Investigation of the resistance and susceptible responses of tomato (Solanum lycopersicum) to tomato yellow leaf curl virus, the invasion biology of tomato begomoviruses in Costa Rica and characterization of two weed-infecting begomoviruses in the Caribbean Basin

The research in this thesis addressed aspects of the tomato-begomovirus interaction: molecular analyses of resistance and susceptible determinants, invasion biology in Costa Rica and the role of weeds as reservoirs. In Chapter II, I used an RNA-Seq approach to identify differentially expressed genes (DEGs) during infection by tomato yellow leaf curl virus (TYLCV) of the near isogenic lines LA3473-R and LA3474-S, which have the Ty-1 (resistant) and the ty-1 (susceptible) alleles, respectively. Differences in gene expression in the resistance and susceptible responses were observed. VIGS analyses of selected DEGs further revealed that genes encoding WRKY transcription factors, nucleotide-binding site and leucine-rich repeat (NBS-LRR) containing proteins, and receptor-like protein kinases (RLKs) may play a role in the resistance and susceptible responses to TYLCV infection. Sequence analyses of the cloned Ty-1 gene provided further insight into how some of the newly identified DEGs participate in the Ty-1 defense response. In Chapter III, I examined the invasion biology of three emergent/introduced begomoviruses in Costa Rica to assess their interactions in terms of disease severity and viral accumulation and genetics. Here, I first completed the molecular and biological characterization of tomato yellow mottle virus (ToYMoV) using full-length infectious DNA-A and DNA-B clones to fulfill Koch’s postulates for the ToYMoD and to show that the virus infects a subset of solanaceous species. Full-length infectious clones of an isolate of tomato leaf curl Sinaloa virus (ToLCSiV) from Costa Rica also were generated, and were used to assess genetic interaction (pseudorecombination) with ToYMoV. The infectious clones of ToYMoV, ToLCSiV and TYLCV were then used to inoculate tomato plants with each virus and all combinations, and symptoms induced and viral DNA accumulation were determined. Tomato plants infected with combinations of two or all three viruses developed more severe symptoms than plants inoculated with each virus alone. Symptoms of ToLCSiV and ToYMoV appeared earlier (~7 d post-inoculation [dpi]) than those of TYLCV (~10 dpi). However, by 14 dpi and beyond, TYLCV symptoms had become dominant in all mixed infections. Virus accumulation was determined by qPCR and revealed that, although symptoms of TYCLV became predominant in mixed infections with the NW bipartite begomoviruses, levels of ToYMoV and ToLCSiV were similar to those in plants infected with the individual viruses. ToYMoV and ToLCSiV DNA levels were negatively impacted early in infection (7 dpi), but later in infection (14 and 21 dpi) a neutral synergistic interaction was observed. These results are discussed in terms of begomovirus invasion biology and implications for begomovirus disease development and management in Costa Rica. In Chapter IV, I describe the molecular and biological properties of two bipartite begomoviruses associated with striking golden/yellow mosaic symptoms in Malachra sp. and Abutilon sp. plants on Hispaniola, and assess their potential to cause disease in crop plants. Sequence and phylogenetic analyses, together with infectivity studies with infectious clones, established that the symptoms in Malachra sp. were caused by the crop-infecting bipartite begomovirus tobacco leaf curl Cuba virus (TbLCuCV), whereas those in Abutilon sp. were caused by a new species of weed-infecting begomovirus, for which the name Abutilon golden yellow mosaic virus (AbGYMV) is proposed. Host range experiments showed that TbLCuCV also induced moderate to severe disease symptoms in Nicotiana benthamiana, tobacco (N. tabacum) and common bean plants (Phaseolus vulgaris) plants. In contrast, AbGYMV induced mild or no symptoms in these plants, indicating a high degree of adaptation to Abutilon sp. in the Dominican Republic, and low potential to cause crop diseases. Pseudorecombinants generated with the infectious clones of these viruses were highly infectious and induced severe symptoms in N. benthamiana and Malachra sp. Furthermore, both viruses co-infected Malachra sp., thereby increasing the probability of virus evolution via recombination and pseudorecombination. Together, these results suggest that TbLCuCV primarily infects Malachra sp. in the Caribbean Basin, and occasionally spills over to infect and cause disease in crops other than tomato, whereas AbGYMV is well-adapted to Abutilon sp. and has not been reported infecting crops