Isolation and characterization of Resistance Gene Analogue (RGA) from Fusarium resistant banana cultivars

Isolation and characterization of resistance genes from local banana cultivars was important in order to support the development of FOC resistant banana cultivars. Resistance gene analogues (RGAs) were isolated and characterized form three fusarium resistant banana cultivars using degenerate primers based on NBS domains. From 91 fragments sequenced, 17 fragments were positively NBS-type sequences and encoded as MNBS1MNBS17. Phylogenetic analysis of MNBS deduced amino classified into three groups; the first group consisted of 14 members (MNBS1-MNBS14) with 97.4% identity, and the other three groups consisted of one member (MNBS15, MNBS16 and MNBS17, respectively) with 28.5% identity. All MNBS sequences were categorized as non-TIR-NBS-LRR. Comparison and phylogenetic analysis of MNBS with other known RGA and R genes showed that deduced amino acid MNBSs shared 91.7-98.8% identity with Musa NBS-LRR and 19.9-35.5% identity with known R genes. Among them, MNBS17 shared 50.5% identity with RGC2 (ABY75802) that assosiated to FOC race 4 resistant Musa species.


Introduction
Banana and plantain are important fruit crops in the world, but they face the complexity of pests and diseases.One of the major fungal disease is Panama disease caused by Fusarium oxysporum f. sp. cubense (FOC).This disease is one of the devastating fungal diseases in banana.It is difficult to control because the innoculum is easily spreads in the field and persists in the soil for over 20 years without losing the virulence (Agrios, 2005).More importantly, the majority of commercial banana cultivars cultivated in the world such as Cavendish and plantain subgroups are reported suceptible to this pathogen (Ploetz, 2006).
Fusarium wilt control in banana plantation by using cultivation practices such as chemical control, soil treatments, crop rotation and organic amendments could reduce the severity of the disease.However, its commercial application is limited (Pegg et al., 1993).The use of resistant cultivars is the best alternative for controlling FOC and conventional banana breeding for FOC resistance has been actively reported (Rowe and Rosales, 1993;Tomekpe et al., 2004;Sebuliba et al., 2006).Technically, it faces the problems of flower sterility and ploidy of commercial cultivars (Roux et al., 2004).Breeding program through genetic engineering is a promising technique, but little information is available on the genetic basis of R gene on banana for resistance to FOC TR4 (Li et al., 2012).However, transgenic banana cultivars have been reported by number of researchers around the world.Paul et al. (2011) reported transformed apoptosis-inhibition-related genes in 'Lady Finger' banana.Based on the evaluation under glasshouse conditions, transformed banana plants showed resistant to FOC race 1. Mohandas et al. (2013) used antimicrobial peptide (Ace-AMP1) gene to confer resistance to FOC race 1 on 'Rasthali'.The evaluation was carried out in the screenhouse.
The conserved NBS-LRR contains at least three domains, such as the variable N-terminal domain, the nucleotide binding site (NBS), and the Cterminal LRR motif.The NBS domain consists of p-loop/kinase-1, kinase-2, kinase-3a and hydrophobic (GLPL) and highly conserved in diverse organisms (Traut, 1994).Based on the Nterminal motifs, the NBS-LRR class of R genes is grouped into two distinct subclasses.The subclass I TIR-NBS-LRR, showed homology to the Drosophila Toll and mammalian Interleukin-1 receptor (TIR) proteins at their N-terminal region (Meyers et al., 1999).The subclass I is restricted to dicot and gymnosperm species (Tarr and Alexander, 2009).The subclass II non-TIR-NBS-LRR, exhibited a coiled-coil (CC) motif at the Nterminal region, instead of the corresponding TIR.This type R gene is widely distributed in both monocot and dicot species (McHale et al., 2006).
Based on NBS-LRR domain from known R genes, degenerate primers have been designed and used to isolate resistance gene analoque (RGA) from various plants such as soybean (Kanazin et.al., 1996), lettuce (Shen et al.,1998), lens (Yaish et al., 2004), wild apple (Baek and Choi, 2013) using PCR approach.The same approach was used to isolate RGAs from banana cultivars and wild species by Pei et al. (2007), Peraza-Echeverria et al. (2008), Azhar andHeslop-Harrison (2008), Sun et al. (2009), and they have isolated and characterized 12 RGAs from China's banana, 5 RGAs from segregated line of M. acuminata ssp.malaccensis, 135 RGA from cultivars and wild species, 5 RGAs from 'Gold Finger', respectively.This present study was based on the PCR amplification and characterization of NBS-LRR class of RGA from three banana cultivars.Evaluation of their nucleotide diversity and phylogenetic relationship with known R genes from other plants were also carried out.

Plant materials
Three banana cultivars were used as sources of genomic DNA.They were Indonesian banana cultivars (Klutuk Wulung [BB] and Rejang [AA]) and introduced from ITC collection (Calcuta-4 [AA]).All selected cultivars were shown to be resistant to FOC tropical race-4 at the previous studies (Molina et al., 2009;Sutanto et al., 2012).The leaves were collected from one-month-old acclimatized planlets obtained from in vitro micropropagation culture.
The suspension was extracted twice with equal volume of chloroform:isoamyl alcohol (24:1).The supernatant was precipitated with two volume of pre chilled (-20ºC) 95% ethanol and sodium acetate (0.3 M).The pellet was washed twice with 70 % ethanol.Dried pellet was dissolved in an appropriate volume of double distilled sterile water.

Degenerate primers and PCR conditions
Four primer combinations of degererate primers designed based on NBS-LRR containing Ploop/kinase-1, kinase-2, kinase-3a and hydrophobic domains were used to amplify genomic fragments of NBS-LRR from banana.The primer combinations tested were: Information on the denerate primers used were presented in Table 1.PCR amplification was carried out in a 25 µL total volume containing 1 µL of genomic DNA, 5.0 µL of 5X KAPA2G buffer, 0.5 µL of 25 mM MgCl 2 , 0.5 µL of 10 mM dNTP mix, 1.25 µL of each forward and reverse primers, 0.5 unit of KAPA2G fast Taq polymerase, and double distilled water.PCR amplification was performed in a thermal cycler (Perkin Elmer) with an initial denaturing step of 95°C for 3 min, followed by 35 cycles of 95°C for 10 sec, 55°C for 10 sec and 72°C for 3 sec, and a final extension at 72°C for 10 min.PCR products were electrophoresed on a 1.5% TBE agarose gel at 80 V for 25 min, stained with ethidium bromide and visualized on a UV transilluminator.

Cloning PCR product and sequencing
Amplification products with clear and distinct band were purified using Gel/PCR DNA Fragments Extraction Kit (Geneaid, Taiwan).Purified products were ligated into the pGEM-T Easy Vector Systems (Promega) overnight and then transformed into DH-5α competent cells by the 42°C heat shock method.Potential transformants were selected on an LB plate containing ampicillin (50 μg/ml), X-gal and IPTG.The cultures were incubated at 37°C overnight.Single white colony was picked and inoculated into liquid LB medium containing ampicillin and grown overnight cultivation with vigorous shaking.Recombinant plasmids were extracted and purified using High-Speed Plasmid Mini Kit (Geneaid, Taiwan) and sent to 1 st BASE for sequencing.

Sequence BLAST, alignment and phylogenetic analysis
Vector sequences were removed manually from the raw sequence data of RGA clones.The clone sequences were compared with the sequences in the non-redundant database at NCBI GenBank using the BLAST. ClustalW (Larkin et al., 2007) was used to align the deduced amino acid sequences of the PCRderived genomic fragments of banana with the known R genes from Arabidopsis thaliana (RPP1 and RPP5), tomato (Prf and Mi-1,2), peper (Bs2), potato (Gpa2), wheat (Yr10), flax (L6 and M), and Nicotiana tabbacum (N).The alignments were graphically displayed using GeneDoc (Nicholas et al., 1997).A neighbour-joining phylogenetic tree, drawn with MEGA 5.0 (Tamura et al., 2011) was subsequently generated to reveal relationship between the compared sequences.Bootstrap analysis was employed with 1000 replications.

Result PCR amplification of targeted RGA
Primer combinations F9(F)+F6(R) produced one distinct clear band with amplification product size of ~550 bp in all three cultivars (Rejang, Calcuta-4 and Klutuk Wulung), while the combination of F9(F)+F10(R) produced two bands, with the product size ranged from ~550 bp to ~1100 bp (Figure 1).Product with the size of ~550 bp was the expected DNA fragment size of the Ploop until GLPL motifs of NBS domain.However, two primer combinations F5(F)+F6(R) and F5(F)+F10(R) produced no amplification product.PCR product obtained from the combination of F9(F)+F6(R) was used for further analysis.

Sequence analysis of banana RGAs
Out of 95 clones sequenced, 91 clones produced readable sequencing results at 68-959 bp in length.However, only 16 sequences with uninterupted open reading frame (ORF) typical of NBS were further analyzed.Those fragments, MNBS1-MNBS16 with the ranged size of 523-628 bp, contained P-loop, kinase-2, kinase-3a and hydrophobic domain (GLPL).The remaining 75 sequences were BLAST analyzed to the Musa genome (http://banana-genome.cirad.fr/)and revealed three remaining sequences with coverage above 60% and sequence identity above 95% were showed in Table 2.One clone D6_K14 with 98.44% homolgy to putative disease resistance protein RGA3.This sequence was truncated NBS with only has P-loop and kinase 2 motifs and encoded by MNBS17 for further analysis.Nucleotide sequence identity among 17 banana RGA range from 52.2% to 98.5%, while identity of their deduced amino acid range from 18.3% to 100%.

Diversity analysis of Musa RGAs.
Phylogenetic analysis of deduced amino acid showed that 17 RGAs resulted from this study were devided into four groups.Fragments with >97% identity of deduced amino acid were consider to be identical.Fourteen RGAs (MNBS1-MNBS14) were in the same group with 97.4% sequence identity and considered as group I.The MNBS15, MNBS16 and MNBS17 showed only 28.5% sequence identity and considered as separate groups (group II, III and IV, respectively) (Figure 2).

Multiple sequence alignment of Musa RGAs and known R genes
Based on multiple alignment of the deduced amino acid of Musa MNBS and other R genes revealed that the P-loop/kinase-1a (G[V/M/I] GKTT), kinase-2 ([L/V][L/I]DDV[W/D]), kinase-3a and hydrophobic domain (GLPL) motifs of the NBS domain were highly conserved among Musa MNBS sequences and the other known Musa NBS-LRR protein such as: Prf , Mi-1,2, Bs2, Gpa2, Yr10, RPP1, RPP5, L6, M, and N proteins (Figure 3).Among them RPP1, RPP5, L6, M, and N belong to TIR-NBS-LRR, while the others belong to non-TIR-NBS-LRR.The TIR-type and non-TIR-type of NBS-LRR proteins were distinguished by specific motif at the N-terminal region and amino acid residue at the end of kinase-2.For non-TIR-type the residue at the end of kinase-2 is tryptophan (W) and for TIR-type is aspartate (D) (Tarr and Alexander, 2009).Multiple alignment analysis of deduced amino acid sequences also generated genetic identity matrix as shown in Table 4.This matrix showed the identity of each accession to others.

Discussion
Plants have developed sophisticated mechanisms to recognize and protect againts pathogens.The interaction between hosts and pathogens elicits both localized and systemic responses (Meyers et al., 2003).Plant disease resistance genes encode R proteins that represent the first plant defense against infection of many pathogens.The pathogens recognized by the R protein are usually highly specialized for host plant specific (Friedman and Baker, 2007).
Disease resistance trait is frequently controlled by a specific recognition between plant disease resistance (R) and pathogen avirulen (Avr) genes (Jones, 2001).Successful recognition of Avr product of pathogen by R poduct of host plant triggers variour defense mechanisms which include the hypersensitif response (HR), and results in a compatible interaction and leading to resistance of the plant (Baker et al., 1993).
In this study, we have sequenced 94 clones derived from amplified PCR products.Sixteen sequences encoded by MNBS1-MNBS16 were typically RGA with uninterrupted ORFs and one sequence, MNBS17, was truncated RGA with only P-loop and kinase-2 motifs.The characteristic of motifs in the known NBS (Meyers et al., 2003) were also found in MNBS proteins.
Two highly conserved motifs were found in MNBS deduced amino acid sequence: kinase-1a and kinase-2.The kinase-1a (GG[V/M]GKTT) was also called P-loop or Walker A motif, formed a glycine (G)-rich flexible loop containing an invariant lysine (K) residue involved in binding the phosphates of the nucleotide (Walker et al., 1982).This motif were shown to bind ATP in NBS-LRR resistace protein I-2 and M1 from potato (Tameling et al., 2002), suggesting that the Musa MNBS protein may bind ATP.The kinase-2 or Walker B motif ([L/V][L/I]DDV[W/D]) has an invariant aspartate (D) that coordinate divalent metal ion (Mg 2+ ) required for phospho-transfer reactions (Traut, 1994).
The absence of TIR-NBS-LRR was not only occured in Musa, but also reported in other monocots such as: rice (Zhou et al., 2004), wheat (Zhang et al., 2011), maize (Xiao et al., 2006), barley (Madsen et al., 2003), sorghum (Cheng et al., 2010), and sugarcane (Que et al., 2009).The TIR-NBS-LRR RGAs have been eliminated from majority monocots over time during evolution, although no mechanism has been described how to explain the elimination process.Pan et al. (2000) proposed a model suggesting that NBS-LRR evolution had involved two stages.During stage 1, plant genom had a few NBS-LRR genes, while during stage 2, both the TIR and the non-TIR groups underwent amplification and diversification in the dicot species.According to sequence analysis done by Cannon et al. (2002), non-TIR-type NBS genes appeared to be much more diverse than TIRtype NBS, and it was therefore tempting to speculate that non-TIR-type NBS genes were more ancient than the TIR-type.However, recent study on NBS evolution done by Yue et al. (2012) showed that the NBS domains found in early land plants (Coleochaetales, liverworts, and bryophytes) were closer to the TIR-type NBS domain at sequence level and they concluded that TIR-type NBS genes had developed earlier than non-TIRtype.
Refer to genetic identity matrix shown in Tabel 3, all deduced amino acid of Musa MNBSs shared 91.7-98.8%identity with Musa NBS-LRR proteins deposited in GenBank database, and 19.9-35.5% identity with known R genes such as Bs2, Gpa2, Prf, Yr10 dan Mi-1.2, suggesting that Musa MNBSs might have the function of resistance gene.Among them, the identity bentween MNBS17 and RGC2(ABY75802) were relatively high (50.5%).RGC2 expression was found to be associated only to FOC race 4 resistance line of M. acuminata spp. malaccensis (Peraza-Echeverria et al., 2008).This indicated that it might be a possible function of resistance to fusarium wilt of banana.

Conclusion
We have isolated and characterized 17 RGAs from FOC resistance banana cultivars, which will add to the existing RGA information in the GenBank, and it would be potentially used for molecular markers for screening resistance genes from banana.Further studies on the expression of RGAs are required to find out if the RGAs play a role in FOC resistance or in other diseases.

Figure 2 .
Figure 2. Phylogenetic tree of deduced amino acid sequences of Musa RGAs based on the neighbor-joining method.The numbers on the branches indicate bootstrap values (1000 replications).Bootstrap (%) refers to the percentage of trees in which the members form a clade.Branches corresponding to Musa RGA families are labeled as I, II, III and IV. ..

Figure 3 .
Figure 3. Multiple alignments of deduced amino acid sequences of MNBSs with other proteins of different plant R genes and Musa NBS-LRR from GenBank DNA database.The location of conserved domain are shown above the sequence (C1: kinase 1a or P-loop, C2: kinase 2, C3: kinase 3a, and C4: hydropobic domain or GLPL).

Figure 4 .
Figure 4. Phylogenetic tree of the deduced amino acid sequences of Musa RGAs and other R genes based on multiple alignment using Clustal W and the tree was generated using neighbor-joining method.The numbers on the branches indicate bootstrap values (1000 replications).Bootstrap % refers to the percentage of trees in which the members form a clade.

Table 1 .
Degenerate primers used for PCR amplification of NBS-LRR fragments from banana genomic DNA.

Table 2 .
BLAST result of three remaining clones to the Musa genome (http://banana-genome.cirad.fr/)with above 60% of querry coverage and above 95% of sequence identity.

Table 3 .
Comparison of deduced amino acid sequences among MNBSs with Musa NBS-LRR deposited in the NCBI GenBank database.