Journal Article•
Molecular cloning, characterization and transcriptional variability study of resistance gene candidates from wild Curcuma spp. for resistance against Pythium aphanidermatum.
TL;DR: A PCR mediated approach has been made using degenerate primers designed on conserved region (P-loop and GLPL) of the NBS domain from R-genes that provides the source for cloning analogous sequences called resistance gene candidates (RGCs) from three wild turmeric plants resistant or susceptible to Pythium aphanidermatum.
Abstract: The genetic base of preferred turmeric (Curcuma longa L.) genotypes has eroded due to their continuous domestication through exclusive vegetative routes thus making them highly susceptible to various pests and pathogens. Molecular cloning of resistance related sequences from wild genotypes can result in efficient turmeric improvement by evolving more effective resistance specificities compared to cultigens. In this study, a PCR mediated approach has been made using degenerate primers designed on conserved region (P-loop and GLPL) of the NBS domain from R-genes that provides the source for cloning analogous sequences called resistance gene candidates (RGCs) from three wild turmeric- Curcuma aromatica, Curcuma angustifolia and Curcuma zedoaria. Twenty-one wild turmeric RGCs were isolated and grouped into four phenetic classes. A strong amino acid identity ranging from 41 to 53% together with presence of internal conserved motifs provided evidence that the isolated RGCs belong to the non-toll interleukin receptor (non-TIR) NBS-LRR R-gene sub-family. Southern hybridization showed a high copy representation of turmeric RGCs. Expression variability of wild turmeric RGCs was analyzed through reverse transcription PCR in root tissues of the three wild turmeric plants resistant or susceptible to Pythium aphanidermatum. Cap12 and Can12 showed a constitutive expression in both resistant and susceptible plants of Curcuma aromatica and Curcuma angustifolia respectively while Czp11 expression was realized only in Pythium aphanidermatum resistant lines of Curcuma zedoaria as well as Curcuma longa L. This result can pave way towards the identification and characterization of a potential Pythium aphanidermatum resistance gene in turmeric.
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TL;DR: Structural and phylogenetic analyses grouped CzR1 within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS-LRR R-genes and semi quantitative RT-PCR analysis showed constitutive expression of CZR1 which gets significantly upregulated in response to infection by different strains of P. aphanidermatum.
18 citations
TL;DR: In this article , degenerate primers were used for cloning and characterization of NBS-LRR type resistant gene from betelvine against anthracnose disease, and a strong amino acid identity ranging from 90.38 to 97.75% and the presence of internal conserved motifs, provided evidence that the identified resistance gene analogs (RGAs) belong to the non-TIR NBS LRR class of R genes.
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TL;DR: The usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean is shown, with genetic linkage shown for eight RGAs with partial resistance to anthracnose, angular leaf spot and Bean golden yellow mosaic virus.
Abstract: A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with re...
115 citations
"Molecular cloning, characterization..." refers background in this paper
...The co-amplification of non-specific DNA fragments from the degenerate primers is a common feature and has been absorbed in various other species (Di Gaspero and Cipriani, 2002; Lopez et al., 2003)....
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TL;DR: The results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa.
Abstract: Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F2 lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa.
109 citations
"Molecular cloning, characterization..." refers methods in this paper
...This strategy have been successfully employed in many plant species for the isolation of homologous resistance sequences by heterologous amplification (Leister et al., 1996; Kanazin et al., 1996; Mago et al., 1999; Xiao et al., 2006; Nair and Thomas, 2007)....
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TL;DR: It is shown that in resistant plants, fungal growth into this region of the vascular tissue is prevented, suggesting a correlation with the I-2-mediated resistance response.
Abstract: Summary
The tomato resistance gene I-2 is one of at least six members of a gene family that are expressed at low levels in the roots, stems and leaves of young tomato plants. Plants transformed with constructs containing a functional I-2 promoter fused to the β-glucuronidase (GUS) reporter gene were used in detailed expression studies. Highest GUS activity was found in stems of young tomato plants. Histochemical analysis revealed that the I-2 promoter drives expression of the reporter gene in vascular tissue of fruits, leaves, stems and mature roots. In younger roots, expression was most abundant at the base of lateral root primordia. Microscopical analysis of young tomato plants revealed expression in tissue surrounding the xylem vessels. We show that in resistant plants, fungal growth into this region of the vascular tissue is prevented, suggesting a correlation with the I-2-mediated resistance response.
104 citations
"Molecular cloning, characterization..." refers result in this paper
...Northern analysis of various known NBS-LRR R-genes also exhibit similar results (Parker et al., 1997; Milligan et al., 1998; Mes et al., 2000)....
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TL;DR: The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process.
Abstract: The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.
99 citations
"Molecular cloning, characterization..." refers background in this paper
...…sequences are considered as RGCs if they exhibit high sequence identities to known R-genes/other RGCs, show the presence of conserved NBS-LRR domain found in R-genes and are represented by open reading frame (ORF) of more than 100 amino acids uninterrupted with stop codons (Noir et al., 2001)....
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...The cloned sequences are considered as RGCs if they exhibit high sequence identities to known R-genes/other RGCs, show the presence of conserved NBS-LRR domain found in R-genes and are represented by open reading frame (ORF) of more than 100 amino acids uninterrupted with stop codons (Noir et al., 2001)....
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TL;DR: Comparison with similar sequences from soybean revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.
Abstract: Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.
93 citations
"Molecular cloning, characterization..." refers background in this paper
...In previous studies, NBS sequences identified in other plant species also showed a comparable range of identities of Rgenes/RGCs such as 30-45% in Phaseolus vulgaris (Rivkin et al., 1999), 31-51% in grape (Di Gaspero and Cipriani, 2002), 27-50% ginger (Nair and Thomas, 2007) and 38 to 82% in Kaempferia galanga (Joshi et al....
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...…NBS sequences identified in other plant species also showed a comparable range of identities of Rgenes/RGCs such as 30-45% in Phaseolus vulgaris (Rivkin et al., 1999), 31-51% in grape (Di Gaspero and Cipriani, 2002), 27-50% ginger (Nair and Thomas, 2007) and 38 to 82% in Kaempferia galanga…...
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