Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

Abstract

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

A unigene catalogue of 5700 expressed genes in cassava

“…The domestication of cassava is a relatively recent event and could explain the limited genetic differences observed between cassava cultivars (Sanchez et al, 1999). This also explains the difficulties in detecting polymorphism in cassava and, consequently, for mapping studies (Fregene, personal communication;Lopez et al, 2003). Nevertheless, we detected significant differences in the in silico expression level of a group of 12 genes that should be confirmed by experimental studies.…”

“…The identification of quantitative trait loci (QTL) associated with resistance to several strains of Xam has contributed to a better understanding of the genetic mechanisms of CBB disease resistance (Jorge et al, 2000(Jorge et al, , 2001. The recent isolation of resistance gene candidates (RGCs) in cassava provided new markers that can be used in marker-assisted selection and high resolution genetic mapping for map-based cloning of resistance genes (Lopez et al, 2003). Nevertheless, it is necessary to characterize other genes that are involved in the downstream signaling cascades in plant defense responses for this crop.…”

Ice recrystallization inhibition proteins of perennial ryegrass enhance freezing tolerance

“…The LRR is also present in many other plant proteins, including the large LRR receptor kinase protein family (LRR-RK), and disease resistance gene proteins which are characterized by the presence of a nucleotide-binding site (NBS) near the N-terminal and a variable number of LRR domains (approximately 10-40) at the C-terminus (Lopez et al 2003). The LRR plays an important role in protein-protein interaction (Diévart and Clark 2004).…”

Development and application of transgenic technologies in cassava

“…It is recognized that progress in the era of post-genomics is resulting in identification and cloning of genes of agronomic interest from model species such as A. thaliana and from cassava itself. Progress towards the latter at CIAT, Colombia, will most likely yield sequences of interest from M. esculenta for resistance to CBB (Lopez et al, 2003; Verdier, this issue), CMD (Akano et al, 2002) and in the longer term to whitefly (Bellotti and Arias, 2001) and post-harvest deterioration (Reilly et al, this issue). By screening for homology, it is also hoped to access genes coding for beneficial traits from cassava's wild relatives.…”