Characterization of Mi1.2 Whitefly (Bemisia tabaci) Resistance Gene

Corresponding Author: Sherin Amin Mahfouze National Research Centre, Genetic Engineering and Biotechnology Division, Genetics and Cytology Department, Dokki, 12622, Egypt E-mail : sherinmahfouze@yahoo.com Abstract: Tomato (Solanum peruvianum) Mi gene provides resistance to whitefly (Bemisia tabaci), potato aphids and nematode making Mi a useful source in integrated pest management program. The aim of this work was to isolate, clone and sequence Mi1.2 gene from S. peruvianum. In addition, physico-chemical identification of amino acids deduced from Mi1.2 gene was done. Secondary (2D) and tertiary (3D) structures of Mi1,2 protein were also predicated. Distinct amplicons of 620, 600, 3300 and 1993 bp were successfully amplified using PCR amplification. The full-length DNA (5.4 kbp) and cDNA (4 kbp) of Mi1.2 gene was isolated using specific primers. Fragments 620 and 600 bp cloned into Escherichia coli XL-1 Blue and sequenced. Sequencing results of both assembled fragments (620 and 600 bp) joined at the overlap region (1440 bp). A BLAST search confirmed that the DNA sequence from the amplified fragments was Mi1.2 gene. It shared 98% identity and deduced amino acids shared 97% identity with Mi1.2 gene published in GenBank. An Open reading frame (ORF) of Mi1,2 protein encoded for 479 amino acid residues with molecular weight 54.59 KDa and isoelectric point (PI) 5.52 was calculated using Expasy’s ProtParam server. 2D and 3D structures of Mi1.2 protein was analyzed using SOPMA and SwissProt software, respectively.


Introduction
Tomato (Solanum lycopersicum L.) is an important vegetable crop and it is produced worldwide under both the glasshouse and open field (Kaur et al., 2014;McDaniel et al., 2016;Alatar et al., 2017). Whitefly (Bemisia tabaci) of Aleyrodidae family (Order: Hemiptera), is among the most harmful insects of tomato and causes significant yield loss. It effects on harvest directly by phloem feeding and indirectly imputable to the plant viruses transmission via their saliva such as Tomato Yellow Leaf Curl Virus (TYLCV) (Momotaz et al., 2010;Chen et al., 2015). A family of Mi genes arising from wild tomato (Solanum peruvianum L.) confers resistance against several of pests such as whiteflies (Bemisia tabaci), potato aphids (Macrosiphum euphorbiae) and root-knot nematodes (Meloidogyne sp.) (Nombela et al., 2003;Pallipparambil et al., 2014).
The Mi genes have three homologues, viz., Mi 1.1 , Mi 1.2 and Mi 1.3 . Only Mi 1.2 provides whitefly, aphid and nematode resistance in tomato (Seah et al., 2004). Mi 1.2 gene produces a transcript of approximately 4 kbp that encodes a putative protein of 1,257 amino acid residues (Rossi et al., 1998). The protein is identified by the presence of a nucleotide binding site (NBS) and a leucine-rich repeat motif (LRR) . Proteins of the NBS-LRR motif structure composed of the largest category of cloned plant resistance genes against viruses, fungi, bacteria, insects and nematodes (Dangl and Jones, 2001). Mi 1.2 is a potential gene in tomato integrated pest management program (Nombela et al., 2003;Mahfouze et al., 2015).
The aim of this work was to isolate, clone and sequence Mi 1.2 gene from S. peruvianum. In addition, physico-chemical identification of amino acids deduced from Mi 1.2 gene was done. Secondary (2D) and tertiary (3D) structures of Mi 1.2 protein were also predicated.

Plant Materials
Young leaves of tomato (Solanum peruvianum) plants were obtained from Indian Agricultural Research Institute (IARI), New Delhi, India. All the collected plant material were kept at -80°C for storage.

Design of Oligonucleotide Primers for Mi 1.2 Gene
A total of four primers with different degrees of specificity were designed according to the public sequence of Mi 1.2 gene (GenBank accession number AF039682.1) using SMS Sequence Manipulation Suite (http://bioinformatics.org/sms2/index.html).TomMi3 and TomMi4 primers containing chimeric regions complementary to one another. These chimeric overlapping sequences, which amplified Mi 1.2 gene were mixed and annealed at the overlaps. Primers used in this study were designed with various factors in consideration: GC content, melting temperature for primer set, formation of hairpin loops and dimerization of oligos. Various oligonucleotides used in this study are listed in Table 1. Primers were synthesized by Sigma Aldrich Chemicals, Bangalore, India.

Reverse Transcriptase (RT-PCR) and Isolation of Full Length of cDNA
Total RNA was extracted from tomato leaves using Trizol reagent according to the manufacture's recommended protocol (Sigma, India). cDNA was synthesized from total RNA (1 µg/sample) using Reverse transcriptase kit and oligo-DT primers (Thermo Scientific, UK) in 10 µL reaction at room temperature for 15 min and then heated at 65°C for 10 min to inactivate DNaseI. The DNaseI-treated total RNA was then reverse-transcribed using the RT-PCR kit (Thermo Scientific, Uk). Mi 1.2 cDNA was PCR amplified using F.P. TomMi3and R.P TomMi4 primers. The β-actin gene-specific primers (Table 1) were added to the same RT-PCR reactions as internal standards for RNA quantity. PCR reactions of Mi 1.2 amplification contained sterile distilled water 36.4, 10 µL 10× PCR buffer HF (Promega Corp.), 1.0 µL dNTPs (10 mM), the two primer combinations (F.P. TomMi3and R.P TomMi4) (0.3 µL each) (0.5 µM), 0.5 µL Phusion Taq DNA polymerase (Thermo Secientific, UK) (0.02 Units/µL). 1.5 µL of DNA template (~400 ng) was added to the reaction. PCR cycles were 98°C for 3 min; 35 cycles of: 98°C for 30s, 64.3°C for 30s, 72°C for 3 min; 72°C for 10 min. Similar PCR conditions were used for the β actin primers, with the exception that the annealing temperature was 55°C.
PCR products for all samples was electrophoresed on 0.8% agarose containing ethidium bromide (0.5 µg mL −1 ) in 1x TBE buffer (89 mM Tris-HCl, 89 mM Boric acid, 2.5 mM EDTA, pH 8.3) at 75 constant volt and determined with UV transilluminator. The size of each fragment was determined with reference to a size marker of 1 kbp DNA ladder (Thermo Scientific, UK).

Cloning of the Mi 1.2 Gene
Fragments of the expected size, 620 and 600 bp for Mi 1.2 gene was excised from the agarose gels and further purified using Gene Jet Gel DNA purification kit (Thermo Scientific, UK). The quality and concentration of the purified products was confirmed by gelelectrophoresis in a 0.8% agarose gel in 1xTBE buffer and by measuring the absorbance ratio at 260 nm wavelength using a NanoDrop ND-1000 spectrophotometer. The purified PCR products were ligated into pGEM®-TEasy vector (Promega, Mannheim, Germany). Ligation reactions were prepared containing the appropriate quantities of vector and insert (1:3), 1 µL of the 2 × ligase buffer and 2.5 U T4 DNA ligase supplied with the kit. The reaction volume was made up to 10 µL with sterile dsH 2 O and the reactions were incubated at 4°C overnight. Ligated plasmids were transformed into E. coli XL-1 Blue competent cells. Isolation of plasmid DNA from E. coli XL-1 Blue was done by the alkaline lysis method according to Sambrook et al. (1989).

Digestion of Plasmid DNA with Restriction Enzyame EcoR1
To confirm the presence of positive intact clones, restriction enzyme digestion of plasmid DNA was also carried out with EcoR1 at 37°C overnight.

Sequencing
Partial nucleotide sequence of Mi 1.2 gene was done by Applied Biosystems (Inst model/Name 3100/3130XL-1468-009, India using gene-specific primers. The sequence was aligned with corresponding sequences from the database using BLAST from the website http://www.ncbi.nlm.nih.gov/blast. Multiple alignments and phylogenetic tree of protein were performed using CLC Main Workbench 5 program, Denamark.

2D and 3D Structures of Mi 1.2 Protein
The primary amino acid sequence of Mi 1.2 protein was subjected to predict its secondary and tertiary structures using SPOMA (Geourjon and Deleage, 1995) and a SWISS-MODEL workspace servers (http://swissmodel.expasy.org/workspace), respectively (Arnold et al., 2006).

Isolation of Full Length Genomic DNA and cDNA of Target Gene
The size of Mi 1.2 gene using the specific primers the F.P. TomMi3 and R.P. TomMi4 was 5.4 kb (Fig. 2). In the present study, the total RNA was isolated using TRIzol reagent. The RNA profile on 0.8% agarose gel indicated the intactness of different subunits of RNA. cDNA was synthesized by RT-PCR using oligodT as 5' and 3' primers. The cDNA product of around 4 kb is shown in Fig. 3.

Cloning of Mi 1.2 Gene Fragments
The fragments of the expected sizes 620 and 600 bp were excised from agarose gels and the DNA products were cleaned up by a Gene Jet Gel DNA purification Kit (Thermal Scientific, UK). The quality and concentration of the purified products was confirmed by gelelectrophoresis in a 0.8% agarose gel by measuring the absorbance. The purified PCR products were ligated into pGEM®-T-Easy vector (Promega, Mannheim, Germany). To screen positive colonies, four or five white colonies were picked from pGEM: Mi 1,2 construct, Mini-preparations were performed with all colonies. To determine the insert orientation within pGEM-T-Easy vector was performed by digestion of EcoR1 ( Fig. 4 and 5) and sequencing. Fig. 4 and 5 indicated that the transformation with pGEM: Mi 1.2 was successful and plasmid with correct insert orientation.

Multiple Sequence Alignments and Phylogenetic Tree
Sequencing results of both assembled fragments (620 and 600 bp) joined at the overlap region (1440 bp) (Fig. 6). The sequence was submitted at the GenBank with the accession number KU886265. BLAST analysis showed that the Mi 1.2 gene under study had the identity ranged 98-82% to the root-knot nematode resistance Mi 1.2 genes recorded in GenBank (Table 2). On the other hand, the deduced amino acids sequence of Mi 1.2 protein gave the homology 97-61% (Table 3). The amino acids sequence of Mi 1.2 protein was aligned with six different accessions of other Mi proteins published in GenBank by CLC Main Workbench 5 program, Denamark (Fig. 7). The phylogenetic tree applied by using CLC Main Workbench 5 program, Denamark with the UPGMA method is presented in Fig. 8. A close relationship was found between our Mi 1.2 protein and other NBS-LRR proteins (Fig. 8).

3D Structure Modeling of Mi 1.2 Protein
The three-dimensional (3D) structure protein was carried out by using SWISS-MODEL workspace server. The predicated structure of Mi 1.2 under study was similar to Solanum lycaopersicum root-knot nematode resistance protein (NP_001234063.1). Both had monomers, consisted of α-helix and β-sheets with a compact structure, as shown in Fig. 10.   Histidine (

Discussion
The whitefly is an important insect pest of many crop plants, including tomato. Many wild tomato species contains Mi gene, which provides resistance to whitefly (McDaniel et al., 2016). The wild tomato (S. peruvianum) resistance gene Mi encodes a protein with CC-NBS-LRR motifs . Mi 1.2 is a single dominant gene in tomato, which provides resistance against certain phloem-feeding herbivores such as whiteflies, aphids, psyllids and root-knot nematodes (Nombela et al., 2003;Pallipparambil et al., 2014;Chen et al., 2015). Schaff et al. (2007) mentioned that Mi gene provides resistance of tomato, glycosyltransferase and extension may play a main role in the cell wall synthesis, which is a fundamental defence against root knot nematode. The NBS-LRR class of R genes could be sub-divided into two main groups depend on existence of domains identical to the Toll and interleukin-1 receptor or coiled-coil (CC) domain at the amino terminal (Bhattarai et al., 2007). In this study, we designed four pairs of primers for the amplification of Mi 1.2 gene from GenBank accession number AF039682.1. Primers TomMi1, TomMi2, TomMi3 and Tom Mi4 amplified 620, 600, 3300 and 1993 bp DNA fragments, respectively. Moreover, primers IMOF1 and IMOR1 amplified 998 bp (Bendezu, 2004). We isolated full length DNA and cDNA of Mi 1.2 gene from S. peruvianum tomato leaves using the primers F.P. TomMi3 and R.P. TomMi4. Sequencing results of both assembled fragments (620 bp and 600 bp) joined at the overlap region confirmed the Mi 1.2 gene sequence.
BLAST analysis showed that the Mi 1.2 gene under study (1440 bp) was homologous to tomato root-knot nematode resistance genes in the GenBank. Nucleotide sequence of Mi 1.2 gene under study was encoded 479 amino acids with molecular weight 54.59 KDa and PI was 5.52, which showed that Mi 1,2 protein was acidic. The PI is significant in protein purification because it represents the pH where solubility is typically minimal. Here, the protein isoelectric point signifies where mobility in an electro-focusing system is zero and in turn, the point where the protein will aggregate (Geourjon and Deleage, 1995). Chen et al., (2006) used the specific primers AM-FW1 and AM-RV1 for the isolation of full length DNA of Mi 1.2 resistance gene of 5.4 kb. They have cloned Mi 1.2 gene into the pDONR201 vector. Recombinant plasmid pDMi was confirmed by digestion by ApaI and NruI restriction enzymes and by sequencing. The results indicated that the amplicon 5367 bp was long. Also, observed that the DNA fragment had two introns, contained on an Open reading frame (ORF) of 3774 bp encoding 1257 amino acids. The BLAST results found that the predicted ORF of Mi 1.2 gene had 99% identity with tomato root-knot nematode resistance gene Mi (AF039682) recorded in GenBank, which is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.
In the present study, we predicated the secondary and tertiary structures of Mi 1,2 protein using SPOMA and SWISS-MODEL workspace servers, respectively (Geourjon and Deleage, 1995;Arnold et al., 2006). The results showed that Mi 1.2 protein composed of 232 αhelix (48.4%), 77 β-sheets (16.1%), 39 Beta-turn (8.14%) and 131 coil (27.4%). The 3D model of the Mi 1.2 is described as consisting of an alpha helix and several beta pleated sheets with a compact structure. It is similar to the 3D crystal structure of S. Lycopersicum root-knot nematode resistance protein (NP_001234063.1). This is the first report describing Mi 1,2 protein isolated from S. peruvianum. In this research, we provided information on the threedimensional structure (3D) of Mi 1.2 protein that associate directly to the corresponding R resistance proteins of the NBS-LRR class. The determination of the crystal structure of Mi proteins will help us to understand the protein-protein interactions between the R protein of the tomato and the Avr protein of the whitefly as confirmed in 'gene for gene' model. In addition, it provides us to comprehend the main role of an intermediary protein complex, which has been visualized in 'guard theory' of plant disease resistance (Chisholm et al., 2006;Chattopadhyaya and Pal, 2008). Dorna et al., (2014) obtained that the threedimensional protein structure (3D) by protein crystallography (X-ray) provides to examine folds and motifs in the proteins, molecular folding, phylogenetic and structure/function relationships. One of the principle research troubles in proteomics is the prediction of the tertiary structures (3D). Proteins are long residues composed of 20 different amino acid sequences that in physiological conditions assume an alone 3-D structure. Information on the protein structure provides the study of biological operations with detail. The sequence-protein-structure paradigm (the "lock-and-key" theory) tells that the protein can perform its biological operation only by folding in to a singular, structured shape estimated by its amino acid residue (Anfinsen, 1973). Presently, it has been known that not all protein operations are linked to a folded shape (Tompa and Csermely, 2004;Dunker et al., 2008). Some proteins are unfolded or disordered to achieve their functions (Gunasekaran et al., 2003). These proteins are known Intrinsically Disordered Proteins (IDP) and act about 30% of the protein sequences.

Conclusion
Up to now, there have been no researches on the secondary (2D) and tertiary (3D) structures of Mi 1.2 protein. This is the first report describing Mi 1.2 protein isolated from S. peruvianum. The prediction of (3D) modeling is among the research troubles in structural bioinformatics. The 3D structure of a protein that has no templates in the Protein Data Bank (PDB) is a very difficult. A Knowing of the 3D structure of the Mi 1.2 protein provides very important data on its the biological function in the plant cell.

Ethics
This research paper is original and contains unpublished data. The corresponding author confirms that all of other authors have read and accepted the manuscript.