepare “stocks” that were stored at -80 °C.

Transformation of A. tumefaciens. The pCAMBIA plasmids with the constructions were grown in 2xYT solid culture medium containing 25 µg mL^-1 of Cap for pCAMBIA2201, or 50 µg mL^-1 of Kan for pCAMBIA 2301 at 37 °C for 12 h, and then the plasmids were extracted using the Qiaprep Spin miniprep kit. The competent EHA 105 and AGL 1 strains of A. tumefaciens (^{Kayim and Koc 2005}; ^{Almeida et al., 2003}) were selected for transformation through electroporation (Bio-Rad, 165-2100) under one pulse conditions at 2.5 kV (Bio-Rad, 165-2100). The bacterial cells of the EHA 105 strain that were supposedly transformed were grown in solid YEP culture medium (10 g L of bactopeptone, NaCl 5 g L, yeast extract 10 g L) with rifampicin (Rif) (50 µg mL^-1) and Kan (50 µg mL^-1), while the cells of the AGL 1 strain were grown in solid YEP culture medium with Rif (50 µg mL^-1), Kan (50 µg mL^-1) and carbenicillin (Carb) (25 µg mL^-1). Aliquots of 50, 100 and 200 µL of the two Agrobacterium strains were tested and incubated for two to three days at 27 °C. The individual colonies that grew were analyzed through end-point PCR using the primers and under the conditions previously described for each gene. The colonies that were positive were sent out for sequencing. The colonies whose transformation and construction were verified through sequencing were grown in liquid YEP culture medium using the corresponding antibiotics before being cryopreserved at -80 °C.

Results and discussion

Identifying genes. The concentration of Arabidopsis DNA was 478.2 ng µL^-1 with a value of optical density (O.D.) units of 2.04. The concentration of C. sinensis DNA was 48.7, and its OD reading was 1.7. The PCR products run in agarose gel showed bands of the expected size, that is, 486 pb, corresponding to the AZI-1 and PR-1 genes, and for the CsNDR-1 gene one 618 pb band was obtained (Figure 1). Sequencing of the products showed at least 99% identity.

Cloning genes in the pUC118-FMV-Poly-2-1 plasmid. The purified genes were inserted into the pUC118-FMV-poly-2-1 vector (Febres et al., 2013). Individual E. coli colonies that grew were analyzed through PCR to detect the transformed colonies. For the AZI-1 gene, 18 colonies were analyzed using the AZI-1 F/AZI-1 R primers, and three positive colonies were obtained (Figure 2A, lanes 2, 4 and 10). For the PR-1 gene, 18 colonies were also analyzed using the PR-1 F/PR-1 R primers, and six positive colonies were obtained (Figure 2B, lanes 2, 9, 11 to 13, and 16). For the CsNDR-1 gene, the transformation was done twice, and a total of 35 colonies were analyzed using the CsNDR-1 F/CsNDR-1 R primers; two positive colonies were obtained, and one of them appears in lane 12 of Figure 2C. All the colonies that were positive by PCR were sent out for sequencing, and the colony of each of the AZI-1, PR-1 and CsNDR-1 genes that showed the greatest similarity with the sequence reported in the database of the NCBI GenBank was selected to continue cloning in vector pUC118-FMV-poly-2-1. For the PR-1 gene, colony three showed the highest level of similarity (99%); in the case of the AZI-1 gene, colony one showed 99% similarity, and colony 14 showed 99% similarity in the CsNDR-1 gene. Three constructions were built using the best colonies of each gene inserted between the FMV promoter and the 35S termination signal of pUC118-FMV-Poly-2-1 (Figure 3A, B and C).

Figure 1 Amplification of the AZI-1, PR-1 and CsNDR-1 genes. 1.5% agarose gels with 5 μl of PCR product (lanes 1-2 and 4-5) plus 3 μl of load buffer. M; molecular weight marker (1 kb DNA ladder). A). Lane 1: amplification of the Ara bidopsis AZI-1 gene. Lane 2: negative control of the AZI-1 gene. Lane 4: amplification of the Arabidopsis PR-1 gene. Lane 5: negative control of the PR-1 gene. B). Lane 1: CsNDR-1 negative control. Lanes 2 and 3: amplification of the CsNDR-1 gene. 

The genes isolated in this study (CsNDR-1, AZI-1, PR-1) have been consistently described as possible candidates for generating resistance to several pathogens (^{Lu et al., 2013}). These genes are part of the 633 genes that are altered after inoculation with CLas, according to ^{Mafra et al. (2013}), and are associated with the plant’s systemic acquired resistance (SAR). Several authors have also identified these genes as part of the plant’s response to CLas and CTV attacks (^{Hu et al., 2017}; ^{Rawat et al., 2017}; ^{Da Graca et al.,2016}; ^{Fu et al., 2016}), and, for this reason, they were selected to develop the constructions.

Figure 2 PCR confirmation of DH5α colonies transformed with the AZI-1, PR-1 and CsNDR-1 genes contained in the pUC118-FMV-Poly-2-1 plasmid. Amplified fragments in 0.8% agarose gel. M: molecular weight marker (1 kb DNA ladder). A). Lane 1: Positive control of the AZI-1 gene. Lanes 2, 4 and 10: Amplification of the expected gene. Lane 19: negative control of the AZI-1 gene. B). lane 1: positive control of the PR-1 gene. Lanes 2, 9, 11, 12, 13 and 16: positive colonies from the amplification of the PR-1 gene. Lane 19: negative control of the PR-1 gene. C). Lanes 1-15: CsNDR-1 gene colonies analyzed. Lane 12: positive sample. Lane 16: negative control. 

The pUC118-FMV-Poly-2-1 plasmid was selected because of its small size, and because it produces a large number of copies (500-700). In addition, its multiple cloning site facilitates the integration of DNA fragments within this vector (Febres et al., 2013), and it is also compatible for subsequent cloning within the pCAMBIA vectors incorporating the CaMV35S and/or NOS promoters and terminators of pCAMBIA.

Figure 3 Schematic representation of the constructions of the genes inserted into the pUC118-FMV-Poly-2-1 plasmid. A). AZI-1, B). PR-1, and C). CsNDR-1. The image shows the AZI-1 (red), PR-1 (yellow) and CsNDR-1 (green) genes cloned under the FMV promoter (orange) and their 35S termination signal (blue). The upper part of each construction indicates the size of the amplicon and the restriction enzymes used during cloning. 

Cloning in pCAMBIA plasmids. The constructions containing the genes of interest were inserted into vectors pCAMBIA 2201 and 2301. The cloning process was repeated twice, modifying DNA concentrations in the ligation reaction because no transformed colonies with the genes of interest were found. Twenty-one colonies were analyzed in each experiment, for a total of 42 colonies analyzed for each gene. For the AZI-1 gene cloned in pCAMBIA 2201, the concentration of DNA-plasmid was modified in the ligation reaction from 31.8 ng µL^-1-108 ng µL^-1 to 63.6 ng µL^-1-72 ng µL^-1; the grown colonies were analyzed by PCR using the AZI-1 F/AZI-1 R primers, and 10 positive colonies were obtained from the second cloning replication (Figure 4A). The AZI-1 gene cloned in pCAMBIA 2301, the DNA concentration in the ligation reaction was from 41.1 ng µL^-1-100 ng µL^-1 to 82.2 ng µL^-1-86.4 ng µL^-1; the grown colonies were also analyzed by PCR using the AZI-1 F/AZI-1 R primers, and four positive colonies were obtained from the second experiment (Figure 4B). For the PR-1 gene cloned in the pCAMBIA 2301 vector, the DNA concentration in the ligation reaction was modified from 25.2 ng µL^-1-86.4 ng µL^-1 to 37.8 ng µL^-1-72 ng µL^-1, and two hours were added to the incubation step after the electric shock; the grown colonies were analyzed using the PR-1 F/PR-1 R primers, and three positive colonies were obtained in the last experiment (Figure 4C). The constructions amplified a product of 486 pb, as expected. For the CsNDR-1 gene in pCAMBIA, a single test was carried out; 32 colonies were analyzed and only two positive colonies were obtained (Figure 4D).

All the constructions containing the AZI-1, PR-1 and CsNDR-1 genes under the FMV promoter and the 35S terminator were cloned between the neomicin fosfotransferasaII (ntptII) selection gene that confers resistance to kanamycin and the β-glucoronidasa (uidA) reporter gene, known as GUS, of the pCAMBIA 2201 and 2301 plasmids. The nptII transformation selection gene remained with the 35S promoter and terminator, and the GUS reporter gene remained with the 35S promoter and the NOS terminator. The AZI-1, PR-1 and CsNDR-1 genes cloned in pCAMBIA 2301 have the nptII gene as the bacterium selection gene (Figure 5A, C and D). The AZI-1 gene inserted in pCAMBIA 2201 is located under the bacterium selection gene to chloramphenicol (Figure 5 B).

Figure 4 PCR confirmation of DH5α colonies with the AZI-1, PR-1 and CsNDR-1 genes inserted into the pCAMBIA 2201 and 2301 plasmids. Amplified fragments in 0.8% agarose gel. M: molecular weight marker (1 kb DNA ladder). A). AZI-1-pCAMBIA 2201. Lane 1: Positive control of the AZI-1 gene. Lanes 4, 6, 7, 8, 11, 12, 14, 16-21: amplification of the expected gene. Lane 23: negative control. B). AZI-1-pCAMBIA 2301. Lane 1: positive control. Lanes 3, 18, 21 and 22: amplification of the expected gene. Lane 23: negative control. C). PR-1-pCAMBIA 2301. Lane 1: positive control of the PR-1 gene. Lanes 6, 7 and 20: positive colonies from the amplification of the PR-1 gene. Lane 23: negative control. D). CsNDR-1 in pCAMBIA 2301. Lanes 20 and 22: positive colonies for the CsNDR-1 gene. Lane 24: negative control. 

The constructions were sent out for sequencing in order to corroborate the identity and orientation of the insertion before being transformed to A. tumefaciens; the ones that were at least 99% homologous were selected. Figure 6 shows 100% identity of colony two in the case of the AZI-1 gene inserted in pCAMBIA2301. The pCAMBIA plasmids are successfully used in the genetic transformation of several plants, including citrus (^{De Oliveira et al., 2015}; ^{Hajeri et al., 2014}; ^{Pinheiro et al., 2014}).

Transformation of A. tumefaciens. Two A. tumefaciens strains were transformed using the three genes cloned in pCAMBIA and then individual colonies were analyzed through PCR to confirm that they have been transformed. The construction of the PR-1 gene in pCAMBIA2301 in the EHA105 strain developed several individual colonies (Figure 7A), as did the construction of the CsNDR-1 gene in pCAMBIA2301 in the Ag11 strain (Figure 7B). The PCR results confirmed that there were positive colonies for both genes, but the verification through sequencing did not show a high level of identity in the case of the construction with the PR-1 gene. The construction of the CsNDR-1 gene with the FMV promoter and the 35S terminator cloned in pCAMBIA 2301 was successfully inserted into the two A. tumefaciens EHA 105 and AGL 1 strains. Eleven colonies were randomly analyzed through PCR using the CsNDR-1 F/CsNDR-1 R primers, and one positive colony was obtained in the EHA105 strain (Figure 8A), while four positive colonies were obtained for the AGL1 strain (Figure 8B). The low transformation efficiency of Agrobacterium and E. coli is somewhat common in gene construction and cloning experiments. Gelvin (2003) mentioned that many factors may be involved, such as the low competence of the bacterial cells, stressed cells, poor quality DNA, low or high DNA concentration during transformation, the conditions during fragment ligation, the conditions during electroporation, the incubation temperature, and the concentration of antibiotics, among others. The A. tumefaciens EHA 105 strain has been used in citrus genetic transformation and has produced excellent transformation percentages because of its virulence (^{Hao et al., 2016}; ^{Dutt et al., 2015}). The AgL1 strain has also shown a high level of transformation in citrus (^{Shi et al., 2016}; ^{Kayim and Koc 2005}).

Figure 5 Schematic representation of the construction in pCAMBIA 2201 and 2301. FMV-AZI-1-35S construction inserted into the plasmids A). pCAMBIA 2301, and B). pCAMBIA 2201. C). FMV-PR-1-35S construction, and D). FMV-CsNDR-1-35S construction inserted into the pCAMBIA 2301 plasmid. The genes of interest (red, yellow and green bars) were inserted between the ntptII selection gene that confers resistance to kanamycin with the CaMV (Cauliflower mosaic caulimovirus) promoter and terminator, and the GUS reporter gene under the 35S promoter of CaMV and the nopaline synthase (NOS) terminator. 

Figure 6 Analysis of the AZI-1 gene cloned in pCAMBIA2301 conducted in the GenBank. The Query sequence is the Ara bidopsis thaliana reported gene and the Sbjct sequence corresponds to the cloned sequence with 100% similarity obtained in this study. 

Conclusions

The AZI-1, PR-1 and CsNDR-1 genes were isolated through PCR using the designed primers using the Arabidopsis thaliana and Citrus sinensis genome that had specific restriction sites for cloning. The isolated and cloned sequences of the AZI-1, PR-1 and CsNDR-1 genes showed 99% similarity compared to the records of the A. thaliana and C. sinensis sequences in the NCBI GenBank database.

Figure 7 A). Transformed colonies of A. tumefaciens EHA 105 with the pCAMBIA 2301 plasmid with the PR-1 gene in YEP culture medium with Rif (50 μl/ml). B). Transformed colonies of A. tumefaciens Agl1 with the pCAMBIA 2301 plasmid with the CsNDR-1 gene in YEP culture medium with Rif (50 μl/ml), Kan (50 μg/ml) and Carb (25 μg/ml). The numbers indicate the colonies that were randomly selected to confirm the presence of the gene using PCR. 

Figure 8 PCR of A. tumefaciens colonies. M: Molecular marker (1 kb DNA ladder). A). EHA 105 transformed with the CsN DR-1 gene. Lane 1: positive control. Lane 8: positive colony with the CsNDR-1 gene. Lane 12: negative control. B). AGL 1 transformed with the CsNDR-1 gene. Lane 1: CsNDR-1 positive control. Lanes 2-5: positive colonies with the CsNDR-1 gene. Lane 6: negative control. 

The three genes (AZI-1, PR-1 and CsNDR-1) were incorporated between the FMV promoter and the 35S CaMV terminator of the pUC118-FMV-poly 2-1 cloning vector. Cloning the genes in the pCAMBIA2301 and pCAMBIA2201 vectors made it possible to incorporate a transformation selection gene (35S-nptII-35S) and one reporter gene (35S-GUS-NOS) into the constructions. The three genes were then independently cloned into pCAMBIA2301 while gen AZI-1 was cloned only into pCAMBIA2201.

The CsNDR-1 gene was incorporated into the A. tumefaciens EHA 105 and AgL1 strains. Genes AZI-1 and PR-1 could not be integrated into any of the Agrobacterium strains. Identifying, isolating and cloning the CsNDR-1, AZI-1 and PR-1 genes involved in the plants defense mechanisms against a pathogen’s attack may be over-expressed through genetic engineering in citrus and used to induce resistance to several pathogens, including the Candidatus liberibacter bacterium, the causal agent of HLB and Citrus tristeza virus.