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Molecular Pathogens
MP2010, Vol.1, No.1
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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.