Study of the interactions between Broad bean wilt virus 1 and its host plants

Broad bean wilt virus 1 (BBWV-1) is the type member of the Fabavirus genus, in the Secoviridae family. BBWV-1 is worldwide distributed and infects important horticultural and ornamental crops causing considerable economic losses. However, information about the biological and molecular characteristics of BBWV-1 isolates is scarce. BBWV-1 is composed of two molecules of positive single-stranded RNA (ssRNA+) that are separately encapsidated in virions of icosahedral morphology. Each ssRNA+ encodes polyproteins which are processed by proteolytic cleavage into functional peptides. RNA1 (~ 5.8 kb) encodes for one polyprotein that renders proteins involved in viral replication: a protease cofactor (PRO-CO), an helicase (HEL), a viral genome-linked protein (VPg), a protease (PRO) and an RNA-dependent RNA polymerase (POL). RNA2 (~ 3.4 kb) has two “in frame” successive AUG initiation codons and encodes for two polyproteins (2a and 2b). Both polyproteins render identical large and small coat proteins (LCPs and SCPs, respectively), and putative movement proteins of different sizes that share the same C-terminal amino acid sequence. The longest putative movement protein, named MP, is encoded by polyprotein 2a, whereas the smallest putative movement protein, named VP37, is encoded by polyprotein 2b and lacks the first 90 amino acids present in MP. To improve the knowledge about BBWV-1, this doctoral thesis was addressed to study the biological properties of different BBWV-1 isolates and the implication of BBWV-1 RNA2-encoded proteins in the plant defence mechanisms and symptom development. For this purpose, four BBWV-1 viral isolates (Ben, PV0548, B41/99 and NSRV) obtained from different countries and clustered in different phylogenetic groups were inoculated in several herbaceous plant species. Each BBWV-1 isolate induced different symptoms on broad bean (Vicia faba) and pepper (Capsicum annum) whereas no differences of induced symptomatology were observed on Nicotiana benthamiana plants. Moreover, tomato (Solanum lycopersicum) plants, infected with all four BBWV-1 isolates, remained asymptomatic. No differences of infectivity between BBWV-1 isolates were found in broad bean, pepper, tomato nor in N. benthamiana plants. None of these BBWV-1 isolates did infect common bean (Phaseolus vulgaris), cucumber (Cucumis sativus) and melon (Cucumis melo) plants, although these herbaceous species had been previously reported as BBWV-1 or BBWV-2 hosts. RNA accumulation over time of BBWV-1 isolates Ben, B41/99, NSRV and PV0548 on broad bean and pepper plants was not correlated with symptom severity. To study the functions of BBWV-1 RNA2 encoded proteins, first a BBWV-1 full-length cDNA infectious clone (pBBWV1-Wt) was generated. Then, a mutant construct lacking the VP37 RNA2-encoded protein (pBBWV1-G492C) was engineered. Agroinfiltration assays in different plant species showed that pBBWV1-Wt had the same biological properties than the original BBWV-1 isolate infecting broad bean, tomato, pepper and N. benthamiana plants whereas pBBWV1-G492C was unable to infect pepper and tomato plants. Moreover, pBBWV1-G492C induced milder symptoms than pBBWV1-Wt in broad bean and N. benthamiana plants. These results suggest that BBWV-1 VP37 protein is related with host specificity and plant symptom development but is not involved in viral multiplication and viral particle formation. Transient expression of BBWV-1 VP37 protein, using a Potato virus X (PVX) viral vector, confirmed the VP37 protein as a pathogenicity determinant since it enhanced PVX-related symptoms and induced systemic necrosis associated with programmed cell death (PCD) in N. benthamiana plants. Moreover, VP37 protein was characterized as a post-transcriptional gene silencing (PTGS) suppressor by two different assays: transient expression in transgenic N. benthamiana 16c plants expressing constitutively the green fluorescent protein (GFP) and movement complementation of a viral construct based on Turnip crinkle virus (pTCV-GFP). The same studies were performed on the other RNA2-encoded proteins and showed that BBWV-1 MP and SCP proteins had PTGS suppressor activity. Transient expression of BBWV-1 SCP protein using the PVX viral vector revealed that it was a determinant of pathogenicity, enhancing the PVX-related symptoms and inducing the PCD-associated systemic necrosis in N. benthamiana plants.