Evolución de los begomovirus del rizado amarillo del tomate en presencia del alelo de tolerancia Ty-1

This thesis has aimed at the evolution of begomoviruses associated to tomato yellow leaf curl disease (TYLCD) in some of their hosts. TYLCD is caused by a complex of viral species belonging to the genus Begomovirus (family Gemiviridae) that creates huge losses in tomato in many countries where their insect vector is found, the whitefly Bemisia tabaci. The most exploited genetic resistance for controlling TYLCD is the one provided by Ty-1 tolerance gene derived from Solanum chilense. Due to the extensive use of this source of resistance, the present study emphasises the evolution of those begomoviruses bearing that gene. In a first step, the evolution of quasispecies of the most relevant begomoviral species in Spain has been studied [including tomato yellow leaf curl virus (TYLCV), tomato leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl Málaga virus (TYLCMaV)] after the inoculation of their infectious clones onto two agricultural species (tomato with or without the Ty-1 gene and common bean) and a wild reservoir weed (Solanum nigrum). The results showed that Ty-1 allele in tomato diminished the fitness of TYLCSV and TYLCV (and allowed a steady accumulation of TYLCMaV), compared to the levels reached in common bean and S. nigrum. The genetic complexity and heterogeneity of the mutant spectra from Rep, C4, IR and CP regions comparable to the one shown by RNA viruses but those were higher in common bean and S. nigrum. The adaptation to the different hosts, taken as changes in fitness, was achieved without variation of the consensus sequence, despite of the high complexity and heterogeinity of the viral quasispecies in each host. Secondly, the evolution of viral quasispecies in long-term co-infections with TYLCV and TYLCSV in tomato with or without the Ty-1 allele have been analysed. The viral progeny at 421 dpi showed a very high frequency of recombinant, many often with several breakpoints. Only in the absence of Ty-1 allele, mutated and recombinant defective genomes have been found, showing deletions, repeats, inversions and translocations with regard to the parental viral genomes. Another objective of the present study was to locate the regions determining the differential biological behaviour found among different TYLCD-like isolates infecting tomato plants bearing the Ty-1 allele. Some infectious clones of chimeric and mutant viruses were inoculated for this task. The results indicated that the different accumulation rate detected between TYLCSV and TYLCV infecting tolerant tomato plants lies along the Rep, C4 and IR regions, probably due to the silencing suppression activity elicited by the Ty-1 allele. TYLCV-IL and the derived chimeric constructs between that and TYLCV-Mld induced mild yellowing in tolerant tomato plants, but we have not been able to locate the corresponding virulence-associated determinants. The C4-deficient mutant TYLCV-Mld showed a significant accumulation rate than the wild type in tomato plants bearing the Ty-1 allele, which supports the role of C4 protein as a pathogenicity factor. Finally, the genetic diversity of begomovirus was artificially increased by two strategies in order to determine if a resistance breaking genetic variant could emerge. The first strategy consisted in the treatment of infected tomato plants with 5-fluorouracil, a mutagenic base analogue. The results showed a very slight increased mutation frequency (attributed to a strong purifying selection in this host) and a very high toxicity in tomato plants. However, through error-prone RCA (epRCA), a mutant library of begomovirus with very high mutation frequency was obtained. After the inoculation of the mutant library in tomato plants and several graft passages, the great diversity reduced to a small group of polymorphic mutations in the consensus sequence of the viral progeny. No case with resistance overcoming was detected at all. This great durability of the Ty-1 allele-associated tolerance may be due to a stronger purifying selection in the tolerant host than the adaptive selection in these specific conditions.