Identification and characterization of a resistance gene analog (RGA) from the Caricaceae Dumort family

Abstract

The majority of cloned resistance (R) genes characterized so far contain a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain, where highly conserved motifs are found. Resistance genes analogs (RGAs) are genetic markers obtained by a PCR-based strategy using degenerated oligonucleotide primers drawn from these highly conserved "motifs". This strategy has the advantage of the high degree of structural and amino acid sequence conservation that is observed in R genes. The objective of the present study was to search for RGAs in Carica papaya L. and Vasconcellea cauliflora Jacq. A. DC. Out of three combinations of primers tested, only one resulted in amplification. The amplified product was cloned in pCR2.1TOPO and than sequenced using M13 forward and reverse primers. Forty-eight clones were sequenced from each species. The 96 sequences generated for each species were cleaned of vector sequences and clustered using CAP3 assembler. From the GENEBANK, one RGA was identified in C. papaya showing a BlastX e-value of 2x10-61 to the gb|AAP45165.1| putative disease resistant protein RGA3 (Solanum bulbocastanum). To the extent of our knowledge this is the first report of a RGA in the Caricaceae Dumort family. Preliminary structural studies were performed to further characterize this putative NBS-LRR type protein. Efforts to search for other RGAs in papaya should continue, mostly to provide basis for the development of transgenic papaya with resistance to diseases.

Genetic diversity analysis in a set of Caricaceae accessions using resistance gene analogues

“…Majority of the plant disease resistance proteins identified to date belong to a limited number of classes, of which those containing nucleotide-binding site (NBS) motifs are the most common. Amaral et al [ 12 ] used the primer combination P1b and RNBS-D [ 13 ] to amplify RGAs in Carica papaya transgenic variety Embrapa PTP18 and Vasconcellea cauliflora. Forty eight clones were sequenced from each of the two species and the only RGA that was identified was from Carica papaya transgenic variety Embrapa PTP18.…”

Relationship between Psidium species (Myrtaceae) by resistance gene analog markers: focus on nematode resistance

“…The NBS domain contains three strictly ordered motifs; a P-loop or kinase-1a, kinase-2, and kinase-3a, which are critical for the binding of various ATP/GTP proteins (Tameling et al, 2002). Conserved domains are also described within NBS and LRRs, and include the hydrophobic domains GLPLAL and RNBS, with the latter consisting of RNBS-A, -B, and -C motifs (Dinesh-Kumar et al, 2000;Tameling et al, 2002;Amaral et al, 2006). GLPLAL is a highly conserved domain found in NBS-LRR resistance genes (Lu et al, 2011), which has also been described as a transmembrane motif of the NBS domain (Baek and Choi, 2013).…”