Banana (Musa spp.) is one of the most important fruit crops in the world in terms of production and consumption. Fusarium wilt is regarded as one of the most devastating diseases of banana, affecting plantations in almost all banana-growing countries of the world (Ploetz et al., 1990). This disease is caused by the soil-borne fungus Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) (Stover, 1962). The fungus, surviving as chlamydospores, will germinate to infect the lateral or feeder roots when they come into contact with banana roots (Beckman, 1990). After infection, the pathogen will colonize and block the plant's vascular system, a process that leads to wilting, and eventually, plant mortality (Ploetz and Pegg, 2000). In recent years, Race 4 of this fungous pathogen (Foc4) has become the most virulent race of this disease. It can infect almost all the banana and plantain cultivars, including those that were resistant to other races of the disease (Ploetz, 1993).

The most common cultivar in worldwide commercial production is the Cavendish cultivar, which has resistance to some isolates of FOC (Pegg et al., 1996). Commercially grown banana plants, which are clonally propagated, sterile triploid plants, are highly susceptible to Fusarium wilt due to many factors (Pegg et al., 1996; Vuylsteke, 2000). Although plant micropropagation leads to the reduction in the spread of FOC, it also results in enhanced susceptibility to FOC for two years after plantlets are removed from tissue culture (Smith et al., 1998). Options for the control of Fusarium wilt are limited by ineffectual chemical control and the lack of commercially suitable resistant cultivars (Smith et al., 2006). And no known fungicide is effective in controlling FOC4 up to now. Hence, the development of new banana cultivars resistant to this disease, FOC4 in particular, holds the key to the successful control of this disastrous disease in banana production.

Unfortunately, cultivated banana varieties are mostly triploid and can only be propagated asexually, making it difficult to improve this crop genetically using conventional plant breeding methods that rely heavily on cross-pollination between plants. As an alternative, the introduction of resistance genes into banana plants via biotechnological means offers a valuable way of developing resistant banana cultivars (Sagi, 2000). To date, however, no R gene/s capable of conferring resistance to FOC4 has been reported. One potential source of Fusarium R gene/s is the ‘Goldfinger’ (AAAB) banana breed in Honduras showing strong resistance to diseases, especially to race 1 and race 4 of Fusarium oxysporum f.sp. cubense (Foc) (Pegg et al., 1996). This kind of banana, therefore, potentially represents an important source of Fusarium R genes for molecular breeding.

The largest class of R genes encodes proteins with a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain. The NBS-LRR genes are unevenly distributed between chromosomes, several being clustered as local multigene families (Meyers et al., 1999). The NBS-LRR class can be divided in two subclasses, the TIR and the non-TIR, depending on the presence of a domain at the N-terminus with homology to the Drosophila Toll and mammalian Interleukin-1 receptors (TIR) (Meyers et al., 1999). Non-TIR-NBS-LRR genes are present in both monocot and dicot plants, whereas TIR-NBS-LRR genes appear to be restricted to dicot plants (Meyers et al., 2003; Zhou et al., 2004).

According to the conservative regions of the nucleotide-binding site and the leucine-rich repeat  (NBS- LRR) in cloned wilt resistance genes, the polymerase chain reaction with degenerate primers was employed to isolate resistance  gene analogues (RGAs) from the genomics DNA of wilt resistance germplasm ‘Goldfinger’ (AAAB) banana. Twenty fragments of resistance  gene analogues (RGAs) were isolated, which were of expected size (about 530 bp). The availability of these sequences opens the possibility of applying different strategies for their functional analysis and for developing disease resistance in this crop.

1 Results
1.1 RGCs of the NBS-type from banana
Twenty fragments of resistance gene analogues were isolated from ‘Goldfinger’, which were of expected sizes (about 530 bp). Analysis of their deduced amino acid sequences revealed the presence of the typical NBS motifs of R genes in the spatially correct locations, leading to the conclusion that all the 20 RGAs, designated GF1~GF20, were resistance related gene analogs associated with fusarium wilt in banana. Sequence identity among the 20 RGAs ranged from 41.1% to 99.3%, while identity of their deduced amino acid sequences ranged from 33.2% to 96.3%. The comparison between the deduced amino acids sequence of banana NBS/LRR resistance gene analogues in GenBank (EU123872) and those of GF3, GF7, GF11 and GF18 showed a sequence identity of 93%, 87%, 93% and 88%, respectively, while the remaining 16 banana NBS resistance gene analogues in this study shared 38%~65% amino acids homology with other R genes. The results indicate that banana NBS resistance genes belong to multigene families.

1.2 Sequence comparisons of banana RGCs with other R genes
Analysis of the deduced amino acids of these RGAs showed that their domain structures were NB-ARC and belonged to non TIR-NBS-class resistance gene candidates, containing 4 conservative amino acid domains of P-loop (GMGGVGKTT), Kinase-2 (LVLDDIW), RNBS-B (CKVLFTTRS) and hydrophobic amino acids (GLPLALKVL) (figure 1). The N-terminal regions of the banana RGCs showed no sequence similarity to the TIR domain, suggesting the RGCs of this species belong to the non-TIR-NBS-LRR type like other monocot R gene and RGC sequences. Similarity searches of the protein databases also revealed that each RGC showed significant similarity to RGCs isolated from other monocots such as Oryza sativa, Saccharum offcinarum and Avena sativa as well as some known non-TIR-NBS-LRR resistance genes. The 20 RGAs showed approximately 28%~54% sequence identity to Fom-2, I2C-1, I2C-2, and I2 which confers resistance to Fusarium in Cucumis and Lycopersicon.
 

Figure 1 Alignment of the deduced amino acid sequences of the NBS regions of 20 RGAs of ‘Goldfinger’ NBS sequences and selected non-TIR-NBS-LRR R genes known to confer disease resistance

1.3 Phylogenetic relationships of the banana RGCs
The phylogenetic analysis for RGA nucleotide sequences and the deduced amino acids revealed that the 20 sequences could be divided into 5 distinct types (figure 2). All of The amino acids deduced from the RGAs shared homology (28%~54%) with those deduced from the known wilt resistance genes such as Fom-2, I2C-1, I2C-2, and I2 which indicate the conservation of disease resistance gene evolution. Technically, these RGAs isolated in the present study would lay a base for the further cloning of wilt resistance genes in banana. And they could also be used as molecular marker for screening of candidate wilt resistance genes in banana.

Figure 2 Phylogenetic tree of Musa RGAs and know plant R genes based on NBS domain

2 Discussion
Resistance (R) genes in plants play a crucial role in disease prevention. Many R genes are dominant, or incompletely dominant, and require specific dominant avirulence (Avr) genes in the pathogen for their function (Flor, 1946). This genetic interaction between plant and pathogen led to the current view that such R genes encode receptors for Avr gene-dependent pathogen molecules (Staskawicz et al., 1995). Upon recognition of these molecules, R gene products activate plant defense mechanisms. These defenses include rapid production of an oxidative burst resulting in cell wall cross-linking, localized cell death (the hypersensitive response), salicylic acid biosynthesis, and induction of genes characteristic of systemic acquired resistance (Levine et al., 1994; Ward et al., 1991). R genes have been shown to encode two broad categories of leucine-rich-repeat (LRR) proteins that can be distinguished by protein domain structure and site of pathogen perception (Jones and Takemoto, 2004). The NBS-LRR-containing R proteins mediate the recognition of an intracellular pathogen-derived signal. Thus far, NBS-LRR proteins have been shown to function in resistance signaling only in response to pathogen. The second category of R proteins is inserted in the plasma membrane and minimally consists of an extracellular LRR domain and a transmembrane (TM) domain (Jones and Takemoto, 2004).

Many studies suggested that there were three main relationships between RGA and resistance genes. First, RGA is part of R gene or pseudo gene. In Arobidopsis, a sort of RGA was tightly linked with Rpp5 and was proved to be part of Rpp5 (Kanazin et al, 1996). Second, RGA was tightly linked with R gene. For instance, RGA were found to be tightly linked with different located R genes in rice (Nori et al., 2001). Third, RGA is unrelated to target gene. For example, some RGA did not link with located R-genes in lettuce (Di Gaspero et al., 2002).

Several features of the banana putative RGCs isolated in this study suggest they are non-TIR-NBS-LRR disease resistance genes. For example, the characteristic motifs of the NBS domain of known resistance genes described (Meyers et al., 1999; Pan et al., 2000;Peraza-Echeverria et al., 2008) are present in each banana RGC at similar positions. One of these motifs, the highly conserved P-loop, has been shown to bind ATP in the NBS-LRR resistance proteins I2 and Mi from tomato (Tameling et al., 2002), suggesting the banana RGC proteins may also bind ATP. The non-TIR (nT) motif (Bai et al., 2002), which is associated only with the non-TIR subclass of NBS sequences, is found in the N-terminal region of the banana RGCs, while none of the motifs associated with the TIR subclass are found in the corresponding region of the banana RGCs. All of the amino acids deduced from the RGAs shared homology (28%~54%) with those from the known wilt resistance genes such as Fom-2, I2C-1, I2C-2 and I2. Among them, the homology between GF7 and I2 are relatively high (about 54%), which indicated that it might be a possible function of resistance to Fusarium wilt of banana.

3 Materials and methods
3.1 Plant material
'Goldfinger’ (Musa spp., AAAB) plantlets resistant to FOC subtropical race 4 (Smith et al., 1998) were obtained from South Subtropical Crop Research Institute, Chinese Academy of Tropical Agricultural Science, and were maintained in a phytotron at 28℃. Roots were harvested from 4-month-old plants, frozen in liquid nitrogen and then stored at -80℃ for use.

3.2 DNA extraction
DNA was extracted from plant tissue using a modified CTAB method based on Doyle and Doyle (1987). Fresh root tissues (2~3g) were ground to powder and transferred to the extraction buffer [2% (w/v) CTAB (cetyltrimethyl-ammonium bromide), 1.4 mol/L NaCl, 20 mmol/L EDTA, 100 mmol/L Tris-HCl (pH 8.0) and 2% (v/v) β-mercaptoethanol]. The suspension was extracted twice with equal volume of chloroform:isoamyl alcohol (24:1). Dried pellet was dissolved in an appropriate volume of double distilled sterile water.

3.3 Degenerate Primers Design
According to the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in cloned wilt resistance genes, five pairs of primers were designed to isolate R gene analogues (RGAs) from the genomic DNA of wilt resistance germplasm ‘Goldfinger’ (AAAB) banana (table 1).

Table1 Degenerate primers used in amplification Note: Codes for mixed bases: R=A/G, W=A/T, M=A/C, K=G/T, Y=C/T, S=C/G, H=A/T/C, D=A/T/G, V=A/C/G, B=C/G/T, N=A/T/G/C, I=hypoxanthine

3.4 PCR and cloning
PCR mixtures (50 μL) contained 5 μL 10×buffer (TaKaRa with MgCl₂), 4 μL 2.5 mmol/L dNTP, 1 μL of each degenerate primer, 5 U Taq polymerase (TaKaRa), 1 μL DNA, and then added ddH₂O to 50μL. Cycling conditions were performed as follows: Hot start; initial denaturation at 95℃ for 3 min, then followed by 35 cycles each with denaturation at 94℃ for 45s, anneal at 52℃ for 30s , and elongation at 72℃ for 60s, with a final elongation step of 72℃ for 10min. Gel-purified amplicons were cloned into the plasmid pGEM-T Easy vector (Promega) and transformed into Escherichia coli JM109 competent cells following the manufacturer's instructions. Plasmids were extracted using the High Pure Plasmid Isolation Kit (TaKaRa).

3.5 Sequence analysis
All the cloned transformants were sent to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. Then the sequences were assembled and edited using the DNAMAN 5.2.9 Demo version software. Similarity searches were performed with the BLAST program (www.ncbi.nlm.nih.gov) using the default settings. Predicted protein sequences were aligned using the ClustalX 1.81 Program. A phylogenetic tree was constructed by the neighbor-joining method implemented in the Molecular Evolutionary Genetics Analysis (MEGA) software with the Poisson correction.

Acknowledgement
This research was supported by the National Public Welfare Scientific Research Institution Foundation (SSCRI200714, SSCRI200804) and the Hainan Provincial Natural Science Foundation (No.807039)

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