Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

The elderly health status and its correlation with ageing biomarkers: the European Project Mark-Age

According to the latest statistics projections formulated by Eurostat, the proportion of elderly EU-27’s population aged over 65 years old is predicted to increase from 17.5 % in 2011 to 29.5 % by 2060. This "population explosion" makes extremely important to identify the different genetic and molecular mechanisms which underpin the morbidity and mortality along with new strategies able to counteract or slow down its progress. In this scenario fits the European Project MARK-AGE whose aim was to identify a robust set of biomarkers of human ageing able to discriminate between chronological and biological ageing and to derive a model for healthy ageing through the analysis of three populations from different European countries, supposed to be characterized by different ageing rate: 1. Subjects representing the “Normal” or “Physiological” aging. 2. Subjects representing the “successful” or “decelerate” aging 3. Subjects representing the “accelerated” aging. The aim of this work was to recruit and characterize volunteers, to perform an accurate analysis of the health status of elderly recruited subjects (60-79 years) verifying any possible dissimilarity in their aging trajectories, to identify a set of robust ageing biomarkers and investigate possible correlations between ageing biomarkers and health status of recruited volunteers. The model proposed by MARK-AGE Project regarding different ageing trajectories has been confirmed and several ageing biomarkers have been identified.

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.