Molecular cloning and characterization of cotton ubiquitin-conjugating enzyme and disease resistance genes involved in programmed cell death

Two cDNAs and their corresponding genes (GhUBC1 and 2) encoding ubiquitin-conjugating enzymes (E2s) have been cloned and characterized from allotetraploid cotton Gossypium hirsutum ((AD) ₁ genome). Three additional E2 genes (GaUBC1, GtUBC2, and GrUBC2) have also been identified from diploid cottons Gossypium arboreum (A₂ genome), Gossypium thurberi (D₁ genome), and Gossypium raimondii (D₅ genome), respectively. The derived amino acid sequences of the five closely related cotton E2s are 77–79% identical to yeast ScUBC4 and ScUBC5. The GhUBC1/2 gene family is composed of two members, and genomic origin analysis indicates that GhUBC1 and 2 are individually present in the A and D subgenomes of G. hirsutum. The transcript levels of GhUBC1/2 increased significantly in leaves and flowers at senescence, suggesting that GhUBC1/2 may play a role in the degradation of target proteins that function in the delay of the senescence program. Correlated with high auxin content and auxin-associated effects, GhUBC1/ 2 are also highly expressed in the youngest leaves, the apical part of lateral roots, and elongating fibers, suggesting that GhUBC1/2 may also function in the degradation of short-lived regulatory AUX/IAA proteins during auxin response. Genetic complementation experiments revealed that GhUBC1 and 2 can substitute for the function of ScUBC4 and 5 required for the selective degradation of abnormal and short-lived proteins in a yeast ubc4ubc5 double mutant. Northern analysis showed that GhUBC1/ 2 expression in immature root galls of susceptible cotton cultivars inoculated with root-knot nematodes (RKN) was down-regulated about two-fold in comparison to that in the non-inoculated roots, suggesting that the down-regulation of ubiquitin-conjugating enzyme genes GhUBC1/2 might be crucial for the formation of giant cells and for the establishment of a susceptible state.; Partial sequences of five disease resistance (R) genes (Gh1-2-1, Gh2-3-1, Gh2-3-2, Gh3-3-2, and Gh6-2-3) have been amplified by PCR from G. hirsutum. The complete sequences of the two I2-like genes, Gh2-3-1 and Gh2-3-1-m, have been subsequently isolated and characterized. Both Gh2-3-1 and Gh2-3-1-m encode R proteins that contain NBS and LRR motifs. A single base mutation occurs within Gh2-3-1-m , which generates a stop codon and interrupts the I2-like protein into two ORFs. Four RFLP markers, cCot5D2/EcoRI-1, cCot5D2/ EcoRI-2, cCot119A6/EcoRI, and cCot33B2/ EcoRI, have been mapped within a linkage group and closely linked to two QTLs for RKN resistance. The RFLP marker, cCot119A6/EcoRI, could be used as a molecular maker for RKN resistance in the cotton breeding program.